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Durene-capped porphyrin complexes of iron(II) and their interaction with imidazoles, isonitriles, CO,… David, Shantha K. 1985

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DURENE-CAPPED PORPHYRIN COMPLEXES OF IRON(II) AND THEIR INTERACTION WITH IMIDAZOLES, ISONITRILES, CO, AND 0 2 BY SHANTHA K. DAVID B.Sc, U n i v e r s i t y of Waikato, New Zealand, 1978 M.Sc, U n i v e r s i t y of Waikato, New Zealand, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED ST^IDARD THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER, 1985 © Shantha K. David, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date NOV/. Z<Z> /9gf DE-6(3/81) i i ABSTRACT Based on well developed procedures, durene-4/4 (la) and durene-7/7 (3a) capped porphyrins containing a t o t a l l y hydrophobic c a v i t y have been prepared: c y c l i z a t i o n of the chain-linked dipyrromethane dimer (4) allows for porphyrin formation despite d i s t o r t i o n of the porphyrin skeleton from p l a n a r i t y caused by the imposition of a t i g h t cap. The c r y s t a l structure of the F e * * * ( l b ) C l complex shows considerable d i s t o r t i o n of the porphyrin from p l a n a r i t y . The u v - v i s i b l e s p e c t r a l trends for the free base porphyrins indicate that some porphyrin d i s t o r t i o n e x i s t s also i n the durene-5/5 free base (2a), while the 7/7-derivative (3a) i s e s s e n t i a l l y f l a t . Proton nmr data suggest that the durene moiety i n the 7/7-base i s suspended c l o s e r to the porphyrin plane than i n the t i g h t e r capped-5/5 and -4/4 analogs. The heme de r i v a t i v e s , Fe^(durene-por), have been studied with respect to t h e i r i n t e r a c t i o n with imidazoles, isocyanides, CO, and The binding constants of 1,5-dicyclohexyl-(Dclm) and 1,2-dimethyl-(1,2-Me2lm)-imidazoles to the unhindered side of the four-coordinate hemes are s i m i l a r w i t h i n the durene seri e s despite differences i n the porphyrin plane d i s t o r t i o n ; f o r s t e r i c reasons, these imidazoles do not coordinate on the capped (or d i s t a l ) side of the heme. The s i z e of the d i s t a l pocket i n the five-coordinate durene-7/7 and -5/5 systems has been probed using the bulky isocyanides, tosylmethylisocyanide (TMIC) and t-butylisocyanide (t-BuNC), which d i f f e r i n t h e i r s p a c i a l requirements on binding to the i r o n . The extremely r e s t r i c t i v e d i s t a l environment of the durene-4/4 system, however, i n h i b i t s the coordination of e i t h e r i i i 4 a . , M = 2 H b_, M = F e isocyanide. The Fe**(durene-7/7)(Dclm) complex ex h i b i t s a reduced o v e r a l l a f f i n i t y f o r CO r e l a t i v e to simple f l a t , open hemes; t h i s i s manifested i n a depressed ass o c i a t i o n rate f o r CO, and i s interpreted as a d i s t a l s t e r i c e f f e c t as a r e s u l t of the durene-cap. The durene-5/5 and -A/A systems also show reduced CO a f f i n i t i e s compared to open hemes, but t h i s r e s u l t s predominantly from increased d i s s o c i a t i o n rates f o r CO from the six-coordinate Fe**(Por)(Dclm)(CO) complexes because of proximal s t e r i c s t r a i n induced by the porphyrin plane d i s t o r t i o n . The carbonyl s t r e t c h i n g frequencies f o r the Fe**(Por)(B)(CO) species (B = methylimidazole or 1,2-Me2lm) of the durene s e r i e s are discussed with respect to r e l a t i v e d e s t a b i l i z a t i o n of the Fe-C-0 moiety. The f i v e -coordinate Fe**(durene-por) (B) systems (B - Dcl-m or 1,2-Me2im) bind O2 r e v e r s i b l y to s i m i l a r extents, implying a n e g l i g i b l e e f f e c t of porphyrin s k e l e t a l d i s t o r t i o n . A 10-fold reduced a f f i n i t y f o r CO by the i v Fe (durene-4/4)(B) complex r e l a t i v e to durene-5/5 (or other less d i s t o r t e d hemes) i s therefore interpreted i n terms of proximal s t e r i c d i s c r i m i n a t i o n of CO r e l a t i v e to O2 within t h i s severely d i s t o r t e d system. Thermodynamic data for CO and O2 binding to the hindered Fe^(durene-4/4)(B) systems are discussed i n comparison to those for planar heme systems. S i g n i f i c a n t l y the five-coordinate durene heme complexes a l l show considerably higher K^/K ^ r a t i o s (M values) r e l a -t i v e to encumbered hemes that incorporate polar amide functions i n t h e i r d i s t a l environments. This i s e n t i r e l y consistent with the concept of e l e c t r o n i c i n t e r a c t i o n s within the d i s t a l binding pocket s t a b i l i z i n g the Fe-02 moiety and increasing the a f f i n i t y of the heme toward O2, r e l a t i v e to CO. The implications of such model . studies f o r binding to the re v e r s i b l e O2 and CO carrying hemoproteins are discussed. V TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables x i L i s t of Figures x i v L i s t of Abbreviations x^- x Acknowledgements x x i i Chapter 1 HEMOPROTEIN STRUCTURE AND FUNCTION 1 1.1 Porphyrin Macrocycle 5 1.2 Iron Spin State and Geometry 6 1.3 Hemoglobin Structure and Cooperativity 8 1.4 Myoglobin Structure 18 1.5 Other Reversible Oxygen Carrying Hemoproteins 19 1.6 Nature of the Bound Dioxygen 20 1.7 Carbon Monoxide Binding i n Hemoproteins 25 CHAPTER 2 MODEL SYSTEMS AND DYNAMICS OF CO AND 0 2 BINDING 27 2.1 Requirement f o r Model Systems 29 2.2 Problems Associated with Model Heme Studies 31 2.3 Early Model Studies 34 2.4 Model Design 34 v i 2.5 D i s t a l S t e r i c E f f e c t s 37 2.5.1 Hemoproteins 37 2.5.2 Anthracene and Adamantane Hemes Al 2.5.3 C o f a c i a l Diporphyrin and Strapped Model Hemes 46 2.5.4 Pyridine-5/5 Cyclophane 47 2.5.5 Picket-Fence and Pocket Systems 49 2.5.6 and C^-Capped Model Hemes 52 2.5.7 C r y s t a l Structure of a S t e r i c a l l y D i s t o r t e d Fe-C-0 Moiety 55 2.5.8 R e v e r s i b i l i t y and S t e r i c "Protection" i n Dioxygen Binding 59 2.6 Proximal S t e r i c E f f e c t s 62 2.6.1 K i n e t i c Expression of Cooperativity i n Hemoglobin 62 2.6.2 Nature of the Proximal Environment 64 2.6.3 CO and 0 2 Binding 65 2.6.4 K i n e t i c Consequences 68 2.6.5 Doming E f f e c t s 70 2.6.6 Base-Elimination Pathway 72 2.7 E l e c t r o n i c E f f e c t s 74 2.7.1 D i s t a l Environment 74 2.7.2 Proximal Ligand B a s i c i t y 77 2.7.3 Porphyrin Peripheral E f f e c t s 81 2.7.4 Solvent P o l a r i t y 82 2.8 Solvation E f f e c t s 84 2.9 Reason f o r Studying the Durene-Capped Hemes 86 v i i CHAPTER 3 THE DURENE-CAPPED PORPHYRINS:'SYNTHESIS AND SPECTRAL COMPARISONS 88 3.1 B r i e f Overview of Porphyrin Synthesis 89 3.1.1 General Porphyrin Synthesis 89 3.1.2 Porphyrins Carrying S t e r i c Encumbrance and/or Chelating Ligands 93 3.2 Synthetic Strategy 98 3.2.1 C y c l i z a t i o n to the Durene-Capped Porphyrins 99 3.2.2 Durene Dia c i d Chain Derivatives 100 3.2.3 Incorporation of the Durene D i a c i d Chain Into the Dipyrromethane Dimer 104 3.3 Spectral Comparisons 108 3.3.1 *H-nmr Spectra of Durene-Capped Porphyrins 108 3.3.2 *^C-nmr Spectra of Durene-Capped Porphyrins 117 3.3.3 E l e c t r o n i c Absorption Spectra of Durene-Capped Porphyrins 122 3 . 4 C r y s t a l Structure of Durene - 4 / 4 Hemin Chloride 129 CHAPTER 4 INTERACTION OF THE DURENE-CAPPED HEMES WITH IMIDAZOLES. ISOCYANIDES. CO AND 0 2 133 4.1 Materials and Apparatus 134 A.1.1 General 134 4.1.2 Thermostatting Equipment 135 4.1.3 E l e c t r o n i c Absorption Spectra 136 4.1.4 Infra-red Absorption Spectra 136 4.1.5 Stopped Flow Apparatus 139 4.1.6 Flash Photolysis Apparatus 139 v i i i 4.2 Solution Preparation 141 4.2.1 Crown Ether-Dithionite Method of Reduction 141 4.2.2 Aqueous D i t h i o n i t e Method of Reduction 144 4.3 Mathematical Analyses 147 4.3.1 Equilibrium Constant Determination 147 4.3.2 Determination of the"Carbon Monoxide Assoc i a t i o n ( k ^ ) and.Dissociation ( k " ^ ) Rate Constants 150 4.3.3 Determination of Equilibrium and Rate Constants f o r Dioxygen Binding Using Flash Photolysis 152 4.4 Results 155 4.4.1 A x i a l Base L i g a t i o n to Four-Coordinate Durene Hemes; K g Values 155 4.4.2 Isocyanide Binding to Five-Coordinate Durene Hemes; K ^ " Values 161 4.4.3 Carbon Monoxide Binding to Five-Coordinate Durene Hemes; K C 0 Values 168 4.4.4 Rate of Carbon Monoxide As s o c i a t i o n to Five-Coordinate Hemes; k c o Values 180 4.4.5 Carbon Monoxide D i s s o c i a t i o n from Six-Coordinate Hemes; k " c o Values 183 4.4.6 Carbon Monoxide Binding to Four-Coordination Durene Hemes; KQQ Values 186 4.4.7 Dioxygen Binding to Five-Coordinate Durene Hemes; K * Values 189 4.4.8 Infra-Red V(C0) Values f o r Six-Coordinate Durene Heme Fe(Por)(B)(CO) Complexes 199 4.5 Discussion 201 4.5.1 E l e c t r o n i c Absorption Spectral Trends Found i n Iron(II) Durene-Capped Systems 201 i x 4.5.2 L i g a t i o n to Four-Coordinate Durene Hemes 203 4.5.3 Isocyanide Binding to Five-Coordinate Durene Hemes 207 4.5.4 Carbon Monoxide Binding 208 4.5.5 Comparison of Carbonyl Stretching Frequencies for Various Heme Systems 214 4.5.6 Dioxygen Binding 217 4.5.7 Thermodynamic Considerations f o r CO and 0 2 Binding 225 4.5.8 Evaluation of CO versus 0 2 Binding i n Model Systems 230 4.5.9 Evaluation of CO versus 0 2 Binding i n Hemoproteins 238 BIBLIOGRAPHY 243 APPENDICES 254 Appendix I Synthesis and Characterization of the Durene-Capped Porphyrins and Hemin Chloride Derivatives 255 1.1 General Methods 256 1.2 Nomenclature of Intermediates 256 1.3 Synthesis of Durene D i a c i d Chain Derivatives 257 1.4 Synthesis of Durene Bis Pyrrole Derivatives 264 1.4.1 The Bis Pyrrole Diketones 264 1.4.2 The Bis Pyrrole Ethyl Esters 266 1.4.3 The Bis Pyrrole Benzyl Esters 267 1.4.4 The Bis Formylpyrroles 268 1.4.5 The Bis Cyanovinylpyrroles 270 1.5 Synthesis of Durene Linked Dipyrromethane Dimers 271 1.5.1 Preparation of 110 271 X 1.5.2 Hydrolysis of 110 273 1.6 C y c l i z a t i o n to the Durene-Capped Porphyrins 274 1.7 Synthesis of the Durene-Herain Chloride Complexes 277 Appendix II Spectral Data f o r the Iron(II) Durene-Capped Systems 279 •v Appendix I I I Raw Data f o r the Binding of Imidazoles, Isocyanides, CO and 0 2 to the Durene-Capped Hemes 284 A Base Binding Constant Determination, Kg 285 B I s o n i t r i l e A f f i n i t y Constant Determination, K R N C 288 C CO A f f i n i t y Constant Determination, K c o 289 D CO A s s o c i a t i o n Rate Constant Determination, kC0 293 E k C 0 Determination Using Varied [Dclm] 295 F CO D i s s o c i a t i o n Rate Constant Determination, k " C 0 296 G CO Binding to Fe(durene-4/4) K c o Determination 298 H O2 A f f i n i t y Constant Determination, K * 299 I 0 2 A s s o c i a t i o n Rate Constant Determination, k z 301 J K i n e t i c Determination of K ^ 302 K Van't Hoff Plots f o r CO and 0 2 Binding to Fe(durene-4/4)(B) Systems 304 x i LIST OF TABLES Table Page 1.1 Properties of the Dioxygen Moiety 21 2.1 Constants for the Binding of CO and 0 2 to Hemoproteins i n Aqueous Media, pH 7.0, 20°C 30 2.2 Isocyanide Binding to Hemoglobin Compared to Chelated Protoheme 38 2.3 Isocyanide Binding to Anthracene - 7/7, -6/6 and Adamantane Hemes i n Benzene/Toluene at 20°C 43 2.4 Constants f o r CO and 0 2 Binding to Pyrrole-Substituted Model Hemes i n Benzene/Toluene at 20°C 45 2.5(a) Constants for CO and 0 2 Binding to R-State "Chelated Picket-Fence" and "Pocket" Hemes i n Toluene at 25°C 51 2.5(b) Constants for CO and 0 2 Binding to T-State "Picket-Fence" and "Pocket" Hemes i n Toluene at 25°C 51 2.6 CO, 0 2 and NO Binding to C 2-, C3- and C4-Capped Hemes i n Toluene 53 2.7 CO Binding to Five-Coordinate "Lacunar" Complexes i n A c e t o n i t r i l e , at 0°C 57 2.8 Dioxygen "Protection" with Increasing S t e r i c Bulk Surrounding the D i s t a l S i t e of Lacunar Complexes with R* - m-xylene, B - Melm 61 2.9 Cooperativity i n 0 2 and CO Binding to " F i t t e d " R-and T-State Chains of Hemoglobin at pH 7.0, 20°C 63 2.10 Comparison of R- and T-State Constants f o r CO and 0 2 Binding to Model Hemes i n Toluene at 20°C 67 2.11 0 2 A f f i n i t i e s f o r C 2-Capped-Strapped Hemes i n Toluene at 20°C 70 2.12 Constants for CO and Oo Binding to "Hanging-Base" Hemes i n Toluene at 20°C 79 2.13 Imidazole versus Pyridine - Influence of Proximal Ligand B a s i c i t y on CO and 0 2 A f f i n i t i e s of Model Hemes 83 x i i 2.14 E f f e c t of Solvent P o l a r i t y on 0 2 A f f i n i t y i n Model Hemes 2.15 CO A f f i n i t i e s of Picket-Fence Systems Relative to Other TPP-Derived Hemes 3.1 ^C-nmr Data on the Durene-Capped Porphyrins Compared to Etioporphyrin II 121 3.2 Comparison of the Square-Pyramidal Coordination Sphere i n Fe(durene-4/4)C1 with Other High Spin Fe(III) Porphyrins 131 4.1 Imidazole Base A f f i n i t y Constants, Kg (M*^), f o r the Durene-Capped Hemes i n Toluene at 23°C 160 4.2 I s o n i t r i l e A f f i n i t y Constants f o r the Durene-Capped Hemes Compared to that f o r Chelated Protoheme 164 4.3 Constants f o r CO Binding to R-State Durene-Capped Hemes Compared to those f o r Chelated Open Hemes i n Toluene at 20°C 177 4.4 Constants f o r CO Binding to T-State Durene-Capped Hemes Compared to those for T-State Open Hemes i n Toluene at 20°C 178 4.5 A f f i n i t y Constants for Dioxygen Binding to the Durene-Capped Hemes i n Toluene at 20°C 194 4.6 Carbonyl Stretching Frequencies for Various Model Hemes 216 4.7 Comparison of R- and T-State Constants f o r CO and 0 2 Binding to Durene-Capped Hemes i n Toluene at 20°C 218 4.8 Comparison of M Values ( K C 0 / K ° 2 ) f o r Model Hemes 220 4.9 K i n e t i c Constants for Dioxygen Binding to Durene and Open Heme Systems i n Toluene at 20°C 221 4.10 L i g a t i o n Free Energy Changes f or CO Binding to Encumbered Hemes Compared with Chelated Mesoheme 226 4.11 L i g a t i o n Free Energy Changes on CO Binding to the Durene-4/4 and -5/5 Hemes 227 4.12 Thermodynamic Constants for CO and 0 2 Binding 229 x i i i 4.13 D i s t a l S t e r i c E f f e c t s Toward CO and 0 2 for Pyrrole-Substituted Systems 231 4.14 0 2 Versus CO Binding for Pyrrole-Substituted Model Hemes 232 4.15 Comparison of CO and 0 2 Association Rates to Four- and Five- Coordinate Hemes 240 x i v LIST OF FIGURES Figure Page 1.1 T e r t i a r y structure surrounding the heme pros t h e t i c group of hemoglobin 4 1.2 d-Orbital populations for five-and six-coordinate ( d 6 ) F e ( I I ) 7 1.3 Saturation curves for dioxygen binding to hemoglobin and myoglobin 8 1.4 Proximal environment of deoxy-hemoglobin 12 1.5 Protein residues that comprise the proximal and d i s t a l environments of the heme 12 1.6 Movement of the ir o n into the porphyrin plane on dioxygen binding 13 1.7 Rotation of the CX\^i dimer with respect to the CX\($2 i n t e r f a c e as the hemoglobin molecule changes i t s quaternary structure from the T- to R- state- 16 1.8 Molecular o r b i t a l diagrams for 0 2 (A) and CO (B) 23 1.9 The Olafson-Goddard bonding model of the Fe-02 moiety 23 2.1 D i s t a l s t e r i c e f f e c t s on isocyanide l i g a t i o n within the heme binding pocket 42 2.2 C r y s t a l structures showing the " t a l l " (A) and "short" (B) binding pockets of the lacunar complexes 32 and 33, res p e c t i v e l y 1 56 2.3 C r y s t a l structure (A) and diagrammatic i l l u s t r a t i o n (B) of a d i s t o r t e d Fe-C-0 moiety within the severely hindered binding s i t e of the lacunar complex, 35 (py) 58 2.4 "T-State s t r a i n " introduced on l i g a t i o n trans to a 2-substituted imidazole 66 2.5 Proximal s t e r i c e f f e c t s on CO binding: (A) to a bulky transition-metal complex; (B) to a "T-state" heme system 69 2.6 Pyrrole-substituted chelated hemes with varying degrees of proximal s t r a i n 72 X V 3.1 Formation of a dipyrromethane from pyrrole precursors 90 3.2 Formation of dipyrromethenes from pyrrole precursors 90 3.3 Formation of a porphyrin v i a the a c i d catalyzed c y c l i z a t i o n of dipyrromethanes 92 3.4 Synthesis of tetraphenylporphyrin-derived systems from pyrrole and substituted benzaldehydes 95 3.5 Strapped porphyrins derived from the "2+2" coupling of chain-linked dipyrromethanes 97 3.6 Schematic i l l u s t r a t i o n of the synthesis of durene d i a c i d chains 101 3.7 Synthesis of durene linked b i s p y r r o l i c intermediates 102 3.8 Transformation of dipyrromethane dimers and c y c l i z a t i o n to the durene-capped porphyrin 103 3.9 H^-NMR spectrum of the durene-4/4 capped porphyrin, 54a 112 3.10 -^H-NMR spectrum of the durene-5/5 capped porphyrin, 55a 113 3.11 H^-NMR spectrum of the durene-7/7 capped porphyrin, 56a 114 3.12 V a r i a t i o n i n chemical s h i f t s of proton signals f o r the 4/4-, 5/5- and 7/7-durene porphyrins compared with that f o r etioporphyrin I I . 115 3.13 ^C-NMR spectrum of the durene-4/4 capped porphyrin, 54a 118 3.14 l^C-NMR spectrum of the durene-5/5 capped porphyrin, 55a 119 3.15 l-^ C-NMR spectrum of the durene-7/7 capped porphyrin, 56a 120 3.16 Trends i n v i s i b l e spectra f or various pyrrole-substituted porphyrins 124 3.17 UV-Visible spectrum of the durene-7/7 porphyrin, 56a 125 3.18 UV-Visible spectrum of the durene-5/5 porphyrin, 55a 126 3.19 UV-Visible spectrum of the durene-4/4 porphyrin, 54a 127 3.20 UV-Visible s p e c t r a l trends f o r the hemin ch l o r i d e d e r i v a t i v e s of the durene-capped porphyrins 128 3.21 C r y s t a l structure of the durene-4/4 hemin ch l o r i d e complex, F e n i ( 5 4 b ) C l 130 x v i A . l Thermostatted c e l l - h o l d e r f o r temperatures above -10°C 137 A.2 6 cm path-length quartz c e l l 138 A.3 (A) Apparatus used for reducing Fe^^^(Por) to Fe**(Por) using crown e t h e r - d i t h i o n i t e 1A3 (B) Tonometer for measuring base binding constants to four-coordinate Fe(II) hemes 1A3 A.A (A) Apparatus used for reducing Fe***(Por) to Fe* 1 (Por) using aqueous d i t h i o n i t e 1A5 (B) Tonometer f o r measuring ligand binding constants to five-coordinate hemes, Fe**(Por)(B) 1A5 A.5 Spectral trends f or four-, f i v e - and six-coordinate F e n ( d u r ene-7/7) systems 156 A.6 Isosbestic s p e c t r a l changes f o r : F e I I ( P o r ) + l,2-Me 2Im^=^Fe I I(Por)(l,2-Me 2Im) at 20°C; Por - durene-5/5; added [l,2-Me 2Im] - 0.45, 1.A8, 6.38 and 13.49 x I O - 4 M i n the 460-300 nm region; f i n a l [l,2-Me 2Im] - 0.03 M 159 4.7 H i l l p l o t f o r : F e I I ( P o r ) + Dclm v ^ Fe 1 1(Por)(Dclm) Por - durene-4/4 160 4.8 Isosbestic s p e c t r a l changes f o r : F e 1 1 (Por) (Dclm) (CO) + TMIC ^ F e 1 1 (Por) (Dclm) (TMIC) + CO at 20°C; Por - durene-5/5; [CO] - 2.4 x IO' 4 M, added [TMIC] - 2.77, 4.84, 6.92, 9.00, 16.90, 23.50 and 33.59 x IO" 4 M ; f i n a l [TMIC] -0.02 M 163 4.9 H i l l p l o t f o r : Fe 1 1(Por)(Dclm)(CO) + T M I C ^ F e 1 1 ( P o r ) ( D c l m ) ( T M I C ) + CO at 20°C; Por - durene-5/5 164 4.10 Spectral trends f o r the l i g a t i o n of isocyanides to the durene-4/4 system 166 4.11 Spectral trends f o r the durene-4/4. -5/5 and -7/7 Fe 1 1(Por)(Dclm)(RCN) systems 167 4.12 Vacuum-line set-up used f o r preparing and admitting C0/N 2 mixtures into a tonometer 168 4.13 Isosbestic s p e c t r a l changes f o r : Fe I I(Por)(l,2-Me 2Im) + C0^=^Fe X I(Por)(1,2-Me 2lm)(CO) at 20°C; Por - durene-5/5; added P c o - 0.0119, 0.0242, 0.0363, 0.0529, 0.0816, 0.117 and 0.240 t o r r i n the 460-300 nm region; f i n a l P c o - 1 atm 170 x v i i 4.14 Isosbestic spectral changes f o r : FeJ1(?or)(l,2-Me2Im) + CO^=iFe 1 1(Por)(1,2-Me 2Im)(CO) at 20°C; ( A ) Por - durene-7/7, ( B ) Por - durene-4/4 171 4.15(a) Spectral trends f or the durene-4/4, -5/5 and -7/7 Fell(?ox)(l,2-Me2Im)(CO) systems 172 4.15(b) Spectral trends for the durene-4/4, -5/5 and -7/7 Fe**(Por)(Dclm)(CO) systems 173 4.16(a) H i l l p l o t f o r : Fe n(Por)(l,2-Me 2Im) + C0^=* F e 1 1 (Por) (1, 2-Me2Im) (CO) at 20°C; Por - durene-5/5 176 4.16(b) H i l l p l o t f o r : Fe 1 1(Por)(Dclm)(Melm) + CO Fe 1 1(Por)(Dclm)(CO) + Melm at 20°C; Por = durene-7/7 176 4.17 Van't Hoff p l o t s f o r : Fe 1 1(durene-4/4) ( B ) + CO 5=^Fe 1 1(durene-4/4) (B)(CO) 179 4.18 Determination of k c o f o r : Fe i : t(Por) (Dclm) + C O ^ F e 1 1 (Por) (Dclm) (CO) at 20°C; Por = durene-5/5 181 4.19 Determination of at va r i e d [Dclm] f o r : F e 1 1 (Por) (Dclm) + C O ^ ^ F e 1 1 (Por) (Dclm) (CO) at 20°C; Por - durene-7/7 181 4.20 Determination of k " c o f o r : Fe I I(Por)(l,2-Me 2Im)(CO)^=iFe I I(Por)(l,2-Me 2Im) + CO at 20°C; Por - durene-4/4 183 4.21 Determination of k " c o f o r : F e n ( P o r ) (Dclm) ( C 0 ) ^ = ± Fe11 (Tor) (Dclm) + CO at 20°C; Por - durene-5/5 185 4.22 Isosbestic s p e c t r a l changes f o r : FeJ1(?or) (CO) + Melm^=±Fe I ] :(Por) (Melm) (CO) at 20°C; Por - durene-4/4; added [Melm] - 0.532, 1.06, 1.59, 2,12, 2.65, 3.89 and 9.04 x 1 0 - 5 M; f i n a l [Melm] -0.02 M 188 4.23 Isosbestic s p e c t r a l changes f o r : F e 1 1 (Por) (Melm) + 0 2 F e 1 1 (Por) (Melm) (0 2) °? at 20°C; Por •= durene-4/4; added P z - 24, 64, 124 t o r r , and 1 atm, i n the 460-300 nm region 191 4.24 H i l l p l o t f o r : Fe i : [(Por)(MeIm) + 0 2 =^2r F e 1 1 (Por) (Melm) (0 2) at 20°C; Por - durene-4/4 192 x v i i i 4.25 Spectral trends for the durene-4/4, -5/5 and -7/7 Fe l : l(Por) (Dclm) (0 2) systems 193 4.26 Van't Hoff plots for: F e 1 1 (durene-4/4) (B) + 0 2 ^ F e 1 1 (durene-4/4) (B) (0 2) 196 4.27 Determination of k ^ for: Fe i : [(Por) (Dclm) + 0 2 F e 1 1 (Por) (Dclm) (0 2) at 20°C; Por = durene-7/7 198 4.28 K i n e t i c determination of K f o r : F e n ( P o r ) (Dclm) + 0 2 ~ * F e 1 1 (Por) (Dclm) (0 2) at 20°C; Por = durene-7/7 198 4.29 (A) Examples of an IR spectrum of Fe(durene-4/4)(Melm)(CO) i n toluene superimposed over that of free CO i n toluene/Melm 200 (B) Subtraction of (A) 4.30 (A) "Squashed" conformation of the durene-7/7 cap 211 (B) "Basket-shaped" conformation of the d i s t o r t e d durene-5/5 and -4/4 systems .211 4.31 Free energy b a r r i e r s f o r CO binding to f i v e -coordinate encumbered and open heme systems 226 4.32 SAG 0 dependencies on ligand bulk for Mb, R- and T-state Hb r e l a t i v e to chelated protoheme i n aqueous suspension at pH 7.0 239 xix LIST OF ABBREVIATIONS Np = Pyrrole nitrogens Cp = Porphyrin carbons Hb = Hemoglobin Mb = Myoglobin SW-Mb = Sperm whale myoglobin AL-Mb = Aplysia myoglobin Gd-Hb = Glycera dibranchiata hemoglobin Dd-Hb = Li v e r fluke hemoglobin CTT-Ery = Chironomous erythrocurorin Lg-Hb = Leghemoglobin HRP = Horseradish peroxidase B = Base L - Ligand: CO or 0 2 Im = Imidazole Py = Pyridine Melm = Methylimidazole 1,2-Me2lm = 1,2-Dimethylimidazole 2-MeIm •= 2-Methylimidazole Dclm = 1,5-Dicyclohexylimidazole THPIm «= 5,6,7,8-Tetrahydroimidazo[1,5-a]pyridine t-BuNC = t e r t - B u t y l i s o n i t r i l e TMIC - T o s y l m e t h y l i s o n i t r i l e CH2CI2 = Dichloromethane THF = Tetrahydrofuran EtOH = Ethanol X X MeOH •= Methanol DMF <= Dimethyl formamide TFA = T r i f l u o r o a c e t i c a c i d *H-nmr - Proton nuclear magnetic resonance l^C-nmr = Carbon-13 nuclear magnetic resonance EPR, ESR •= Electron paramagnetic (spin) resonance t i c •= Thin layer chromatography TMS •= Tetramethylsilane t — D i e l e c t r i c constant L P]y2 " P a r t i a l pressure of gas (L) required to convert h a l f the reactant species to product Abbreviations i n UV-Visible and NMR. Assignments s i n g l e t X(nm) — wavelength i n nanometers doublet A - absorbance t r i p l e t £ — e x t i n c t i o n c o e f f i c i e n t quartet br - broad m u l t i p l e t sh - shoulder xx i Equilibrium and K i n e t i c Constants Equilibrium constant f o r ligand coordination to the f i f t h a x i a l s i t e (unhindered side) of an encumbered heme. Equilibrium constant for ligand coordination to the sixth a x i a l s i t e (encumbered side) of a heme with the f i f t h s i t e already occupied. Asso c i a t i o n rate constant f o r ligand coordination to the s i x t h a x i a l s i t e (encumbered side) of a heme with the f i f t h s i t e already occupied. D i s s o c i a t i o n rate constant f o r ligand d i s s o c i a t i o n from the s i x t h a x i a l s i t e (encumbered side) of a heme with the f i f t h s i t e occupied. xx i i ACKNOWLEDGEMENTS 1 would l i k e to express my sincere gratitude to Professor David Dolphin and Professor Brian James for t h e i r guidance, patience and encouragement throughout the "evolution" of this t h e s i s . I also wish to take t h i s opportunity to thank Professor T. Traylor at the University of C a l i f o r n i a , San Diego, for the extremely rewarding opportunity to work in h i s laboratory. I would l i k e to extend special.thanks to Marco Lopez, who not only made i t possible to complete experiments i n the shortest a v a i l a b l e time, but who's patience and sense of humour also provided a very enjoyable working environment i n San Diego. I am indebted to Dr. Toshio Mashiko who's vacuum-line alone was an i n s p i r a t i o n , not to mention the extremely useful discussions, regarding the manipulation of a i r s e n s i t i v e compounds, which I f e e l p r i v i l e g e d to have had with him. I also wish to thank s i n c e r e l y Dr. T i l a k Wijesekera for taking the time to provide me with valuable d e t a i l s on the synthesis of the durene-capped porphyrins. The c r y s t a l structure of the durene-4/4 hemin chloride was determined by Dr. F. E i n s t e i n and T. Jones at Simon Fraser Uni v e r s i t y , Vancouver. Thanks also to David Thackeray for showing me how to use the FT-IR spectrometer. Some of the diagrams i n t h i s thesis have been adapted from the Ph.D. theses of Drs. Brian Morgan and T i l a k Wijesekera. Thanks are also due to Rani Theeparajah for the f a s t and competent typing of t h i s t h e s i s . Last but by no means le a s t , I would l i k e to thank those people, over the years, who made i t an enjoyable experience for me to work i n the Chemistry Department at U.B.C. - 1 -CHAPTER 1 HEMOFROTEIN STRUCTURE AND FUNCTION 2 1. HEMOPROTEIN STRUCTURE AND FUNCTION Iron-containing proteins are e s s e n t i a l for numerous and varied forms of l i f e , performing v i t a l functions both enzymatic and non-enzymatic i n nature. Typical non-enzymatic functions are electron transport (cytochromes)-'-, and oxygen transport and storage (hemoglobins, myoglobins and erythrocruorin)^. Enzymatic processes catalyzed by hemoproteins include molecular oxygen a c t i v a t i o n (dioxygenases eq. (1) and monooxygenases eq. (2)), hydrogen peroxide destruction (catalases eq. (3)) and hydrogen peroxide u t i l i z a t i o n (peroxidases eq. (4))*. AH 2 + 0 2 > A(OH) 2 (1) AH + 0 2 + 2H + + 2e" > AOH + H 20 (2) 2H 20 2 > 2H20 + 0 2 (3) AH 2 + H 20 2 > A + 2H20 (4) The oxygen transport and storage hemoproteins (of primary consideration here) occur i n a l l vertebrates, some invertebrates and root nodules of leguminous plants. Within t h i s series t h e i r ligand binding behaviour varies tremendously. For instance, the difference i n dioxygen a f f i n i t y between Ascaris Lumbricoides hemoglobin (found i n the p e r i e n t r i c f l u i d of p i g roundworm) with a very high a f f i n i t y ^ " and some f i s h hemoglobins, i s 25,000 f o l d . The e s s e n t i a l difference between these various hemoglobins i s the structure of the pro t e i n surrounding the active s i t e where l i g a t i o n occurs, which evidently has a strong influence on ligand binding. The active s i t e of a l l oxygen carrying hemoproteins contains a heme moiety l a which consists of a ferrous iron coordinated to the four nitrogen atoms of a protoporphyrin IX prosthetic group. The heme i s embedded i n a polypeptide chain of 136 to 153 residues, folded into eight h e l i c a l segments (A-H), F i g . 1.1. Helices F and E constitute the "proximal" and " d i s t a l " sides of the heme, res p e c t i v e l y . The residues i n the immediate environment of the heme are predominantly hydrophobic i n nature, while those on the outer surface of the molecule are hyd r o p h i l i c rendering the p r o t e i n soluble i n water but impermeable to i t . The only linkage between the heme and surrounding p r o t e i n (or globin) i s a coordinate bond between the ferrous i r o n and an imidazole nitrogen of a proximal h i s t i d i n e F8 residue ( l b ) . Oxygen carrying hemoproteins may e x i s t as an i n d i v i d u a l subunit (monomer) of heme and globin, or as an aggregate of two (dimer) or four (tetramer) subunits l i n k e d together v i a strong dipolar and hydrophobic i n t e r -actions, which determine the o v e r a l l quaternary structure of the molecule-'. Small molecules such as dioxygen, carbon monoxide, n i t r i c oxide and some b u l k i e r i s o n i t r i l e ligands a l l r e v e r s i b l y bind to the heme by coordinating to the ferrous i r o n i n the av a i l a b l e s i x t h s i t e opposite the bound imidazole^. 5 1.1 PORPHYRIN MACROCYCLE 2 A l l porphyrins are derived from porphine, 2. In the Fischer system of nomenclature the peripheral positions are numbered 1-8 and the methine posi t i o n s (meso) are designated (X, (3, 8 and y . Various s u b s t i t u t i o n patterns at the peripheral positions give r i s e to a number of porphyrins occurring i n nature. Protoporphyrin IX (la) i s most common, and i s the prosthetic group of hemoglobins, cytochromes, peroxidases and oxidases. The porphyrin r i n g i s highly conjugated with 18 electrons involved i n any one d e l o c a l i z a t i o n pathway. Unlike smaller aromatics however, highly d i s t o r t e d porphyrin systems are known to e x i s t ^ . S k e l e t a l d i s t o r t i o n occurs i n two ways: doming or r u f f l i n g . Doming of the macrocycle r e s u l t s when the mean plane of the four pyrrole nitrogens i s displaced from the mean plane of the porphyrin r i n g , with a l l four pyrrole nitrogens on one side. R u f f l i n g occurs when opposite pyrrole nitrogens are out of the porphyrin plane on the same side and adjacent pyrrole nitrogens are out of the plane on opposite sides. In a s t r a i n induced system these deformations tend to minimize angular bond tension at the (X,(3, 6 and y meso-positions . Localized p l a n a r i t y i n each pyrrole r i n g , however, i s preserved so that there i s l i t t l e loss of aromatic s t a b i l i t y . Removal of the two inner hydrogens gives the porphyrin dianion which i s capable of coordinating various metal(II) - 6 -ions within i t s inner core. Due to the high degree of electron d e r e a l i z a t i o n , both porphyrins and metalloporphyrins are highly coloured compounds with c h a r a c t e r i s t i c u v - v i s i b l e absorption spectra a r i s i n g from 7T—>7T e l e c t r o n i c t r a n s i t i o n s i n the porphyrin macrocycle. 1.2 IRON SPIN STATE AND GEOMETRY9 The neutral four-coordinate Fe(II)-porphyrin moiety can coordinate a v a r i e t y of ligands i n the two remaining a x i a l p o s i t i o n s . These include carbon, nitrogen, phosphorus, oxygen and sulphur ligands, which possess a lone p a i r of electrons f o r donation to the metal centre. In the oxidized f e r r i c ( I I I ) state, harder donors such as cyanide and chloride ions also coordinate to the metal. S i g n i f i c a n t l y , only the ferrous form of the heme i s capable of binding CO and 0 2 . The ferrous iron(d^) can ex h i b i t three spin states (S>=0 low spin, S=l intermediate spin, and S=2 high spin), depending on coordination number and type of ligand. The d - o r b i t a l populations f o r the f i v e - and six-coordinate states seen i n hemoproteins are shown i n F i g . 1.2. With one a x i a l imidazole coordinated, the five-coordinate i r o n i s displaced from the pyrrole nitrogen plane toward the imidazole, r e s u l t i n g i n a square pyramidal geometry with respect to the porphyrin plane. An adequate explanation f o r t h i s l i e s i n the larger e f f e c t i v e radius of the high spin(d^) i r o n with electrons occupying the d e s t a b i l i z e d d x2_ v2 and - 7 -J _ x 2 - y 2 2 2 2 x - y , z _1_ JL x z , y z JL xy x y , x z , yz Coordination number : F i v e S i x Spin S t a t e : H i g h - s p i n L o w - s p i n G e o m e t r y : S q u a r e - p y r a m i d a l O c t a h e d r a l F i g . 1.2 d-Orbital populations f o r five-and six-coordinate ( d b ) F e ( I I ) d z2 o r b i t a l s whose lobes point i n the d i r e c t i o n of the coordinating ligands. On l i g a t i o n of a strong f i e l d s i x t h l i g a n d (such as imidazole, CO, 0 2 , RNC,etc.) the i r o n r e a d i l y assumes a low spin state due to the large c r y s t a l f i e l d s t a b i l i z a t i o n energy achieved i n a d^ octahedral environment. The resultant low spin i r o n i s generally i n the porphyrin plane. 8 1.3 HEMOGLOBIN STRUCTURE AND COOPERATIVITY In vertebrates, the tetrameric hemoglobin found i n red blood c e l l s serves to transport oxygen from the lungs to the muscle tissues, where i t i s stored by monomeric myoglobin. The oxygen uptake c u r v e s ^ for these two hemoproteins (Fig. 1.3) show the f r a c t i o n of heme s i t e s occupied by oxygen as a function of oxygen concentration i n s o l u t i o n (or F i g . 1.3 Saturation curves for dioxygen binding to hemoglobin and myoglobin p a r t i a l pressure of oxygen over the s o l u t i o n ) . For myoglobin the curve i s hyperbolic, and for hemoglobin i t i s sigmoidal. Studies on l i g a t i o n to myoglobin*^ indicate a f i r s t order dependence on oxygen concentra-Mb Mb + 0 2 v ^ Mb02 (5) t i o n , where the equilibrium constant i s defined by: 9 [Mb02] Mb [Mb] [0 2: (6) The sigmoidal curve for hemoglobin*^, having four active s i t e s per molecule, indicates a greater than f i r s t order dependence on oxygen concentration, K 2 Hb (Hb) n + n0 2 ^ (Hb0 2) n (7) with the a f f i n i t y constant defined by: 0, [Hb0 2] n K 1 = (8) H b [ H b ] n [ 0 2 ] n Experimental determination shows n-2.8. The o v e r a l l a f f i n i t y i s determined by the i n d i v i d u a l equilibrium constants for the consecutive binding of one mole of dioxygen to each subunit from K]_ through to , where the r e l a t i v e a f f i n i t i e s are found to be*^ a: ^ ( 1 ) < K 2 (1.76) ~ K 3 (1.31) « K 4 (17.7) (9) at pH 7, 20°C These are r e f e r r e d to as the Adair constants for oxygen binding to hemoglobin. At low oxygen pressure, as i n the t i s s u e s , hemoglobin has a much lower a f f i n i t y for oxygen than myoglobin which allows the gas to be transferred e f f e c t i v e l y from blood to muscle. The property of hemoglobin which allows for release of about h a l f of the bound oxygen at 10 venous pressure involves i n t r i c a t e changes i n the intersubunit contacts within the molecule. This form of subunit to subunit communication, as oxygen i s bound or released, i s known as cooperativity^. The r i s e i n oxygen a f f i n i t y of human hemoglobin between zero and f u l l oxygen saturation i s ca. 5 0 0 f o l d , corresponding to a 3.6 kcal/mol free energy difference involved i n the heme-heme interaction 4'-^. Owing to the ingenious and laborious work of Kendrew^3-, Perutz^ and o t h e r s ^ ^ , the c r y s t a l structure of the hemoglobin molecule was elucidated. S t r i k i n g differences were found to e x i s t between the conformations of deoxyHb (with no bound O2) and oxyHb ( f u l l y saturated with 0 2) c r y s t a l s . This l e d to the recognition that cooperativity i s accomplished v i a a t r a n s i t i o n between two quaternary structures: namely, the low a f f i n i t y deoxy- and the high a f f i n i t y oxy-conformations, termed the "tense"(T) and "relaxed"(R) states, r e s p e c t i v e l y . The hemoglobin tetramer i s composed of four hemes, with two Of and two )3 polypeptide chains, r e s u l t i n g i n two Ct and two |3 subunits with one heme per subunit-*. The f o l d i n g of a polypeptide chain around a heme gives each subunit a t e r t i a r y structure s i m i l a r to that found i n myoglobin 4. Each CV subunit i s linked to a 3^ u n i t to give two sets of dimers, Ct^fii and Q^j^. These dimers are i n turn held together so as to form (Xi j3 2 a n c * OL2 fi\ contacts which make up the o v e r a l l quarternary structure of the tetramer, within which the hemes are separated by distances of 23-39A5. - 1 0 o In the deoxy quaternary s t r u c t u r e ^ the ir o n i s displaced 0.4A from the plane defined by the four pyrrole nitrogens (Np), with accompanied doming of the porphyrin skeleton toward the coordinated proximal imidazole such that the separation between the mean planes of Np and Cp (porphyrin carbons) i s 0.16 and 0.10A i n the CV and (3 subunits, re s p e c t i v e l y . This displacement of the i r o n from the porphyrin plane might be expected from spin-state considerations^ (section 1.2). The plane of the imidazole r i n g eclipses the N]_—Fe—N3 axis and the imidazole i s t i l t e d -10° from the normal, so that C^ i s closer to N]_ than C^ i s to N3 (Fig. 1 . 4 ) * 2,13_ Quantitative energy c a l c u l a t i o n s * ^ indicate that high spin Fe(II) can be accommodated within the porphyrin core with only a small expenditure of energy (<0.5 k c a l ) , whereas s t e r i c repulsion between the asymmetric a x i a l imidazole and porphyrin r i n g accounts for a greater degree of s t r a i n energy (-3 kcal) that prevents Fe from s i t t i n g i n the porphyrin plane. Therefore, both i r o n spin state and globin r e s t r a i n t s may contribute to t h i s displacement of the high spin i r o n atom^'*^. Globin residues surround the proximal imidazole which forms a hydrogen bond between Ng and a main chain Leu F4 residue (Fig. l . S ) * 2 ' * ^ 3 . In the s i x t h coordination ( d i s t a l ) s i t e , v a l i n e E l l , h i s t i d i n e E7, and phenylalanine CD4 residues are i n close proximity to the heme, and occupy the space that would be required for a s i x t h ligand to coordinate. This unligated deoxy quaternary structure i s e s s e n t i a l l y without s t r a i n * ^ " * ^ . In the "relaxed" quaternary structure of the hemoglobin molecule the i r o n atom i s i n (or closer to) the heme plane, which i s now essen-t i a l l y f l a t * ^ . The attached proximal imidazole occupies a more symmetric p o s i t i o n r e l a t i v e to the heme plane, the geometry of which reduces s t e r i c repulsions between the imidazole and porphyrin -1 o o nitrogens 1--'. The Fe-N^ (imidazole nitrogen) bond i s -2. OA, shortened by 0 19 1^ -0.1A r e l a t i v e to the high spin deoxy case 1 '•LJ; the Fe-Np -distance i s the same within experimental error*^. The d i s t a l binding pocket i s more F i g . 1.5 Protein residues that comprise the proximal and d i s t a l environments of the heme 1 3 open, where t i l t i n g of the heme has moved the binding s i t e out, away from the Val E l l and His E7 r e s i d u e s ^ . This t e r t i a r y change at the active s i t e occurs when the quaternary structure changes to that of the l i g a t e d form, i r r e s p e c t i v e of the presence of bound ligand i n a p a r t i c u l a r subunit-1- . This "relaxed" quaternary structure then favours coordination of a s i x t h ligand which binds to i t with higher a f f i n i t y - ' ' ^ . The low a f f i n i t y of the T-state of hemoglobin, and the t e r t i a r y changes within a subunit that eventually lead to quaternary changes of the e n t i r e molecule, are the primary considerations for the function of hemoglobin^. Ligand binding to the d i s t a l s i t e i n hemoglobin requires the i r o n to take up a more cen t r a l p o s i t i o n with respect to both a x i a l groups i n order to reduce repulsive i n t e r a c t i o n between porphyrin and eith e r one. Consequently the most favourable p o s i t i o n for the six-coordinate, low spin Fe(II) i s i n the plane of the porphyrin r i n g . Perutz 4'^ has suggested that t h i s required movement of the ir o n into the porphyrin plane upon l i g a t i o n introduces s t r a i n p r i m a r i l y Fig. 1.6 Movement of the iron into the porphyrin plane on dioxygen binding 14 associated with the Fe-Ng (imidazole nitrogen) bond (Fig. 1.6), and th i s i s responsible for the low a f f i n i t y of the T-structure. Evidence to support t h i s suggestion was gained from studies with nitrosylhemoglobin (HbNO) i n the R-quaternary structure, where the addition of i n o s i t o l hexaphosphate ( I H P ) , which switches the molecular structure into that of the liganded T-state, resulted i n breaking (or severely stretching) the Fe-Ngbond leaving e s s e n t i a l l y a five-coordinate n i t r o s y l bound i r o n * ^ . During the consecutive binding of molecules to deoxyHb, Perutz^ proposed that the quaternary switch to the R-structure i s "triggered" by movement of the ir o n and attached imidazole i n each liganded subunit; t h i s motion i s then transmitted v i a protein s t r u c t u r a l changes to the unligated subunits causing the release of proximal h i s t i d i n e r e s t r a i n t upon ir o n motion i n these remaining subunits, and thus e f f e c t i n g the o v e r a l l quaternary change. The conformational change also reduces blocking of the d i s t a l s i t e . S t r a i n i n the Fe-N^ bond i s considered to r e s u l t from s t e r i c repulsion between the e c l i p s i n g , asymmetric imidazole r i n g and porphyrin nitrogens as the i r o n moves toward the porphyrin plane i n the deoxy quaternary structure*-^ • . Non-bonded close contacts between the proximal h i s t i d i n e and surrounding globin residues have been considered p a r t l y the source of constraint that prevents the imidazole from a t t a i n i n g a more symmetric, non-eclipsing conformation with respect to the porphyrin r i n g * ^ ' * ^ . In addition, c r y s t a l structure determinations indicate that heme t i l t occurs on ligand binding*-^, which may reduce repulsive contacts between proximal imidazole and porphyrin*^. Thus, globin residues i n close proximity to the porphyrin side chains i n the T-state, may influence ligand binding by the extent of heme t i l t - 15 -allowed-'-4. Theoretical c a l c u l a t i o n s ^ 4 have implicated Val FG5, in contact with pyrrole(3), i n a feedback loop s t a b i l i z i n g the proximal h i s t i d i n e and thus contributing to t i l t i n g of the heme on l i g a t i o n (Fig. 1.5). The hydrogen-bond between the side chain of Leu FA and the imidazole r i n g supports the geometry and prevents the imidazole from t i l t i n g backwards or r o t a t i n g to diminish i t s i n t e r a c t i o n with pyrrole(l)-*- 4. When Val FG5 i s displaced by heme t i l t on ligand binding, the movement pushes Leu F4 toward His F8 thereby completing the loop-'-4. Experimentally, s u b s t i t u t i o n of the v i n y l side chain on pyrrole(3) greatly reduces cooperativity, while s u b s t i t u t i o n of the v i n y l on pyrrole(2) has no e f f e c t * 4 ' ^ . Aside from globin r e s t r a i n t s on the deoxy heme, a s i g n i f i c a n t c o n t r i b u t i o n to the low a f f i n i t y of the T-state may a r i s e d i r e c t l y from obstruction of the s i x t h coordination s i t e by d i s t a l r e s i d u e s ^ . In the /3subunit of deoxyHb, the d i s t a l Val E l l residue completely blocks access to the heme centre i n the d i s t a l pocket, and may well play a major r o l e i n c o n t r o l l i n g ligand a f f i n i t y ^ . In the h a l f saturated structure of hemoglobin-^, with two oxygen molecules bound, the CV subunits are the f i r s t to bind oxygen, the j8 subunits remaining deoxygenated. The o v e r a l l molecular structure retains the T-quaternary conformation-^ i n t h i s p a r t i a l l y bound state. The Fe-N^ bond i n the l i g a t e d CV subunit appears longer than i n HbC^ (R-state), corresponding T 7 to a high spin iron- 1-'. The CV hemes are markedly buckled with the ir o n o displaced 0.2A from the plane of the four pyrrole nitrogens, toward the proximal imidazole, whereas i n the deoxy /3 subunits, the hemes are o -• 7 planar with the ir o n 0. 3A from the Np plane 1-'. In the CV -subunit of f u l l y saturated R-state HbO^-^, the imidazole r i n g i s s t i l l i n an 16 e c l i p s e d o r i e n t a t i o n with respect to the N ^ — F e — N 3 axis and the ir o n remains s i g n i f i c a n t l y out of the heme plane ( 0 . 1 6 A ) . (In the / 3 subunit the imidazole r i n g has rotated r e l a t i v e to the porphyrin nitrogens, allowing the iron to move into the Np plane.) Thus non-bonded inte r a c t i o n s associated with the Fe-Ng linkage may be more important i n c o n t r o l l i n g ligand a f f i n i t y i n the CV subunit*^. Propagation of the t e r t i a r y s t r u c t u r a l changes into quaternary subunit changes i s thought to involve a f a i r l y l o c a l i z e d pathway of residues*-^ • ^ , the d e t a i l s of which are as yet not understood. The ^lfil' s u h u n i t linkage i s composed of numerous (17 to 19) hydrogen bonds which make the subunits cohere so strongly that t h e i r contact i s hardly a l t e r e d as the molecule switches from the T- to R-state^. The CVj/^ type contact, however, involves fewer linkages with two a l t e r n a t i v e stable pos i t i o n s for the T- and R-quaternary structures, each braced by a d i f f e r e n t set of hydrogen bonds (Fig. 1.7)^. Quaternary change i s effe c t e d when changes i n t e r t i a r y structure within each subunit A X I S O F S Y M M E T R Y ^ Fig. 1.7 Rotation of the CVTJS]. dimer with respect to the C V ^ interface as the hemoglobin molecule changes i t s quarternary structure from the T- to R- state - 17 -introduces s t r a i n at the interface, thus i n i t i a t i n g the switch to the more f l e x i b l e , high a f f i n i t y R-conformation^. In the absence of ligands the T-quaternary state i s the more stable of the two due to i t s greater hydrophobic energy, having less surface area i n contact with the surrounding water 4. The major contribution to the buried surface area i s associated with c e r t a i n carboxy-terminal residues at the end of the polypeptide chains, which i n deoxyHb form s a l t bridges that are absent i n liganded Hb^. (A s a l t bridge i s a bond between an e l e c t r o p o s i t i v e nitrogen and electronegative oxygen atom). Several s a l t bridges or hydrogen bonds add to the s t a b i l i t y of either the T- or R-quaternary structures. Certain heterotropic agents such as H +, CO2, CI", diphosphoglyerate (DPG), and i n o s i t o l hexaphosphate (IHP), i n t e r a c t with and s t a b i l i z e the T-state by adding to or r e i n f o r c i n g the already e x i s t i n g s a l t bridges i n the structure-'- 9. Thus the cooperativity of hemoglobin i s heavily dependent on the presence of these heterotropic e f f e c t o r s ^ 0 • * 9 . In r e s p i r i n g tissues, the CO2 released dissolves i n the surround-ing aqueous medium to give carbonic acid: C0 2 + H 20 v HC0 3 _ + H + (9) The protons formed i n high concentration bind to hemoglobin, causing a quaternary change to the T-state and thus allowing release of some bound oxygen. This i s known as the Bohr E f f e c t * 9 and has important consequences i n determining the p h y s i o l o g i c a l function of hemoglobin. The extent to which the Bohr e f f e c t i s linked to quaternary change i s uncertain. Conversely, at high pH or low [pH], the s a l t bridges of the 18 T-structure are rendered weaker and the s t r a i n introduced on ligand binding i s relaxed more e a s i l y - ' 0 ' * 9 . 1.4 MYOGLOBIN STRUCTURE Myoglobin(Mb), being a monomeric hemoprotein 2^, binds only one molecule of oxygen, has no intersubunit i n t e r a c t i o n and thus does not show cooperative binding. Its oxygen a f f i n i t y i s about 3-fold l e s s than that of R-state Hb and -50-fold greater than that of T-state Hb 6. The t e r t i a r y changes that take place on ligand binding to deoxy Mb are s i m i l a r but smaller i n magnitude than those for the T- to R-state switch o i n Hb. In deoxyMb the i r o n i s situated -0.47A below the average heme plane toward the proximal h i s t i d i n e 2 ^ . On dioxygen binding the imidazole r i n g rotates into an even more ecl i p s e d p o s i t i o n with respect to the N^—Fe-N3 axis, so that unfavourable i n t e r a c t i o n between C^-H o and N 3 (Fig. 1.4) requires the i r o n to remain 0.2A out of the heme plane i n Mb02 2*. s i m i l a r to the s i t u a t i o n found i n the OJ-subunit 1 o o of Hb02 . (In Mb02, these close contacts decrease from 3.6 and 3.5A o to 3.32 and 3.20A, respectively, while the Fe-N^ bond i s shortened from o 9 , 2.22 to 2.07A)^J-.*» The heme i t s e l f remains s i g n i f i c a n t l y non-planar with doming toward the d i s t a l side. A hydrogen bond e x i s t s between His F8 o (N^) and Leu F4(C0) as i n hemoglobin, which i s lengthened by 0.2A upon oxygenation 2*. The d i s t a l s i t e i s more crowded than i n the R-state of Hb, with the side chains His E7, Val E l l and Thr E10 blocking access into the heme pocket 2*. - 19 1.5 OTHER REVERSIBLE OXYGEN CARRYING HEMOPROTEINS Hb Zurich(|3) i s a modified version of normal human Hb i n which the d i s t a l residue His E7 i s replaced by an arginine i n the (3 s u b u n i t s 2 2 . The side chain of arginine E7 cannot be accommodated within the d i s t a l pocket, however, and so protrudes towards the surface leaving a large c a v i t y on the d i s t a l side of the heme 2 2. This r e s u l t s i n a more "open" ligand binding s i t e i n Hb Zurich(j3)^ compared to that i n normal Hb. Mb A p l y s i a . i s o l a t e d from a common gastropod mollusc, i s s i m i l a r to sperm whale or horse Mb (section 1.4) except that i t lacks the His E7 residue i n the d i s t a l binding pocket 2-^. CTT-Erythrocruorin (Ery) i s a monomeric hemoprotein from the l a r v a of the f l y Chironomus tummi. On comparison with Mb, the d i s t a l Val E l l residue i s replaced by an isoleucine, and the d i s t a l His E7 residue no longer l i e s i n the heme pocket but protrudes into the surrounding water 2^. In the c r y s t a l structure of deoxyEry 2^ a ' p a r t i a l l y o coordinated' water molecule i s located 3.2A away from the Fe centre and o o the i r o n i s displaced only 0.19A from the heme plane as opposed to 0.47A i n deoxyMb2^>2*. On coordination of dioxygen, the Fe i n E r y ( 0 2 ) 2 ^ b moves o even further away from the heme plane, 0.29A, toward the proximal imidazole. In E r y ( C 0 ) 2 ^ a ' 2 ^ , however, the Fe takes up a more cen t r a l o p o s i t i o n , 0.01A, with respect to both a x i a l ligands. Lephemoglobin (Lg-Hb), a monomeric hemoprotein found i n the root nodules of leguminous plants, i s considered to possess an unusually large d i s t a l c a v i t y i n which a more 'mobile' His E7 residue can move more f r e e l y than i n normal Hb or Mb2^. - 20 -Liv e r Fluke Hb (Dd-Hb) i s a monomeric hemoprotein i n which d i s t a l His E7 i s replaced by a glycine r e s i d u e 2 ^ . Glvcera dibranchiata (Gd-Hb), a monomeric hemoprotein i n which His E7 i s replaced by a leucine residue, i s also considered to have a 0 9 more 'open' binding pocket'' . Ascaris Hb i s found i n the p e r i e n t r i c f l u i d of p i g roundworm;3 very l i t t l e i s known about the binding pocket environment of this hemoprotein. 1.6 NATURE OF THE BOUND DIOXYGEN The MO d e s c r i p t i o n for dioxygen shows two unpaired electrons i n the doubly degenerate 7T antibonding o r b i t a l s , leaving 0 2 i n a t r i p l e t ground state configuration with a formal bond order of two (Fig. 1.8 A). Although thermodynamically 3 0, (eqs. (10)-(13), pH 7, 25°C) , 0 2 + e - ^ -» 0 2 _ E° - -0.4 v (10) 0 2" + 2H + + e" ^ v H 20 2 E° - +0.90 v (11) H 20 2 + 2H + + 2e" v ^ 2H 20 E° - +1.35 v (12) 0 2 + 4H + + 4e" ^ v 2H20 E° - +0.80 v (13) the add i t i o n of one electron with formation of a superoxo species (0 2") - 21 -is unfavourable, an overall four electron reduction is highly exergonic (AG° - -73 kcal/mol, eq (13)), this rendering molecular oxygen a powerful oxidizing agent. In biological systems this reduction of dioxygen to water is u t i l i z e d in energy requiring processes. Addition of one or two electrons into the 7T* antibonding orbitals of a neutral dioxygen molecule results in superoxo (O2") eq. ( 1 0 ) , and peroxo ( C ^ 2 " ) , eq. ( 1 1 ) , anions, respectively; the 0-0 bond lengths and bond energies for these species are consistent with reduction in bond order according to 02>02">02^", leaving O2* essentially with a bond order of 1.5 and O2 2" with a bond order of 1.0 (Table 1.1). The reduction in bond order correlates also with a reduced infra-red stretching frequency for the 0-0 bond,iv(0-0) . Table 1.1: Properties of the Dioxygen Moiety Species Bond Compound 0-0 Bond ^(0-0) Order Distance Energy cm"1 (A) (kcal/mol) 0 2 + 2.5 0 2AsF 6 1.123 149.4 1858 0 2 2 0 2 1.207 117.2 1554.7 0 2" 1.5 K0 2 1.28 — 1145 0 2 2' 1 Na 20 2 1.49 48.8 842 The geometry of the bound dioxygen in hemoglobin -remained a subject of considerable controversy, u n t i l the crystal structure determination of a heme model compound Fe(TpivPP)(Melm)(O2) , 3 (see section 2.4), confirmed the suggestion that dioxygen was bound to the heme i r o n with an 'end-on' mode of coordination. The e l e c t r o n i c nature of t h i s bent Fe-0" 0 bond i s s t i l l a matter under debate. The V(0-0) values f o r Fe-C>2 adducts f a l l into the range for a superoxo (O2") type species-^ < J J; i n addition, oxidation of an oxygen-bound i r o n can r e s u l t i n the release of oxygen as superoxide 3 4. However, Fe-02 complexes are e s s e n t i a l l y diamagnetic 3-'. There have been three bonding models proposed to account f o r these observations 3^. The Pauling model 3^ i s as follows: approach of dioxygen to the Fe centre leads to spin p a i r i n g of the electrons i n the 7T o r b i t a l s , following t h e i r loss of degeneracy. The Fe-02 D o n d then involves Odonation from dioxygen into the empty d z2 o r b i t a l of Fe, which i s balanced by d7T back donation from Fe into the antibonding (7T*) molecular o r b i t a l s of both 0* and 0 2. This accounts for the diamagnetism of the Fe-02 adduct. The Olafson-Goddard model3** i s s i m i l a r to that f or ozone where low spin Fe(II) i s considered to e x i s t i n an excited state while dioxygen retains a t r i p l e t configura-t i o n . E x c i t a t i o n of one ele c t r o n from the d^y into the d z2 o r b i t a l of Fe( I I ) , p o i n t i n g toward the t r i p l e t dioxygen, leads to a CT bond (Fig. 23 -B lone-pair on carbon w 2 molecule CO molecule non-bonding pair on oxygen O F i g . 1.8 Molecular o r b i t a l diagrams fo r 0 2 (A) and CO (B) T Q 0 r O o ' o 0^ o-overlap 7r-overlap B F i g . 1.9 The Olafson-Goodard bonding model of the Fe-02 moiety 24 1.9 (A), while 7T-overlap r e s u l t s from i n t e r a c t i o n between the remaining electron i n the d^y o r b i t a l with that i n the 7T* MO of 0 2 (Fig. 1.10 B). This model i s also consistent with a diamagnetic Fe-02 moiety. F i n a l l y , o q the Weiss model jy suggests some net transfer of electron density from i r o n to dioxygen, i n which a dipolar, superoxo-type formulation, Fe—O2", accounts for the J^(O-O) value. I t i s argued, however, that back dona-t i o n from Fe to 7T (0 2) o r b i t a l s could also weaken the 0-0 bond and s t i l l maintain an e s s e n t i a l l y covalent Fe-02 l i R k o v e r a l l 3 ^ . The extent of O bonding i s maximized at (Fe-'O-O) 120°, whereas 7Tbonding tends to increase t h i s angle toward 180° 38b Recent c r y s t a l structure refinements of Mb022* and Hb02*** have confirmed the e a r l i e r descriptions derived from neutron d i f f r a c t i o n data 4^, concerning the end-on o r i e n t a t i o n of the Fe-0-"*-1 l i n k , 3. In Mb02 the Fe-0-0 angle of 115-120° i s more acute than that i n Hb0 2 (Fe-f3-0 - 153-160°) . Extended Huckel c a l c u l a t i o n s indicate that t h i s v a r i a t i o n i s within the error l i m i t s predicted f or t h i s bond angle 120-160° ***. The difference r e s u l t s from l o c a t i o n of the d i s t a l residues i n the respective binding pockets, which, i n the more crowded d i s t a l s i t e of Mb02, are presumably closer to the dioxygen, and compress i t * * * . In addition, the close proximity of these residues i n Mb02 causes the Fe-0-0 plane to e c l i p s e the Fe-N3 axis, a s t e r i c a l l y unfavourable s i t u a t i o n 2 * which i s also found i n the Q! subunit of Hb02 ***. A strong hydrogen bond i s formed between the terminal oxygen 0 2 and the d i s t a l h i s t i d i n e E7 residue i n the binding pockets of Mb02 and the O! subunit of Hb0 2, an i n t e r a c t i o n that i s considered to s t a b i l i z e the bound O2 * & > 2 * . In the (3 subunit of Hb, however, the Val E l l and His E7 residues have been pushed further away from the dioxygen moiety; the d i s t a l His E7 i s - 25 -equidistant to both oxygen atoms and may form a much weaker hydrogen bond with ei t h e r of them***. In t h i s less crowded environment the Fe-O-0 pr o j e c t i o n has greater f l e x i b i l i t y , and does not e c l i p s e the Fe-Np axis but b i s e c t s i t x . In Ery(C>2) , the terminal oxygen atom forms a strong hydrogen-bond with a nearby water molecule i n the d i s t a l pocket 2**. 1.7 CARBON MONOXIDE BINDING IN HEMOPROTEINS Carbon monoxide, a neutral molecule with a bond order of three (Fi g . 1.8 B), binds to a ferrous i r o n with much higher a f f i n i t y than dioxygen**; t h i s has been a t t r i b u t e d to a greater degree of multiple bonding i n the Fe-C bond^*. ^-Donation from CO to Fe i s enhanced by 7Tback donation from Fe to CO, which i s maximized at angle of 180°. The resultant Fe-C-0 un i t i s l i n e a r and close to e l e c t r i c a l n e u t r a l i t y . S imilar bonding character i s involved i n the Fe-C=N-R adduct. Interest i n CO binding to hemoproteins a r i s e s from i t s ro l e i n b i o l o g i c a l systems where the catabolism of heme i s known to produce 1 mole of CO per heme^*^ 2. This causes -1% of the body's Hb and Mb to be bound as HbCO and MbCO, even i n the presence of a i r with zero p a r t i a l pressure of CO, P ^ - 0 ) ^ . A comparative i n t e r e s t between the binding of CO to that of 0 2 i n hemoproteins increased when i t was found that, i n several hemoproteins 2-' >^ • bound carbon monoxide (and cyanide) appeared t i l t e d to the heme normal, whereas i n simple t r a n s i t i o n metal complexes^ the M-C-0 l i n k i s i n v a r i a b l y l i n e a r . (The c r y s t a l structures of these CO-bound hemoproteins had been elucidated even p r i o r 26 to the oxy counterparts, because of the i n t r i n s i c s t a b i l i t y of the former toward oxidation.) The structures of Ery(CO)* , sperm whale MbC0 4 3 D and human HbCO 4 4, although the systems vary greatly i n t h e i r a f f i n i t i e s toward CO, a l l indicate a Fe-C-0 axis t i l t at an angle of -11-13° from the heme normal 4^. This o f f - a x i s t i l t i s thought to a r i s e from s t e r i c interference between globin residues and the l i n e a r l y coordinated ligand i n the d i s t a l binding pocket 3 3 , 4^» 4**, 4. This " d i s t a l s t e r i c hindrance" i s suggested to reduce the CO a f f i n i t y of some hemoproteins 3 3 * 4 9 • compared to that of simple Fe(II) complexes, and is considered responsible f o r the d e t o x i f i c a t i o n of CO i n b i o l o g i c a l systems-'-' where i t s primary source of occurrence i s endogeneous. Dioxygen, however, with favoured bent geometry*^' 2*' 3*, w i l l be less a f f e c t e d by s t e r i c crowding i n the d i s t a l s i t e 3 3 , 4 * * . This proposed s t e r i c d i s c r i m i n a t i o n of CO r e l a t i v e to 0 2 has received considerable a t t e n t i o n over recent years and has l e d to contradictory conclusions regarding i t s s i g n i f i c a n c e 4 ^ * . 4 CHAPTER 2 MODEL SYSTEMS AND DYNAMICS OF CO AND 0 2 BINDING - 28 -2. MODEL SYSTEMS AND DYNAMICS OF CO AND 0 2 BINDING S t a t i c c r y s t a l structure descriptions (sections 1.3 and 1.4) have shown that the most s i g n i f i c a n t changes occurring on ligand binding involve t e r t i a r y changes i n the immediate v i c i n i t y of the heme. These encompass: 1. D i s t a l E f f e c t s : S t e r i c obstruction may serve to lower the a f f i n i t y f o r the s i x t h l i g a n d * ^ * ^ >^ m A l t e r n a t i v e l y , hydrogen-bond formation to a d i s t a l residue may e l e c t r o n i c a l l y s t a b i l i z e or d e s t a b i l i z e the bound moiety***' 2*. 2. Proximal E f f e c t s : Non-bonded globin constraints on the proximal imidazole can prevent the required movement of the i r o n into the porphyrin plane on ligand binding-* • • *^. Further, changes i n b a s i c i t y of the proximal imidazole with strength of hydrogen-bond formation to.a nearby residue (Leu F4) may a f f e c t e l e c t r o n density at the i r o n centre*^' 2*. 3. Peripheral E f f e c t s : Non-bonded repulsions between the heme p e r i -phery and surrounding globin might be expected to influence heme t i l t * ^ • ***' -**. In addition, e l e c t r o n i c i n t e r a c t i o n s (hydrophobic or polar) between the porphyrin r i n g and p r o t e i n residues i n close proximity could a l t e r e lectron density of the heme moiety-**. In order to evaluate the r e l a t i v e importance of these various e f f e c t s on ligan d a f f i n i t y , i t i s necessary to assess t h e i r energy contributions to the dynamics of ligand binding at the heme.,. 29 2.1 REQUIREMENT FOR MODEL SYSTEMS Dynamic equilibrium studies with a l l o s t e r i c proteins are extremely complicated owing to the influence of both t e r t i a r y and quaternary changes on ligand binding. Furthermore, t h e o r e t i c a l c a l c u l a t i o n s ^ 2 coupled with experimental data on monomeric subunits indicate that each ligand encounters several b a r r i e r s during movement through the protein matrix, i n a d d i t i o n to the f i n a l b a r r i e r i n v o l v i n g Fe-ligand bond formation. Constants for CO and 0 2 binding for several hemoproteins reveal that subtle changes i n the p r o t e i n environment surrounding the heme give r i s e to large differences i n the rate of ligand e q u i l i b r a t i o n between the protein systems (Table 2.1); the o v e r a l l equilibrium constant (K L) for ligand binding to the five-coordinate heme, eq. (14). X L „ J (14) k k L i s given by K L = (15) where kL = the second order rate constant for ligand association, and k'^ = the f i r s t order rate constant for ligand d i s s o c i a t i o n . In order to investigate the influence of the immediate p r o t e i n environment (heme pocket) on the various aspects of ligand binding, modified or r e c o n s t i -tuted hemoglobins have been used for comparison with myoglobin and i s o l a t e d normal Oi and /3 chains of Hb. However, d i f f i c u l t i e s a r i s e i n Table 2.1 Constants f o r the Binding of CO and 0 2 to Hemoproteins i n Aqueous Media, pH 7.0, 20°C Protein ,C0 -CO (M _ 1 s' 1) ( s _ 1 ) °9 k 2 k 2 (M" 1 s" 1) ( s " 1 ) ,C0 (M"1) K K C 0 / K ° 2 Ref. Chelated Protoheme^ 3. .6 X 10 6 0. ,009 2. ,6 X 10? 47 4. ,0 X 10 8 5. .5 X 10 5 727 50, Hb 4. .0 X 10 6 0. .013 5. .0 X 10? 28 3. ,0 X 10 8 1. .7 X 10 6 176 c 4, .5 X 10 6 0, .008 6, .0 X 10? 16 6. .0 X 10 8 3. .8 X 10 6 158 0 SW-Mb 5. .0 X 105 0, .015 1, .5 X 10? 10 3. .3 X 10? 1. .5 X 10 6 22 6 AL-Mb 5, .0 X 10 5 0. .02 1, .5 X 10? 70 2. .5 X 10? 2. .1 X IO 5 119 6 Hb (Zh , /3) 2. .5 X 10? - 7. ,0 X 10 7 34 - 2. .1 X IO 6 22 Gd-Hb 2. .2 X 10? 0, .055 1. ,9 X 10 8 2800 4. .0 X 10 8 6. .8 X 10 4 5900 91 Dd-Hb 1. .1 X 10 8 0. .65 3, ,0 X 10 8 30 1. .7 X 10 8 1. .0 X 10? 17 80 CTT-Ery 2. .7 X 10? 0. .095 3. .0 X 10 8 218 2. .8 X 10 8 1. .4 X IO 6 200 92 Lg-Hb 1, .3 X 10? 0. .012 1. ,5 X 10 8 11 1. .1 X IO 9 1. .4 X 10 7 79 119 Ascaris-Hb 1, .7 X 10 5 0. .018 1. .5 X 10 6 0.004 9. .4 X 10 6 3, .8 X IO 8 0.025 119 [HRP 3. ,4 X IO 3 0. ,000016 5. 8 X 10 4 0.007 2. ,1 X 10 8 8. .3 X 10 6 25 ] | 120 o In 2% aqueous MTAB suspension at pH 7. 31 attempting to reconstruct these complicated protein molecules, whereby replacement of one residue i n the active s i t e , leads to other unforseen changes r e l a t e d to the t e r t i a r y structure as a whole^ 2 c. For instance i n Hb Sydney, s u b s t i t u t i o n of Val E11Q3) by alanine r e s u l t s i n a water molecule blocking access to coordination i n the d i s t a l s i t e . Moreover, hemoproteins with modified porphyrins reconstituted into the apoprotein also give r i s e to complicated k i n e t i c s because of globin interactions with the porphyrin peripheral substituents- 3*. Proton nmr studies with myoglobin have indicated that r e c o n s t i t u t i o n gives r i s e to changes i n heme o r i e n t a t i o n within the binding pocket which evidently have a s i g n i f i c a n t e f f e c t on oxygen binding a f f i n i t y - 5 ^ . j n order to i s o l a t e systematically the various contributions of pr o t e i n e f f e c t s on ligand binding, which may serve to d i f f e r e n t i a t e between the favoured binding of some ligands over others, researchers have long sought simple model oxygen c a r r i e r s that can mimic the r e v e r s i b l e binding behaviour of the hemoproteins-^. The advantage with these simpler systems lacking the prot e i n environment i s that they may be f u l l y characterized, and v a l i d comparisons within a series of s i m i l a r models c a r r i e d out to assess the r e l a t i v e importance of a p a r t i c u l a r e f f e c t on ligand a f f i n i t y . More complete information gained from studies on these simpler systems may then be applied to the more complex protein molecules. 2.2 P R O B L E M S A S S O C I A T E D W I T H M O D E L H E M E S T U D I E S Synthetic heme models a l l nec e s s a r i l y incorporate the -active s i t e of hemoglobin that has an ir o n atom coordinated to a porphyrin r i n g and - 32 -a x i a l imidazole, l b . The models are generally derived e i t h e r from porphyrins substituted i n the pyrrole p o s i t i o n s , (e.g. 5-8), as i n the natural systems, or from the meso-substituted tetraphenylporphyrin,9. U n t i l r e l a t i v e l y recently the use of simple ferrous porphyrins as models for r e v e r s i b l e oxygen carrying hemoproteins was thwarted for two r e a s o n s ^ . F i r s t , addition of a nitrogen-donor base to four-coordinate ferrous i n s o l u t i o n usually leads predominantly to a six-coordinate, low spin Fe(Por)(B)2 complex: Fe(Por) + B ~* Fe(Por)(B) ^ Fe(Por)(B) 2 (16) where K B B » Kg . For instance, deuteroheme dimethylester,8b,binds imidazole with Kg -4.5 x 103 M"1 and K f i B - 6.8 x 104 M"1, i n benzene at 25°C 5 5 . The reason for t h i s unusual sequence i n K values i s the large c r y s t a l f i e l d - 33 -s t a b i l i z a t i o n energy achieved i n going from a f i v e - to six-coordinate species i n the presence of a strong f i e l d ligand ( F i g . 1.2). Secondly, i n the absence of a large excess of base, B, addition of C>2 to these heme complexes at room temperature leads r a p i d l y and i r r e v e r s i b l y to formation of a bridged oxo-dimer:^^ F e n ( P o r ) ( B ) 2 ^ ^ - F e 1 1 (Por) (B) + (B) (17) F e I I ( P o r ) ( B ) + 0 2 v s F e 1 1 ( P o r ) ( B ) ( 0 2 ) (18) F e n ( P o r ) ( B ) ( 0 2 ) + Fe i : [(Por) (B)^=i(B) ( P o r ) F e I I I - 0 2 - F e I I I ( P o r ) (B) (19) ( B ) ( P o r ) F e I I I - 0 2 - F e I I I ( P o r ) ( B ) _if£i^ _> 2 F e I V ( P o r ) ( B ) ( 0 2 _ ) (20) F e I V ( P o r ) (B) (0 2-) + Fe I i ;(Por)(B) r a p i d > ( P o r ) F e I I I - 0 - F e I I I ( P o r ) + 2B (21) Autoxidation of the i r o n centre proceeds v i a i n i t i a l formation of a monomeric dioxygen adduct, eq. (18), followed by "dimerization" of two i r o n centres to give a peroxo-bridged complex, eq. (19). Evidence suggests that rapid decomposition of t h i s d i i r o n species to the stable ^l-oxo dimer proceeds v i a a f e r r y l Fe*^-0 2" i n t e r m e d i a t e ^ • . Alternate pathways are a v a i l a b l e i n the presence of protons- 5^. In hemoprotein systems, where dimerization of two i r o n centres cannot occur, autoxida-t i o n of the i r o n has been shown to be anion promoted with formation of the Fe***-anion (Met) species-^b. - 34 -2.3 EARLY MODEL STUDIES I n i t i a l "studies with open heme model complexes (such as 5b-9b) u t i l i z e d two methods that allowed for r e v e r s i b l e oxygenation: use of low temperature (-45 to -80°C)-* 4 • -*8, where /X-oxo dimer formation was s u f f i c i e n t l y retarded, so that oxygen binding occurred according to.eqs. (17) and (18); and coordination of the ferrous complex to a r i g i d polymeric surface containing a covalently attached donor ligand. This l a t t e r s o l i d state method prevented close approach of two i r o n centres so that dimerization could not occur-* 4. Q u a l i t a t i v e low temperature e q u i l i b r i a studies with v a r i e d solvent systems showed that autoxidation was slow i n non-polar solvents (toluene) but ra p i d i n p r o t i c media (methanol)^. In addition, within a serie s of apr o t i c solvent systems, oxygenation was enhanced i n more polar media such as dimethylforma-mide- 5^ 3. These e a r l i e r studies-* 4 were the f i r s t to indicate that the pr o t e i n environment i n hemoproteins was e s s e n t i a l to maintain f i v e -coordinate i r o n ( I I ) , provide a hydrophobic binding pocket f o r oxygena-t i o n to be r e v e r s i b l e ^ , and prevent the close approach of two i r o n centres. 2.4 MODEL DESIGN Continued e f f o r t s to overcome the problems associated with synthetic oxygen c a r r i e r s l e d to the development of more elaborate model hemes. One such model design involves covalent attachment of the desired base to a side chain of the porphyrin p e r i p h e r y ^ , 10-13. This - 35 -method obviates the necessity for large concentrations of exter n a l l y added base. In d i l u t e solutions, the close proximity of the chelated imidazole gives a five-coordinate heme, which at low temperature, r e v e r s i b l y binds o x y g e n i c . At room temperature, however, dimerization between two open faces again leads to autoxidation (eqs. (19)-(21)). 10 ,Cht lot»d m«»ohtm», R ^ C H j C H , . R ^ C H j C H ^ O j C H , t ! ,Ch» l a t«d p r o t o h t m e . R ' = C H = C H t . R * = C H , C H j C 0 2 C H , 1 2 , C h e l o t t d d i o c e t y l d e u l e r e h e m e . R*= C O C H , , R ^ C H , C H j C O j C H , ^ 3 , C h « l o t t d pyrreh«m», R ' = C H a C H t . R * - H Attempts to achieve r e v e r s i b l e binding at ambient temperature gave r i s e to a model design incorporating s t e r i c bulk on only one face of the porphyrin. On addi t i o n of low concentrations of a x i a l base B, formation of Fe(Por)(B)2 can be retarded because of s t e r i c bulk on one side, so that f i v e - c o o r d i n a t i o n i s predominant at room temperature. With both faces now e f f e c t i v e l y blocked, dimerization i s prevented and r e v e r s i b l e oxygenation at the s i x t h s i t e can be achieved. The f i r s t such model to appear was the "picket-fence" porphyrin*'*, 14a, with pivaloylamide groups providing the bulk on one porphyrin face. A five-coordinate heme 8, M s 2 H b, M r Fe 36 -complex, lAb(Melm), derived from t h i s model, bound 0 2 r e v e r s i b l y at room temperature: (22) The oxy complex thus formed was stable and allowed for the f i r s t c r y s t a l structure determination of a heme with bound d i o x y g e n 3 * * . A v a r i a t i o n of t h i s design was developed i n the capped porphyrin system 15b° , where straps containing ester and ether functions connect the cap to porphyrin periphery over one face. A d i s t i n c t advantage with the cap model system i s that the si z e of the cap may be v a r i e d by a l t e r i n g the number of methylene carbons i n the anchoring straps, 15-17. By t h i s design binding of a second base may be more e f f e c t i v e l y blocked, and the e f f e c t s of s t e r i c crowding with varying cap s i z e studied. Yet 37 another advantage with a l l models incorporating ' d i s t a l ' bulk i s that by changing the e l e c t r o n i c nature of the ' d i s t a l ' functions, the p o l a r i t y of the binding s i t e may also be al t e r e d . As well, a number of other systems have appeared which adopt the design with d i s t a l encumbrance alone, or i n combination with a covalent-l y attached coordinating N-donor base on the opposite side. These systems have been used to model the v a r i e t y of possible factors a f f e c t -ing l i g a n d binding to hemoproteins, i n e f f o r t s to understand the r e l a t i v e c o n t r i b u t i o n of these factors i n energy terms. 2.5 DISTAL STERIC EFFECTS 2.5.1 Hemoproteins ( i ) Isocyanide binding and k i n e t i c consequences of d i s t a l s t e r i c e f f e c t s . Olson's group 0 J have c a r r i e d out d e t a i l e d studies on isocyanide binding to hemoproteins. Isocyanides bind tenac i o u s l y to iron(II) centres with a l i n e a r mode of coordination, s i m i l a r i n nature to that of CO, but are s t e r i c a l l y more demanding ligands that involve three or more atoms i n a v e r t i c a l o r i e n t a t i o n r e l a t i v e to the porphyrin plane. In hemoproteins, a f f i n i t y f o r these ligands decrease with increasing s i z e of the a l k y l group ( K ^ ^ : methylisocyanide > isopropylisocyanide > t-butylisocyanide)° 3. Since the nature of the Fe-CNR bond i s the same 38 -i n a l l cases, t h i s change i n a f f i n i t y should be a r e f l e c t i o n of s t e r i c i n t e r a c t i o n between the bulky R group and pro t e i n residues within the d i s t a l binding pocket. The k i n e t i c consequence of the o v e r a l l reduction i n a f f i n i t y with increasing ligand bulk was revealed i n these studies to be manifested predominantly i n decreased a s s o c i a t i o n rates, with r e l a t i v e l y l i t t l e change i n d i s s o c i a t i o n rates even f o r the b u l k i e s t isocyanide (Table 2.5). This was interpreted i n terms of the free Table 2.2 Isocyanide Binding to Hemoglobin Compared to Chelated Protoheme^ 3 (11) Chelated a-Subunit Hb^ Rate Protoheme- Ratios CH.NC , , 7 c k 3 M"1 s ' 1 1.0 x 10 7 3.9 x 10 5 26 -CH,,NC , k 3 s ' 1 0.9 3.9 4 ki-PrNC M - l s - l 6.9 x 10 7 1.2 x 10 4 6 x 10 3 k-i-PrNC s - l 1,5 0.1 15 kt-BuNC M - l s - l 1.4 x i o 8 1.2 x 10 3 1 x 10 5 k-t-BuNC s - l 1 - 8 0.06 30 — In aqueous suspension, pH 7, 20°C — In aqueous micelles, pH 7, 20°C energy (AG assoc.) required to disrupt protein residues, i n order to e s t a b l i s h correct ligand o r i e n t a t i o n , p r i o r to Fe-L bond f o r m a t i o n 0 3 . I t is now widely accepted 0 3~°^ that a d i s t a l side s t e r i c i n t e r a c t i o n between the ligand and d i s t a l environment i s expressed predominantly by change i n the association rate at constant d i s s o c i a t i o n rate f o r a given system. ( i i ) CO and 0 2 binding The d i f f e r e n t nature of the Fe-L bond (for L = CO or 0 2) i s i l l u s t r a t e d by the asso c i a t i o n rate constant f o r the ligands to a 'bare' model heme complex (11), which i s -10-fold higher f o r 0 2 than f o r CO (Table 2.1). For these diatomics the d i f f e r e n t modes of coordination ( l i n e a r and bent f o r CO and 0 2, respectively) are considered to r e s u l t i n the s t e r i c d i s c r i m i n a t i o n of CO, leading to a lowered a f f i n i t y r e l a t i v e to 0 2 i n some hemoproteins (Section 1 . 7 ) 3 3 ' 4 9 • 5°. For instance, i n HbZurich(/3) , the more 'open' binding pocket i n the /3-sub-units c o r r e l a t e s with a higher a s s o c i a t i o n rate f o r CO with l i t t l e change i n as s o c i a t i o n rate f o r 0 2 4 9 (Table 2.1). S i m i l a r l y , i n other hemoproteins such as Dd-Hb 2 8, Gd-Hb 2 9, E r y 6 6 and LegHb 6 7, high CO a f f i n i t i e s r e l a t i v e to normal Hb are a t t r i b u t e d to replacement of the d i s t a l His E7 residue, which leads to a more 'open' or f l e x i b l e binding pocket (Section 1.5). However, i n contrast, the CO a f f i n i t y of normal R-state Hb i s much the same as that of the simple open chelated protoheme complex (11) i n aqueous suspension (Table 2.1) 4°•^ 3•° 8. 40 -In other hemoproteins such as deoxyMb, however, which shows a reduced CO a f f i n i t y r e l a t i v e to deoxyHb, d i s t a l s t e r i c i n t e r a c t i o n s may play a r o l e i n ligand binding 4*". A n i n v e s t i g a t i o n into the r e l a t i v e binding a f f i n i t i e s of 0 2, NO and CO i n horse Mb compared to the model protoheme complex, 6b(MeIm), i n aqueous suspension, showed reduction r a t i o s of 1.6, 1.9 and 15.0 for these gases, r e s p e c t i v e l y , i n the hemoprotein*^. (The s t e r i c requirement o f - n i t r i c oxide, which has a stable bent geometry when coordinated to an i r o n centre, should be s i m i l a r to that of 0 2 ) . These o v e r a l l a f f i n i t y r a t i o s indicate that binding of CO i s depressed 10-fold r e l a t i v e to 0 2 and NO i n the pro t e i n compared to the model heme. This study therefore supports the concept of d i s c r i m i n a t i o n against l i n e a r l y coordinated ligands r e l a t i v e to those with preferred non-linear geometry, purely on s t e r i c grounds **9. However, i n the absence of k i n e t i c information, caution should be exercised i n in t e r p r e t a t i o n . ( i i i ) T i l t i n g of the Fe-C-0 linkage The o f f - a x i s t i l t of the Fe-C-0 moiety observed i n hemoprotein c r y s t a l structures Is at present a subject of some contro-v e r s y 3 3 • 4**• 4 8* 68 . Heme t i l t , a r i s i n g from side chain-globin interac-tio n s , i s considered to play an important r o l e i n ligan d b i n d i n g * 4 . I t has been suggested 7 0 that a t i l t e d Fe-C-0 linkage may r e s u l t from heme t i l t rather than actual d i s t o r t i o n of the l i n e a r Fe-C-0 moiety, given the l i m i t a t i o n s of pr o t e i n s t r u c t u r a l r e s o l u t i o n . In support of t h i s i s a Fourier difference s t r u c t u r a l determination of Im(Met)Hb, - A l -which i n r e l a t i o n to H20(Met)Hb, reveals a considerable degree of heme t i l t corresponding to a r o t a t i o n of -10° i n the Im bound hemoprotein 7*; the bulky Im appears to be accommodated p r i m a r i l y by heme t i l t rather than by movement of d i s t a l residues within the binding pocket 7*. T h e o r e t i c a l c a l c u l a t i o n s also indicate that such heme t i l t i s of low 1A energy cost . Furthermore, hemoglobin with no s i g n i f i c a n t reduction i n CO a f f i n i t y r e l a t i v e to a model heme, and myoglobin with a 25-fold reduction i n CO a f f i n i t y , both show o f f - a x i s t i l t of the Fe-C-0 moiety i n t h e i r respective c r y s t a l s t r u c t u r e s 4 ^ . In e f f o r t s to evaluate systematically the importance of s t e r i c d i s c r i m i n a t i o n against CO, which may contribute to i t s d e t o x i f i c a t i o n i n hemoprotein systems, several simple model systems incorporating s t e r i c bulk into t h e i r d i s t a l s i t e s have been studied. 2.5.2 Anthracene and Adamantane Hemes The anthracene-7/7 , 18, and -6/6, 19, cyclophanes possess a large anthracene cap jo i n e d v i a amide linkages to the porphyrin p e r i p h e r y 7 2 . The adamantane heme, 20, has a bulky, non-aromatic group held close above the porphyrin r i n g 7 3 with a t o t a l number of cap linkages one atom less than that i n the 6/6-anthracene heme. - 42 -18 , Fe ^Anihracene-7,7) , n = 2 12, Fe (Anthracene-6,6) , n=1 P . -CK,CM,?-OCHj-Pn 20, F e (Adomontane) ( i ) Isocyanide binding The binding pocket shape and si z e i n these heme models was i n i t i a l l y probed using the bulky t-butylisocyanide and tosylmethyl-isocyanide ligands (Fig. 2 . 1 A and B , r e s p e c t i v e l y ) , which vary i n F i g . 2.1 Central Steric Effect / C W C H \ I N III C _ A _ A_ t-Butylisocyanide Peripheral Steric Effect C H , I N III c I Fe JB. Tosylmethyl-isocyanide D i s t a l s t e r i c e f f e c t s on i s o c y a n i d e l i g a t i o n w i t h i n the heme b i n d i n g p o c k e t t h e i r respective s t e r i c requirements for binding. t-Butylisocyanide (t-BuNC), with branching i n the t h i r d atom away from the metal centre, i s expected to s u f f e r a c e n t r a l s t e r i c e f f e c t from d i s t a l groups d i r e c t l y above the porphyrin macrocycle 7 3. Tosylmethylisocyanide (TMIC), with branching i n the fourth atom, would experience s t e r i c i n t e r a c t i o n with - 43 -d i s t a l groups o f f to one side of the binding pocket - t h i s isocyanide i s therefore regarded as being subject to a peripheral s t e r i c e f f e c t 7 3 . The binding a f f i n i t i e s of the model hemes 18-20 towards these two isocyanides, r e l a t i v e to that for chelated protoheme, having no d i s t a l r e s t r i c t i o n , leads to the following i n t e r p r e t a t i o n . The 7/7- and 6/6-anthracene hemes show a considerable c e n t r a l s t e r i c e f f e c t towards the binding of t-BuNC0-''73, an e f f e c t that i s more pronounced i n the t i g h t e r cap system (Table 2.3). This has been a t t r i b u t e d to the large anthracene "roof" being unable to swing to one side of the binding p o c k e t 7 3 . The adamantane heme shows les s s t e r i c hindrance toward the binding of t-BuNC compared to the 6/6-anthracene heme, but a s l i g h t l y Table 2.3 Isocyanide Binding to Anthracene-7/7, -6/6 and Adamantane Hemes i n Benzene/Toluene at 20°C ^ Compound Kt-BuNC Reduction K T M I C Reduction (M"1) Factor (M"1) Factor (11) Chelated Protoheme 1.7 x 10 8 1 7.0 x 10 9 1 (18) Fe(anthracene-7/7)(Dclm) 8.0 x 10 4 2 x 10 3 7.0 x 10 8 10 (19) Fe(anthracene-6/6)(MeIm) 1.5 x 10 2 1 x 10 6 7.0 x 10 5 1 0 4 (20) Fe(adamantane)(MeIm) 3.0 x 10 4 5 x 10 3 2.0 x 10 5 3 x 10 4 - 44 -greater s t e r i c e f f e c t toward TMIC 7 3. The c r y s t a l structure of the free base adamantane p o r p h y r i n 7 3 indicates that a cap a l k y l hydrogen, which o i s c l o s e s t to the porphyrin (-3.5 A away), i s o f f to one side of the binding pocket thus presumably i n t e r f e r i n g with lig a n d binding v i a a periph e r a l s t e r i c e f f e c t . Thus, the anthracene-6/6 heme i s considered to show a severe c e n t r a l s t e r i c e f f e c t , while the adamantane heme a peripheral s t e r i c e f f e c t toward ligand binding. ( i i ) CO and 0 2 binding Table 2.4 shows the k i n e t i c data for CO and 0 2 binding to these hemes (18-20). N-Methylimidazole (Melm, 39) was found to coordinate under the 7/7-anthracene cap, and therefore the b u l k i e r 1,5-dicyclo-hexylimidazole (DCIm, 40) was used as a proximal base**-*•73. I t can be c l e a r l y seen that both CO and 0 2 s u f f e r considerable s t e r i c hindrance to binding as evidence by t h e i r reduced ass o c i a t i o n rate r e l a t i v e to that f o r binding to chelated mesoheme 0^• 7 3. The r a t i o s of the ass o c i a t i o n rate constants f o r the s t e r i c a l l y hindered heme compared to an open heme represents the extent of s t e r i c d i s c r i m i n a t i o n toward CO r e l a t i v e to 0 2. The la r g e s t discriminatory e f f e c t on CO, seen i n the adamantane heme, i s less than 3-fold r e l a t i v e to chelated mesoheme. This factor i s s r n a l l 0 ^ and does not account f o r larger differences seen i n the hemoproteins. Therefore, from studies with these model hemes e x h i b i t i n g both c e n t r a l and peripheral s t e r i c e f f e c t s , Traylor has concluded that d i s t a l side s t e r i c e f f e c t s play only a minor r o l e i n any dis c r i m i n a t i o n against CO i n hemoproteins 0-*. Table 2.4 Constants f o r CO and 0 2 Binding to Pyrrole at 2 0 ° C 6 5 - 7 A » 7 5 Compound k o u k ' u u (M" 1 s" 1) ( s ' 1 ) (10) Chelated Mesoheme 1. .1 X 10 7 -0. .05 (18) Fe(anthracene-7/7)(Dclm) 6. .0 X 10 6 0, ,05 (18) Fe(anthracene-6/6)(Melm) 3. .0 X 10 4 0. .05 (20) Fe(adamantane)(Melm) 9 .2 X 10* 0. .05 (26) Fe(pyridine-5/5)(Melm) 6, .3 X IO 2 0. .24 (22) Fe-Cu-5(THPIm) 9 .0 X 10 4 0. .02 (21) Fe-Cu-4(MeIm) 2, .0 X 10 4 0. .02 (25) Fe-SP-15(DcIm) 9. .0 X 10 4 0. .04 (24) Fe-SP-14(DcIm) 8 .0 X 10 3 0. .04 (25) Fe-SP-13(MeIm) 6. .0 X 102 0, .07 Unpublished data i n parentheses from Chang's systems Substituted Model Hemes i n Benzene/Toluene KC0 k 2 k 2 0, K 2 k° 2/k' (M'1 ) (M" 1 s" 1) ( s " 1 ) (M"1) 2. .2 x 10 8 8. 4 x 10 7 4800 1. .7 x 10 4 7.6 1. .2 x 10 8 6. 0 x 10 7 1000 6. ,0 x 10 4 11 6. .0 x 105 1. 0 x 10 5 800 1. 3 x IO 2 3 1. .8 x 105 1. 5 x 10 5 690 2. 2 x 10 2 16 2. .6 x 10 3 1. 1 x 10 4 68 1. 6 x 10 2 17 4. .5 x 10 6 1. 8 x 10 6 91 2. 0 x 10 4 20 1. .0 x 10 6 5. 2 x 10 5 160 3. 0 x 10 3 26 2. .2 x 10 6 1. 7 x 10 6 250 6. 8 x 10 3 19 2. .0 x 105 3. 0 x 10 5 (120) 2. 5 x 10 3 38 8. ,5 x i o y (2 .4 x 10 4) (130) 1 .8 x 10 2 (40) p r i v a t e l y communicated by T.G. Traylor. 2.5.3 C o f a c i a l Diporphyrin and Strapped Model Hemes R 21 , F e - C u - 4 . n - I 22. < F e - C u - 5 . n • 2 R-n-pentyl 23 , Fe SP-l3.n • 5 24 . Fe SP-I4. n - 6 25. , Fe SP-I5. n« 7 S t e r i c a l l y hindered models studied by Ward et a l . ' ^ include mixed metal c o f a c i a l diporphyrins and cyclophane strapped porphyrin systems. The c o f a c i a l diporphyrin complexes, 21 and 22, have an i n e r t copper porphyrin l i n k e d to the heme thereby providing d i s t a l compression toward a s i x t h ligand, while the strapped systems 23-25 provide a "shearing" stress towards l i g a t i o n 7 4 . For the larger cap systems Melm was found to coordinate on the " d i s t a l " side thus forming six-coordinate b i s base complexes, Fe(Por)(B)2. Therefore more bulky imidazoles THPIm (Al) and DCIm (40) were used, for which only five-coordinate complexes were formed i n solutions containing excess b a s e 7 4 . ( i ) CO and 0 2 binding The k i n e t i c s of CO and 0 2 binding to these five.-? coordinate s t e r i c a l l y hindered hemes show . a general reduction i n l i g a n d associa-- 47 -t i o n rate compared to chelated mesoheme, (Table 2 . 4 ) 7 4 . The i n i t i a l i n t roduction of s t e r i c hindrance i n the Fe-Cu-5 and FeSP-15 systems r e s u l t s i n a 3-fold discriminatory e f f e c t toward CO r e l a t i v e to 0 2. I n t e r e s t i n g l y , further decrease i n pocket si z e provides l i t t l e further s t e r i c d i f f e r e n t i a t i o n : 4-fold i n the Fe-Cu-4(MeIm) complex, and 6-fold i n the FeSP-14(DCIm) and FeSP-13(Melm) systems. 2.5.4 Pyridine - 5 / 5 Cyclophane Another v a r i e t y of a p y r r o l e - s u b s t i t u t e d capped heme i s found with the pyridine-5/5 cyclophane system 26, i n which a polar pyridine moiety constitutes a 'cap' over the d i s t a l environment of the heme7-*. The r i g i d amide linkages of the strap apparently clamp the pyridine i n such a manner with respect to the porphyrin plane so as to prevent chelation of the attached pyridine, e i t h e r i n t e r n a l l y or e x t e r n a l l y , to the four-coordinate i r o n c e n t r e 7 ^ , eq. (23). - 48 -(23) Even i n the presence of CO, the pyridine s t i l l f a i l s to l i g a t e , despite the increased a f f i n i t y of a five-coordinate, Fe(Por)(C0) species toward an a x i a l base r e l a t i v e to a four-coordinate heme (Section 4.5.2(a)). In contrast, the r e l a t e d TPP-derived "hanging-pyridine" heme, 45, which also incorporates amide linkages into a short strap that attaches pyridine to meso-phenyl periphery, e f f e c t i v e l y permits i n t e r n a l coordination of the base to form a five-coordinate system. Under high concentrations of ex t e r n a l l y added base, the f i v e -coordinate pyridine-5/5 cyclophane allows coordination of CO and 0 2 under the cap, on the ' d i s t a l ' side of the heme. The a f f i n i t i e s f o r these ligands are greatly reduced r e l a t i v e to an open heme, because of the s u b s t a n t i a l s t e r i c hindrance imposed by such a t i g h t , r i g i d cap (Table 2.4) 7-\ The discriminatory factor with respect to ligand association, derived from a comparison of k ° 2 / k ^ f o r the pyridine heme with that f o r an open heme, however, reveals only a 3-fold d i s t a l s t e r i c e f f e c t toward CO binding r e l a t i v e to 0 2. Once again, t h i s model heme exemplifies a s i t u a t i o n i n which d i s t a l s t e r i c d i s c r i m i n a t i o n of CO i s s l i g h t , despite enormous hindrance to ligand association 7-*. - 49 -2.5.5 Picket-Fence and Pocket Systems Ligand binding i n the s t e r i c a l l y encumbered "pocket" porphyrin s e r i e s 27-29 7° has been compared to that i n the more open "chelated picket- fence" heme, 3 0 7 ° . The pocket porphyrins, that possess a c e n t r a l benzene cap of varying s i z e , might be expected to provide a c e n t r a l s t e r i c e f f e c t toward l i g a t i o n within the d i s t a l c a v i t y . The Fe(TalPoc) pocket , 29, was not useful within t h i s comparison since the cap was too large to preclude binding of Melm on the d i s t a l side; other b u l k i e r imidazoles were not used. ( i ) CO and 0 2 binding Comparing liga n d a f f i n i t i e s alone (Table 2.5(a)), those for 0 2 are very s i m i l a r within the s e r i e s whereas CO a f f i n i t y i s dramatically r e d u c e d 0 4 i n the smallest binding pocket: 3 - f o l d reduction for Fe(MedPoc)(Melm) and 147-fold reduction for Fe(PocPiv)(Melm) r e l a t i v e to chelated picket-fence. This reduction i s r e f l e c t e d p r i m a r i l y i n - 50 -decreased CO association rates, with r e l a t i v e l y constant d i s s o c i a t i o n rates, consistent with the k i n e t i c expression of d i s t a l side s t e r i c e f f e c t s 0 ^ for l i g a t i o n i n other systems0-* • 7^. Dioxygen binding a f f i n i t y i s , however, influenced by decreased a s s o c i a t i o n rates as well as  decreased d i s s o c i a t i o n rates r e l a t i v e to the more open chelated picket-fence heme. Since the e l e c t r o n i c nature of the binding s i t e i s considered very s i m i l a r within the s e r i e s 0 ^ , i t has been suggested that the k i n e t i c v a r i a t i o n i n dioxygen binding i n the s t e r i c a l l y encumbered pockets i s of s t r u c t u r a l o r i g i n 0 ^ . I t i s speculated that "dioxygen i s forced to squeeze into and out of the pocket i n an arrangement not favoured r e l a t i v e to optimal t r a n s i t i o n state geometry". This may serve to disrupt both the a s s o c i a t i o n (k°2) and d i s s o c i a t i o n (k"°2) rates, tending to lower them both to a s i m i l a r extent^ 4. Table 2.5(a) shows the k i n e t i c reduction factors for k^2 and k"^2 i n the pocket systems r e l a t i v e to chelated picket fence; the factors are s i m i l a r i n magnitude with i n a given system. Analogous but l e s s pronounced behaviour with regard to d i s t a l s t e r i c i n t e r a c t i o n s i s observed for the "T-state" pocket-heme s e r i e s , (Table 2.5(b)), compared to the "T-state" picket-fence system, with 1,2-Me2lm as base (see section 2.6.2)° 4. The above i n t e r p r e t a t i o n of CO and 0 2 binding to these pocket complexes therefore lends strong support to the suggestion that CO can be discriminated against r e l a t i v e to 0 2 v i a d i s t a l side s t e r i c e f f e c t s 0 ^ . Table 2.5(a) Constants f o r i n Toluene at CO and 0 2 25°C 6 4 Binding to R-State "Chelated Picket-Fence" and "Pocket" Hemes Compound ,C0 .-CO (M" 1 s' 1) ( s _ 1 ) rCO k 2 Reduction k Reduction (M"1) (M" 1 s' 1) Factor ( s _ 1 ) Factor (M'1) (30 ) Chelated Pf (28) Fe(MedPoc)(MeIm) (27) Fe(PocPiv)(MeIm) 3.6 x 10 7 0.0078 4.6 x 10 9 4.3 x 10 8 1 1.5 x 10 6 0.0094 1.6 x 10 8 1.7 x 10 7 25 5.8 x 10 5 0.0086 6.7 x 10 7 2.2 x 10 6 195 2900 1 71 41 9 322 1.5 x 10-2.4 x 10-2.4 x 10 5 Table 2.5(b) Constants f o r CO and 0 2 Binding to T-State "Picket-Fence" and "Pocket" Hemes i n Toluene at 25°C 6 4 Compound rC0 .-CO (M" 1 s".1) ( s ' 1 ) rCO (M"1) ° 9 k 2 (M" 1 s" 1) Reduction k z Reduction K z Factor ( s " 1 ) Factor (M"1) (14b) Fe(TpivPP)(l,2-Me 2Im) 1. .4 x 10 6 0. .14 1. .0 x 10 7 1. .06 x 10 8 1 46000 1 2. .3 x 10 3 (28) Fe(MedPoc)(l,2-Me 2Im) 2. .1 x 10 5 0, .053 4. .0 x 10 6 5. .2 x 10 6 20 800 58 6. .5 x 10 3 (27) Fe(PocPiv)(l,2-Me 2Im) 9. ,8 x 10 4 0. .055 1. .8 x 10 6 1, .9 x 10 6 56 280 164 6. .8 x 10 3 - 52 2.5.6 C 2- and C3-Capped Model Hemes Comparison of the c r y s t a l structures of the H 2C 2-cap free base p o r p h y r i n 7 7 3 15a, and C 2-cap hemin c h l o r i d e 7 7 * 3 complex, F e m ( C 2 - c a p ) C l , reveals considerable buckling of the porphyrin plane i n the free-base o o porphyrin (mean 0.135 A, max 0.354 A displacement from porphyrin plane), which i s r e l i e v e d to some extent i n the Fe(C 2cap)Cl complex (mean 0.076 o o 77 A, max 0.208 A displacement)''. The separation between the cap to o porphyrin centre i n the hemin chloride i s -4.0 A i n the s o l i d state, a distance too short to accommodate comfortably an Fe-C-0 moiety 7 7' 3. In s o l u t i o n , the larger C3-cap does not prevent b i s base complexation for B «• Melm, and therefore Dclm was used as external base f o r t h i s system (16b). Dioxygen a f f i n i t y measurements on the capped heme models 15-17b, were c a r r i e d out at temperatures below 0°C, where /i-oxo-dimer forma-t i o n was retarded and r e v e r s i b l e oxygenation achieved. A comparison of binding a f f i n i t i e s f o r the five-coordinate hemes, with B - l,2-Me2Im, r e l a t i v e to that for the open Fe(TPP)(1,2-Me 2Im) system shows that CO 70 a f f i n i t i e s are e s s e n t i a l l y unaffected by the presence of the cap'° (Table 2.6); considerable expansion of the d i s t a l c a v i t y must therefore occur i n s o l u t i o n , r e l a t i v e to the s o l i d s t a t e 7 7 a . The 0 2 a f f i n i t y measurements indi c a t e a small s t e r i c e f f e c t (5-fold) toward binding i n the Fe(C 2-cap)(l,2-Me 2Im) heme r e l a t i v e to Fe(T(p-OCH 3)PP)-(1,2-Me 2Im) 7 8 a. The C 2-capped system was therefore considered to impose per i p h e r a l s t e r i c hindrance toward the incorporation of a bent Fe-0 2 moiety, with l i t t l e c e n t r a l s t e r i c e f f e c t toward a l i n e a r Fe-CO arrangement 7 8 3. 53 Table 2.6 CO, 0 2 and NO Binding to C 2-, C 3 - , and C^-Capped Hemes i n Toluene 7 8 P c o (torr) P ° 2 (torr) P N 0 (torr) 1/2 1/2 1/2 25°C -45°C 25°C (9b) Fe(TPP)(l,2-Me 2Im) 0.14 5.3^ 1.1 x IO" 7 (15b) Fe(C 2-cap)(l,2-Me 2Im) 0.20 2.7 2.0 x 10" 6 (16b) Fe(C 3-cap)(l,2-Me 2Im) 0.14 6,000 3.3 x IO" 6 (17b) Fe(C 4-cap)(l,2-Me 2Im) 4.1 - 4.1 x IO' 5 £ Value quoted for Fe(T(p-OCH 3)PP)(1,2-Me 2Im), 51 (l,2-Me 2Im) More recently, the binding of n i t r i c o x i d e 7 8 c to the capped heme ser i e s has been investigated, the data lending further support to the existence of a perip h e r a l s t e r i c i n t e r a c t i o n afforded by the caps i n these systems. Examination of the NO a f f i n i t i e s to the C 2- and C 3-cap models indicates considerable reduction i n NO binding to these hemes, where the v a r i a t i o n i n a f f i n i t y i s i n the order: TPP (1) > C 2-cap - C 3-cap (20) > C 4-cap (40) with r e l a t i v e reduction i n NO binding represented i n brackets. 54 -The very large reduction i n 0 2 a f f i n i t y f o r the Fe(C3-cap)-(1,2-Me2lm) complex has been a t t r i b u t e d to interference from coordina-t i o n of a second 1,2-Me2lm a x i a l base under the la r g e r cap at low temperature 7 8 0. Ligand a f f i n i t i e s are i n v a r i a b l y higher at lower temperatures, where even the s t e r i c a l l y hindered 1,2-Me2lm ligand w i l l form a b i s base complex. Experiments studying the e f f e c t of base concentration on O2 a f f i n i t y , used to determine 1^2 f o r Fe(T(p-0CH3)PP)-(1,2-Me 2Im)2^ 8 a, were not , however, c a r r i e d out on the Fe(C3-cap)-(1,2-Me2lm)2 complex. Moreover, d e t a i l e d s p e c t r a l studies i n v o l v i n g t i t r a t i o n s of the six-coordinate Fe(C3-cap)(Melm)2 system with C^ 7 9, seemed to indicate that the dioxygen molecule was not d i s p l a c i n g the second imidazole, but i n fac t adding d i r e c t l y to form a pseudo-seven-coordinate o x y - s p e c i e s 7 9 . A presumed side-on coordination of the second a x i a l base i n the d i s t a l environment was considered to be weak, as increasing [B] d i d not a l t e r O-2 a f f i n i t y s i g n i f i c a n t l y . Further evidence i n support of t h i s was gained from magnetic s u s c e p t i b i l i t y measurements, resonance Raman and IR s t u d i e s 7 9 . The 'corrected' O2 a f f i n i t y of the five-coordinate Fe(C3-cap)(Melm) complex was s t i l l very low r e l a t i v e to that f o r Fe(C2-cap)(Melm) 7 9 - a s i t u a t i o n analogous to that seen with l,2-Me 2Im as proximal base 7 8* 1. On replacement of the methyl-substituted Imidazoles with the bulky Dclm, coordination under the C^-cap i s precluded even down to 0°C. I n t e r e s t i n g l y , at t h i s temperature, the five-coordinate Fe(C3-cap)(Dclm) complex appears to bind O2 with only a 3 - f o l d reduced a f f i n i t y r e l a t i v e to the C2-cap s y s t e m 7 8 0 . The low O2 binding a f f i n i t y of the Fe(C3-cap)-(Melm) and (1,2-Me2lm) systems at low temperature, thus remains poorly understood. - 55 The sudden drop i n a f f i n i t y toward CO and NO for Fe(C 4-cap)-(1,2-Me2lni) was a t t r i b u t e d 7 8 0 to the large cap of t h i s system, which i s considered t o achieve a "squashed" or "twisted" configuration (31) that r e s t r i c t s ligand binding to the s i x t h coordination s i t e . Studies on the heme systems with B - Dclm are not considered here because o f i n s u f f i c i e n t data for useful comparison. o 21 2.5.7 C r y s t a l Structure of a S t e r i c a l l y Distorted Fe-C-0 Moiety A family of non-porphyrinic, five-coordinate i r o n ( I I ) "lacunar" complexes 32-35 capable of r e v e r s i b l y binding 0 2 and CO, has been synthesized by Busch et al.°^. The ferrous i r o n i s situated within a c e n t r a l core of four nitrogens with an a x i a l base, B - CI, CH3CN, py, etc., l i g a t e d . The bridging group, R*, forms a c a v i t y within which a coordinated s i x t h ligand i s accommodated. The complexes are i s o l a b l e as five-coordinate protonated c h l o r o - s a l t s 8 ^ 3 . The c r y s t a l structures of two such models 32 and 3380b^ v i t h R* - m.-xylylene, reveal that a simple replacement of R 2 - H by CH 2 r e s u l t s i n a s i g n i f i c a n t modification i n the size of the vacant ce n t r a l cavity. In the -(NH) 2- complex 32, the 56 B L a c u n a r Complexes m-xylylene (N-H^ 22 m-xylylene N - C H - 3 3 — 3 2 - ( C H 2 ) 6 (N - C H 3 ) - 2A •(CH2) ( N - H ) 2 - 3_5 R m-xylyl m-x y I y I - ( C H 2 ) 5 -B = CI R 2 R 3 H C H 3 C H 3 C H 3 C H 3 C H 3 H C H 3 B F i g . 2.2 C r y s t a l structures shoving the " t a l l " (A) and "short" (B) binding pockets o f the lacunar complexes 32 and 33, r e s p e c t i v e l y o o d i s t a l c a v i t y i s t a l l and narrow, 7.57 A high i n front and 5.46 A high at the rear, while i n the -(N -CH3) - case 33, the opening i s short and o o wide with corresponding dimensions of 5.02 A and 3.94 A ( F i g . 2.2 A and B, r e s p e c t i v e l y ) . The displacement of the i r o n from the N 4 plane i s s i m i l a r to that found i n other five-coordinate, high spin, heme o o complexes, being 0.65 A and 0.54 A i n the -(N-H) 2- and - ( N - C H 3 ) 2 -- 57 -d e r i v a t i v e s , r e s p e c t i v e l y . S i g n i f i c a n t l y , the CO a f f i n i t y for these two complexes, with B - CI, decreases with contraction i n c a v i t y height, where the -(N-CH 3) 2- system shows a greater than 10-fold decrease i n K^ 0 r e l a t i v e to that f o r the -(N-H) 2- complex (Table 2 . 7 ) 8 0 b . Table 2.7 CO Binding to Five-Coordinate "Lacunar" Complexes i n A c e t o n i t r i l e , at 0 ° C 8 0 b Bridge A x i a l base [CI"] K C 0 mM t o r r " * (33) (N-CH 3) 2-m-xylyl CH3CN, CI" 1 0.0012 (32) (NH) 2-m-xylyl CH3CN, CI" 1 0.010 (35) (N-CH 3) 2(CH 2) 6 CH3CN, CI" 1 >100 (34) (NH) 2(CH 2) 5 CH3CN, CI" 1 <10"3 A d i f f e r e n t type of s t r u c t u r a l a l t e r a t i o n found between complexes 35 and 34, i n v o l v i n g a shortening of the bridging methylene chain (R*) by one carbon, r e s u l t s i n an enormous reduction i n CO a f f i n i t y of more than 1 0 5 - f o l d (Table 2 . 7 ) 8 0 b . A c r y s t a l structure of the CO bound -(CH 2) 5(NH 2)- complex with B - p y r i d i n e 8 0 b reveals a t i l t e d Fe-C-0 moiety r e l a t i v e to the normal to the N 4 plane ( F i g . 2.3 A ) , accompanied by bending i n the Fe-C-0 angle. F i g 2.3 B depicts the extent of t h i s d i s t o r t i o n which i s considered to r e s u l t from " s t e r i c i n t e r a c t i o n - 58 -Fi g . 2.3 C r y s t a l structure (A) and diagramatic i l l u s t r a t i o n (B) of a di s t o r t e d Fe-C-0 moiety within the severely hindered binding s i t e of the lacunar complex, 35 (py). between the bound CO and pentamethylene bridging group" 8 nb. ^he c i o s e s t o methylene hydrogen atoms pointing into the c a v i t y are -2.62 and 2.92 A from the oxygen of CO, the former of which i s less than the predicted Van der Waals contact distance of 2.7 A ° ^ b . Other s t r u c t u r a l parameters o of Fe geometry, including displacement from the N 4 plane (0.05 A), are s i m i l a r to those seen i n six-coordinate low spin heme complexes. This non-porphyrinic complex i s the only known model structure to show a d i s t o r t e d bound CO. Resonance Raman studies on the CO complexes of the strapped FeSP heme se r i e s (23-25) also i n d i c a t e increased t i l t of the Fe-C-0 moiety with decrease i n strap l e n g t h 8 * : the Soret e x c i t a t i o n r e s u l t e d i n an enhancement i n the i n t e n s i t y of the Fe-CO bending mode r e l a t i v e to that of the Fe-CO s t r e t c h i n g frequency. Furthermore, a bending mode could not be detected i n a simple, open heme model which possesses no d i s t a l hindrance to CO b i n d i n g 8 * . The enhancement of t h i s v i b r a t i o n a l mode was suggested to a r i s e from possible charge transfer from porphyrin (7T*) to C0( TT*) o r b i t a l s , f a c i l i t a t e d by i n t e r a c t i o n between the CO and pyrrole nitrogens, which i n turn increases with greater Fe-C-0 t i l t 8 * . These studies lend support to he suggestion that a d i s t o r t e d - 59 -Fe-C-0 linkage may well be c o r r e l a t e d with severe non-bonded i n t e r a c t i o n between CO and d i s t a l functions of the binding c a v i t y , which serves to lower CO a f f i n i t y 8 0 * 0 , 8 * . For the lacunar complexes, whether there i s a discriminatory e f f e c t toward CO r e l a t i v e to 0 2 binding, however, awaits 0 2 a f f i n i t y determination under s i m i l a r conditions of base and solvent p o l a r i t y . 2.5.8 R e v e r s i b i l i t y and S t e r i c "Protection" i n Dioxygen Binding For the various s t e r i c a l l y encumbered systems studied, i t i s u s e f u l to consider the extent to which r e v e r s i b l e oxygenation i s achieved during e q u i l i b r a t i o n with 0 2. A l l trans-doubly-strapped d e r i v a t i v e s such as 44-46 and 49 are stable toward oxidation at room temperature, regardless of dioxygen a f f i n i t y . In these systems, incorporation of a coordinating base into one of the diagonal strapping groups provides added pr o t e c t i o n against dimerization since both porphyrin faces are e f f e c t i v e l y blocked. This Is c l e a r l y i l l u s t r a t e d by the oxidation h a l f - l i f e times ( t j y 2 ) f o r the four-coordinate "basket-handle" hemes 36 a and b corresponding to 11 min and 6 s for the c r o s s - t r a n s - l i n k e d and adjacent-cis l i n k e d isomers, r e s p e c t i v e l y 8 2 . - 60 -Within the class of compounds that possess s t e r i c hindrance over only one porphyrin face, the picket-fence (14b and 30)^4, pocket-hemes (27-29)64 a n d c o f a c i a l - d i p o r p h y r i n systems (21-22) 7 4 a l l su c c e s s f u l l y undergo r e v e r s i b l e oxygenation at room temperature. Dioxygen binding data for other strapped (23-25) 7 4 and capped (18-20, 2 6 ) 6 5 - 7 5 heme systems, however, were not obtained by d i r e c t t i t r a t i o n with 0 2, but instead, v i a k i n e t i c methods u t i l i z i n g f l a s h photolysis of the stable Fe-CO adduct i n the presence of CO /O2 mixtures (section 4.3.3(b)). A l t e r n a t i v e l y , studies at low temperature, allowing extrapolation to room temperature, provided K°2 constants f o r the C 2- and C3-capped systems (15-16) 7 8. Iron(II) complexes of the unique lacunar systems, 32-35, also undergo r e v e r s i b l e oxygenation at low temperature (<0°C) i n acetone-Melm-water mixtures, with Melm as coordinated base 8 0 3'**. Within the s e r i e s R* - jn-xylyl, increasing the s t e r i c bulk of R 2 and R 3 groups i s seen to a f f o r d more stable Fe-0 2 adducts. When both R 2 and R 3 are bulky benzyl and phenyl groups, re s p e c t i v e l y , oxygenation i s f u l l y r e v e r s i b l e even at room temperature, and the a f f i n i t y Is higher r e l a t i v e to other complexes of the same serie s (Table 2.8) 8 0 (*. On the basis of t h i s and other studies, i t was suggested that 8 0 0'' 3', where dimerization between an oxy-species and a five-coordinate heme, according to eqs. (19)-(21), i s not p o s s i b l e , oxidation of Fe(II) may occur v i a two other mechanisms: (1) D i s s o c i a t i o n of 0 2" from the Fe-0 2 adduct, t h i s process being promoted In p r o t i c media by s t a b i l i z a t i o n of the polar t r a n s i t i o n state, F e " I - 0 2 - 3 4 b . 8 0 c . - 61 -(2) Outer-sphere electron transfer from i r o n ( I I ) to free i n v o l v i n g p a r t i c i p a t i o n of small anionic molecules that f a c i l i t a t e formation of superoxide (O2") eit h e r by close approach or coordination to the deoxy i r o n centre 3 4 0» 8 ^ c • Table 2.8 Dioxygen "Protection" with Increasing S t e r i c Bulk Surrounding the D i s t a l S i t e of Lacunar Complexes, with R* = in-xylene, B - Melm 8 0 d R 2 R 3 t l / 2 ( a t * a t m ®2) Amax u CH-: C H 3 <1 min •35 CH 2C 6H 5 CH 3 CH 2C 6H 5 CH 3 C 6H 5 C 6«5 - 3 min - 15 min - 24 h -15 -5 20 Solvent i s acetone/Melm/^O - 3:1:1 by volume & Highest temperature at which r e v e r s i b i l i t y may be achieved A s t e r i c a l l y "protected" d i s t a l c a v i t y might be expected to hinder oxidation v i a the l a t t e r mechanism 8^ 0» d. A l t e r n a t i v e l y , a hig h l y polar d i s t a l c a v i t y , which s i g n i f i c a n t l y increases dioxygen a f f i n i t y , can also lead to ra p i d i r r e v e r s i b l e oxidation of the i r o n by promoting loss of 62 superoxide by mechanism (1); the high 0 2 a f f i n i t y and subsequent rapid oxidation of the camphonyl picket-fence heme (37) may exemplify such a s i t u a t i o n , that r e s u l t s from the "strong p o l a r i t y of the amide protons 1 183 _ 31 2.6 PROXIMAL STERIC EFFECTS 2.6.1 K i n e t i c Expression of Cooperativity i n Hemoglobin Examination of the independent r a t i o s f o r CO and 0 2 binding to the T- and R-quaternary states of H b ° 3 ' 8 4 , reveals that, while cooperative binding for 0 2 i s expressed v i a change i n d i s s o c i a t i o n rate, enhanced l i g a t i o n of the slower reacting CO molecule i s manifested p r i m a r i l y i n increased a s s o c i a t i o n rate to the R-state r e l a t i v e to the T-state of Hb 8 4 (Table 2.9). With respect to CO binding, the sequential a s s o c i a t i o n rate constants (k^ — > k 4) for l i g a t i o n to tetrameric Hb, show no monotonic increase for the T- to R-quaternary change 8^: k x (1.0) < k 2 (7.0) > k 3 (2.0) k 4 (48.0) 10 5 M"* s " 1 at pH 7.0, 20°C Although k i n e t i c cooperativity i s apparent for the binding of a second CO to form a Hb(C0)2 species, the rate of CO a s s o c i a t i o n to the t h i r d subunit (k 3) indicates a T-state type t e r t i a r y environment. Thus ligand a s s o c i a t i o n rates are influenced p r i m a r i l y by the l o c a l environment, determined by t e r t i a r y s t r u c t u r a l features of a p a r t i c u l a r binding s i t e , which may be changed both by ligand binding to a neighbouring subunit without change i n quaternary structure, or by the quaternary Q c O C s t r u c t u r a l change itself°. This and other s i m i l a r r e p o r t s 0 0 i n trends for CO binding to the hemoglobin tetramer, add support to the usefulness of ligand binding studies to i n d i v i d u a l heme units i n the absence of quaternary e f f e c t s . Table 2.9 Cooperativity i n 0 2 and CO Binding to " F i t t e d " R- and T-State Chains of Hemoglobin at pH 7.0, 20°C 6 3 L L -L -L Ligand k^/k^ k j /k^ K^/K^ 0 2 Ot 20 15 300 |8 5 120 600 CO (CVand/3) 50 10 500 Subscripts R and T denote the R- and T-states, r e s p e c t i v e l y - 64 -2.6.2 Nature of the Proximal Environment Sub s t i t u t i o n of N-methylimidazole (Melm), 39, as proximal base by 1,2-dimethylimidazole (l,2-Me 2Im), 42, or 2-methylimidazole (2-MeIm) 43, i n model heme systems leads to a substantial reduction i n ligan d a f f i -n i t y at the s i x t h a v a i l a b l e s i t e 8 7 . For the 2-substituted imidazoles, binding of a second ligand i s retarded to such an extent that Kg > Kg B, eq. (16), and at low base concentrations f i v e - coordination pertains about the heme i n simple open s y s t e m s 8 7 " 8 9 . For instance, with Fe(TPP), 9b, and l,2-Me2Im as base i n toluene at 20°C, log Kg - 4.43 and log Kg B - 0.39 7 8 a. The five-coordinate states of both types of systems B -Melm, or l,2-Me2Im, are e s s e n t i a l l y without s t r a i n , as indicated by the Kg equilibrium constants eq. (16), with s l i g h t differences i n a f f i n i t y r e s u l t i n g from v a r i a t i o n i n the base pK a values; e.g. for Fe(PocPiv), 27, log K M e I m - 4.46, pK a - 7.25, and log K 1 | 2 . M e 2 I m - 4.73, pK a -7.85 76. Thus, s t r a i n i s introduced into the "2-substituted" systems only on binding of a second a x i a l ligand. R-state Proximal Bases: 8 IV H CHj 38 39 40 N 41 T-state Proximal Bases; tr CH, 42 43 H 65 -A comparison of the c r y s t a l structure of the 5-coordinate model systems Fe(TpivPP)(2-Melm) 9 0 and Fe(TPP)(2-MeIm) 7 a with those of the hemoproteins (deoxyHb and deoxyMb)* 2 reveals remarkable s i m i l a r i t i e s i n o the Fe-Nj m bond distances (-2.1-2.2 A) and Fe displacement from the heme plane (0.40-0.55 A ) 7 . This adds further support to the suggestion of a lack of any globin induced s t r a i n i n the deoxy conformations of the hemoproteins* 4 >9*. 2.6.3 CO and 0 2 Binding The reductions i n CO and 0 2 a f f i n i t i e s f o r some R- and T-type model systems are given i n Table 2.10. For the f l a t "open" meso- and deuteroheme d e r i v a t i v e s , a 180-fold and 160-fold reduction i n CO a f f i n i t y i s observed 8 9, r e s p e c t i v e l y . Analogous binding behaviour i n the more encumbered systems gives r i s e to a f f i n i t y reduction r a t i o s of 30-50 f o l d f o r both CO and 0 2. The picket-fence systems, 14b(l,2-Me 2Im) and 30, seen to be somewhat anomalous among model hemes, are considered more f u l l y i n Section 2.8. This reduction i n a f f i n i t y f o r model hemes, although diminished i n magnitude r e l a t i v e to the KR/KJ r a t i o of -500 i n hemoglobin (Table 2.9), has allowed models with bases B - 38-41, and 42-43, to be l a b e l l e d R- and T-state systems, r e s p e c t i v e l y 8 7 , 8 9 . In the low a f f i n i t y , six-coordinate systems with bound 0 2 or CO, the presence of a methyl (or larger) substituent i n the 2- or 5-position of imidazole r e s u l t s i n s t e r i c a l l y unfavourable close contact between the substituent and porphyrin r i n g as the i r o n attempts to take up a more c e n t r a l p o s i t i o n with respect to both a x i a l l i g a n d s 8 7 , 8 8 (Fig. 66 N N R-H.CH, F i g . 2 . 4 "T-State s t r a i n " introduced on l i g a t i o n trans to a 2-substituted imidazole 2.4). The c r y s t a l structure of the 'strained' Fe(TpivPP)(2-MeIm)(0 2) n o O complex shows the i r o n to remain 0.1 A out of the heme plane toward the proximal imidazole, compared to the r e l a t i v e l y unstrained s i t u a t i o n i n Fe(TpivPP)(Melm)(0 2) where the i r o n i s i n the porphyrin plane 3*. This r e s t r i c t e d movement of the i r o n i n models i s analogous to the eclipsed o r i e n t a t i o n of the imidazole plane with respect to the N^-Fe-^ axis, seen i n T-state hemoglobin (Fig. 1.4). The resultant lowering i n ligand a f f i n i t y i n models thus lends support to the suggestion that such e c l i p s i n g i n T-state hemoglobin gives r i s e to unfavourable non-bonded repulsion between imidazole and porphyrin plane on ligand binding and i s thus p a r t i a l l y responsible for the low a f f i n i t y of the T - s t a t e 8 7 . S i g n i f i c a n t s t r e t c h i n g of the Fe-Nj m bond i s not evidenced, however, i n e i t h e r of the six-coordinate model systems 9* Fe(TpivPP)(Melm)(0 2) or Fe(TpivPP)(2-MeIm)(0 2). By comparison, a recent c r y s t a l structure refinement of deoxyHb shows the non-bonded -N^ distance to be - 0.1-O - i o 0.3 A shorter i n R-state Hb0 2 than i n T-state deoxyHb i Z. This i s only Table 2.10 Comparison of R- and T-State Constants f o r CO and 0 2 Binding to Model Hemes i n Toluene at 20°C Compound kCO k"2 k-C0 k""2 KC0 R"2 R / R / T / T / R / R / / k C 0 / k ° 2 / k - C 0 / k - ° 2 / K C 0 / K ° T T R R T T ° 9 k 2 Ref. (8b) Deuteroheme(Im)/(2-MeIm)^ 10 16 160 89 (7b) Mesoheme(MeIm)/(2-MeIm)^ 12 15 180 89 (19) Fe(anthracene-6/6)(Melm)/-(l,2-Me 2Im)£ 3.5 12 50 65 (18) Fe(anthracene-7/7)(Melm)/-(l,2-Me2Im) 10 5 50 70 (28) Fe(MedPoc)(Melm)/(1,2-Me2Im) 7 3 3 11 40 35 64 (27) Fe(PocPiv)(MeIm)/(l,2-Me 2Im) 6 1 6 31 40 35 64 (15b) Fe(C 2-cap)(MeIm)/(l,2-Me 2lm) 37 170^ 78 (16b) Fe(C 3-cap)(DcIm)/(l,2-Me 2Im) 34 78 £ In benzene at 20°C k- In 2% aqueous CTAB suspension at 20°C £ Unpublished, r e f . 70 & K ° 2 determined by extrapolation from low temperature studies; problem with base binding under cap (section 2.5.6). - 68 -o s l i g h t l y less than the difference of (0.3-0.5 A) corresponding to movement of the ir o n toward the porphyrin nitrogens on oxygenation of -1 o Hb x . Since the reduction i n a f f i n i t y for the R- to T-state change i n hemoglobin i s generally s i g n i f i c a n t l y greater than i n model hemes, the non-bonded repulsion between a x i a l base and porphyrin, which serves to lower ligand a f f i n i t y i n model systems, i s not the sole cause of lowered a f f i n i t y (-500 fold) i n the T-state of hemoglobin* 2. Low a f f i n i t y i n the hemoprotein i s l i k e l y to ari s e from a v a r i e t y of constraints by globin on the geometry of the ent i r e heme complex* 2. 2.6.4 K i n e t i c Consequences The "proximal e f f e c t " observed when switching from an R- to T-state model system i s r e f l e c t e d about equally by decrease i n the asso c i a t i o n rate ( k c o ) and increase i n the d i s s o c i a t i o n rate ( k " C 0 ) f o r CO binding, eq. (24), (Table 2.10). kC0 Fe(Por)(B) + CO ^ ~* Fe (Por) (B) (CO) (24) k-C0 Traylor et a l . , have drawn an analogy between the s t r a i n introduced on CO binding i n common t r a n s i t i o n metal carbonyls and that found i n T-state heme complexes 0^. In F i g . 2.5 A, as R s i z e increases the cone angle (/) increases, introducing a greater degree of repulsion between the bound CO and R groups; t h i s r e s u l t s i n an increased CO d i s s o c i a t i o n 69 rate. In f i v e - coordinate heme complexes, i t i s suggested that s t e r i c hindrance i n the proximal group, B, w i l l likewise induce bending i n the N-Fe-N angle (Fig. 2.5 B), and increase s t e r i c repulsion between d i s t a l ligand and pyrrole nitrogens, thus r e s u l t i n g i n an increased ligand d i s s o c i a t i o n rate . That such porphyrin doming does occur i n the five-coordinate state of T-state systems i s shown by i n the c r y s t a l 7 0 structure of Fe(TPP)(2-Melm) / a, where a displacement of 0.15 A i s found between Np (porphyrin nitrogens) and the heme plane. This increase i n d i s s o c i a t i o n rate i s a r e f l e c t i o n of constraint i n the bound complex, and i s d i s t i n c t from hindrance introduced by s t e r i c obstruction of the ligand p r i o r to Fe-L bond formation 0^ (see section 2.5.1). The decreased as s o c i a t i o n rate for the T-state heme i s presumably caused by diminished access to the i r o n centre because of i t s increased displacement toward the proximal imidazole i n the five-coordinate heme; there i s , however, no comparative s t r u c t u r a l evidence f o r a five-coordinate R-state model system to support t h i s . With respect to 0 2 binding, an R- to T-state change, influenced by both "proximal" s t e r i c and " d i s t a l " e l e c t r o n i c e f f e c t s of the heme environment, does not follow a general k i n e t i c pattern a r i s i n g from proximal e f f e c t s alone (see Section 4.5.6(b)). e A B F i g . 2.5 Proximal s t e r i c e f f e c t s on CO binding: (A) to a bulky transition-metal complex; (B) to a "T-state" heme system 70 -2.6.5 Doming E f f e c t s Several proximal s t r a i n e f f e c t s are found i n the C 2-capped-strapped model hemes of Baldwin et a l . 9 3 , 44a and b. These pyridine-strapped models show s i g n i f i c a n t l y lower a f f i n i t y toward 0 2 than the five-coordinate Fe(C2-cap)(py) analog with exte r n a l l y added base (Table 2.11), even though a l l three systems have the same C 2-cap i n t h e i r Compound ^1/2 ®2 ( t o r r ) (15b) Fe(C 2-cap)py 26 (44b) FeC 2-cap-C 5-strap 110 (44a) FeC 2-cap-C 4-strap 1080 - 71 -d i s t a l environment and, i n addition, e l e c t r o n i c e f f e c t s of the proximal ligand b a s i c i t y should be s i m i l a r within the s e r i e s 9 3 . The low a f f i n i t y i n the strapped hemes was thought to a r i s e from two other e f f e c t s 9 3 : porphyrin doming and/or non-bonded s t e r i c i n t e r a c t i o n s between the proximal chelate arms and porphyrin r i n g . These e f f e c t s are independent of • that which would r e s u l t from s u b s t i t u t i o n i n the 2- or 6-position of the pyridine r i n g (see Section 2.6.2). The strapped proximal ligand was considered to perhaps "lock" the complex into a domed configura-t i o n 9 3 during coordination of the base. Undoming required on 0 2 (or CO) binding, i n order that the i r o n may move into the centre of the porphyrin plane, would involve movement of the whole strapping group and 0 ^ p o s s i b l y more energy' . Also considered noteworthy, i s the difference i n the proximal environment between these models and those of the "hanging-pyridine" hemes, 45-46 (section 2.7.1). The s i t e of attachment of the strap to the porphyrin aromatic r i n g i n the former i s meso to the porphyrin, i n a p o s i t i o n that i s perhaps more l i k e l y to give r i s e to unfavourable " f o l d i n g of the side arms" i n order f o r the proximal group to move cl o s e r to the porphyrin plane on s i x t h l i g a n d b i n d i n g 9 3 . Either or both of these e f f e c t s may contribute to the reduced 0 2 a f f i n i t y , and are expected to be more pronounced i n the C3-cap-C4-strapped system, 44a, with i t s more r i g i d , t i g h t e r strap, where the a f f i n i t y i s much lower (10-fold) r e l a t i v e to the C5-strapped case, 4 4 9 3 . 72 -2.6.6 Base-Elimination Pathway I NH I NH I (CH2)2 | JCHJz A Unstrained B Ring Stroin • (CH2)3>^ C_ Foce Strain D_ Springboord Slroin R-(CH2>3-N0N Fig. 2 . 6 Pyrrole-substituted chelated hemes with varying degrees of proximal strain Single chelate model hemes with induced proximal s t r a i n toward binding of CO have been studied by Geibel et a l . 9 4 . These modified models may possess: " r i n g s t r a i n " that r e s u l t s from a shortened chelate attachment, "face s t r a i n " because of a substituent placed i n the 2-position of the attached proximal base, and "springboard s t r a i n " induced by a bulky substituent on the proximal base side-chain connection nearer to the heme edge, (Fig. 2.6 B, C and D, r e s p e c t i v e l y ) . A d e t a i l e d k i n e t i c analysis f o r these strained systems has c l e a r l y demonstrated that CO binding occurs v i a a "base-elimination" pathway 9 4 i n v o l v i n g displacement of the chelated base p r i o r to CO binding, eq. (25), steps 2-4: 73 -(25) This results i n a larger CO association rate for the four-coordinate heme94, where k c o > k B C 0 , and i s attributed to the fact that the four-coordinate iron(II) i s in the porphyrin plane as opposed to being displaced toward the proximal imidazole as i n the five-coordinate state'-*. In addition, the increased potential for approach to the iron centre from either face of the heme, after base displacement, contributes a rate enhancement factor (see also Section 4.5.2(a)). It was suggested that this mechanism of "base-elimination" may provide an alternative explanation to that involving dis t a l steric effects, by which hemoproteins increase their ligand a f f i n i t i e s 0 8 , 9 0 . Base displacement in hemoproteins may arise via protonation of the proximal imidazole, which has been shown spectrally to promote dissociation of the imidazole from the iron centre i n simple heme models 9 0. Investigation of the pH vs. CO association rate profile for - 74 -sperm whale Mb indicated that below pH 5, the CO "on-rate" increased to a value s i m i l a r to that f o r Erythrocruorin (Ery) 9**. The l a t t e r hemoprotein shows an increased a s s o c i a t i o n rate for both CO and 0 2, although c r y s t a l structure evidence reveals a s i m i l a r s i z e binding p o c k e t 2 4 " 2 ^ , compared to that of normal Hb (Table 2.1). The s i m i l a r i t y between the absorption spectrum of Mb at pH -4 and that of a simple four-coordinate heme indicated that, at low pH, protonation of the proximal imidazole had resulted i n a breaking of the Fe-Im bond, leaving a " f a s t e r reacting" four-coordinate i r o n i n the hemoprotein 96. More r e c e n t l y 9 7 , however, an analogous experiment c a r r i e d out with Ery showed no change i n CO association rate between pH 4-9, i n d i c a t i n g that the base displacement mechanism was probably not operative i n t h i s hemoprotein at p h y s i o l o g i c a l pH. The higher CO and 0 2 a s s o c i a t i o n rates to Ery are now suggested 9 7 to r e s u l t from a smaller displacement of the o five-coordinate i r o n from the heme plane i n t h i s hemoprotein, -0.2 A, o compared to that i n normal Hb, 0.4 A (see Section 4.5.9). 2.7 ELECTRONIC EFFECTS 2.7.1 D i s t a l Environment S t a b i l i z a t i o n of the bound dioxygen moiety v i a hydrogen-bonding with the His E7 residue was f i r s t suggested 3 7 i n view of the e l e c t r o n i c p i c t u r e of an end-on coordinated geometry i n the d i s t a l pocket. X-ray c r y s t a l structure refinements have since confirmed the existence of a - 75 -hydrogen-bond in sperm whale MbO-22* and in the Q!-subunit of human HbO-2*8; in the subunit, the distal histidine N^-H is further away and equidistant from both 0 atoms suggesting a weaker interaction with either or both 0 atoms*0. The absence of such hydrogen-bonding to the oxygen in CO of HbCO is consistent with electroneutrality of the Fe-C-0 unit 4 4. The electronically different nature of the Fe-0-0 and Fe-C-0 adducts is well demonstrated in heme model studies by examining the kinetics of dioxygen and carbon monoxide binding. Lavalette et a l . 9 8 , have compared O2 and CO binding to two model hemes that differ solely in the electronic nature of their distal environment, with amide functions in one, 45, and less polar ether linkages in the other, 46. These "pyridine-hanging base" hemes incorporate the proximal base into a strap across one face of the porphyrin, while a strap over the other face prevents dimerization of two heme units and determines the electronic nature of the distal environment. The distal cavity size in these hemes is considered very similar with only slight differences in shape owing to the more rigid amide linkages 9 8. The CO affinity is clearly - 76 -u n a l t e r e d w i t h i n these f i v e - c o o r d i n a t e systems, w i t h only minor changes i n a s s o c i a t i o n and d i s s o c i a t i o n r a t e s ; the 0 2 a f f i n i t y , however, i s s t r i k i n g l y higher (10-fold) f o r the amide-heme complex, the d i f f e r e n c e being predominantly r e f l e c t e d i n a decreased d i s s o c i a t i o n r a t e f o r dioxygen (Table 2.12). The presence of p o l a r amide l i n k a g e s i n the cap of 45 th e r e f o r e s t r o n g l y increases the s t a b i l i t y of the bound oxy-species by reducing the r a t e of d i s s o c i a t i o n of 0 2 from the heme 9 8. Table 2.12 Constants f o r CO and 0 2 Binding t o "Hanging-Base" Hemes i n Toluene at 20°C 9 8 Compound k C 0 k" C 0 K C 0 k ° 2 k'° 2 K ° 2 (M - 1 s" 1) ( s " 1 ) (M _ 1) (M'1 s' 1) ( s " 1 ) (M"1) (46) Fe-Ether-Py 6.8 x 10 7 0.069 9.9 x 10 8 3.0 x 10 8 4 x 1 0 4 7.3 x 10 3 (45) Fe-Amide-Py 3.5 x 10 7 0.03 1.2 x 10 9 3.0 x 1 0 8 5 x 10 3 7.2 x 10 4 (49) Fe-Amide-Im 4.0 x 10 7 0.0067 6.0 x 10 9 3.1 x 10 8 6.2xl0 2 5.0 x 10 5 The Fe-CO adduct, on the other hand, i s r e l a t i v e l y u n a f f e c t e d by the change i n p o l a r i t y of the b i n d i n g c a v i t y . The s i g n i f i c a n c e of comparing dioxygen d i s s o c i a t i o n r a t e s at constant CO d i s s o c i a t i o n r a t e should be r e a l i z e d , as both these o f f - r a t e s are subject to v a r i a t i o n from other e f f e c t s that are proximal i n n a t u r e ^ ( s e c t i o n 2.6.4). Proton nmr - 77 -s t u d i e s " indicate that a proton of a d i s t a l amide function points toward the centre i n the free base derivative of 45, which suggests that a hydrogen-bond may be formed i n the bound 0 2 complex of Fe-Amide-Py(0 2 ) . In the c r y s t a l structure of Fe(TpivPP)(Melm)(0 2), 3, however, the amide hydrogens of the 'pickets' prove to be too fa r from the bound dioxygen for favourable hydrogen-bonding to occur 3*. In the hemoproteins, sperm whale MbD and a p l y s i a Mb 2 3 provide a d i r e c t comparison. The absence of the d i s t a l His E7 residue i n a p l y s i a Mb 2 3 r e s u l t s i n a 7-fold increase i n 0 2 d i s s o c i a t i o n rate r e l a t i v e to that f o r horse Mb, where His E7 forms a hydrogen-bond with the bound 0 2 i n the c r y s t a l l i n e s t a t e 2 * . 2.7.2 Proximal Ligand B a s i c i t y The influence of proximal ligand b a s i c i t y on coordination a f f i n i t i e s of CO and 0 2 has been investigated i n many model systems. Studies c a r r i e d out with substituted p y r i d i n e s 7 9 using Fe(C 2-cap) indicated that oxygen a f f i n i t i e s were e s s e n t i a l l y i n s e n s i t i v e to changes i n pK a of the proximal base. However, s u b s t i t u t i o n of pyridine by an imidazole was found to increase s u b s t a n t i a l l y (8-fold) 0 2 a f f i n i t y i n t o l u e n e 7 9 . The bett e r 7T-donating a b i l i t y of the imidazole evidently enhances 0 2 a f f i n i t y , while varying C donor e f f e c t s within the substituted pyridines have r e l a t i v e l y l i t t l e e f f e c t 7 9 . Similar behaviour with other systems, such as the Im/Py-chelated picket-fence hemes*^0 (47 and 48) and the Im/py-chelated mesohemes*^*, i s evidenced i n 40-fold and 20-fold changes, res p e c t i v e l y , f o r 0 2 binding (Table - 78 -2.13). The c o n s i s t e n t l y higher 0 2 a f f i n i t y observed with increased e l e c t r o n density at the i r o n centre supports the idea of charge separation ( F e 1 1 1 - 0 2 " ) i n the bound a d d u c t 1 0 1 * 1 0 2 . Inconsistent r e s u l t s are found i n the case of CO binding, where only a 2-fold decrease i n a f f i n i t y i s observed with replacement of Im with Py i n the chelated mesohemes 1 0 1 i n aqueous suspension (Table 2.13). Data from a deuteroheme (4-cyano-Py)/(Im) system had e a r l i e r revealed a 9-fold lower CO a f f i n i t y with Py as b a s e 8 8 , and more recently a 13-fold change has been observed f o r the chelated picket-fence hemes 1 0 0 i n toluene. The s e n s i t i v i t y of bound CO to the greater 7T-basicity of the imidazole a x i a l group i s i n any case c e r t a i n l y diminished compared to the dioxygen case (within each model system), but some charge separation i n the Fe-C-0 adduct, enhanced by increased b a s i c i t y , cannot be ruled o u t 1 0 0 . One exception i s found within the "hanging-base" systems, 45 and 49, where rather s i m i l a r changes, 7-fold and 5-fold are observed for 0 2 and CO, r e s p e c t i v e l y (Table 2.12). In t h i s case, other proximal e f f e c t s due to the strapping group may come into play also. - 79 -Table 2.13 Imidazole versus Pyridine - Influence of Proximal Ligand B a s i c i t y on CO and 0 2 A f f i n i t i e s of Model Hemes ,C0 -CO CO 3 l / 2 -0, * l / 2 (M' 1 s" 1) ( s " 1 ) (torr) (M' 1 s' 1) ( s " 1 ) ( t o r r ) (47) Im-chelated 3-Picket-Fence 2.9xl0 7 0.014 4.9xl0" 5 2.6xl0 8 3.9xl0 3 1.26 (48) Py-chelated 3-Picket-Fence 4.8xl0 7 0.33 6.4xl0" 4 3.0xl0 8 1.9xl0 5 52.2 (10) Im-chelated^ Mesoheme l . l x l O 7 0.02 1.3xl0" 3 2.2xl0 7 23 • 0.57 Py-chelated^ Mesoheme 1 . 2 x l 0 7 0.035 2 . 1 X 1 0 ' 3 1 . 7 x l 0 7 380 1 2 . 2 £ In toluene at 25°C 1 0 0 ^ In aqueous suspension at 2 0 ° C 1 0 1 49. Fe -Amide-lm - 80 -Further s i g n i f i c a n c e of an imidazole as proximal base i s seen i n the c r y s t a l structures of deoxy and liganded hemoglobin, where a hydrogen-bond between the proximal imidazole N j . -H and leucine F4 residue i s indicated i n both f o r m s 1 3 , 1 8 (Figs. 1.4 and 1.5). I t was s u g g e s t e d x u z that v a r i a t i o n i n the strength of t h i s hydrogen-bond could be a means of c o n t r o l l i n g hemoprotein r e a c t i v i t y . Following t h i s suggestion Mincey and Traylor q u a l i t a t i v e l y investigated CO binding to protoheme dimethyl ester-diimidazolate, 6b(Im")2 and -diimidazole, 6b(Im)2, under i d e n t i c a l concentrations of added base (0.1 M) i n aqueous suspension. The o v e r a l l a f f i n i t y f o r CO decreased by ca. 75-fold f o r the imidazolate complex. In a more quantitative study, the CO associa-t i o n rates i n toluene were compared within the five-coordinate heme systems Fe(TPP)(Im)/(Im") and deuteroheme (Im)/(Im*) 1 0 4. The observed CO as s o c i a t i o n rate decreased by factors of 168 and 120 f o r the tetraphenyl- and deutero- porphyrin systems, re s p e c t i v e l y , when imidazole was replaced by the imidazolate anion. This was i n t e r -p r e t e d 1 0 4 as r e s u l t i n g from greater s t a b i l i z a t i o n of the f i v e -coordinate anionic complex Fe(Por)(InT) r e l a t i v e to the t r a n s i t i o n state on the CO-blnding pathway. A much smaller e f f e c t (2-3-fold) i s found with p a r t i a l hydrogen-bonding that occurs when 1,10-phenanthroline i s added to a s o l u t i o n of the neutral complex 1 0 4. This reagent, which i s known to strongly hydrogen-bond with the N-H of imidazoles, has been used also more r e c e n t l y 1 0 5 i n a study with Fe(TTPPP)(2-MeIm), 50(2-MeIm). In t h i s case, however, v i r t u a l l y no change i n o v e r a l l CO a f f i n i t y could be detected a f t e r the addi t i o n of 1,10-phenanthroline. A comparative study with dioxygen has not been c a r r i e d out q u a l i t a t i v e l y , although the greater s e n s i t i v i t y toward oxidation f o r the 81 -an ion ic imidazolate complexes to [l-oxo dimers corroborates the suggest ion that hydrogen-bonding of the proximal imidazole may p lay a s i g n i f i c a n t r o l e i n r e g u l a t i n g hemoprotein l i g a n d a f f i n i t y ^ 3 . 2.7.3 Porphyr in Per iphera l E f f e c t s The e l e c t r o n i c e f f e c t s of subst i tuents on the protoporphyr in IX p r o s t h e t i c group l a i n hemoprotein s tudies are compl icated because of s t e r i c i n t e r a c t i o n s between the porphyr in s ide chains and those of the surrounding g l o b i n ^ ° • . T ray lo r et a l . ^ 6 have s tud ied the model heme s e r i e s : che la ted meso- (10), and proto- (11) and diacetyldeuteroheme (12) i n order to inves t iga te the e f f e c t of changing e l e c t r o n densi ty at the i r o n centre v i a porphyr in s i d e - c h a i n s . In t h i s s tudy, 0 2 a f f i n i t y was observed to increase i n the s e r i e s e thy l > v i n y l > a c e t y l s u b s t i -tuents , cons is ten t with inc reas ing e l e c t r o n dens i ty at the heme. This was r e f l e c t e d i n a decreased d i s s o c i a t i o n rate f o r 0 2 over a range of - 82 -ca. 2 0 - f o l d . The binding of CO, however, was r e l a t i v e l y i n s e n s i t i v e to side-chain e l e c t r o n i c e f f e c t s as evidenced by much smaller changes i n k i n e t i c constants. Thus side chain 0~ e f f e c t s are considered to have l i t t l e influence on the thermodynamic s t a b i l i t y of the carbonyl adduct which i s governed by both O as well as 7T-interactions within the Fe-CO m o i e t y 1 0 6 . Since the F e - 0 2 adduct, however, i s considered to involve some net transfer of electron density from metal to ligand, the greater s e n s i t i v i t y of O 2 binding to electron density at the i r o n i s r e a d i l y r a t i o n a l i z e d . These observations thus support greater charge separation i n the F e - 0 2 i n t e r a c t i o n , r e l a t i v e to Fe-CO 1 0 6. 2.7.4 Solvent P o l a r i t y Further support for the F e 1 I 1 - 0 2 " formulation i s gained from e f f e c t s of solvent p o l a r i t y on O 2 binding. A l l the open heme systems show increase i n 0 2 a f f i n i t i e s of ca. 10-30 f o l d with increase i n solvent p o l a r i t y , consistent with the greater s t a b i l i z a t i o n of charge separation i n the F e - 0 2 s P e c i e s i n the more polar s o l v e n t 7 8 3 (Table 2.14). One exception i s found with the Fe(C 2-cap)(Melm) system, where changing the solvent from toluene to dimethylformamide r e s u l t s i n only a 3-fold increase i n O 2 a f f i n i t y r e l a t i v e to a 1 0 - f o l d e f f e c t f or the Fe(T(p-OMe)PP)(l,2-Me 2Im) s y s t e m 7 8 3 . This was interpreted i n terms of a r e s t r i c t i v e environment within the d i s t a l pocket of the C 2-cap, that does not r e a d i l y allow for correct o r i e n t a t i o n of solvent molecules for e f f e c t i v e s o l v a t i o n of the dipolar F e - 0 2 m o i e t y 7 8 3 . Therefore polar 83 -Table 2.14 E f f e c t of Solvent P o l a r i t y on 0 2 A f f i n i t y i n Model Hemes 7 8 a °2 Solvent system D i e l e c t r i c P 1/2 Constant (torr) (30) Chelated Pf Toluene 2.4 1:1 toluene/MeOH 17.6 0.58^ 0.059 3-(10) Chelated mesoheme 90% toluene/CH 2Cl 2 3.1 2% aqueous CTAB 80.4 suspension 3 x l 0 4 ( M ' 1 ) ^ l x l 0 6 ( M - 1 ) ^ (51) Fe(T(p-0CH 3)PP)-(l,2-Me 2Im) Toluene DMF 2.4 36.7 5.3°-0.41°-(15b) Fe(C 2-cap)(MeIm) Toluene DMF 2.4 36.7 4.3d-3.3 d £ At 25°C. k At 20°C. £ At -45°C. ^ At 0°C. C H . O 51, FeT(p-OCH,)PP OCHf CH,0 - 84 -solvents are considered to lose advantage over those l e s s polar i n such cases, with l i t t l e difference i n 0 2 binding a f f i n i t y between d i f f e r e n t solvent systems. Contrasting r e s u l t s are obtained for CO binding to various hemes i n solvents of varying p o l a r i t y , ranging from d i s c r i m i n a t i o n a g a i n s t 1 0 7 CO binding to that of enhanced 6 4 CO binding i n more polar solvents. The v a r i a t i o n i s however s l i g h t , r e l a t i v e to the enhanced 0 2 a f f i n i t i e s , and i s therefore again consistent with the more covalent nature of the Fe-CO un i t . 2.8 SOLVATION EFFECTS Comparison of the CO a s s o c i a t i o n rate of Fe(TpivPP)(1,2-Me 2Im) with that of Fe(TPP)(l,2-Me 2Im) reveals a -10-fold increase for the picket-fence system (Table 2.15). An i n t e r p r e t a t i o n 6 4 of t h i s behaviour derives from the r e l a t i v e s o l v a t i o n of the five-coordinate complexes, where the 'open' TPP system i s considered to be subject to stronger solvent s t a b i l i z a t i o n r e l a t i v e to the 'protected' picket-fence heme, p r i o r to l i g a t i o n . This suggestion i s supported by the higher CO a f f i n i t i e s of the more 'protected' Fe(T-MesP)(l,2-Me 2Im) 6 4 and Fe(TTPPP)(l,2-Me 2Im) 1 0 5 systems. Furthermore, the strong s o l v a t i o n of the 'open' unligated species must be reduced i n the t r a n s i t i o n state for l i g a n d association, so that the r e l a t i v e d i s s o c i a t i o n rates f o r both open and protected CO bound complexes should be s i m i l a r 6 4 . This behaviour i s also found i n the R-state, with an enormously high CO 85 Table 2.15 CO A f f i n i t i e s of Picket-Fence Systems Relative to Other TPP-Derived Hemes° 4 kC0 (M" 1 s' 1) k-C0 CO P l / 2 (torr) T-State (9b) Fe(TPP)(l,2-Me 2Im) 1.6 x 10" (50) Fe(TTPPP)(l,2-Me 2Im) (52) Fe(T-MesP)(l,2-Me 2Im) (14b)Fe(TpivP)(l,2-Me 2Im) 1.4 x 10I 0.24 0.14 0.15 0.009 0.008 0.0089 R-State (53) Chelated TPP (30) Chelated Pf (49) Fe-Amide-Im 4.4 x 10 6 3.6 x 10 7 4.0 x 10 7 -0.04 0.0078 0.0067 -1.0 x 10" 3 2.2 x IO" 5 1.7 x IO' 5 86 a f f i n i t y (45-fold) for chelated picket-fence r e s u l t i n g from 10-fold increase i n accompanied by a 4.5 f o l d decrease i n k"(-'(-) r e l a t i v e to chelated TPP, 53. S i g n i f i c a n t l y , t h i s anomalous behaviour r e l a t i v e to an 'unencumbered' analog i s only observed with TPP-derived systems. The o r i g i n of the high ligand a f f i n i t y appears to be i n t r i n s i c to these synthetic systems and i s not expressed i n the p y r r o l e - s u b s t i t u t e d model hemes (see section 4.5.8(b)). 2.9 REASON FOR STUDYING THE DURENE CAPPED HEMES D i f f e r e n t i a t i o n between CO and 0 2 i n model systems may a r i s e from e l e c t r o n i c and/or s t e r i c e f f e c t s , where various studies have led to contradictory i n t e r p r e t a t i o n with regard to d i s t a l d i s c r i m i n a t i o n of CO. A simple reference series of systems where the influence of d i s t a l s t e r i c e f f e c t s on CO and 0 2 binding may be evaluated i n the absence of s t a b i l i z i n g e l e c t r o n i c i nteractions i s not, however, a v a i l a b l e . The durene-capped hemes, 54-56b, provide such a s e r i e s . V a r i a t i o n i n cap s i z e within the ser i e s covers the e n t i r e range i n model systems studied. The durene-7/7 strap has the same number of atoms l i n k i n g cap to porphyrin periphery as i n the anthracene-7/7 heme (18), and may be expected to be even more f l e x i b l e with methylene carbons replacing the more r i g i d amide linkages. The number of atoms i n the durene-4/4 cap i s even less than i n the extremely s t e r i c a l l y hindered FeSP-13 (23) and pyridine-5/5 (26) systems, although the difference may be o f f - s e t by greater f l e x i b i l i t y of the durene-4/4 methylene chains. 87 -Moreover, the incorporation of a t i g h t bulky strap over the porphyrin face may be expected to r e s u l t i n s i g n i f i c a n t doming of the porphyrin r i n g from p l a n a r i t y . Such porphyrin doming e x i s t s i n the c r y s t a l structures of deoxy- and oxyMb 2 1, and thus may well play a role i n hemoprotein ligand a f f i n i t y . Thus within the completely hydrophobic d i s t a l c a v i t y of the durene s e r i e s , the r e l a t i v e contribution of s t e r i c e f f e c t s , both proximal and d i s t a l , may be evaluated for both R- and T-state heme systems. 5 5 , 5 6 5 4 , JL. Mr 2 H Jb, M= Fe Durene - 4 , 4 ,n»4 Durene - 5 , 5 ,n» 5 Durene - 7 , 7 ,n» 7 - 88 -CHAPTER 3 THE DURENE CAPPED PORPHYRINS: SYNTHESIS AND SPECTRAL COMPARISONS - 8 9 -3. THE DURENE CAPPED PORPHYRINS: SYNTHESIS AND SPECTRAL COMPARISONS 3.1 BRIEF OVERVIEW OF PORPHYRIN SYNTHESIS This section provides a b r i e f covering both simple and s t e r i c a l l y reviews dealing with these topics summary on porphyrin synthesis, encumbered systems. Several recent i n d e t a i l are a v a i l a b l e 1 0 8 " 1 1 0 . 3.1.1 General Porphyrin Synthesis Meso substituted tetraphenylporphyrin (9a) i s e a s i l y prepared by r e f l u x i n g equimolar amounts of pyrrole and benzaldehyde i n propionic acid, with y i e l d s of - 2 0 % 1 1 1 . The successful synthesis of pyrrole-substituted porphyrins, however, requires much experience and Ingenuity, the challenge of which has i n t r i g u e d porphyrin chemists f o r a long t i m e 1 0 8 . Such synthesis has generally been d i r e c t e d by one of three d i f f e r e n t routes, represented below, that depend on the type of periph e r a l s u b s t i t u t i o n desired; the / 3-substituents i n every case become the porphyrin periphery: (1) "One-pot" condensation of m o n o p y r r o l e s 1 0 8 a (2) "2+2" ad d i t i o n of d i p y r r o l i c p r e c u r s o r s 1 0 8 1 1 (3) Intramolecular c y c l i z a t i o n of a l i n e a r t e t r a p y r r o l e 1 0 8 c . - 90 -The f i r s t of these a l t e r n a t i v e s has l i t t l e synthetic value, being useful only i n the synthesis of symmetrically substituted porphyrins. For instance, i n the synthesis of octaethylporphyrin (5a), an improved route developed by Paine et a l . ^ 2 involves condensation of the monopyrrole, pyrrylmethylamine (57), i n r e f l u x i n g a c e t i c a c i d ( y i e l d 50%). The "2+2" coupling of d i p y r r o l i c intermediates provides a method by which peripheral s u b s t i t u t i o n may be v a r i e d i n a c o n t r o l l e d manner. Commonly used d i p y r r o l i c precursors are dipyrromethanes (61) and dipyrromethenes 64,66,67. The former of these i s prepared from the condensation of intermediates such as 58 with an CV-free pyrrole, 60, i n a c i d i c media, v i a formation of the resonance s t a b i l i z e d p y r r y l c a r b i n y l cation, 59 (Fig. 3.1)^^ 8 b. Dipyrromethenes may be synthesized by r e a c t i o n of an CV-formyl pyrrole 62, with an CV-free pyrrole, 63, i n strongly a c i d i c conditions, or by the s e l f condensation of an CV-methyl-CV-free pyrrole, 65, i n the presence of bromine (Fig. 3 . 2 ) ^ 8 d ; they are most stable i n the form of bromide s a l t s . Porphyrins may be obtained from dipyrromethanes by reaction between CV-formyl and CV-free functions (Fig. 3.3). Condensation proceeds v i a formation of a bilene-b to a porphodimethene intermediate, which undergoes oxidation to a p o r p h y r i n ^ 8 0 . For dipyrromethenes also, the required a i r oxidation for porphyrin formation suggests a porphodimethene i n t e r m e d i a t e ^ 0 * 3 . The synthesis of porphyrins from d i p y r r o l i c intermediates i s l i m i t e d , however, owing to symmetry r e s t r i c t i o n s imposed by the nature of these coupling reactions. The intramolecular c y c l i z a t i o n of l i n e a r t e t r a p y r r o l e s enables other types of unsymmetrically substituted porphyrins to be synthesized. 91 • i c F i g . 3.1 Formation of a dipyrromethane from pyrrole precursors F i g . 3.2 Formation of dipyrromethenes from pyrrole precursors P a r t i c u l a r i n t e r e s t i n these intermediates derives from t h e i r involvement i n biosynthetic pathways to n a t u r a l l y occurring p o r p h y r i n s 1 1 3 . In the laboratory, reaction between dipyrromethenes of type 68 and 69 i n the presence of stannic c h l o r i d e , followed by treatment with hydrogen bromide, gives 1,19-dideoxybiladiene-ac (70) i n 70-95% y i e l d . Heating the tetrapyrrole i n o-dichlorobenzene for 15 - 92 minutes affords the corresponding proto-, meso-, or deuteroporphyrin dimethyl ester, 6a, 7a or 8a, respectively 1 0 8 0. Por p h o d i m e t h T i e Porphyrin F i g . 3 .3 Formation of a porphyrin v i a the acid-catalyzed c y c l i z a t i o n of dipyrromethanes 93 3 . 1 . 2 Porphyrins Carrying S t e r i c Encumbrance and/or Chelating Ligands Attachment of extraneous functional groups at the porphyrin periphery may be accomplished b y ^ 4 : (a) r e a c t i o n with a preformed porphyrin skeleton, or (b) incorporation of such groups into the molecule p r i o r to porphyrin formation, where intramolecular c y c l i z a t i o n to the porphyrin i s the f i n a l step of the synthesis. The majority of s t e r i c a l l y encumbered porphyrins studied have been prepared by the f i r s t method. For instance, coupling between the porphyrin diamine, 7 1 , and an anthracene d i a c i d c h l o r i d e , 7 2 , under conditions of high d i l u t i o n , r e s u l t s i n the free base porphyrin - 94 -d e r i v a t i v e s of the systems, 18 or 1 9 1 0 7 . Traylor's (18-20 and 2 6 ) 7 3 > 7 5 - 1 0 7 , and Chang's (21-25) 7 4 porphyrin systems were a l l prepared by t h i s approach. Condensation between pyrrole and substituted benzaldehydes r e s u l t s i n modified tetraphenylporphyrins, carrying substituents on the meso phenyl rings, 73 (Fig. 3.4). In the case of ortho-substitution, an equilibrium mixture of atropisomers 74a-d i s obtained, from which the desired porphyrin isomer i s separated by chromatography. This approach, allowing subsequent chemical transformation of the R group, has been popular i n the synthesis of several s t e r i c a l l y hindered and chelated systems derived from t e t r a p h e n y l p o r p h y r i n 6 1 * 5 • 7 6 • 8 3 • 1 0 0 « 1 1 5 • 1 1 6 . The f i r s t porphyrin to be synthesized by t h i s method was the picket-fence porphyrin 14a of Collman et a l . 6 1 * 5 , formed by r e a c t i o n of the aminophenylporphyrin, 75, with p i v a l o y l c h l o r i d e . The configuration of the Q!, CX ,Ct,Q!-atropisomer (see 74a) Is frozen by formation of the bulky, r i g i d amide "pickets". Collman's group also adapted the same strategy to prepare a serie s of pocket p o r p h y r i n s 7 6 , 27-29 -and chelated h e m e s 1 0 0 - 1 1 6 30, 47 and 48. 95 -d F i g . 3.4 Synthesis of tetraphenylporphyrin-derived systems from pyrrole and substituted benzaldehydes In a modification of t h i s approach, Momenteau et a l . ^ - * have attached a diagonal strapping group across both faces of an ortho-s u b s t i t u t e d tetraphenylporphyrin d e r i v a t i v e . Incorporation of an a x i a l base Into one of the straps affords various doubly-strapped "hanging-base" porphyrins. For instance, condensation of 1,12-dibromododecane with the o-hydroxyphenylporphyrin 76 gives a mixture of s i n g l y - l i n k e d porphyrins 77 and 78; further r e a c t i o n of the desired trans-cross linked isomer 77 with 3,5-bis(3-bromopropyl)pyridine, followed by i r o n - 96 -i n s e r t i o n , r e s u l t s i n the ether-linked hanging pyridine heme, 46 i i - > . Analogous re a c t i o n with the o-aminophenylporphyrin, 75, and appropriate d i a c i d c h l o r i d e chain derivatives gives the amide-linked pyridine, 45, and imidazole, 49, "hanging-base" p o r p h y r i n s " ^ . Baldwin's group was the f i r s t to design an a l t e r n a t i v e synthetic route to s t e r i c a l l y encumbered porphyrins, i n v o l v i n g the attachment of s t e r i c bulk to the porphyrin precursors p r i o r to porphyrin r i n g forma-117 t i o n . In t h i s modified version of obtaining a tetraphenylporphyrin d e r i v a t i v e , the incorporation of a capping group to give the t e t r a -97 aldehydes 79a-c, followed by t h e i r condensation with four equivalents of pyrrole i n r e f l u x i n g propanoic acid, r e s u l t s i n the capped porphyrins 15-17a, r e s p e c t i v e l y * * 7 . A recent extension of t h i s idea allows attachment of a pyridine ligand, following formation of the C^-capped porphyrin, to give the doubly-strapped porphyrins 44a and b 9 3 . A p p l i c a t i o n of Baldwin's approach to pyr r o l e - s u b s t i t u t e d derivatives has been s u c c e s s f u l l y accomplished by W i j e s e k e r a 8 , 1 0 9 i n the synthesis of the CQ to C ^ i strapped porphyrins 81a-c. In t h i s case, the primary o b j e c t i v e 8 , 1 0 9 was to d i s t o r t d e l i b e r a t e l y the porphyrin skeleton from p l a n a r i t y by diagonally l i n k i n g a very short strap across one face. This elegant design avoids the imposition of a t i g h t strap over a preformed porphyrin skeleton, which would be an unfavourable r e a c t i o n that demanded d e s t a b i l i z a t i o n of the planar system i n the f i n a l step of the synthesis. The o v e r a l l scheme i s based on the "2+2" coupling of the two dipyrromethane u n i t s , which i n t h i s case are li n k e d together v i a the C r>* 9 d n« 6 Fig. 3.5 Strapped porphyrins derived from the "2+2" coupling of chain-linked dipyrromethane* 98 -already incorporated methylene carbon strap 80a-d (Fig. 3.5). A nine-carbon methylene chain was found to be the lower l i m i t , as the attempted synthesis of a porphyrin from an eight-carbon chain precursor, 80d, proved unsuccessful 1^* 9. Once established for the Cg-Cii s e r i e s , the same plan was e a s i l y extended to synthesize the durene-5/5 capped porphyrin 55a'^ 9. The bulky durene moiety was expected to hinder" more e f f e c t i v e l y coordination of a second a x i a l base to a five-coordinate heme, compared to a system having only a diagonal methylene chain over the s i x t h s i t e . The desire to compare the CO and 0 2 a f f i n i t i e s within a given s e r i e s of capped porphyrins (section 2.9), possessing varying degrees of s k e l e t a l d i s t o r t i o n and size of " d i s t a l " pockets, prompted the synthesis of the durene-4/4, 54, and -7/7, 56, analogs. 3.2 SYNTHETIC STRATEGY 1 0 9 A d e t a i l e d "retrosynthetic approach to the target dipyrromethane dimer," 112, has been described by Wijesekera 1^ 9. The following sections attempt only to h i g h l i g h t the important features at each stage of the synthesis of the durene-capped porphyrins. The experimental procedure and c h a r a c t e r i z a t i o n of compounds i s provided i n Appendix I. - 99 -3.2.1 C y c l i z a t i o n to the Durene-Capped Porphyrins Porphyrin formation r e l i e s on the intramolecular c y c l i z a t i o n of the dipyrromethane dimer, 112, v i a formation of a porphodimethene intermediate (see F i g . 3 .8) . Three important features contribute to the success of t h i s f i n a l r e a c t i o n 1 0 9 . F i r s t l y , the s t r a i n introduced by imposition of a short strap has less of an adverse e f f e c t on the more f l e x i b l e porphodimethene intermediate, which i s not a planar system, than on a porphyrin, so that the i n i t i a l c y c l i z a t i o n step should proceed more smoothly. Once formed, however, the porphodimethene undergoes ra p i d autoxidation, where the net gain i n resonance s t a b i l i z a t i o n energy i s the d r i v i n g force f o r porphyrin formation. Secondly, choice of a dipyrromethane rather than a dipyrromethene precursor i s c r u c i a l . Formed from the dipyrromethane dimer, the bilene-b can accommodate the diagonal strap, while also allowing f o r correct o r i e n t a t i o n of the remaining O f o r m y l and Of-free functions to undergo a second intramolecular coupling to the porphodimethene ( F i g . 3 .3 ) . F i n a l l y , CV-formyl and Cf-free functions must combine i n an intramolecular h e a d - t o - t a i l fashion w i t h i n each molecule. Intermolecular r e a c t i o n between these functions on d i f f e r e n t molecules w i l l lead only to polymerization without porphyrin formation. An important requirement of the acid-catalyzed condensation then i s that i t should be c a r r i e d out under high d i l u t i o n conditions so that intermolecular coupling may be minimized. Toluene-p-sulphonic a c i d was c a r e f u l l y s e l e c t e d 1 0 9 as c a t a l y s t for the c y c l i z a t i o n . The dipyrromethane dimer, 112 ( d i l u t e d i n THF) was added slowly and s t e a d i l y , by means of a syringe pump, into a reaction f l a s k containing the a c i d c a t a l y s t i n a large volume of C H 2 C I 2 . 100 -3.2.2 Durene Diacid Chain Derivatives The methylene side chains on commercially a v a i l a b l e b i s ( c h l o r o -methy1)durene,. 82, were extended simultaneously, two carbons at a time, v i a a s e r i e s of malonate syntheses (Fig. 3.6). The t e t r a e s t e r s (e.g. 83) formed from the a l k y l h a l i d e s were not i s o l a t e d , but saponified i n s i t u d i r e c t l y to the tetraacids (84, 91 or 96). Subsequent decarboxylation i n r e f l u x i n g DMF afforded the corresponding d i a c i d 85, 92, or 97. At each stage, the a c i d functions required transformation into the a l k y l halides (88 or 95) before a subsequent malonate re a c t i o n could be performed. This was accomplished v i a a diborane r e d u c t i o n 1 1 8 to give the corresponding d i o l (87 or 94), followed by hydrobromination to give the dibromides (88 or 95, r e s p e c t i v e l y ) . The diborane reduction of carboxylic acids i s generally more f a c i l e compared to reduction of the ester d e r i v a t i v e s . However, the low s o l u b i l i t y of the durene diacids i n THF l e d to only p a r t i a l r e a c t i o n 1 ^ 9 . For t h i s reason the reduction was c a r r i e d out on the more soluble d i e s t e r d e r i v a t i v e (86 or 93) which smoothly underwent complete re a c t i o n to the d i o l . Two or three such malonate syntheses accomplish preparation of the durene-5/5 (92) or -7/7 (97) d i a c i d d e r i v a t i v e s , r e s p e c t i v e l y . Formation of the durene-4/4 d i a c i d (90) required extension of the methylene side chains by only one carbon, following the 3-carbon stage (88). This was e a s i l y e f f e c t e d by preparation of the n i t r i l e , 89, which then hydrolyzed i n basic media to give 90. Fig. 3.6 Schematic i l l u s t r a t i o n of the synthesis of durene d i a c i d chains - 102 -F i g . 3.7 Synthesis of durene linked b i s p y r r o l i c intermediates - 103 -112 Fig. 3.8 Transformation of dipyrromethane dimers and cyclization to the durene-capped porphyrin - 104 -3.2.3 Incorporation of the Durene Diacid Chain into the Dipyrromethane Dimer The f i r s t step toward synthesis of the dipyrromethane dimer required the formation of a chain-linked d i p y r r o l i c intermediate. This was achieved by coupling the durene d i a c i d chain with two equivalents of a monopyrrole unsubstituted at one of the j 3-positions ( F i g . 3 . 7 ) 1 1 9 . 2-Ethoxy-3,5-dimethylpyrrole, 99, was s e l e c t e d 1 0 9 as the / 3 -free pyrrole because of i t s ease of preparation and appropriate s u b s t i t u t i o n pattern (to be modified l a t e r on i n the synthesis). The condensation, c a r r i e d out i n two stages, involved the i n i t i a l a c t i v a t i o n of the a c i d function to the a c i d c h l o r i d e , 98, by r e f l u x i n g i n t h i o n y l c h l o r i d e . A c y l a t i o n was then c a r r i e d out at room temperature, i n the presence of a mild c a t a l y s t , stannic c h l o r i d e , to give the d i p y r r o l i c intermediate, 100. The rather low y i e l d obtained i n t h i s step could not be improved by ei t h e r increasing the amount of c a t a l y s t or a l t e r i n g the reaction temperature. The diketone was i s o l a t e d i n highest y i e l d (70%) f o r the 4/4-derivative, 100a, because of the ease at which the product p r e c i p i t a t e d out of the quenched s o l u t i o n . Subsequent reduction of the keto function to 101 f i x e s the capping group with a methylene chain of desired length, to the flanking pyrr o l e s . The diborane reduction, performed at room temperature f or a l l three d e r i v a t i v e s , proceeded with no complications. The next objective i n the synthetic plan was to achieve the modification of the CV-ethylester function into the desired Q!-formyl d e r i v a t i v e . This multi-step transformation would f i r s t require decarbethoxylation of the Qf-ethylester, 101, to give the a -free pyrrole, - 105 -104, on which a V i l l s m e i e r formylation reaction may be performed to y i e l d the a-aldehyde, 106^®. Direct s a p o n i f i c a t i o n of an OL-ethylester pyrrole to an Ct-acid pyrrole, 103, however, occurs only slowly, r e q u i r i n g prolonged r e f l u x i n basic media, with r i s k of subsequent decarboxylation to the a i r s e n s i t i v e C¥-free pyrrole. This reluctance to undergo hydrolysis i s presumably caused by deactivation of the carbonyl function of the ester group toward n u c l e o p h i l i c attack, since i t i s attached to an electron r i c h pyrrole nucleus. Moreover, i s o l a t i o n of the pyrrole acid, a gelatinous p r e c i p i t a t e , i s a rather laborious task. Hydrogenolysis of the benzylester d e r i v a t i v e (102) was therefore chosen as the preferred r o u t e 1 ^ 9 to the pyrrole acid, 103, since the product i n t h i s case could be e a s i l y i s o l a t e d as a residue by simple evaporation of the solvent (THF). T r a n s e s t e r i f i c a t i o n to the benzylester was c a r r i e d out i n r e f l u x i n g benzyl alcohol with .sodium benzyloxide as c a t a l y s t 1 ^ 0 - , 121 During t h i s reaction, prolonged r e f luxing i n benzyl alcohol (b.p. 205°C) l e d to decomposition, p a r t i c u l a r l y i n the case of the 7/7 -de r i v a t i v e , 102c. Therefore, care was taken to minimize r e a c t i o n time, by immediate add i t i o n of the c a t a l y s t once r e f l u x temperature was attained, and quenching of the r e a c t i o n mixture within 2-3 minutes a f t e r the addition of the c a t a l y s t was complete. Successful i s o l a t i o n of the benzylester product was dependent on the volume of benzyl alcohol used. The 7/7-derivative was the most soluble, o i l i n g out of the quenching s o l u t i o n i f too much benzyl alcohol was used, while the 4/4-derivative (102a) was the l e a s t soluble, r e q u i r i n g a larger volume of solvent to ensure that the pyrrole e t h y l e s t e r dissolved completely i n the r e f l u x i n g reaction s o l u t i o n . Hydrogenolysis of the pyrrole benzylester, 102, to the acid, 103, - 106 -proceeded smoothly and cleanly with l i t t l e complication. I n i t i a l reluctance of the 7 / 7-derivative to hydrogenate was observed on occasion. This was probably caused by the presence of a small amount of impurity that poisoned the c a t a l y s t , since f i l t r a t i o n , followed by a d d i t i o n of fresh c a t a l y s t seemed to remedy the problem. (The 7 /7-benzylester d e r i v a t i v e p r e c i p i t a t e d out of the quenching s o l u t i o n slowly, and was perhaps more apt to become contaminated with impurity compared to the A / A -intermediate). Decarboxylation of the a c i d function was accomplished conveniently i n r e f l u x i n g DMF 1 0 9. The reaction was followed by uv spectroscopy to the stage where the 285 nm carbonyl peak was reduced to a shoulder. Continued r e f l u x i n g , however, di d not remove the shoulder completely. The V i l l s m e i e r f o r m y l a t i o n 1 0 8 * 5 • 1 2 0 was performed d i r e c t l y on the Cf-free p y r r o l e , 1 0 4 , i n DMF, without i s o l a t i o n of t h i s compound. A s o l u t i o n of phosphorus oxychloride and DMF, added to the tt-free p yrrole, i n i t i a l l y forms the iminium s a l t , 1 0 5 1 0 8 b • 1 2 0 • 1 2 2 . Subsequent hydrolysis of t h i s intermediate i n m i l d l y basic media affords the Ct-formyl d e r i v a t i v e . A grey s o l i d containing a high degree of impurity (accumulated from three reactions) was i s o l a t e d . A f t e r formation of the b i s formylpyrrole, the next stage of the synthetic scheme involved attachment of a second pyrrole to each end of the chain l i n k e d d i p y r r o l i c intermediate i n order to form the desired dipyrromethane dimer. This necessitated the conversion of 1 0 6 into an intermediate containing an Qf-chloromethyl function (at the other Ct-position), i n preparation f o r coupling with a second pyrrole (Fig. 3 . 8 ) 1 2 1 . The Cf-formyl group, however, which i s s e n s i t i v e to attack by a c i d i c reagents, cannot be expected to survive such a c h l o r i n a t i o n - 107 -reaction 1*- 1 9. Protection of the aldehyde as a more stable cyanovinyl f u n c t i o n 1 2 3 was therefore e s s e n t i a l from t h i s point on. Moreover, the cyanovinyl may be e a s i l y cleaved i n basic media to regenerate the 1 aldehyde (when required) • L Z J. Reaction to form the dicyanovinyl d e r i v a t i v e , 107, occurred very r e a d i l y i n r e f l u x i n g toluene with malononitrile, using cyclohexylamine base as c a t a l y s t 1 ^ 9 . A major advantage i n forming 107 was i n i t s ease of p u r i f i c a t i o n 1 ^ 9 . The dicyanovinyl i s a bri g h t yellow compound which moves well on s i l i c a gel, unlike the dialdehyde counterpart. Therefore, conversion of the crude dialdehyde to the dicyanovinyl intermediate allowed p u r i f i c a t i o n by column chromatography; the p u r i f i c a t i o n was b a s i c a l l y a f i l t r a t i o n process using s i l i c a gel and C H 2 C I 2 as e l u t i n g solvent. With the aldehyde group protected, the next major concern was the p o s s i b i l i t y of c h l o r i n a t i n g the al k y l a t e d benzene r i n g 1 ^ 9 . Since the pyrrole nucleus i s known to be more reactive toward e l e c t r o p h i l i c attack, however, i t was expected that c h l o r i n a t i o n of the CV-methyl group would s u c c e s s f u l l y compete with s u b s t i t u t i o n at the benzene r i n g 1 ^ 9 . Within the pyrrole nucleus i t s e l f , the CV-methyl p o s i t i o n i s known to be s e l e c t i v e l y c h l o r i n a t e d over the j3-methyl group when using sulphuryl c h l o r i d e 1 ^ 8 * 3 . Moreover, the presence of an e l e c t r o n withdrawing dicyanovinyl function should deactivate the r i n g toward a second c h l o r i n a t i o n 1 ^ 9 . Indeed, with j u s t two equivalents of sulphuryl chloride added, only Cx-chlorination at the pyrrole nucleus took place; a high y i e l d of dipyrromethane dimer (110) was i s o l a t e d from the subsequent step. Best r e s u l t s were obtained when the moisture s e n s i t i v e CV-chloromethyl d e r i v a t i v e , 108, was i s o l a t e d as a residue, following evaporation of the dichloroethane, and used d i r e c t l y i n the pyrrole - 108 -coupling reaction. Under the c a r e f u l l y chosen a c i d i c medium for the coupling r e a c t i o n 1 ^ 9 , formation of the dipyrromethane dimer, 110, proved to be f a c i l e and clean for a l l three durene analogs. Subsequent base hydrolysis conveniently cleaved the cyanovinyl and ethylester functions simultaneously. Because of the greater s o l u b i l i t y of the dipyrromethane i n r e f l u x i n g n-propanol, t h i s solvent was chosen 1^ 9 over ethanol f o r the reaction. Although cleavage of the cyanovinyl function can be followed by uv spectroscopy, complete formation of the ac i d from the ethylester was ensured only by prolonged r e f l u x i n the basic medium. A f t e r a c i d i f i c a t i o n of the reaction mixture with a c e t i c acid, the gelatinous p r e c i p i t a t e , 111, was i s o l a t e d by f i l t r a t i o n (very slow), and washed with water to remove the excess aqueous acid. F i n a l l y , decarboxylation of the CV-acid was c a r r i e d out i n DMF to form the desired CV-free function. The dipyrromethane dimer, 112, thus obtained was used d i r e c t l y i n the c y c l i z a t i o n step to give porphyrin (section 3.2.1). 3.3 SPECTRAL COMPARISONS 3.3.1 1H-nmr Spectra of Durene-Capped Porphyrins The *H nmr spectra were recorded at 400 MHz i n C D C I 3 (Figs. 3.9-3.11). Porphyrin sample concentrations were maintained below 0.05 M i n order to minimize any v a r i a t i o n i n chemical s h i f t caused by porphyrin - 109 -a g g r e g a t i o n 1 ^ . The a l k y l s u b s t i t u t i o n pattern surrounding the porphyrin periphery of the durene capped systems i s e s s e n t i a l l y i d e n t i c a l to that of etioporphyrin I I , 113, except that the C2H5 groups at p o s i t i o n s 3 and 13 are replaced by methylene chain linkages of the durene cap. As for several other s i m i l a r s t r a p p e d 1 0 9 and c a p p e d 1 1 0 porphyrins that have been synthesized, the simple 1H-nmr spectrum of e t i o II i s used as a suitable reference. 4 The diamagnetic r i n g current of the h i g h l y d e l o c a l i z e d porphyrin macrocycle causes protons associated with the periphery to experience a deshielding e f f e c t , and resonate further downfield from TMS. For e t i o I I , the peripheral group resonances appear as four d i s t i n c t s i g n a l s : the protons from a l l four equivalents methyl groups resonate at 8 3.62, the e t h y l groups appear as a t r i p l e t and quartet at 5 1.87 .and 5 4.11, r e s p e c t i v e l y , and the four methine protons (5, 10, 15, 20), although expected to give r i s e to a doublet, occur as a s i n g l e t at 8 10.11. 1 2 5 - 110 -Placement of a strap diagonally across one face of the porphyrin introduces asymmetry into the molecule, and r e s u l t s i n a s p l i t t i n g of the methyl and methine proton s i g n a l s 1 0 9 , 1 2 6 . The s p l i t t i n g of the methyl proton resonances for the long chain, unstrained durene-7/7 porphyrin, i s s l i g h t (Fig. 3.12 A ) . As the chain length i s decreased, however, an u p f i e l d s h i f t i s observed for the higher f i e l d s i g n a l , that corresponds to methyl groups at the 2 and 12 p o s i t i o n s . This s h i f t , that increases with decreasing cap s i z e , i s i n t e r p r e t e d 1 0 9 , 1 1 0 i n terms of increasing d i s t o r t i o n of rings A and C from the porphyrin plane, toward the t i g h t connecting strap. Such d i s t o r t i o n would r e s u l t i n diminished deshielding by the methyl protons attached at the 2 and 12 p o s i t i o n s , thereby giving r i s e to an u p f i e l d s h i f t . A s i m i l a r trend i s found i n the methine proton resonances (Fig. 3.12 B). With decreasing chain length, both meso signals are s h i f t e d u p f i e l d , with the peak at higher f i e l d being s h i f t e d more. I r r a d i a t i o n of the m u l t i p l e t due to the 7 and 17 methylene protons (at 6 4.09 and 64.06 for the durene-5/5 and -4/4 d e r i v a t i v e s , respectively) causes an enhancement i n the i n t e n s i t y of the higher f i e l d meso-proton resonance, i n d i c a t i n g that t h i s s i g n a l corresponds to the protons at the 5 and 15 p o s i t i o n s . I t i s these protons that are closer, i n terms of carbon-carbon bonds, to the chain-linked 3 and 13 r i n g p o s i t i o n s . D i s t o r t i o n caused by a strap l i n k i n g p o s i t i o n s 3 and 13 i s therefore expected to a f f e c t the 5 and 15 meso protons more than the 10 and 20 protons, thereby g i v i n g r i s e to the increasing separation between t h e i r respective resonances. This observation for the durene porphyrins i s consistent with findings i n other s y s t e m s 1 0 9 , 1 1 0 . A r e v e r s a l of t h i s trend i s displayed f or the N - H protons i n a I l l -downfield s h i f t , r e l a t i v e to e t i o I I , the s h i f t increasing with decreasing cap si z e (Fig. 3.12 C). Situated within the porphyrin core, these N-H protons experience extensive s h i e l d i n g from the porphyrin r i n g current that causes them to resonate at high f i e l d ( 6-3.78 for e t i o I I ) . A d i s r u p t i o n of the electron d e r e a l i z a t i o n , caused by increasing d i s t o r t i o n of the macrocycle from p l a n a r i t y , i n t h i s case r e s u l t s i n a diminished s h i e l d i n g e f f e c t and thus a gradual s h i f t to lower f l e l d l 0 9 . H 0 . Inequivalence of the CH 2 protons of the e t h y l groups at p o s i t i o n s 7 and 17 i s revealed as a complex mu l t i p l e t resonance instead of a quartet ( 5 4.11) as i n e t i o I I . The inequivalence i n unmetallated systems has been interpreted i n terms of i n t r i n s i c asymmetry i n the porphyrin molecule^O^,127_ A s t r i k i n g feature of the ^H-nmr spectra i s the large u p f i e l d s h i f t s observed f o r some of the proton resonances of the methylene strap (up to 5 -1.74 f o r the durene-4/4 system). Such u p f i e l d s h i f t s have been observed f o r various strapped porphyrin d e r i v a t i v e s ^ ^ , 110,128 Those protons belonging to functional groups positioned above the porphyrin macrocycle experience a strong s h i e l d i n g e f f e c t from the diamagnetic r i n g current, which i s responsible f o r the u p f i e l d s h i f t ( r e l a t i v e to that of the free chain d e r i v a t i v e ) . The protons associated with carbon atoms attached to the porphyrin periphery, however, experi-ence the deshielding e f f e c t of the porphyrin r i n g current and thus resonate further downfield . r e l a t i v e to those above the porphyrin macrocycle. A s e r i e s of double resonance experiments c a r r i e d out on the C^g-strapped porphyrin 81b!09 revealed that a p a r t i c u l a r m ultiplet F i g . 3.10 H^-NMR spectrum of the durene-5/5 capped porphyrin, 55a F i g . 3. 11 H^-NMR spectrum of the durene-7/7 capped porphyrin, 56a 115 -A: Porphyrin Methyl Protons B: Methine Protons 4 / 4 j j 5/5 I j j 7/7 II II etioll j 1 1 1 1- —1 1 1 1 1 1-3 . 6 3 . 5 3 . 4 3 . 3 10.1 10-0 9 . 9 9 - 8 9 . 7 9 . 6 C: Nitrogen Protons D: Durene Methyl Protons 4 / 4 | | 5 / 5 | 7/7 I I etioE 1 1 1 1 — —i 1 1 1 1 1 1— - 3 8 - 3 . 7 - 3 . 6 - 3 5 0 . 4 0 . 3 0 . 2 0 . 1 0 - 0 . 1 - 0 . 2 Fig. 3.12 Variation in chemical shifts of proton signals for the 4/4-, 5/5- and 7/7-durene porphyrins compared with that for etioporphyrin II. 116 -(assigned to two chain CH2 protons) arises not from protons on the same carbon, but from equivalent protons attached to opposite carbons of the diagonal methylene chain. In t h i s r i g i d C i o _ s t r a P P e d system, two protons attached to the same carbon resonated as much as 6 3.64 apart, an i n d i c a t i o n of the influence of a r i n g current e f f e c t that depends on the d i r e c t i o n i n which a proton p o i n t s 1 0 9 . Those that point toward the porphyrin macrocycle experience a stronger s h i e l d i n g e f f e c t than those pointing away. Further s i g n i f i c a n t information can be gleaned from the signals of the durene methyl groups (Fig. 3.12 D). In the 7/7- and 5/5- systems a l l four methyls are equivalent, appearing as a s i n g l e t i n each case. This indicates that r o t a t i o n of the c e n t r a l durene m o i e t y 1 0 9 i s possible to the extent that a l l the methyl protons experience a s i m i l a r environment. In the 4/4-system, however, t h i s r o t a t i o n i s presumably r e s t r i c t e d owing to the r i g i d i t y of a shorter l i n k i n g chain, so that the s i g n a l a r i s i n g from the four methyl groups becomes s p l i t . High temperature ^-nmr spectra run i n toluene-dg showed coalescence of t h i s doublet into a s i n g l e t at >80°C. Low temperature studies on the 7/7-and 5/5- systems i n CD2CI2 down to -70°C r e s u l t e d i n gradual broadening of the durene methyl proton s i g n a l , without s p l i t t i n g and indicates considerably greater f l e x i b i l i t y associated with the larger straps. The chemical s h i f t f o r these methyl protons shows no consistent trend within the durene systems, with the s i n g l e t f o r the 7/7-cap occurring at higher f i e l d r e l a t i v e to those for the 4/4- and 5/5-durene caps. An analogous s i t u a t i o n i s observed within the C^Q (81b) and C9 (81c) strapped p o r p h y r i n s 1 0 9 . The highest f i e l d methylene chain resonance i s observed with the C^o'derivative, despite the (possibly) - 117 -clo s e r proximity of the smaller strapped Cq-chain protons to the porphyrin macrocycle. This once again i l l u s t r a t e s that the sh i e l d i n g depends on the p a r t i c u l a r o r i e n t a t i o n of a proton with respect to the porphyrin r i n g current i n such r i g i d , strapped systems. In the 7/7-durene system, however, since the f l e x i b i l i t y of the cap allows free r o t a t i o n of the durene moiety, an u p f i e l d s h i f t i n the methyl resonance ( r e l a t i v e to the 5/5-system) may indicate that the preferred conforma-t i o n f o r t h i s l a rger cap requires the durene moiety to be suspended cl o s e r to the porphyrin plane than i n the smaller strapped systems, thus experiencing a stronger s h i e l d i n g e f f e c t . Conclusive information on the 7/7-cap conformation, however, awaits a c r y s t a l structure determination. 3.3.2 •LJC-nmr Spectra of Durene Capped Porphyrins The 1 3C-nmr spectra were recorded i n 10% T F A - C D C I 3 , and are therefore of the porphyrin dications 1 2° (Figs. 3.13-3.15). The peak assignments are based on the already e x i s t i n g data f o r etioporphyrin I I , 113109 a n c j durene-5/5 porphyrin s y s t e m 1 ^ _ -rh e signals are divided up into f i v e groups: ( i ) the a- and/3- p y r r o l i c carbons, ( i i ) the meso carbons, ( i i i ) the r i n g a l k y l substituents, ( i v ) the chain methylene carbons and (v) the durene r i n g and methyl carbons. The O! - and j3-pyrrolic carbons of e t i o II appear as four d i s t i n c t resonances (Table 3.1), each corresponding to two Q! and two j3 carbons. For the capped systems, the eight d i f f e r e n t types of p y r r o l i c carbons give r i s e to the eight observed s i g n a l s 1 ^ _ Porphyrin meso carbons are p a r t i c u l a r l y s e n s i t i v e to the 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 1 1 F i g . 3.13 C-NMR spectrum of the durene-A/4 capped porphyrin, 54a f4 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm F i g . 3.14 13C-NMR spectrum of the durene-5/5 capped porphyrin, 55a 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 p p m F i g . 3.15 13C-NMR spectrum of the durene-7/7 capped porphyrin, 56a 121 -TABLE 3.1: 1 3C-nmr Data on the Durene-Capped Porphyrins Compared to Etioporphyrin I I . Eti o II (113) 7/7 , (56a) 5/5 1 ( 5 5 a ) 4/4 (54a) (X- and jS-pyrrolic 143.54 145 .45 146 .07 146.54 carbons 143 .45 143 .08 144.87 142.29 142 .68 142 .90 143.00 142 .46 142 .71 140.94 141.53 141 .65 141 .25 140.31 140 .85 140 .31 139.57 136.92 139 .71 (140 • 31) 139.42 138 .78 139 .17 138.62 Durene r i n g 135 .52 134 .79 135.69 130 .60 130 .25 129.49 129.17 Meso carbons 98.39 99 .28 99 .87 99.93 97.95 98 .91 98.84 Chain methylene 31 .19 30 .80 28.66 29 .85 29 .46 28.00 29 .26 (29 .46) 27.55 28 .44 (29 .46) 25.20 28 .37 27 .20 27 .76 26 .82 CH 3CH 2 20.11 20 .15 20 .16 20.07 CH 3CH 2 16.42 16 .43 16, .67 16.38 Durene CH3 15. .20 14. .65 14.46 Porphyrin CH3 11.67 12. .19 12, .28 12.00 11. .66 11, .68 11.59 122 -s u b s t i t u t i o n pattern at the pyrrole periphery, to the extent that two signals are observed for the two d i f f e r e n t meso carbon environments i n e t i o I I . S i m i l a r l y , the durene-5/5 and -4/4 porphyrins, also exhibit two resonances i n the same region. In the 1 3C-spectrum of the 7/7-system, however, the two resonances coincide, so that only one s i g n a l corres-ponding to the four meso carbons i s observed. The peripheral methyl carbon resonances are s p l i t i n a l l three durene systems because of the i n t r i n s i c asymmetry introduced into the molecule with the presence of a cap. Separate resonances corresponding to benzene carbons 1-,4-C and 2-,3-,5-,6-C, as well as a single peak due to the durene methyl carbons, are observed for the durene-7/7 and -5/5 systems. In the 4/4-system a further s p l i t t i n g of the r i n g 2-,3-,4-,6-C and methyl carbons r e s u l t s , caused by the r i g i d i t y of the smaller cap; t h i s i s consistent with ^H-nmr data observed for the durene methyl protons. F i n a l l y , the chain methylene carbons give r i s e to separate signals that correspond to the number of equivalent carbons from ei t h e r side of the l i n k i n g chain. As i n the case with other s y s t e m s 1 ^ ( a n c j u n i i k e the proton s i g n a l s , the resonances of the methylene carbons do not undergo the pronounced u p f i e l d s h i f t s caused by the diamagnetic r i n g current. 3.3.3 E l e c t r o n i c Absorption Spectra of Durene-Capped Porphyrins The two hydrogens i n the centre of the free-base porphyrin strongly reduce the conjugated r i n g symmetry, and give r i s e to two possible tautomeric f o r m s 1 2 9 . The intense (Soret) band found at -400 nm - 123 -i s due to a strongly allowed 7T—> 7T t r a n s i t i o n , which i s s i m i l a r for both tautomers, while the four weaker bands (I, I I , III and IV) i n the 650-480 nm region a r i s e i n p a i r s , two from each tautomer, from a second forbidden 7T—> 7T , t r a n s i t i o n which i s two-fold degenerate- 1-^. The r e l a t i v e i n t e n s i t i e s of the four v i s i b l e bands are s e n s i t i v e to the nature of substituents at the porphyrin, p e r i p h e r y 1 2 9 . For a l k y l substituted porphyrins, a t y p i c a l etio-type spectrum i s observed with the i n t e n s i t i e s of the four bands varying i n the order IV>III>II>I (Fig. 3.16 A ) 1 2 9 b . The presence of strongly electron-withdrawing groups such as aldehydes, carboxylic acids, esters, a c r y l i c acids etc., causes band III to become more intense than IV, a f f o r d i n g a rhodo-type spectrum (Fig. 3.16 B ) 1 2 9 b . Two rhodofying groups on adjacent 'pyrrole' subunits cancel out each other's e f f e c t r e s u l t i n g i n an etio-type spectrum, while such substituents on diagonally opposite pyrrole rings r e s u l t In an enhancement of the rhodofying e f f e c t and gives r i s e to an oxorhodo spectrum (Fig. 3.16 C ) 1 2 9 b . A fourth v a r i a t i o n i n free base spectra i s found for unsymmetrically substituted porphyrins, with four or more periph e r a l p o s i t i o n s unsubstituted, as for l,4-diethyl-2,3-dimethyl-porphyrin; such porphyrins e x h i b i t a phyllo-type spectrum (Fig. 3.16 D )129b Aside from porphyrin substituent e f f e c t s , s k e l e t a l d i s t o r t i o n can also a l t e r porphyrin free-base spectra. This i s exemplified i n the Cg-Cn porphyrin s e r i e s , 8 1 a - c , 8 , 1 0 9 where the d i s t o r t e d Cg-system -exhibits a rhodo-type of spectrum, although the p e r i p h e r a l s u b s t i t u t i o n pattern i s e s s e n t i a l l y that of an etioporphyrin system. This has been i n t e r p r e t e d 8 , 1 0 9 as a r i s i n g from the extreme porphyrin plane d i s t o r t i o n found i n t h i s system; t h i s appears to have a s i m i l a r e f f e c t on the - 124 -F i g . 3.16 Trends i n v i s i b l e spectra f o r various p y r r o l e - s u b s t i t u t e d porphyrins F i g . 3.17 UV-VIsIble spectrum of the durene-7/7 porphyrin, 56a F i g . 3.18 UV-Visible spectrum of the durene-5/5 porphyrin, 55a 1 1 1 1 1 1 1 1 300 350 400 450 500 550 600 650 700 Wavelength (n m) F i g . 3.19 UV-Visible spectrum of the durene-4/4 porphyrin, 54a (CH 2 ) , ' (CH 21, i • . . . i • - — i 350 400 450 500 550 600 650 700 Wavelength (n m) F i g . 3.20 UV-Visible spectral trends f o r the hemin chloride d e r i v a t i v e s of the durene-capped porphyrins 1 2 9 molecular o r b i t a l s of the macrocycle as that caused by e l e c t r o p h i l i c substituents attached to the porphyrin periphery. A s i m i l a r trend i s found within the durene-series, where shorten-ing of the l i n k i n g methylene chain of the 'durene cap', correlates with a gradual increase i n i n t e n s i t y of band I I I r e l a t i v e to that of I V (Fig. 3 . 1 7 - 3 . 1 9 ) . Therefore, for the durene systems also the observed increase i n i n t e n s i t y of band I I I i s interpreted i n terms of increased porphyrin plane d i s t o r t i o n i n the smaller strapped 5 / 5 and 4/4-d e r i v a t i v e s . Notably, the enhancement i n the i n t e n s i t y of band I I I i s much less pronounced i n the durene-4/4 porphyrin than i n the extremely d i s t o r t e d Co-system. A trend i n spectral change i s also observed for the four d i f f u s e bands of the hemin chloride derivatives within the durene series (Fig. 3.20). For the smallest capped durene-4/4 system, porphyrin plane d i s t o r t i o n i s indeed c l e a r l y evidenced i n the c r y s t a l structure of the hemin chloride (section 3.4). 3.4 CRYSTAL STRUCTURE OF DURENE-4/4 HEMIN CHLORIDE 1 3 0 Crystals of the t i t l e complex suitable f or X-ray d i f f r a c t i o n were grown by slow evaporation of a s o l u t i o n of the hemin chloride, F e 1 1 1 -(54b)Cl, i n CH2Cl2/MeOH. The structure determination was c a r r i e d out by Dr. F. E i n s t e i n and T. J o n e s X J U at Simon Fraser U n i v e r s i t y , Vancouver. The r e s u l t s of the analysis are presented below. The molecular structure of Fe(durene-4/4)Cl (Fig. 3.21) indicates a very d i s t o r t e d porphyrin core which r e s u l t s d i r e c t l y from the carbon 130 C(171) Cl (D C(182) C(81) C(82) C{251) C(24) yV\ CJZooJ C(56D F i g . 3.21 Cr y s t a l structure of the durene-4/4 hemin chloride complex, F e 1 I ] [ ( 5 4 b ) C l 'strap'. Nevertheless, the square pyramidal coordination environment found about the Fe atom i s t y p i c a l of other high spin Fe(III) porphyrins which do not contain a ' s t r a p ' 1 3 1 . The F e ( l ) - C l ( l ) distance of 2.232 A o T T T i s close to the value of 2.218 A found for FeJ-J--L (protoporphyrin) Cl and o the Fe••••CR (centroid of the phenyl ring) distance of 5.613 A indicates no i n t e r a c t i o n between the phenyl group of the strap and F e ( l ) . The d i s t o r t i o n of the porphinato skeleton found here i s not as marked as found i n the re l a t e d free-base Cg porphyrin 81c, that 8 possesses a shorter methylene carbon chain across the porphyrin • The - 131 -angle between the best planes of pyrrole rings 1 and 3 i s 43.0° compared with 68.5° i n Cp, (on the basis of a coplanar angle of 0°). The corresponding angle between rings 2 and 4 i s 17.2°, compared with 13.5° i n Cg. The s t r a i n induced by the strap appears to be d i s t r i b u t e d throughout the en t i r e molecule rather than being l o c a l i z e d within the two 'strapped' pyrrole rings. Bond distances and angles around the porphinato skeleton are remarkably s i m i l a r to those found i n other 'undistorted' nonstrapped XFe(III) porphyrins (Table 3 . 2 ) 1 3 1 . The bond parameters for the 'strap' Table 3.2: Comparison of the Square-Pyramidal Coordination Sphere i n Fe(durene-4/4)Cl with Other High Spin F e ( I I I ) Porphyrins.. Fe(durene-4/4)C1 Average Range Fe-Np 2.051-2.062 2.069 2.060-2.087 Fe-Ct N 0.485 0.47 0.39-0.54 C t N - N p 1.981-2.010 2.015 Fe-Ct p 0.642 0.51 0.39-0.62 Np •= porphinato nitrogen C t N - centroid of porphinato N atoms Ct_ - centroid of best plane of 24-atom porphinato skeleton - 132 -and a l k y l substituents are also normal. Transannular parameters indic a t e that the cen t r a l porphyrin c a v i t y has contracted as a r e s u l t of binding to the iron, e.g. N ( l ) , N(3), and N(2), N(4) are 3.965 and 4.016 o o A, respectively, compared with 4.003 and 4.241 A i n the Cq-porphyrin. In the c r y s t a l structure of the TPP-derived C2-capped system, 15a, such 'contraction' r e s u l t s i n less r u f f l i n g of the porphyrinato skeleton i n the hemin chloride d e r i v a t i v e Fe(C2-cap)Cl r e l a t i v e to that i n the free-base porphyrin. 133 CHAPTER 4 INTERACTION OF THE DURENE-CAPPED HEMES WITH IMIDAZOLES, ISOCYANIDES, CO, AND 0 2 134 -4. INTERACTION OF THE DURENE-CAPPED HEMES WITH IMIDAZOLES, ISOCYANIDES, CO, AND 0 2 4.1 MATERIALS AND APPARATUS 4.1.1 General Toluene (1 L) was washed with C.H2SO4 (2 x 200 mL) followed by water (2 x 100 mL), and d i s t i l l e d over CaH 2 immediately p r i o r to use. o-Pichlorobenzene ( A l d r i c h , s p e c t r a l grade) was used d i r e c t l y without further p u r i f i c a t i o n . Sodium D i t h i o n i t e (sodium hydrosulphite) and 18-crown-6 (1,4,7,-10,13,16-hexaoxacyclooctadecane) were purchased from A l d r i c h . 1.2-Dimethylimidazole (Aldrich) was r e c r y s t a l l i z e d from heptane, while 1-methylimidazole (Aldrich) was d i s t i l l e d under reduced pressure from KOH. 1.5-Picvclohexylimidazole (40) was prepared according to a procedure described by T r a y l o r 6 5 . ^H-nmr (5, C D C I 3 ) : 1.2-2.0 (m, 20H, c y c l o h e x y l - C 5 H 1 0 ) , 2.43 (m, IH, 5-C-CH), 3.73 (m, IH, 1-N-CH), 6.73 (s, IH, 4-CH), 7.47 (s, IH, 2-CH); m.p. - 113-114°C. t-Butvlisocvanide. tosylmethvlisocyanide. and n-butylisocyanide were purchased from A l d r i c h and used d i r e c t l y . Gas-lipht syringes with Luer-lock t e f l o n t i p s and teflon-coated plungers were purchased from Hamilton. Carbon monoxide (99.3%) and dioxypen (99.99%) were passed through KOH columns p r i o r to use. Nitrogen (99.99%) was I n i t i a l l y passed 135 through a Ridox column to remove any trace oxygen, and dried over o molecular sieves (3 A). The s o l u b i l i t i e s of CO and 0 2 i n toluene used were 1.0 x 10" 5 M t o r r " 1 and 1.2 x IO"-* M t o r r " 1 respectively, i n toluene, and 6.8 x 10" 6 M t o r r " 1 and 8.0 x 10" 6 M t o r r " 1 respectively, i n CH 2C1 2, at 2 0 ° C 1 3 2 . 4.1.2 Thermostatting Equipment (a) For temperatures down to -10°C A Haake (model FK) c i r c u l a t i n g thermostatting bath f i l l e d with MeOH was used to maintain a constant temperature. The Haake was connected to a c e l l - h o l d i n g Dewar (Fig. 4.1), constructed i n the Chemistry Department Mechanical Shop, U.B.C., by means of rubber leads attached to a copper c o i l . The copper c o i l was immersed i n another s o l u t i o n of methanol contained within the c e l l - h o l d i n g Dewar. This methanolic s o l u t i o n thermally e q u i l i b r a t e d a copper block i n the c e l l -holding compartment. A tonometer (with c e l l path length 1 mm or 10 mm) could be suspended i n the Dewar using a rubber seal between the , central tube and tonometer. The s o l u t i o n i n the tonometer was e q u i l i b r a t e d to temperature by the surrounding copper block. During the course of a t i t r a t i o n , the e n t i r e Dewar was placed within the Cary spectrometer, while the tonometer was c a r r i e d from vacuum-line to Dewar. When equilibrium experiments below 0°C were performed, the s t a i n l e s s s t e e l jacket f i t t e d with quartz windows could be evacuated and f i l l e d with argon, i n order to prevent condensation on the windows. 136 In addition, the central tube was flushed with argon each time the tonometer was replaced for temperature e q u i l i b r a t i o n . (b) For low temperatures down to -80°C A 6 cm path length quartz c e l l mounted inside a small Dewar (Fig. 4.2) was used for low temperature work. The Dewar was f i l l e d with a slush bath of appropriate temperature to e q u i l i b r a t e the s o l u t i o n contained within the quartz c e l l . The slush baths used were: chloro-benzene-liquid N 2 (-45°C), bromobenzene-liquid N 2 (-30°C), carbon t e t r a -chloride - l i q u i d N 2 (-23°C) o-dichlorobenzene-liquid N 2 (-18°C), ethylene glycol-dry ice (-15°C), and cyclohexane-liquid N 2 (+6°C). 4.1.3 E l e c t r o n i c Absorption Spectra A Cary 17 spectrophotometer was used to obtain a l l u v - v i s i b l e spectra i n the 700-250 nm region. 4.1.4 Infra-red Absorption Spectra Carbonyl stretching frequencies of Fe 1 1(Por) ( B )(CO) complexes were recorded on a N i c o l e t 5DX Fourier transform IR spectrometer. Solutions of the heme complex i n toluene were transferred into IR s o l u t i o n c e l l s with KBr windows (volume 0.25 ml, path length 0.5 cm). - 137 -tonometer septum cap CENTRAL TUBE ^ — in from Haake RUBBER STOPPER Copper coil EVACUATED STAINLESS STEEL JACKET EVACUATION VALVE CONSTANT TEMPERATURE BATH 1 -WffoMiViim-t COPPER BLOCK OPTICAL CELL -QUARTZ WINDOW COPPER BLOCK Fig. A.l Thermostatted cell-holder for temperatures above -10°C 138 -"> joint Q To VACUUM LINE D E W A R F L A S K C O N S T A N T T E M P E R A T U R E B A T H *J S E A L E D - I N Q U A R T Z W I N D O W S 4.2 6 cm path-length quartz c e l l 139 -4.1.5 Stopped Flow Apparatus Carbon monoxide d i s s o c i a t i o n rates were studied using a Durrum Stopped Flow (model 110) apparatus. Toluene solutions of TMIC and Fe I X(Por)(B)(CO) of equal volume, were pr e - e q u i l i b r a t e d to 20°C and ra p i d l y mixed i n a 2 cm path length c e l l compartment. The change i n absorbance versus time f o r the formation of Fe 1 1(Por)(B)(TMIC) was monitored spectrophotometrically (see section 4.4.5(b)). 4.1.6 Flash Photolysis Apparatus Photolysis of a (d 6) M(Por)L n (n-1 or 2) adduct gives r i s e to ( 7T—>7T*) excited states which are strongly antibonding f o r IT acceptor ligands such as CO. Consequently Fe 1 1(Por)(CO) complexes are highly photolabile ( 0 - l ) 1 3 4 . Nitrogenous ligands, however, which act pr i m a r i l y as O donors, are stable toward photolysis from the FeH(Por) c e n t r e 1 3 5 . Systems other than d 6, Fe I J-N0 (d 7) and F e 1 1 ^ ( d 8 ) , also photo-dissociate the gaseous ligand to a much l e s s e r extent than Fe 1 1-C0 (<t>Q2 -10"2,</>NQ - 1 0 * 3 ) 1 3 5 , 1 3 6 . I t Is s u g g e s t e d 1 3 5 that photoexcitation i n these systems i s followed by rapid r a d i a t i o n l e s s processes that populate states lower i n energy than that of 7T o r b i t a l s , and the M-axial li g a n d bond strength i s not s i g n i f i c a n t l y reduced. Flash photolysis under e i t h e r CO or CO/O2 mixtures was used to determine CO k i n e t i c constants, and equilibrium and k i n e t i c constants f o r 0 2 binding. The experiments were performed i n the Chemistry Department, U n i v e r s i t y of C a l i f o r n i a , San Diego. A d e s c r i p t i o n of the - 140 -apparatus has been provided e l s e w h e r e 1 3 7 . The sample i n a tonometer was thermostatted to i0.1°C by a c l o s e l y f i t t i n g aluminum block. P h o t o l y s i s was accomplished u s i n g a Phase-R DL2100D tunable, flashlamp pumped dye l a s e r w i t h a r a t i n g of 0.5 I/pulse; the decay ra t e constant of the l a s e r was -1.7 x 10° s" 1, w i t h a pulse width of 400 ns. The p h o t o l y s i n g pulse enters the apparatus at r i g h t angles to the monitoring beam that f o l l o w s the sample absorbance at a f i x e d wavelength. Data from s e v e r a l p h o t o l y s i s events were s t o r e d i n a microprocessor and l a t e r computer analyzed. - 141 -4.2 SOLUTION PREPARATION A l l s o l u t i o n studies were performed i n toluene unless otherwise noted. In the past, s e l e c t i o n of a suitable solvent has been determined p r i m a r i l y by i t s a b i l i t y to sustain a six-coordinate heme-02 adduct, during oxygen equilibrium studies. Toluene, providing a r e l a t i v e l y non-polar environment of d i e l e c t r i c constant 2.38, increases the s t a b i l i t y of the oxygen adduct by reducing the rate at which i t autoxidizes (section 2.5.8). For the sake of consistency, t h i s solvent has been used i n the majority of heme-ligand binding studies, i n order that data from various systems may be compared d i r e c t l y . Because of the tendency for i r o n ( I I ) to become oxidized to i r o n ( I I I ) i n the presence of trace 0 2 (section 2.2), preparation of a l l heme solutions and subsequent binding experiments were necessa r i l y c a r r i e d out with c a r e f u l exclusion of a i r . Two d i f f e r e n t methods were used f o r the chemical reduction of the hemin chl o r i d e complexes, depending on the type of experiment being performed. 4 . 2 . 1 Crown Ether-Dithionite Method of Reduction (a) Crown e t h e r - d i t h i o n i t e s o l u t i o n A s o l u t i o n of crown-ether d i t h i o n i t e was prepared according to the method described by Mincey and T r a y l o r 1 3 8 . The s o l i d complex was not i s o l a t e d i n t h i s case, but used d i r e c t l y as a s o l u t i o n 7 ^ . - 142 -Methanol (1.0 mL) and 18-crown-6 (0.1 g, 0.38 mmol) were placed i n a v i a l and purged with N 2. A large excess of sodium d i t h i o n i t e (0.42 g, 2.3 mmol) was added into the solution, a septum inserted over the opening, and the mixture s t i r r e d magnetically for - 30 min. The s o l i d sodium d i t h i o n i t e was allowed to s e t t l e to the bottom of the v i a l , l eaving a cl e a r s o l u t i o n on top, from which small aliquots ( 5 0 f l L ) were withdrawn by syringe. The s o l u t i o n was found to r e t a i n i t s reducing a c t i v i t y overnight, although fresh samples were always prepared immediately p r i o r to use. (b) Hemin chloride reduction The hemin chloride dissolved i n dry toluene (4 mL) was placed i n a round-bottom f l a s k , clamped h o r i z o n t a l l y as shown i n F i g . 4.3.A. The so l u t i o n was freeze-thaw degassed three times, CO admitted, and tap B closed o f f . A f r e s h l y prepared s o l u t i o n of crown e t h e r - d i t h i o n i t e complex i n methanol (10 flL) was then syringed i n v i a tap A, and the mixture s t i r r e d f o r 15 min to ensure complete reduction of the heme to the ferrous state. The f l a s k was then connected v e r t i c a l l y to a tonometer (Fig. 4.3.B, v i a j o i n t s P and Q) which was subsequently evacuated with taps C and D open. Then, with tap D closed, tap B was opened and the s o l u t i o n allowed to flow into the tonometer. F i n a l l y , N 2 at 1 atm was admitted v i a tap D, tap C closed o f f , and the f l a s k disconnected. With the use of t h i s system of transfer, the f a i n t l y cloudy s o l u t i o n , formed a f t e r the i n j e c t i o n of reducing agent, could be f i l t e r e d through the sintered 143 F i g . 4 . 3 (A) Apparatus used f o r reducing F e m ( P o r ) to Fe I ] :(Por) using crown e t h e r - d i t h i o n i t e joint Q (B) Tonometer f o r measuring base binding constants to four-coordinate Fe(II) hemes •eptum plug B - 144 -glass f r i t to give i n the tonometer a clea r s o l u t i o n of reduced henie under N2/CO. This method was used i n preparing solutions of four-coordinate hemes for t i t r a t i o n with substituted imidazoles. With the tonometer connected to a vacuum-line, a small volume of toluene (0.5 mL) was pumped away to ensure complete removal of the methanol (10 piL) and CO, leaving in situ the four-coordinate heme complex. The tonometer was c a l i b r a t e d so that the s o l u t i o n volume could be measured inside the c e l l . Where trace amounts of CO were found to i n t e r f e r e with the imidazole-binding e q u i l i b r i a , as i n the Fe 1 1(durene-5/5) and F e 1 1 -(durene-7/7) t i t r a t i o n s with Dclm, the hemin chl o r i d e was reduced i n the absence of CO, under N 2 atmosphere. U t i l i z a t i o n of CO was preferred whenever possib l e , however, because of the added protection against oxidation o f f e r e d by formation of an iron(II)-C0 system. By t h i s technique of reduction and transfer, the four-coordinate heme could be r e l i a b l y generated and was found to be stable i n s o l u t i o n , with no trace of oxidation over at l e a s t 3 h. 4.2.2 Aqueous D i t h i o n i t e Method of Reduction A s o l u t i o n of the hemin chloride and appropriate imidazole base, of required concentration, i n toluene (6 mL), was placed i n f l a s k A of a set up as shown i n F i g . 4.4 A. The e n t i r e three f l a s k system and hemin s o l u t i o n was deoxygenated, N 2 admitted and tap 4 closed. A small volume (0.5 mL) of deoxygenated aq. Na 2S 20 4 (-2 M) was cannulated into the hemin s o l u t i o n v i a tap 1, by means of a 2' long double-ended needle and - 145 -connection to line Flask C Flask A septum plug CaH 2 — 4 stirrer F i g . 4.4 (A) Apparatus used f o r reducing F e I I I ( P o r ) t o F e 1 1 (Por) usi n g aqueous d i t h i o n i t e Joint 0 (B) Tonometer f o r measuring l i g a n d b i n d i n g constants to f i v e - c o o r d i n a t e hemes, Fe**(Por)(B) to Itn* B 146 -a stream of N 2. The mixture was s t i r r e d f o r 15 min, within which time reduction to the five-coordinate, Fe**(Por)(B), complex was complete. The toluene layer was then decanted into f l a s k B and p a r t i a l l y dried over anhydrous Na2SO^ for a few minutes. The s o l u t i o n was f i n a l l y t i l t e d into f l a s k C and dried over CaH 2 for 30 min. At t h i s stage, tap 3 was closed and f l a s k A disconnected f o r convenience. The f l a s k system was then connected to a tonometer ( F i g . 4.4.B) by attaching j o i n t s P and Q, and the tonometer evacuated completely, keeping taps 5 and 6 open. With tap 6 closed, tap 4 was then opened and the s o l u t i o n allowed to f i l t e r through the f r i t i nto the tonometer. Tap 5 was closed o f f , the fl a s k s removed, and N 2 admitted i n through tap 6. A septum plug was inserted into the out l e t of tap 6 and the "dead space" purged with N 2. Aliquots of ligand, e i t h e r s o l u t i o n or gas could be syringed i n by opening tap 6, when desired. This technique of s o l u t i o n preparation was preferred over that of the 'crown e t h e r — d i t h i o n i t e ' method, because the heme s o l u t i o n i n t h i s case contained no excess reducing agent or methanol which could p o t e n t i a l l y i n t e r f e r e with heme-ligand binding behaviour. - 147 4.3 MATHEMATICAL ANALYSES 4.3.1 Equilibrium Constant Determination The equilibrium constants f o r reactions shown i n eqs. (26) and (27), F e I I ( P o r ) + B " Fe i : [(Por)(B) (26) B -= Dclm, 1,2-Me2lm or Melm Fe I T ( P o r ) ( B ) + L _ F e 1 1 ( P o r ) ( B ) ( L ) (27) L = i s o n i t r i l e , CO, or 0 2 are defined by: [Fe(Por)(B)] K B - (28) [Fe(Por)][B] [Fe(Por)(B)(L)] and K L - (29) [Fe(Por)(B)][L] The constants are a measure of the a f f i n i t y of the reactant heme complex fo r the ligan d B or L, and are given by the inverse of the concentration of B or L required to convert h a l f of the reactant heme into p r o d u c t 1 3 9 . Kg denotes the binding constant of a base, B, to the unhindered side of the four-coordinate capped heme, where coordination on the capped side i s i n h i b i t e d f o r s t e r i c reasons, and the re a c t i o n stoichiometry i s therefore 1:1 f o r heme:base. In a separate experiment K1, i s determined. 148 In t h i s case excess of base, B, blocks the unhindered side of the heme, so that the binding constant (K*") corresponds e x c l u s i v e l y to the coordination of L on the capped face. Equations (28) or (29) may be re-written i n a form such as: f r a c t i o n of product y K B[B] or K L[L] - = (30) f r a c t i o n of reactant l - y I f followed spectrophotometrically, y can be evaluated from: A - A Q Aeo " A o where A Q - i n i t i a l absorbance of reactant A^ — f i n a l absorbance of product and A - absorbance at a s p e c i f i c [B] or [L] The logarithmic conversion of eq. (30) gives the H i l l equation 140. (31) y log( ) - log[B] + log Kg (32a) l - y or log[L] + log K L (32b) y A p l o t of log( ) versus log [B] or log [L] should r e s u l t i n a s t r a i g h t l - y l i n e of slope 1.0 for the coordination of one extra l i g a n d at the heme, and the equilibrium constants Kg or K L may be determined from the x- or y- axis intercept of the log:log p l o t . In general, when the value of the slope i s not exactly 1.0, the x-axis intercept has been used to give 149 -1 T 1 a d i r e c t estimation of K R - , or - . For the gases, with L -= [B] [L] CO or 0 2 eq. (27), the ligand concentration [L] i s commonly expressed i n terms of p a r t i a l pressure of the gas over the s o l u t i o n (P^). The equi-l i b r i u m constant f o r the binding of a gas can then also be written as: 1 K ( t o r r " 1 ) - (33) • L p l / 2 L where Pi/2 ~ t n e p a r t i a l pressure of gas ( L ) required to convert h a l f the five-coordinate heme into the six-coordinate l i g a t e d species. Concentrations of ligand (B or L) and heme are chosen so that an excess of [ligand] r e l a t i v e to [heme] e x i s t s i n s o l u t i o n at a l l times, i n order that equilibrium may be established independent of [heme]. I f the a f f i n i t y of a heme complex for a given lig a n d i s so high such that, f o r example, a [ligand] of -1 x 10"° M with a [heme] -2 x 10"° M, converts e s s e n t i a l l y h a l f the reactant heme into product, then the [ligand] and the [heme] must be correspondingly lowered i n order to f i n d conditions i n which excess ligand i s present at equilibrium. This would mean a reduction i n [heme] and r e s u l t i n absorbance values too low for spectrophotometric changes to be monitored accurately. In such instances, the a f f i n i t y constants cannot be determined by d i r e c t t i t r a t i o n with the ligand, but are instead measured by using competitive l i g a t i o n techniques (see sections 4.4.2(b) and 4.4.3(b)). Equilibrium constant determinations for 0 2 and CO binding to some durene-4/4 heme systems were c a r r i e d out at v a r i e d temperatures, where Van't Hoff p l o t s of l n K versus 1/T(°K), eq. (34), afforded the thermo-dynamic constants A H 0 and A s ° from the slope and y-intercept, 150 -r e s p e c t i v e l y 1 3 9 . _ A H ° A S 0 In K - + ( 3 4 ) RT R 4.3.2 Determination of the Carbon Monoxide Asso c i a t i o n ( k c o ) and D i s s o c i a t i o n ( k " c o ) Rate Constants Subsequent to photolysis of the Fe-CO bond, the rate at which the heme-CO complex i s re-formed, eq. (35), may be followed spectrophoto-m e t r i c a l l y 1 4 1 . hV Fe(Por)(B)(C0) > Fe(Por)(B) + CO % Fe(Por)(B)(CO) (35) v-CO At a constant, known [CO], where [CO] » [heme], pseudo f i r s t - o r d e r conditions e x i s t , and the observed rate constant ( k Q ^ s ) Is given by: <obs " k c o[CO] + k " C 0 ( 3 6 ) where k^ u and k"^° are the a s s o c i a t i o n and d i s s o c i a t i o n rate constants, r e s p e c t i v e l y 1 4 2 . Monitoring at a p a r t i c u l a r wavelength i n the 460-300 nm region r e s u l t s i n traces of absorbance versus time. For each a l i q u o t of CO added, the data were computer analyzed to give f i r s t - o r d e r p l o t s of ln(A B-A) versus t, and the observed rate constant ( k 0 ^ s ) derived from - 151 -the s l o p e 1 3 7 . The k0^,s values at varying [CO] were then p l o t t e d to obtain the k C 0 and k " c o rate constants, according to eq. (36). For most heme systems studied i n the present work, k " ^ was n e g l i g i b l e as judged by l i n e a r p l o t s that went through, or very close to the o r i g i n , i . e . kobs - k c o [ C O ] , where k c o[CO] » k" c o. The k"<-'(-) rate constant may be calculated from the as s o c i a t i o n rate and equilibrium constants according to eq. (37) XO .-CO (37) For those systems with s u f f i c i e n t l y high CO o f f - r a t e s , k " u u i s experi-mentally determined from the y-intercept of a p l o t of k D ^ s vs. [CO] (see above). A l t e r n a t i v e l y , the method of CO displacement by an i s o n i t r i l e l i g a n d has been used s u c c e s s f u l l y to determine very low CO o f f -r a t e s 6 5 , 7 * ^ . Five-coordinate hemes bind i s o n i t r i l e s with much higher a f f i n i t y than CO, so that f o r appropriately chosen [CO] and [RNC] the replacement of CO by RNC, within Fe(Por)(B)(CO) complexes, follows f i r s t order k i n e t i c s that are determined by the rate at which CO dissociates from the heme. The absorbance changes with time are monitored spectro-photometrically using a stopped flow apparatus f o r systems with 0.1 < k " c o < 1 s " 1 , or a Cary spectrometer f o r systems with k " c o < 0.1 s" 1. A f i r s t order p l o t of log (A^-A) vs. time gives a s t r a i g h t l i n e from the slope of which the V.'^® value i s r e a d i l y estimated. 152 4.3.3 Determination of Equilibrium and Rate Constants f o r Dioxygen Or, Or, -Or, Binding Using Flash Photolysis; K l , k l , k 1 (a) Photolysis of Fe(Por)(B)(0 2) species Direct photolysis of an Fe(Por)(B)(0 2) complex under varying pressures of 0 2 gives an observed rate f o r the re-formation of the dioxygen complex determined by: k o b s - k 2 [ 0 2 ] +. k 2 (38) from which k and k z may be estimated using a p l o t of k ^ g vs. [ 0 2 ] 1 4 2 . The success of t h i s method f i r s t depends on the s t a b i l i t y of the oxygen complex toward i r r e v e r s i b l e oxidation. Secondly, because of the low quantum y i e l d f o r the ph o t o l y t i c d i s s o c i a t i o n of a Fe-0 2 adduct (0Q^-1O"2), only those systems with a high o v e r a l l a f f i n i t y toward 0 2 have been s u c c e s s f u l l y studied by t h i s method 6 4. (b) Photolysis Using C0/0 2 Mixtures An a l t e r n a t i v e procedure f o r determination of rate constants involves use of C0/0 2 competition kinetics 6 0* 5» 1 4 3. This technique u t i l i z e s the usual 10-fold difference i n 0 2 and CO as s o c i a t i o n rate constants, where k 2 > k c o , so that f o r appropriately chosen [0 2]/[C0] mixtures, photolysis of the Fe(Por)(B)(CO) complex leads i n i t i a l l y to - 153 the f a s t a ssociation of 0 2, followed by slow O2/CO e q u i l i b r a t i o n back to the heme-CO complex, according to eq. (39): hV k u2, f a s t + O 9 Fe(Por)(B)(CO) -> Fe(Por)(B) Fe(Por)(B)(0 2) A + CO slow 1 Fe(Por)(B)(CO) (39) The high o v e r a l l a f f i n i t y towards CO, K C O[CO] > K 2 [ 0 2 ] , ensures that the equilibrium eventually l i e s toward that of the fully-formed CO species. The two rates " f a s t " and "slow" are s u f f i c i e n t l y well separated so that independent f i r s t - o r d e r traces may be obtained and analyzed. The f a s t rate affords a d i r e c t determination of k z , while the slow rate allows f o r the ext r a c t i o n of pertinent data f o r oxygen equilibrium constant determination, K . The observed rate constant for the slow return of Fe(Por)(B)(CO) i s given by the Gibson e q u a t i o n 1 4 3 a : ( k o b s ) s l k" 2 . k c o[CO] ow k c o[CO] + k " c o + k° 2[0 2] + k " ° 2 (40) Since k " c o i s small r e l a t i v e to a l l the other f a c t o r s , eq. (41) re s u l t s from inversion of eq. (40), 154 -°9 1 I K 2 [ 0 2 ] + + (41) (kobs)slow k"°2 k C O[CO] k C O[CO] and may be re-written i n the form of eq. (42) k c o[CO] o k c o[CO] - K 2 [ 0 2 ] + 1 + (42) *°2 ( kobs)s low ^ A p l o t of k u u [ C O ] / ( k 0 b s ) s i o w versus [0 2] then gives a slope of value K ° 2 and y-intercept of (1 + k C O[CO]/k ° 2 ) . A d i s t i n c t advantage of the method u t i l i z i n g C0/02 mixtures i s that the favoured equilibrium toward the Fe(Por)(B)(CO) species e f f e c t -i v e l y protects the heme from oxidation even i n the presence of high [0 2]. This k i n e t i c method has therefore been generally p r e f e r r e d 6 5 • 7 4 • 9 9 over that using pure 0 2 6 4, for obtaining oxygen k i n e t i c data with a number of model systems. 155 -4.4 RESULTS 4.4.1 A x i a l Base L i g a t i o n to Four-Coordinate Durene Hemes; Kg Values (a) Solution preparation Four-coordinate capped heme complexes are generally extremely a i r -s e n s i t i v e . With one face unprotected (the uncapped s i d e ) , dimerization between two i r o n centres can occur very r e a d i l y , i n the presence of trace oxygen (eqs. 19-21). The method for preparation and transfer of these complexes i s described i n section 4.2.1. A s i g n i f i c a n t advantage i n u t i l i z i n g the 'crown-ether-dithionite' method of reduction i s that the excess reducing agent, that remains i n the c l e a r toluene s o l u t i o n a f t e r removal of methanol, minimizes the r i s k of oxidation 7*-* during subsequent t i t r a t i o n with a N-base. (b) Q u a l i t a t i v e observations Figure 4.5 shows the s p e c t r a l properties of the four-, f i v e - , and six-coordinate, Fe(durene-7/7), Fe(durene-7/7)(Dclm), and Fe-(durene-7/7)(Dclm)(Melm) systems, re s p e c t i v e l y . The spectral appearances of a l l three species are comparable with those observed for other p y r r o l e - s u b s t i t u t e d s y s t e m s 1 4 4 (see section 4.5.2). The d i s t i n c t differences i n shape and i n t e n s i t y between the f i v e - and six-coordinate spectra (460-700 nm region) proved highly advantageous f o r estimating the extent of bis-base complexation, according to eqs. (26) and (43). n- 7 , . . . . • • • • • . - . i — i — i — . — • — ' — • — • — • — ' — 1 — 1 400 450 500 550 600 650 W a v e l e n g t h ( n m) F i g . 4.5 Spectral trends for four-, f i v e - and six-coordinate Fe 1 1(durene-7/7) systems Fe I ] :(Por)(B) + B Fe i : [(Por)(B)2 (43) I n i t i a l q u a l i t a t i v e observations using the r e a d i l y a v a i l a b l e Melm (39) as base indicated appreciable binding of a second a x i a l base to the larger capped durene-7/7 and -5/5 systems. For the 7/7-system p a r t i -c u l a r l y , a t y p i c a l six-coordinate, • hemochrome 1 4 4, spectrum was immediately formed even with [Melm] as low as 5 x 10" 4 M f o r [heme] -5 x IO" 5 M. In the case of the 5/5-durene heme, binding under the cap was much weaker but, at the high [B] required for studying CO and 0 2 binding e q u i l i b r i a (see sections 4.4.3 and 4.4.7), a considerable amount of s i x -coordination was observed, rendering Melm unsuitable as an a x i a l base for study with these larger capped systems. With the durene-4/4 heme, however, the smaller cap e f f e c t i v e l y prevented coordination of a second base even i n neat Melm. Based on studies with other s y s t e m s 6 5 • 7 4 • 7 8 a , the bulky Dclm (40) was considered u n l i k e l y to coordinate under the larger durene-7/7 and -5/5 caps because of s t e r i c reasons, and indeed t h i s was found to be the case. Less than 10% of the bis-base species was formed with these hemes i n solutions saturated with Dclm (1.2 M). For comparison, 1,2-Me2lm was also used i n order to provide a T-state system with respect to trans l i g a t i o n (section 2.6.3). In t h i s case, coordination of a second 1,2-Me2lm i s poor because of s t e r i c repulsion introduced between the 2-methyl group and porphyrin plane (Fig. 2.4), as the i r o n attempts to take up a more ce n t r a l p o s i t i o n with respect to both a x i a l ligands. Even with the 'open' system, Fe(OEP)(1,2-Me 2Im), with added [l,2-Me 2Im] -0.3 M, c h a r a c t e r i s t i c bands at 547 nm and 518 nm i n d i c a t i v e of 158 coordination of a second b a s e 1 4 4 , were not observed. The Dclm and 1,2-Me2lm bases were therefore chosen as R- and T-state bases, res p e c t i v e l y , for a l l three durene systems. (c) Equilibrium Constant Determination, Kg Values The tonometer used (Fig. 4.2 B) was c a l i b r a t e d so that the so l u t i o n volume could be measured (—0.05 mL) following transfer, p r i o r to base addition. Aliquots of the appropriate substituted-imidazole, dissolved i n deoxygenated toluene, were then added through the septum plug using a gas-tight m i c r o l i t r e syringe that had been flushed with N 2. Concentrations of base (B) and heme were maintained to give at le a s t a 10-fold excess of [base] r e l a t i v e to [heme] -6 x 10" 6 M, i n s o l u t i o n at a l l times, so that equilibrium conditions were established independent of [heme]. In order to minimize error due to trace oxidation of the heme complex, the t i t r a t i o n s were performed at room temperature (~23°C); t h i s reduced the experimental time required, since temperature e q u i l i b r a t i o n was unnecessary. Excellent i s o s b e s t i c points could be obtained for equilibrium changes i n a l l cases (see Fig. 4.6), even for the 'open' Fe(OEP) complex, i n d i c a t i n g clean conversion to the five-coordinate heme with no trace of oxidation. Spectral analyses according to the H i l l equation 1 4 1-' (see Appendix HIA) gave the Kg values (— 20%) quoted i n Table 4.1. F i g . 4.7 shows a t y p i c a l p l o t of log (k0-A/A-Aa) vs. log [B], corresponding to the binding of Dclm to the Fe(durene-4/4) complex (Appendix IIIA (1)). ICH.L, V (CH,). J B (CH,). n • 5 B = CH, 1,2-Dimethylimidazole 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 Wavelength (n m) Isosbestic spectral changes for: Fe i :(Por) + l,2-Me2Im ^-^-Fe 1 1(Por)(1,2-Me 2Im) at 20°C; Por = durene-5/5; added [l,2-Me 2Im] = 0.45, 1.48, 6.38 and 13.49 x I O - 4 M in the 460-300 nm region; f i n a l [1,2-Me2'lm] = 0.03 M - 160 -Table A . l Imidazole Base A f f i n i t y Constants, Kg (M" 1), f o r the Durene-Capped Hemes i n Toluene at ~23°C Compound 1,2-Me2lm Dclm Melm (5b) Fe(OEP) 1.2 x 10 4 (56b) Fe(durene-7/7) 1.0 x 10 4 3.3 x 10 3 (55b) Fe(durene-5/5) 5.8 x 10 3 2.0 x 10 : (56b) Fe(durene-4/4) 7.5 x 10 3 6.0 x 10 3 5.7 x 10-Fig. 4 . 7 H i l l plot for: Fe I 3 :(Por) + Dclm Por - durene -4/4 ^ Fe-^CPor) (Dclm) 161 4.4.2 Isocyanide Binding to Five-Coordinate Durene Hemes, K K N ( j Values Analogous to the comparative studies c a r r i e d out by Traylor et a"L 65,70^ (section 2.5.2), the s t e r i c nature of the s i x t h coordination s i t e i n the durene heme series was probed using t-butylisocyanide (t-BuNC) and tosylmethylisocyanide (TMIC) ligands. (a) Solution Preparation The five-coordinate complex Fe(Por)(Dclm) i n toluene was prepared and transferred into a tonometer (Fig. 4.4 B ) according to the method described i n section 4.2.2. Aliquots of the appropriate i s o n i t r i l e i n deoxygenated toluene were then added through the septum plug using a gas-tight m i c r o l i t r e syringe, and the heme s o l u t i o n e q u i l i b r a t e d to 20°C i n the spectrophotometer. (b) Equilibrium Constant Determination ( i ) Durene-7/7 and -5/5 systems The binding constants f o r the coordination of t-BuNC to the durene-7/7 and -5/5 five-coordinate hemes were determined by d i r e c t t i t r a t i o n . During t i t r a t i o n , a large excess of Dclm was maintained i n t-BuNC solut ion, so that KJ^Qjjjj[DcIm] » K^ -_ g^j^Q [ t - BuNC ] ; furthermore, K > Dclm Kt-BuNC^> s o the equilibrium constant K^-BUN C obtained f o r each heme 162 corresponds e x c l u s i v e l y to the binding of t-BuNC on the capped side of the heme, without any complication caused by displacement of the Dclm a x i a l base. The a f f i n i t i e s of TMIC for both hemes were too high to measure by d i r e c t a d d i t i o n of the i s o n i t r i l e to the five-coordinate heme (at [heme] , TMIC -3 x 1 0 " ° ) , so that K determinations had to be c a r r i e d out by com-Dclm J p e t i t i v e l i g a t i o n m e t h o d s 6 5 u s i n g both TMIC and CO. In a t y p i c a l experiment, Fe(Por)(Dclm)(CO) was t i t r a t e d with TMIC. The binding constant f o r TMIC i s s u f f i c i e n t l y higher than that f o r CO ( K T M I C > K C 0 ) , so that li g a n d concentrations may be chosen where TMIC e f f e c t i v e l y displaces CO from under the cap, and forms Fe(Por)(Dclm)(TMIC) according to eq. ( 4 4 ) : K C 0 Fe(Por)(Dclm)(CO) ' ^ Fe(Por)(Dclm) K ™ i c I" ( 4 4 ) KC0,TMIC " " ^ Fe(Por)(Dclm)(TMIC) KTMIC where KC0,TMIC _ ( 4 5 ) K c o • [CO] Once again, conditions were met so that KD ci m[DcIm] » KCQ[C0] or KTMIC[TMIC], ensuring that neither TMIC nor CO displaced the a x i a l base, and thus the observed equilibrium was indeed that of exchange under the cap. The s p e c t r a l change obtained for the replacement of CO by TMIC to the Fe(durene-5/5)(Dclm)(CO) system i s i l l u s t r a t e d i n F i g . 4.8, and the corresponding p l o t of log (AQ-A/A-A^) V S . log[TMIC] shown i n F i g . 4.9. 1 6 3 F i g . A.8 Isosbestic s p e c t r a l changes f o r : Fe i : r(Por)(DcIin)(CO) + TMIC ;===± F e 1 1 (Por) (Dclm) (TMIC) + CO 20°C; Por «= durene-5/5; [CO] = 2.A x I O - 4 M, added [TMIC] = 2.77, A.8A, 6.92, 9.00, 16.90, 23.50 and 33.59 x IO* 4 M; f i n a l [TMIC] -0.02 M - 164 e.i F i g . 4 . 9 H i l l p l o t f o r : Fe i : c(Por)(DcIm)(CO) + TMIC ^ = ^ F e I : t ( P o r ) (Dclm) (TMIC) + CO at 20°C; Por - durene-5/5 Data analyses f o r a l l systems are given i n Appendix IIIB, and determined KRNC v a i u e s (± 20%) quoted i n Table 4.2. Table 4.2 I s o n i t r i l e A f f i n i t y Constants f o r the Durene-Capped Hemes Compared to that f o r Chelated Protoheme Compound KTMIC 'Peripheral' K t _ B u N C 'Central' reduction reduction (M"1) factor (M"1) factor (10) Chelated Protoheme 7 x 10 9 1.7 x 10 8 (56b) Durene-7/7(DcIm) 9.2 x 10 8 7.6 2.5 x 10 5 680 (55b Durene-5/5(Dclm) 6.4 x 10 6 1.1 x 10 3 2.1 x 10 3 8.1 x 10 4 165 ( i i ) Durene-4/4 System: For the five-coordinate Fe(durene-4/4)(B) systems, B - Dclm or Melm, an extremely high concentration of added isocyanide (0.02 M) was necessary before any spe c t r a l change was observable at a l l . The blue s h i f t (to 400 nm, Fig. 4.10) observed during such a t i t r a t i o n i s , however, u n c h a r a c t e r i s t i c of the t y p i c a l changes (red s h i f t ) seen for the coordination of isocyanides under the larger 7/7- and 5/5-caps (Fig. 4.11). Instead, the blue s h i f t i s s i m i l a r to that obtained when TMIC i s added to a four-coordinate durene-4/4 heme i n the absence of any other liga n d (Fig. 4.10). To investigate further the nature of t h i s s pectral deviation, a q u a l i t a t i v e binding experiment was performed with n-butylisocyanide (n-BuNC). Addition of t h i s s t e r i c a l l y less demanding isocyanide to the five-coordinate, Fe(durene-4/4)(Dclm) complex gave r i s e to the expected r e d - s h i f t e d type of s p e c t r a l change; t h i s corresponds to isocyanide l i g a t i o n with formation of a six-coordinate Fe(Por)(B)(n-BuNC) complex (Fig. 4.11). This strongly indicates that the absence of any s p e c t r a l change at intermediate added [t-BuNC] r e s u l t s from s t e r i c r e s t r i c t i o n s imposed by the small durene-4/4 cap, which prevents the bulky t-BuNC ligand from coordinating at the s i x t h a v a i l a b l e s i t e . At s u f f i c i e n t l y high [t-BuNC], however, displacement of Dclm must occur with t-BuNC coordinating on the unhindered side of the heme. Unfortunately, because of the rather low s o l u b i l i t y of TMIC i n toluene, s u f f i c i e n t l y high [TMIC] required to observe coordination of t h i s i s o n i t r i l e under the 4/4-cap could not be obtained i n the tonometer, thus precluding determination of K ™ * c for t h i s system. - 166 -F i g . 4.10 Spectral trends f o r the l i g a t i o n of isocyanides to the durene-4/4 system F i g . 4.11 Spectral trends f or the durene-4/4, -5/5 and -7/7 Fe I I(Por)(DcIm)(RCN) systems - 168 -4.4.3 Carbon Monoxide Binding to Five-Coordinate Durene Hemes; K C 0 Values (a) Solution Preparation and Method of CO a d d i t i o n The five-coordinate heme complexes Fe(Por)(B), prepared according to the method described i n section 4 . 2 . 2 , were found to bind CO too strongly to allow for determination using pure CO ~ 0.001-1.0). Therefore, d i l u t e d CO/N2 mixtures prepared i n a glass bulb, attached to a manometer, were used for the t i t r a t i o n s . I n i t i a l l y - 2 0 t o r r CO was admitted into an evacuated bulb, and N 2 added to barometric pressure. The t o t a l pressure i n the bulb was then reduced roughly to h a l f by evacuation (measured accurately on the manometer), and the bulb again f i l l e d with N 2 . This evacuation procedure was repeated several times u n t i l a desired p a r t i a l pressure of CO remained i n the bulb. With a connected tonometer containing the five-coordinate heme, the system was degassed completely (tap 1 closed, F i g . 4.12), and the F i g . 4.12 Vacuum-line set-up used f o r preparing and admitting CO/N2 mixtures into a tonometer - 169 -l i n e and manometer allowed to e q u i l i b r a t e to the p a r t i a l pressure of toluene (tap 4 closed). Appropriate pressures of the prepared CO/N2 mixture were admitted into the tonometer from the bulb, by slowly opening and c l o s i n g tap 1, and were measured on the manometer. The heme so l u t i o n was s t i r r e d b r i e f l y and the tonometer disconnected. By this method, CO/N2 mixtures could be prepared i n advance, allowing very low p a r t i a l pressures of CO (-10"2 t o r r , within — 10% error) to be admitted into the tonometer. (b) Equilibrium Constant Determination, K t u Except for the Fe(durene-7/7)(Dclm) and Fe(durene-5/5)(Dclm) systems, whose a f f i n i t i e s toward CO were too high to be followed by d i r e c t t i t r a t i o n , a l l other K^ 0 values were obtained v i a the simple addition of CO/N2 mixtures to the five-coordinate hemes, according to eq. (46): K c o Fe(Por)(B) + CO v s Fe(Por)(B)(CO) (46) Examples of the i s o s b e s t i c s p e c t r a l changes observed f o r the B=l,2-Me2lm system are shown i n Figs. 4.13 and 4.14. The s p e c t r a l trends for the Fe(Por)(B)(CO) complexes of the durene s e r i e s , f o r B - 1,2-Me2lm and Dclm, are i l l u s t r a t e d i n Figs. 4.15(a) and (b), r e s p e c t i v e l y . The CO extent of CO binding to the heme, as measured by Pi/2> * s dependent on the p a r t i a l pressure of the gas, P^0, over the toluene s o l u t i o n , so that f o r the durene-5/5, -7/7 and OEP Fe(Por)(1,2-Me2lm) complexes and o b < ' • • •—. I • •—.—.—• . . •— __. I . • • . • 350 400 450 500 550 600 650 700 Wavelength (n m) F i g . 4.13 Isosbestic spectral changes f o r : Fe I ] :(Por)(l,2-Me2lm) + CO ^  *- Fe i : [(For) (1,2-Me2Im) (CO) at 20°C; Por = durene-5/5; added P c o = 0.0119, 0.0242, 0.0363, 0.0529, 0.0816, 0.117 and 0.240 t o r r i n the 460-300 nm region; f i n a l P c o = 1 atm rt < 450 500 550 600 650 700 450 500 550 600 650 700 Wavelength (n m) Wavelength (n m) A _B F i g . 4.14 Isosbestic spectral changes f o r : Fe I I(Por)(l,2-Me 2Im) + C O ^ i F e 1 1 (Por) (1, 2-Me2Im) (CO) at 20°C; (A) Por - durene-7/7, (B) Por - durene-4/4 350 400 450 500 550 600 650 700 W a v e l e n g t h (n rn) F i g . 4.15(a) Spectral trends f or the durene-4/4, -5/5 and -7/7 Fe I I(Por)(l,2-Me 2Im ) ( C O ) systems —'—1—"—•—1—» ' • I—-*-—I—'—•—I—•—I—i—.—.—.—I—. . , , I I 350 400 450 500 550 600 650 700 W a v e l e n g t h (n m) F i g . 4.15(b) Spectral trends f or the durene-4/4, -5/5 and -7/7 Fe i : i :(Por)(DcIm)(CO) systems - 174 -the Fe(durene-4/4)(Dclm)systems, determination of P could be achieved for i n i t i a l values of P ^ as low as 0.01 t o r r (corresponding to a concentration of free CO i n s o l u t i o n of -10" 7 M, although [heme] was as high as 3 x 10" 6 M). For the range of P c o - 0.01-0.1 t o r r , log ( A - A Q / A Q J - A ) was found to be l i n e a r l y dependent on log with a slope -1.0. A f t e r each addition of CO, the s o l u t i o n i n the tonometer was shaken vigorously for 5-10 min to ensure e q u i l i b r a t i o n of the gas, and the tonometer then thermostatted to 20°C for a further 10 min. The solution:tonometer volume was small (3:300 mL), so that changes i n P ^ with d i s s o l u t i o n i n the toluene phase were considered n e g l i g i b l e . A d e t a i l e d analysis for the Fe(durene-5/5)(1,2-Me2lm)(CO) system, corresponding to the s p e c t r a l changes shown i n F i g . 4.13 (460-360 nm), i s given i n Appendix I I I C ( l ) and the H i l l p l o t i s shown i n F i g . 4.16(a). K i n e t i c a l l y derived and k " ^ constants provided an independent confirmation of K ^ (see sections 4.4.4 and 4.4.5); there was reasonable agreement within a f a c t o r of two for the two methods. In a l l cases, the added [B] was s u f f i c i e n t l y high such that Kg[B] » K^o [CO], i n order to ensure that base displacement by CO on the unhindered side of the heme complex was not occurring. Under these added base conditions, K ^ values were consistent within ±20%, over a 10-fold range i n [base], 0.1 < [B] < 1.0 M. The K c o values for the two systems Fe(7/7)(Dclm) and Fe(5/5)(Dclm) were n e c e s s a r i l y determined by competitive binding methods 6 5. The t i t r a t i o n s were c a r r i e d out by following the displacement of e i t h e r Melm or t-BuNC from under the durene cap, by CO, according to eqs. (47) and (48), r e s p e c t i v e l y . - 175 KMeIm KC0 Fe(7/7) (Dclm) (Melm) ^ ^ Fe(7/7) (Dclm) Fe(7/7) (Dclm) (CO) (47) Kt-BuNC KC0 Fe(5/5)(Dclm)(t-BuNC) Fe(5/5)(Dclm) _ Fe(5/5)(Dclm)(CO) (48) K M e l m[MeIm] where K c o -= (49) [CO] K t- B u N C[t-BuNC] [CO] or K c o - (50) In each case K " E I M or K 1 - " 0 ^ 0 was independently determined by d i r e c t a d d i t i o n to the respective five-coordinate hemes (see Appendices IIIA(9) and I I I B ( l ) ) . Concentrations were maintained where Kn ci m[DcIm] » Kj4ej_m[MeIm] , K t.g u^Q[t-BuNC] , or K^ofCO] so that displacement of Dclm by ei t h e r Melm, t-BuNC or CO d i d not occur. In a l l cases data were analyzed according to the H i l l e q u a t i o n 1 4 0 (Appendix IIIC) and the values are quoted i n Tables 4.3 and 4.4. A H i l l p l o t corresponding to the Fe(durene-7/7)(Dclm)(Melm)(CO) system i s shown i n F i g . 4.16(b). - 176 -F i g . 4.16(b) H i l l p l o t f o r : Fe i : [(Por) (Dclm) (Melm) + CO ^ = ± F e I X ( P o r ) (Dclm) (CO) + Melm at 20°C; Por - durene-7/7 - 177 Table 4.3 Constants f o r CO Binding to R-State Durene-Capped Hemes Compared to those f o r Chelated Open Hemes i n Toluene at 20°C Compound ,C0 (M - 1 s' 1) •CO a ( s " 1 ) .-CO b ( s " 1 ) ,co CM'1) (10) Chelated Protoheme 1.1 x 10 7 0.025 4 x 10c (11) Chelated Mesoheme 1.1 x 10 7 -0.05 8 x 10 6 °-2 x 10c (56b) Durene-7/7(Dclm) 9.5 x 10 5 0.02 1.1 x 10 6 d 0.014 6.6 x 10' (55b) Durene-5/5(Dclm) 1.1 x 10 7 0.29 0.13 8.6 x 10 7 (54b) Durene-4/4(DcIm) 3.0 x 10 6 0.7 4.3 x 10 s (54b) Durene-4/4(Melm) 4.9 x 10 6 1.9 x 10 6 & & Measured experimentally 0.7 6.9 x 10c fe Derived from k c o and K rCO °- In 10% CH 2C1 2-toluene at 20°C £ In CH 2C1 2 at 20°C 178 Table 4.4 Constants f o r CO Binding to T-State Durene-Capped Hemes Compared to those f o r T-State Open Hemes i n Toluene at 20°C Compound k C 0 k _ C 0 ^ k _ C 0 ^ K C 0 (M' 1 s" 1) ( s " 1 ) ( s " 1 ) (M"1) (8b) Deuteroheme(2-MeIm)£ 1.0 x 10 6 0.45 2.2 x 10 6 ^ (5b) OEP(l,2-Me2Im) 0.56 - 2.2 x 10 6 (56b) Durene-7/7(l,2-Me 2Im) 1.3 x 10 5 0.05 0.04 3.3 x 10 6 (55b) Durene-5/5(l,2-Me 2Im) 1.1 x 10 6 1.1 0.52 2.1 x 10 6 (54b) Durene-4/4(l,2-Me 2Im) 6.3 x 10 5 4.8 4.2 1.5 x 10 5 - Measured experimentally. - Derived from k^° and K^°. ^ Ref. 137. £ Calculated from k c o and k' c o. (c) Temperature V a r i a t i o n E q u i l i b r i a ; A H C O , ASQQ Values For the Fe(durene-4/4)(B) systems, B - Dclm and l,2-Me2Im, K c o values were measured at d i f f e r e n t temperatures within the range 0-30°C (Appendix IIIC). Plots of In K c o vs. 1/T(°K), shown i n F i g . 4.17, A ° A ° provided the thermodynamic constants, AHQQ and A S ^ Q (see Appendix IIIK and Table 4.12, p. 229) . - 179 -F i g . A.17 Van't Hoff pl o t s f o r : Fe I I(durene - V^)(B) + CO F e 1 1 (durene-4/4) (B) (CO) 180 -4.4.A Rate of Carbon Monoxide Association to Five-Coordinate Hemes; k c o Values (a) Solution Preparation and CO addition Solutions of five-coordinate heme i n toluene were prepared and tr a n s f e r r e d into a tonometer containing a s t i r r e r bar, according to the method described i n section 4.2.2. Tonometer and s o l u t i o n were degassed, and aliquots of CO syringed i n through the septum plug of tap 6 (Fig. 4.4 B). The en t i r e tonometer was clamped h o r i z o n t a l l y i n a water-bath for 15 min, thermostatted to 20°C, while the heme solu t i o n was s t i r r e d magnetically. (b) Determination of k o u Subsequent to f l a s h photolysis of the heme-CO adduct, the rate of CO a s s o c i a t i o n to the five-coordinate heme, eq. (35), was monitored separately at X M A X of both Fe(Por)(B)(CO) and Fe(Por)(B) species, i n the 460-350 nm region. I d e n t i c a l k^° rate constants (^15%) were obtained from slopes of k O D S versus CO pressure using data at both wavelengths. A t y p i c a l p l o t i s shown i n F i g . 4.18 for the Fe(durene-5/5) system, and complete data analyses are given i n Appendix III D. To confirm that CO binding was indeed occurring at the sixth-coordination s i t e without base displacement, experiments were c a r r i e d out where [B] was v a r i e d at constant CO pressure for a l l three durene-heme systems with B - Dclm. A p l o t of k^° versus [B] i s shown i n F i g . 4.19 f o r the durene-7/7 system. 1 8 1 -in o o o "I 1 1 r- "i 1 1 — 1 1 — 1 r CO 5 _1 -1 k Dclm= 9 5 x 1 0 M s at ["Dclm] s 0.9 + X 412 nm > X 425 nm ' • ' • ' • i i i i i i ' • ' 0 B.J B.P B.3 0.1 0.5 0.E B.7 0.8 0.9 J.B [DclmJ M F i g . A.19 Determination of k C 0 at var i e d [Dclm] f o r : Fe 1 1(Por)(Dclm) + CO Fe 1 1(Por)(Dclm)(CO) at 20°C; Por - durene-7/7 - 182 -At P C 0 ~ 200 t o r r , k c o (-15%) i s seen to be independent of [B] down to 0.3 M (Appendix III E). As [B] becomes < 0.1 M, the k C 0 rate constant increases, suggesting CO association v i a the mechanism of base elimination, as described by White et a l 1 3 7 . ( s ection 2.6.6). As [B] i s decreased, the observed rate at which the base binds to the four-coordinate heme, kg[B][heme], i s also lowered. Under these conditions, CO a s s o c i a t i o n e f f e c t i v e l y begins to compete with base a s s o c i a t i o n to the four-coordinate heme (kQQ[ c°] ~ kg[B]). The observed rate, a f t e r p h o tolysis, i s therefore increased by CO ass o c i a t i o n to the four-coordinate heme (since k^g > kg*^) t and t h i s i s followed by rapid base binding to the five-coordinate CO complex; eq. (51), steps 2-4. Fe(Por)(B) CO ® Fe(Por) ® K CO Fe(Por)(B)(CO) © * Fe(Por)(C0) (51) This study conclusively shows that at the high [B] > 0.3 M used i n a l l CO binding experiments, the k g ^ values obtained do indeed corres-pond to CO coordination on the capped side of the five-coordinate heme complex. - 183 -4.4.5 Carbon Monoxide D i s s o c i a t i o n from Six-Coordinate Hemes, k ~ c o Values (a) Flash Photolysis For the Fe(durene-4/4)(1,2-Me2lm)(CO) system, k"^° was determined from a p l o t of k o b s vs. [CO] according to eq. (36). The experimental method i s i d e n t i c a l to that described i n section 4.4.4(b), except that for determination of k " ^ very low pressures of CO were admitted into the tonometer so as to allow a more r e l i a b l e extrapolation to the y-intercept at [CO] -0, F i g . 4.20. The concentrations used ensured that CO [CO] > i / ^ l ^ - M e Im > [heme] at a l l times, so that pseudo f i r s t - o r d e r T 1 1 1 1 1 1 1 1 ; 1 1 1 1 1 1 1 1 r [CO] x 10 M F i g . 4.20 Determination of k" c o f o r : Fe I 1(Por)(l,2-Me 2Im)(CO)^=^Fe I I(Por)(l,2-Me 2Im) + CO at 20°C; Por - durene-4/4 - 184 conditions were maintained. When carrying out the analysis (Appendix III F), the [CO] was estimated (more accurately) by incl u d i n g the small amount of free CO released into s o l u t i o n following photolysis of the Fe(Por)(1,2-Me 2Im)(CO) complex. A p l o t of k o b s vs. [CO] afforded a value of k"<-'(-) -4.8 s" 1, within an acceptable range of the value (4.2 s" 1) c a l c u l a t e d using the and Vp® values (see Table 4.4). (b) CO Displacement by I s o n i t r i l e s I n i t i a l l y , s u f f i c i e n t CO was added to form f u l l y the s i x -coordinate Fe(Por)(B)(CO) complex (as described i n section 4.4.4(a)); t h i s was then r a p i d l y treated with a deaerated s o l u t i o n of TMIC, and the rate of formation of the six-coordinate Fe(Por)(B)(TMIC) complex followed spectrophotometrically using ei t h e r a Cary spectrometer or Stopped Flow apparatus (see section 4.1). Relative concentrations of CO and TMIC were chosen such that K T M I C[TMIC] > K C O[CO] (by >10-fold) when TMIC e f f e c t i v e l y displaced CO from the s i x t h coordination s i t e . High [B] of -1.0 M was maintained i n a l l cases so that base displacement by ei t h e r CO or TMIC d i d not occur to a s i g n i f i c a n t extent. Under these conditions, the rate of Fe(Por)(B)(TMIC) formation was determined e x c l u s i v e l y by the rate at which CO dissociates from the heme, since k R N C[RNC] » k" c o. The f i r s t - o r d e r rate constants ( k " c o ) obtained were found to be independent over a 10-fold v a r i a t i o n i n [CO]/[TMIC], and i d e n t i c a l rates were obtained when monitoring at ^ m a x corresponding to e i t h e r Fe(Por)(B)(CO) or Fe(Por)(B)(TMIC) species; a t y p i c a l p l o t of l n ( A 0 S -A) vs. time i s shown i n F i g . 4.21 for the Fe(durene-5/5)(Dclm) - 185 -system, and data analyses for a l l systems given i n Appendix III F. The k " C 0 values obtained (±25%) are quoted i n Tables 4.3 and 4.4. In a l l cases, values of k " c o determined by experiment and c a l c u l a t i o n from K C 0 CO and k o u are considered to be i n reasonable agreement (within 2-fold) for the d i f f e r e n t methods used, and t h i s further confirms the v a l i d i t y of r e s u l t s obtained. Unfortunately, because of the poor a f f i n i t y of the durene-4/4 hemes toward a l l i s o n i t r i l e s , including n-BuNC, the CO o f f - r a t e s for these systems with B-Dclm or Melm could not be obtained. F i g . 4.21 Determination of k * L U f o r : F e I I ( P o r ) (Dclm) (CO) ^ ^ F e n ( P o r ) (Dclm) + CO at 20°C; Por - durene-5/5 - 186 4.4.6 Carbon Monoxide Binding to Four-Coordinate Durene Hemes, K c o Values Four-coordinate "open" hemes react with CO to form five-coordinate CO Fe(Por)(CO) c o m p l e x e s 7 2 - 8 8 • 1 4 5 , where K C O > K C O (section 4.5.2). For the four-coordinate durene hemes, a knowledge of KQQ> l n a d d i t i o n to Kg, seemed necessary i n order that appropriate r a t i o s of [C0]/[B] could be maintained to prevent displacement of B by CO on the unhindered side of the heme, during reaction of Fe(Por)(B) with CO. Additio n of 1 atm of CO to the Fe(durene-4/4) system resulted i n formation of a new single Soret band at 400 nm; t h i s i s s i m i l a r to the type of s p e c t r a l change observed f o r other severely s t e r i c a l l y hindered systems a l s o 7 2 . Unlike "open" hemes or those with more spacious " d i s t a l " s i t e s , coordination of a second CO to a hindered heme i s retarded even further f o r s t e r i c reasons r e l a t i v e to that to a unhindered system 7 2, so that even In the presence of high [CO], only one band appears i n the Soret region f o r a hindered heme, which corresponds to a clean formation of Fe(Por)(C0). Thus, the severely hindered durene-4/4 heme was selected f o r convenience f o r a KQQ determination. The KgQ value was measured i n d i r e c t l y , v i a base addition to the Fe(Por)(CO) complex, with f i n a l formation of Fe(Por)(B)(CO), eq. (52). The s p e c t r a l changes observed are shown i n F i g . 4.22 for B - Melm. P r i o r knowledge of K^ and K g ^ , coupled with experimental K values, allows c a l c u l a t i o n of KQQ according to eq. (53). - 187 -(52) (53) K The estimated KQQ values were 0.95, 5.3 and 6.5 x 10 3 M"1 with the added bases 1,2-Me2lm, Melm and Dclm, res p e c t i v e l y , i n toluene at 20°C (Appendix IIIG). Although subject to considerable error, the procedure depending on determination of three d i f f e r e n t constants, t h i s method of t i t r a t i o n was experimentally convenient since CO was present at a l l times during t i t r a t i o n , and thus protected the heme from oxidation. Di r e c t t i t r a t i o n of the a i r - s e n s i t i v e four-coordinate heme with CO (as fo r Kg determinations) gave K^o ~2 x 10 5 M"1, i n reasonable agreement with the values obtained v i a the competitive t i t r a t i o n s ; the average value f o r KQQ i s taken as -4.3 x IO 5 M"1. - 188 -• i t i i i i i — 350 400 450 Wavelength (n m) F i g . 4.22 Isosbestic s p e c t r a l changes f o r : Fe I I(Por)(CO) + M e l m ^ i F e 1 1 (Por) (Melm) (CO) at 20°C; Por - durene-4/4; added [Melm] - 0.532, 1.06, 1.59, 2,12, 2.65, 3.89 and 9.04 x 1 0 - 5 M; f i n a l [Melm] -0.02 M 189 4.4.7 Dioxygen Binding to Five-Coordinate Durene Hemes, K *• (a) Ambient Temperature ( i ) Solution Preparation Five-coordinate heme Fe(Por)(B) solutions i n toluene (6 mL) were prepared and transferred into a tonometer (Fig. 4.4.B) by the method described i n section 4.2.2. In order to ensure that no moisture remained, a small volume of toluene (-0.75 mL) was pumped o f f , while the s o l u t i o n was s t i r r e d inside the tonometer, thus removing any trace H2O. Appropriate pressures of ei t h e r d i l u t e d (O2/N2) or pure dioxygen were admitted into the tonometer using the procedure outlined i n section 4.2.2. The s o l u t i o n was shaken vigorously and e q u i l i b r a t e d at 20°C. ( i i ) Equilibrium Constant Determination by Spectrophotometric T i t r a t i o n Reversible oxygenation at room temperature could be achieved for a l l the Fe(durene-Por)(B)(O2) complexes with B=DcIm. Within the s e r i e s , the oxy-complexes formed with the t i g h t e r capped durene-4/4 systems (B = Dclm or Melm) were found to be the most stable toward oxidation ( t i / 2 ~2 h), reminiscent of the s i t u a t i o n seen i n the 'lacunar' systems with 'protected' d i s t a l s i t e s (section 2.5.7). A greater tendency toward oxidation noted i n the durene-5/5 and -7/7 systems may r e s u l t from a more f l e x i b l e cap being able to swing to one side thus allowing for fl-oxo dimer formation; a l t e r n a t i v e l y , approach of an extraneous agent - 190 -(OH" or C l " ) , that can p o t e n t i a l l y promote oxidation of the ir o n v i a an "outer-sphere" mechanism , i s more e f f e c t i v e l y hindered i n the tig h t e r capped d i s t a l environment of the 4 / 4-systera (section 2 . 5 . 7 ) . D i r e c t t i t r a t i o n with 0 2 for the five-coordinate durene-4/4 complexes, B=DcIm or Melm, at room temperature gave absorbance changes with sharp i s o s b e s t i c points i n d i c a t i n g clean conversion to the oxy-species according to eq. ( 5 4 ) (shown i n F i g . 4 . 2 3 for B=MeIm). K 2 Fe(Por)(B) + 0 2 ^ F e ( P o r ) ( B ) ( 0 2 ) ( 5 4 ) Removal of dioxygen by pumping regenerated the five-coordinate heme with n e g l i g i b l e accompanying heme oxidation. The data were analyzed accord-ing to the H i l l e q u a t i o n 1 4 0 (Appendix III H) and a t y p i c a l p l o t of log (A-AQ /A^-A) versus P * i s shown i n F i g . 4 . 2 4 . Reasonable estimates . 0, ( — 2 5 % ) of K z for durene - 7 / 7 and - 5 / 5 hemes, B-Dclm, could also be obtained at room temperature (Table 4 . 5 ) ; deoxygenation a f t e r f u l l formation of F e ( P o r ) ( B ) ( 0 2 ) indicated l e s s than 2 0 % oxidation of heme, during a period of ca. 2 0 min under 0 2 . A high [B] of - 1 . 0 M had to be maintained i n a l l cases to thwart e f f e c t i v e l y i r r e v e r s i b l e oxidation to the ^i-oxo dimer. Trends i n oxy-complex spectra f o r the durene seri e s are shown i n F i g . 4 . 2 5 . The a f f i n i t y constants f o r 0 2 binding to the durene systems with B=l ,2-Me 2Im were too low ( ? i / 2 t 0 ° high) to allow measurement at room temperature, but such values could be determined k i n e t i c a l l y (section (d)). i i 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 Too W a v e l e n g t h ( n m) F i g . A. 23 Isosbestic spectral changes for: F e 1 1 (Por) (Melm) + 0 2 F e 1 1 (Por) (Melm) (0 2) at 20°C; Por - durene-A/A. added P 1 - 2A, 6A, 12A t o r r , and 1 atm, i n the A60-300 nm region 192 -log P 2 1.4 1.5 1.6 1.7 1.8 — i 1 1 1 \ — 1.9 H 2.0 X 424 nm Slope = 1.2 0 2 P1|2 = 8 3 t 0 r r 4.24 H i l l p l o t f o r : Fe I I(Por)(MeIm) + 0 2 ; = ± F e 1 1 (Por) (Melm) (0 2) at 20°C; Por - durene-4/4 t < » • - • • • • • • - • • I • • • • ' I ' 350 400 450 500 550 600 650 700 W a v e l e n g t h (n m) F i g . 4.25 Spectral trends f o r the durene-4/4, -5/5 and -7/7 Fe I I(Por)(Dclm) ( O 2 ) systems 19A Table A.5 A f f i n i t y Constants for Dioxygen Binding to the Durene-Capped Hemes i n Toluene at 20°C Compound B - Dclm K 2 & K ° 2 fe 10 3 ( t r 1 ) 10 3 (M"*) B - l,2-Me2Im a (M"1) K ° 2 Oo K 2 £ (56b) Fe(durene-7/7)(B) 0.55 -1.3 38 (55b) Fe(durene-5/5)(B) 1.0 -1.3 36 (5Ab) Fe(durene-A/A)(B) 0.60 1.2^ 0.5 1.0^ 3A 95 - K i n e t i c determination. - Equilibrium determination. - Extrapolation from equilibrium determination at low temperature. £ B - Melm. (b) Low Temperature E q u i l i b r i a ; AH ° , A S ° values ( i ) Solution Preparation A s o l u t i o n of five-coordinate heme Fe**(durene-A/A)(B) i n toluene (17 mL) was prepared as described i n section A.2.2 and transferred into a c e l l mounted inside a Dewar (Fig. A.2). During the transfer, the f l a s k system i n F i g . A.A was attached to the c e l l by connecting j o i n t s P and Q, and the system evacuated with taps 5 and 6 open (tap A closed). 195 -With tap 6 then closed and tap 4 opened, the s o l u t i o n was allowed to flow into the c e l l . F i n a l l y , taps 5 and 6 were closed o f f and the f l a s k s disconnected. The Dewar f l a s k was f i l l e d with a slush bath of appropriate temperature, and the s o l u t i o n degassed completely by opening tap 6. D i l u t e d mixtures of O2/N2 were admitted into the c e l l v i a a f l e x i b l e s t a i n l e s s s t e e l tube attached to j o i n t Q. The pressures of dioxygen were measured according to the method out l i n e d i n section 4.4.3(a). The volume of s o l u t i o n was maintained well below the narrow neck of the c e l l , i n order that shaking the c e l l agitated the s o l u t i o n s u f f i c i e n t l y (15 min), to e f f e c t e q u i l i b r a t i o n of O2 between the s o l u t i o n and gas phase. The Dewar was then disconnected from the vacuum-line (taps 5 and 6 closed), mounted inside a Cary 17 spectrometer and s p e c t r a l changes recorded. Data analyses f o r both systems are given i n Appendix III H. Van't Hoff p l o t s of l n K°2 vs. 1/T(°K) shown i n F i g . 4.26 provided A H Q ^ arid A S Q ^ values for both Fe(durene-4/4) (B) systems, B-=DcIm and l,2-Me2Im (see Appendix IIIK and Table 4.11). (c) A s s o c i a t i o n Rate Constant Determination; k°2 Values Solutions of Fe(Por)(B)(O2) under pure 0 2 were flashed while monitored at the X m a x corresponding to re-formation of the same species, i n an attempt to determine the f a s t rate constant k^2 according to eq. (37), ( s ection 4.3.4). A l t e r n a t i v e l y , a s o l u t i o n of Fe(Por)(B)(CO) was prepared under a known [CO], and aliquots of O2 then syringed into the tonometer. A f t e r e q u i l i b r a t i o n , the s o l u t i o n was flashed while being - 197 -monitored at the X m a x for Fe(Por)(B)(O2). Unfortunately, the low a f f i n i t y of the durene heme systems toward O2 necessitated the addition of high O2 pressures to achieve any noticeable change i n absorbance during re-formation of the Fe(Por)(B)(O2) species (fast r a t e ) . At these high [O2], however, the observed rate constants ( k 0 ^ s ) for the 4/4- and 5/5-systems approached that of the decay rate constant of the l a s e r beam. The lower k 0 D S values f o r the 7/7-heme, however, provided a r e l i a b l e determination of k ° 2 -9.8 x 10 6 (± 20%) M"1 s " 1 f o r t h i s system (Appendix I I I , I ) . F i g . °9 A. 27 shows the p l o t of k0^,s vs. P c . (d) Equilibrium Constant Determination Using CO/O2 Mixtures Solutions of precursor five-coordinate hemes under mixtures of [C0]/[02] (see previous section (c)) were flashed while being monitored separately, at X m a x values corresponding to both Fe(Por)(B) and Fe(Por)(B)(CO) species. The k i n e t i c determination of K * from the 'slow rate' (section 4.3.4(b)) afforded consistent r e s u l t s (±25%) at both X m a x p o s i t i o n s ; the K * values were i n reasonable agreement (within 2-fold) with those measured by equilibrium t i t r a t i o n , once again confirming the v a l i d i t y of r e s u l t s obtained from both methods (Table 4.5). A t y p i c a l p l o t of k C 0 [ C 0 ] / k o b s v s - p ° 2 i s s h o w n i n F i g - 4 - 2 8 f o r t h e Fe(durene-7/7)(Dclm) system, and data analyses for a l l systems are given i n Appendix III J ) . For a l l durene systems k 2 » k C 0 [ C 0 ] , which ren-ders values of 1 + k C 0 [ C 0 ] / k 2 from the y-intercept, eq. (42), too - ° 2 small f o r a r e l i a b l e determination of k . 198 -* 8 IE 16 S0 ?f EB 3P 36 !0 Oo P z torr Fig. 4.27 Determination of k° 2 for: Fe 1 1 (Por) (Dclm) + 0 2 ^ r ^ F e 1 1 (Por) (Dclm) (02) at 20°C; Por - durene-7/7 199 -4.4.8 Infra-Red KCO) Values f o r Six-Coordinate Durene Heme Fe(Por)(B)(CO) Complexes (a) Solution Preparation Five - coordinate heme solutions i n toluene were prepared as described i n section 4.2.2. Aft e r drying over CaH 2, the s o l u t i o n was transferred into a 25 mL round-bottom f l a s k containing a septum plug. The volume of toluene was reduced to -0.3 mL by evacuation, and CO at 1 atm admitted into the f l a s k to generate the Fe(Por)(B)(CO) complex. Concentrations were estimated to give [heme] -0.013 M, [B] -1 M and [free CO] -7.4xl0" 3 M, ensuring that base displacement by CO d i d not occur on the unhindered side of the heme. Approximately 0.25 mL of so l u t i o n was withdrawn using a gas-tight m i c r o l i t r e syringe and in j e c t e d into an IR s o l u t i o n c e l l purged with CO. An IR spectrum of a toluene s o l u t i o n , containing base at —1 M and free CO at ~7.4 x 10" 3 M was subtracted from that of each Fe(Por) (B) (CO) complex, to obtain J^(CO) for the B=MeIm and 1,2-Me2lni systems. Such a spectrum i s shown i n F i g . 4.29 f o r the Fe(durene-4/4)(Melm)(CO) system. The V(C0) values for a l l the durene complexes are given i n Table 4.6 (p. 216). Attempts to measure the oxygen st r e t c h i n g frequency of the stable Fe**(durene-4/4)(Melm)(0 2) complex, i n toluene, proved unsuccessful. F i g . 4.29 (A) Examples of an IR spectrum of Fe (durene-4/4) (Melm) (CO) i n toluene superimposed over that of free CO In toluene/Melm (B) Interactive substraction of (A) 201 -4.5 DISCUSSION 4.5.1 ELECTRONIC ABSORPTION SPECTRAL TRENDS FOUND IN IRON(II) DURENE-CAPPED SYSTEMS Subs t i t u t i o n of the hydrogens with a metal(II) i n the ce n t r a l core of the porphyrin gives r i s e to exact f o u r - f o l d symmetry; the four low i n t e n s i t y bands i n the v i s i b l e region of the free base spectrum are now replaced by two bands, Q! and (3, which are r e l a t e d to bands I and I I I , and II and IV of the free-base porphyrins, r e s p e c t i v e l y 1 2 6 > 1 4 6 . The i n t e n s i t i e s of the CX- and (3- bands remain much weaker (10-fold) r e l a t i v e to that of the Soret peak i n metalloporphyrin systems also. Electron-withdrawing substituents at the porphyrin periphery tend to s h i f t the (Vand ft bands to lower wavelength, and also cause an increase i n the i n t e n s i t y of the /3-relative to the CX b a n d 1 2 6 , 1 4 6 . Furthermore, with respect to the c e n t r a l metal, l i g a t i o n and formal charge a f f e c t the energy l e v e l s and occupation of the d - o r b i t a l , and t h i s can give r i s e to new charge t r a n s f e r and d-d t r a n s i t i o n s that mix and s h i f t the observed porphyrin 7T—> 7T t r a n s i t i o n s 1 - ' 0 . The four-coordinate i r o n ( I I ) durene-capped systems e x h i b i t spectra very s i m i l a r to those of other p y r r o l e - s u b s t i t u t e d hemes60b,144 j-^e appearance of multiple bands i n the Soret region has been interpreted i n terms of departure from f o u r - f o l d (D^) symmetry, which may r e s u l t from displacement of the high spin Fe(II) from the centre of the porphyrin p l a n e 1 4 4 (Fig. 4.5). Furthermore, the s i t u a t i o n may be complicated by the occurrence of equivalent states of d - o r b i t a l f i l l i n g 1 4 4 . The f i v e -coordinate high spin Fe(II) system i s characterized by one broad - 202 -absorption i n both the v i s i b l e and Soret region ( F i g. 4 . 5 ) 1 4 4 . These spectra of the four- and five-coordinate species are i d e n t i c a l for a l l three durene-capped hemes (Appendix I I ) . On coordination of a s i x t h ligand, however, s t r i k i n g v a r i a t i o n s are found i n the appearance of spectra i n the v i s i b l e region, within the durene s e r i e s . For the planar durene-7/7 system, coordination of an imidazole, i s o n i t r i l e , CO and 0 2 gives r i s e to sharp Ot,(3 and Soret bands, the r e l a t i v e i n t e n s i t i e s of which are c h a r a c t e r i s t i c of each type of ligand (Figs. 4.5, 4.11, 4.15 and 4.25). (No attempt i s made here to discuss i n t e r a c t i o n s between the a x i a l ligand, iron, and porphyrin o r b i t a l s , which give r i s e to the various 7T — > 7T* t r a n s i t i o n s o b s e r v e d 1 4 6 . ) With the 5/5- and 4/4- six-coordinate systems, a progressive decrease i n 'cap' size appears to cor r e l a t e with an increase i n the j3 band i n t e n s i t y at the expense of the ct band,which decreases i n i n t e n s i t y f o r a l l of the i s o n i t r i l e , CO and 0 2 bound systems (Figs. 4.11, 4.15(a), 4.15(b) and 4.25). These changes i n the v i s i b l e region are accompanied by a r e d - s h i f t and decrease i n i n t e n s i t y of the Soret band. S i m i l a r to the explanation proposed to account f o r the observed s p e c t r a l differences of the free-base porphyrins, these gradual s p e c t r a l changes f o r the six-coordinate systems may be interpreted i n terms of increased porphyrin s k e l e t a l d i s t o r t i o n i n the s e r i e s : 4/4 > 5/5 > 7/7. A l t e r n a t i v e l y , the added presence of a metal could also i n d i r e c t l y a f f e c t the porphyrin 7T—>7T* t r a n s i t i o n ; displacement of the Fe(II) from the porphyrin plane, with increased s k e l e t a l d i s t o r t i o n , may a l t e r i n t e r a c t i o n between the metal d - o r b i t a l s and porphyrin 7T-orbitals. I n t e r e s t i n g l y , the observed 7T—>7T* t r a n s i t i o n s are s e n s i t i v e to such e f f e c t s only i n the low spin six-coordinate complexes, and not in - 203 -the high spin four- or five-coordinate systems, f o r which a l l three durene hemes e x h i b i t i d e n t i c a l spectra. An explanation for this p o s s i b l y derives from the greater f l e x i b i l i t y of the four- and five-coordinate systems with respect to the Fe p o s i t i o n , i n the absence of a trans s i x t h ligand. On coordination of a s i x t h a x i a l ligand, the f a c t that the increase i n the (3 band becomes more pronounced for a 'T-state' system, with B - l,2-Me2Im (Fig. 4.15(a)), r e l a t i v e to that for an 'R-state' analog, B - Dclm (Fig. 4.15(b)), suggests that the Fe  p o s i t i o n , which i s further from the heme plane i n the former, may be the dominating influence on the s p e c t r a l trends obtained f o r the six-coordinate systems of the durene s e r i e s . 4.5.2 L i g a t i o n to Four-Coordinate Durene Hemes (a) General comparison of a x i a l l i g a t i o n constants A knowledge of rate and a f f i n i t y constants f o r l i g a t i o n to four-coordinate hemes i s e s s e n t i a l f o r the discussion of subsequent displace-ment e q u i l i b r i a i n the presence of various ligands. In the presence of C-donor ligands such as imidazoles, pyridines, amines etc., four-coordinate i r o n ( I I ) porphyrin complexes i n v a r i a b l y favour an octahedral environment, because of the CFSE achieved on forming a six-coordinate B low-spin Fe from a five-coordinate high-spin state; Kg » Kg, eqs. (26) and (43). In the presence of strong f i e l d 7T-acceptor ligands such as CO, however, five-coordinate low-spin complexes can be f o r m e d 1 4 5 , where t r a n s - l i g a t i o n of a second CO i s disfavoured because of competition for - 2 0 4 -F e - C O (7T—> 7T*) b o n d i n g 8 8 - 1 4 5 ; i n t h i s case, K ^ Q < K C O , e.g. KQQ = 5 X C O 1 0 4 M"1 and K C O - = 2 1 . 0 M _ 1 for deuteroheme i n toluene at 2 5 ° C 8 8 . This trans-coordination of C O should be distinguished from the high a f f i n i t y C O of a five-coordinate base-ligated heme for C O , where Kg >KQQ- Although the a s s o c i a t i o n rate constant for C O binding to a four-coordinate heme ( K Q O ) i s higher by about 1 0 - f o l d than that to a five-coordinate C O Fe(Por)(B) system (kg ), k c o - 1 0 8 M"1 s " 1 for 'open' hemes 4 6 (see sec-t i o n 2 . 6 . 6 ) , the presence of an N-donor base trans to C O increases electron density at the Fe centre so that C O d i s s o c i a t e s from the six-coordinate system to a much les s e r extent, k"^° « k.^g- The l a t t e r B e f f e c t dominates over the former so that t y p i c a l l y the o v e r a l l a f f i n i t y constants reveal that K C 0 > K c o by - 1 0 4 - f o l d , i n toluene at 2 0 ° C . I s o n i t r i l e ligands are also strong f i e l d 7T-acid ligands s i m i l a r to C O ; t h e i r enhanced (J-donor power r e l a t i v e to C O , however, i s evidenced by the formation of b i s - i s o n i t r i l e complexes with four-coordinate R N C , S hemes, i . e . K ^ Q » K R J J C 6 3 . For simple open hemes i n toluene at 2 0 ° C , rate constants kg and B kg f o r the a s s o c i a t i o n of an imidazole according to eq. ( 5 6 ) are both - 1 0 8 M"1 s " 1 1 4 , R B k \ k B ^ Fe(Por) + B v s Fe(Por)(B) s v F e ( P o r ) ( B ) 2 (56) while that for an i s o n i t r i l e binding to a five-coordinate heme , k ^ c B (eq. 5 7 ) , i s about 1 0 8 - 1 0 9 M _ 1 s" 1, B - Melm or Dclm 6 5. RNC kB . Fe(Por)(B) • v Fe(Por)(B)(RNC) ( 5 7 ) - 205 -(b) Base Binding, Kg values The extent of a x i a l base l i g a t i o n to an i r o n centre i s determined both by the 0"-donor a b i l i t y , measured by pK a, and 7T-acceptor property of the base. In general, Kg values for coordination of a s i n g l e a x i a l base have been shown to c o r r e l a t e with pK a, f o r bases with s i m i l a r bonding character. For instance, the a f f i n i t i e s of F e ^ ^ C ^ c a p ) f o r the substituted pyridines: 3-Clpy (pK a 2.84), py (pK a 5.27), and 3,4-Me2py (pK a 6.46), vary i n the order Kg - 34, 76, and 182 M"1, respect-i v e l y 1 4 0 * 5 . Comparing the heme systems derived from mesoheme 6 5• 6 8 with those from T P P 7 6 * 1 4 7 , base binding constants for the l a t t e r are usually higher owing to the weaker basic properties of the tetraphenylporphyrin system; e.g. f o r Fe(anthracene-6/6) 6 5, K M e I m - 3 x IO 3 M"1, and for F e ( T P P ) 1 4 7 , K M e I m -6 x 10 4 M"1 i n toluene at -20°C. An exception to t h i s trend i s found within the s e r i e s of capped porphyrins (15-17b) studied by Basolo et a l . 1 4 0 ^ , f o r which binding of a f i f t h a x i a l base i s retarded s i g n i f i c a n t l y compared to that for other TPP-derived systems: e-6- Kl,2-Me Im - 1 x 1 q 3 i n toluene at 20°C f o r F e ( C 2 - c a p ) 7 8 a ' 1 4 0 b . For these capped hemes, r e s t r i c t e d movement of the i r o n toward the a x i a l base, caused by porphyrin plane d i s t o r t i o n 7 7 , i s suggested to be responsible f o r t h i s reduced a f f i n i t y f o r a f i f t h l i g a n d 7 8 a . In the case of the durene-4/4 heme, considerable porphyrin plane d i s t o r t i o n toward the capped face i s revealed i n the c r y s t a l structure of the hemin chloride d e r i v a t i v e (section 3.4). The Kg values for a l l three durene hemes, however, f a l l i nto the range expected for undistorted p y r r o l e - s u b s t i t u t e d d e r i v a t i v e s 6 5 (Table 4.1). In t h i s s i t u a t i o n , the four-coordinate i r o n may already be s l i g h t l y displaced - 206 -from the porphyrin plane toward the f i f t h a x i a l s i t e , so that ligand coordination on t h i s side i s not appreciably a f f e c t e d by porphyrin d i s t o r t i o n . For a p a r t i c u l a r base, no apparent trend i s seen within the durene s e r i e s , although a c o r r e l a t i o n does e x i s t between the Kg value and pK a of a base with each of the durene systems. The differences i n Kg values between the durene hemes are i n any case s l i g h t (within 2-3-fold), and more importantly indicate that d i s t o r t i o n of the porphyrin skeleton, p a r t i c u l a r l y pronounced i n the durene-4/4 case, has no s i g n i f i c a n t e f f e c t on coordination of a base to the unhindered side of the heme. (c) CO Binding, K C O Comparison of KQQ X 10 5 M"1 for the durene-4/4 heme with that fo r open hemes, K C O -5.7 x 10 4 M _ 1 i n toluene at 20°C, 8 8 . 1 4 5 reveals a s i g n i f i c a n t increase (-10-fold) f o r the binding of CO to the durene heme despite the presence of a cap on one side. Such enhanced affinity,shown by the durene heme f o r CO r e l a t i v e to that of an undistorted system, i s not evidenced i n the formation of a five-coordinate Fe(Por)(B) complex where B i s a substituted imidazole (section 4.5.2(b)); t h i s difference, presumably i s a r e f l e c t i o n of the respective 0*/7Tbonding character of CO r e l a t i v e to an imidazole base when coordinating as a f i f t h a x i a l ligand. While complexes of the type Fe(Por)(B) formed with strong Odonors such as substituted imidazoles/pyridines are considered to be paramagnetic, those formed with CO, Fe(TPP)(C0) f o r i n s t a n c e 1 4 5 , have been shown by proton nmr studies and s o l u t i o n e q u i l i b r i a to be diamagnetic; i n the - 207 -l a t t e r case, a strong 7T-acceptor ligand l i k e CO can be expected to favour a low-spin Fe(II) with f i l l e d d X y y 2 o r b i t a l s , which w i l l lead to enhanced metal d7T donation. For the d i s t o r t e d durene-4/4 heme, displacement of the Fe toward the coordinating CO (on the uncapped side) may serve to improve d7T donation from metal to CO, which would be r e f l e c t e d i n an enhanced a f f i n i t y f o r CO compared to that of a planar four-coordinate heme where the Fe i s i n the porphyrin plane. 4.5.3 Isocyanide Binding to Five-Coordinate Durene Hemes, Yr^c The reduction factors i n Table 4.2, and shown diagrammatically below, show the extent to which binding of the isocyanides, t-BuNC and TMIC, to the durene-7/7 and -5/5 complexes i s diminished r e l a t i v e to that f o r chelated protoheme^ 0•° 5: Compound t-BuNC TMIC 80 Durene-7/7 Durene-5/5 145 70 The most s i g n i f i c a n t r e l a t i o n s h i p between these factors i s found i n the su b s t a n t i a l reduction f o r the binding of t-BuNC r e l a t i v e to TMIC for - 208 -both hemes (70-80-fold). According to Traylor's d i f f e r e n t i a t i o n for the r e l a t i v e s t e r i c requirements of these l i g a n d s 6 5 ' 7 3 , the r e s u l t s obtained here are r e a d i l y interpreted i n terms of a " c e n t r a l " s t e r i c e f f e c t imposed by the durene moiety i n both heme systems. Examining the con-secutive reduction factors i n the series:chelated protoheme — > 7/7 —> 5/5 systems, the binding of t-BuNC i s s i g n i f i c a n t l y retarded by the i n i t i a l i n troduction of the c e n t r a l durene cap i n the 7/7-complex (680-fold), with further reduction i n a f f i n i t y not as pronounced (120-fold) for the 5/5-system. In the case of TMIC, however, peripheral s t e r i c i n t e r a c t i o n l a r g e l y arises within the more enclosed environment of the 5/5-capped heme, 145-fold, r e l a t i v e to an a f f i n i t y reduction of 7.5-fold f o r the 7/7-complex. The t i g h t e r 4/4-cap severely retards binding of both bulky i s o n i t r i l e s ; t h i s system i s discussed i n section 4.5.3(c) . 4.5.4 Carbon Monoxide Binding, Kou, k u u and k _ o u (a) R-State Comparisons The data i n Table 4.3 c l e a r l y exemplify the necessity f o r obtain-ing and analyzing both k i n e t i c and equilibrium constants. Although the values f o r the durene-7/7 and -5/5 systems are almost i d e n t i c a l , the magnitudes are i n fac t equally o f f s e t by s i g n i f i c a n t differences i n ass o c i a t i o n and d i s s o c i a t i o n rate constants f o r these systems. (With respect to the r e l a t i o n s h i p s between constants discussed below, the k " c o values determined from Xp® and k ^ w i l l be used throughout.) 209 Compared with the 'open' chelated meso- and protoheme systems, the durene-7/7 heme exhibits a 10-fold reduction i n CO a s s o c i a t i o n rate, whereas the durene-5/5 and -4/4 systems both unexpectedly show higher values r e l a t i v e to that of the larger capped analog, despite t h e i r reduced o v e r a l l a f f i n i t y toward bulky i s o n i t r i l e s . To a s c e r t a i n whether t h i s reduction i n k^1® was c h a r a c t e r i s t i c of the 7/7-system and not caused by some o r i e n t a t i o n of toluene molecules obstructing coordination at the s i x t h s i t e , k^° determinations were also c a r r i e d out i n CH2CI2, a solvent medium of a d i f f e r e n t nature. Under otherwise i d e n t i c a l conditions, however, no s i g n i f i c a n t change i n k^° was observed, for the 7/7-system i n CH2CI2 r e l a t i v e to that i n toluene (Appendix I I I , D). The p o s s i b i l i t y of solvent p a r t i c i p a t i o n i n t e r f e r i n g with CO as s o c i a t i o n to the 7/7-system i s therefore ruled out. This observed reduction i n CO a s s o c i a t i o n rate with l i t t l e change i n d i s s o c i a t i o n rate f or the 7/7-heme, r e l a t i v e to the open heme systems, i s i n accordance with d i s t a l side s t e r i c hindrance toward the binding of CO (section 2.5.1a); t h i s hindrance i s imposed by the presence of the durene 'cap', and r e s u l t s i n diminished a c c e s s i b i l i t y to the s i x t h coordination s i t e of the heme. The c e n t r a l s t e r i c e f f e c t of the durene-7/7 cap i s c l e a r l y i l l u s t r a t e d by a reduction i n the binding constant for the bulky t-BuNC ligand r e l a t i v e to an open heme, but seems u n l i k e l y f o r the binding of a less s t e r i c a l l y demanding diatomic to a 'large-capped' system. A p l a u s i b l e explanation i s that the 7/7-cap, with 14 methylene carbon linkages, may prefer a "squashed" conformation i n which the r o t a t i n g durene moiety i s suspended close to the centre of the porphyrin plane (Fig. 4.30 A). The u p f i e l d s h i f t of the durene methyl s i g n a l i n the proton nmr spectrum of the free base porphyrin provides some evidence 210 for t h i s suggestion (section 3.3.1). Ligand coordination to the sixth s i t e would then be retarded by the "pendulous" durene moiety i n close i proximity to the i r o n atom. A s i m i l a r explanation has been proposed to account for the low a f f i n i t y of the Fe(C^-cap)(B) system toward the binding of CO, NO and 0 2 r e l a t i v e to the smaller C3- and C 2-capped 7 ft analogs' 0 (section 2.5.6). For the 7/7-system, however, t h i s suggestion i s substantiated further by some relevant k i n e t i c data. The most s t r i k i n g feature of the CO k i n e t i c s within the durene series i s the exceptionally high CO d i s s o c i a t i o n rate for the 5/5- and 4/4-hemes, p a r t i c u l a r l y for the l a t t e r complex, compared to that for the durene-7/7 and chelated systems. An explanation for t h i s deviant behaviour derives from the extreme porphyrin plane d i s t o r t i o n inherent i n the two smaller capped durene systems. Pronounced d i s t o r t i o n i s evidenced i n the c r y s t a l structure of the durene-4/4 hemin chloride. Furthermore, u v - v i s i b l e s p e c t r a l trends indicate that d i s t o r t i o n also e x i s t s i n the 5/5-porphyrin, although to a l e s s e r degree than i n the 4/4-case, while the s i m i l a r i t y between the spectrum of the 7/7-free base with that of e t i o II suggests that the larger 7/7-capped system remains e s s e n t i a l l y planar (section 3.3.3). To consider the 5/5-complex f i r s t , placement of a more r i g i d diagonal strap, r e l a t i v e to that i n the 7/7-case, presumably induces doming of the porphyrin skeleton i n such a manner as to provide an incoming ligand greater a c c e s s i b i l i t y to the Fe atom (Fig. 4.30 B). This would e f f e c t i v e l y r e s u l t i n an enhanced a s s o c i a t i o n rate (10-fold) compared to that for the 7/7-heme. Subsequent to Fe-CO bond formation, however, porphyrin plane d i s t o r t i o n presumably causes d e s t a b i l i z a t i o n of - 211 -F i g . A.30 (A) "Squashed" conformation of the durene-7/7 cap (B) "Basket-shaped" conformation of the d i s t o r t e d durene-5/5 and -4/4 systems the bound carbonyl and r e s u l t s i n an increased CO d i s s o c i a t i o n rate by 5-10-fold. The doming e f f e c t becomes even more pronounced for the t i g h t e r cap durene-4/4 system, giving r i s e to ca. a 5-fold increase i n CO o f f - r a t e r e l a t i v e to the 5/5-heme. The lower a s s o c i a t i o n rate for CO (4-fold) i s reasonable considering the reduced a c c e s s i b i l i t y to the s i x t h coordination s i t e because of the smaller cap. Porphyrin plane doming of t h i s type may be viewed also as imparting considerable "T-state" character on the five-coordinate heme complex, p a r t i c u l a r l y i n the 4/4-system, where an increase i n d i s t o r t i o n r e s u l t s i n a decrease i n k^ 0 accompanied by an increase i n k"^° r e l a t i v e to the 5/5-complex. This i s e s s e n t i a l l y the same k i n e t i c consequence as switching from an R- to T-state l i g a t e d system (section 2.6.4). The analogy drawn 6 5 between an increase i n cone angle </> i n simple t r a n s i t i o n metal carbonyls and porphyrin plane d i s t o r t i o n found i n T-state hemes (section 2.6.4) i s c l e a r l y depicted by the durene hemes, where increase i n k"^° i s well c o r r e l a t e d with d i s t o r t i o n of the porphyrin skeleton from p l a n a r i t y . This i s p r i m a r i l y a proximal e f f e c t on CO a f f i n i t y i n the durene-5/5 and -4/4 systems, unlike the 7/7-complex which e x h i b i t s d i s t a l s t e r i c hindrance to CO binding, r e l a t i v e to the chelated open hemes. A B - 212 -(b) T-State Comparisons Coordination of a s t e r i c a l l y hindered base at the f i f t h a x i a l p o s i t i o n r e s u l t s d i r e c t l y i n a 160-fold reduction i n CO a f f i n i t y for simple open hemes (Table 2.10). These 'open' T-state systems now c l o s e l y resemble the i n t r i n s i c a l l y d i s t o r t e d 5/5-complex, as evidenced by t h e i r s i m i l a r k i n e t i c constants f o r CO " l i g a t i o n (Table 4.A). For both the 5/5- and A/A-h ernes, the "T-state e f f e c t " of introducing a hindered a x i a l base simply causes a further decrease i n asso c i a t i o n as well as increase i n d i s s o c i a t i o n rate f o r CO r e l a t i v e to that f o r the corresponding R-state complex. The Fe(durene-7/7)(1,2-Me2lm) system provides an i n t e r e s t i n g case. Compared to the simple open T-state systems, deuteroheme (1,2-Me2lm) and Fe(OEP)(1,2-Me2lm), the 7/7-heme displays a 10-fold reduction i n both as s o c i a t i o n as well as d i s s o c i a t i o n rate. This unusual drop i n k " ^ can be accounted for by reluctance of such a large strap to undergo a conformational change. The s p e c i a l arrangement of the strap, which i n i t i a l l y hinders coordination of CO to the five-coordinate heme, presumably adopts a d i f f e r e n t o r i e n t a t i o n i n the six-coordinate complex, to accommodate the bound CO; conversely, the energy now required to disrupt the conformation of t h i s state w i l l tend to re t a r d CO di s s o c i a -t i o n from the heme. This e f f e c t i s also observed f o r the hemoprotein, horse-radish p e r o x i d a s e 2 8 • 1 4 8 • 1 4 9 , where an extremely hindered s i x t h coordination s i t e i s evidenced i n a 1300-fold reduction i n k ^ accompanied by a 500-800-fold reduction i n k " ^ ° r e l a t i v e to R-state hemoglobin; the high energy cost of rearranging p r o t e i n residues within the crowded d i s t a l pocket r e s u l t s i n a diminished tendency for both - 213 -ligand a s s o c i a t i o n and d i s s o c i a t i o n . In the durene-7/7 model, the rate of CO d i s s o c i a t i o n from a T-state heme i s apparently more s e n s i t i v e to the conformational demands of the 'cap', than that from an R-state complex where ligand d i s s o c i a t i o n , i n general, i s much slower. Thus, as a r e s u l t of two e n t i r e l y separate properties found within the durene systems, the 4/4-complex exhibits the highest, and the 7/7-heme the lowest, CO o f f - r a t e f o r any T-state heme observed to date. (c) General r e l a t i o n s h i p s with other py r r o l e - s u b s t i t u t e d hemes On comparing the larger capped durene-7/7 and anthracene-7/7 (18) systems, the former shows s l i g h t l y reduced (-2-fold) a f f i n i t y toward CO, but greater a f f i n i t y (3-fold) f o r t-BuNC (see Tables 2.3, 2.4, 4.2 and 4.3). These differences are s l i g h t , but are an i n d i c a t i o n of the greater ' p o t e n t i a l ' f l e x i b i l i t y of the durene cap toward accommodating a bulky ligand, where the o v e r a l l a f f i n i t y f o r the liga n d i s increased, presumably by slower d i s s o c i a t i o n from the durene-7/7 heme r e l a t i v e to d i s s o c i a t i o n from the anthracene-7/7. An analogous comparison between the durene-4/4 and anthracene-6/6 (19) hemes reveals an 8 - f o l d higher a f f i n i t y f o r CO, but extremely reduced a f f i n i t y toward i s o n i t r i l e s (>600-fold f o r n-BuNC), for the durene-4/4 heme. Contrary to the durene-7/7 case, t h i s behaviour of the 4/4- system r e f l e c t s an extremely r e s t r i c t i v e d i s t a l c a v i t y , imposed by a very r i g i d , small cap, which i n h i b i t s the required o r i e n t a t i o n for coordination of a bulky ligand. The pronounced d i s t o r t i o n of the porphyrin skeleton toward the coordinated base, on the other hand, - 214 -presumably r e s u l t s i n a 'basket-shaped' five-coordinate heme (Fig. 4.26 B), where access to the Fe i s not obstructed s i g n i f i c a n t l y f o r a smaller diatomic (as shown by the high rates of CO a s s o c i a t i o n ) , despite the presence of a t i g h t f i t t i n g cap. In the case of the anthracene - 6/6, the amide linkages presumably add r i g i d i t y to the d i s t a l hindrance provided by the cap, to the extent that a c c e s s i b i l i t y to the Fe i s diminished even for diatomics; the 'pot e n t i a l ' f l e x i b i l i t y of t h i s cap, however, having a larger number of adjoining atoms than the durene-4/4 heme, allows for coordination to some degree of even the b u l k i e s t i s o n i t r i l e , t-BuNC. S i g n i f i c a n t v a r i a t i o n i s also found between the s p a c i a l conformations of the adamantane and anthracene-6/6 caps (section 2.5.2(i)). Within the range of py r r o l e - s u b s t i t u t e d model hemes, the FeSP-13 (23) and pyridine-5/5 cyclophane (26) systems e x h i b i t the lowest a f f i n i t y toward CO, manifested almost e x c l u s i v e l y i n very low a s s o c i a t i o n rates, as a r e s u l t of a si n g l e , short diagonal strap with r i g i d amide linkages i n each case. Unfortunately, i s o n i t r i l e a f f i n i t i e s f o r these extremely hindered models have not been reported. 4.5.5 Comparison of Carbonyl Stretching Frequencies f o r Various Heme Systems I t has been suggested 3 3 that the lowered V(CO) values exhibited by hemoproteins r e l a t i v e to model systems, Fe(Tpiv)(Melm)(CO) for example, r e s u l t from d i s t o r t i o n of the bound Fe-C-0 moiety, which translates to reduced o v e r a l l a f f i n i t y of the hemoproteins toward CO. For instance, the lower CO a f f i n i t y expressed by MbCO cor r e l a t e s with lowered V(CO) - 215 values, while the hemoprotein HbZurichCO, which i s considered to possess a more 'open' binding pocket, correspondingly shows a higher carbonyl s t r e t c h i n g frequency (Table 4.6). The e x i s t i n g compilation of J^(CO) values for a v a r i e t y of model hemes, however, now i n d i c a t e s 6 5 that previous i n t e r p r e t a t i o n of CO s t r e t c h i n g frequencies may be too s i m p l i s t i c . For instance, the open heme chelated protoheme shows a J^(CO) value i n Me2S0 as low as that observed for the carbonyls i n 'hindered' hemoproteins i n aqueous media, while V(CO) values within the encumbered model hemes show no consistent c o r r e l a t i o n with CO a f f i n i t y . Instead, carbonyl s t r e t c h i n g frequencies appear to be subject to s u b s t a n t i a l v a r i a t i o n with change i n solvent medium among the various model hemes. Although no d i r e c t c o r r e l a t i o n e x i s t s between V(CO) and CO a f f i n i t y f o r the durene systems, some consistency i s found allowing i n t e r p r e t a t i o n to be attempted. Of note, carbonyl stretches f o r the 5/5- and 4/4-systems are very s i m i l a r , while those for the R- and T-state 7/7-systems are r e l a t i v e l y lower i n frequency. This observed d i s t i n c t i o n i s consistent with the concept of weaker Fe-CO bonding i n the smaller capped systems, that r e s u l t s perhaps from d i s r u p t i o n of Fe — > CO (7T-7T*) back-bonding with d i s t o r t i o n of the porphyrin skeleton from p l a n a r i t y ; t h i s would c o r r e l a t e with the observed increase i n d i s s o c i a t i o n rates for CO from these complexes, r e l a t i v e to the 7/7-case. A l t e r n a t i v e l y , porphyrin d i s t o r t i o n i n the 5/5- and 4/4-systems may r e s u l t i n c l o s e r proximity between the porphyrin nitrogens and bound CO, where some transfer of electron density from Np into the C-0 bond contributes to an increased V(CO) value. Detailed resonance Raman and IR studies c a r r i e d out on the FeSP hemes suggested that such charge - 216 -Table 4.6 Carbonyl Stretching Frequencies f o r Various Model Hemes Compound Solvent J>C0 Kco Ref cm"1 M" -1 MbCO aqueous 1931,1945,1970 3 X 10 7 150a HbCO aqueous 1951 -4 X 10 8 47 HbCO Zurich aqueous 1951,1958 -10 8 150b EryCO aqueous 1959 2.8 X 66 (9b) Fe(TPP)(l,2-Me 2Im)(CO) toluene 1972 7 X IO* 151 (14b)Fe(TPivPP)(Melm)(CO) 1969 -4 X 10 9 a 33 (27) Fe(Poc.Piv)(MeIm)(CO) 1964 1 .6 x 10 8 76 (15b)FeC 2-cap(MeIm)(CO) toluene 2002 1 X 10 7 151 (15b)FeC 2-cap(l,2-Me 2Im)(CO) toluene 1999 5 X 10 5 151 (16b)FeC 3-cap(MeIm)(CO) toluene 1984 2 X 10 7 151 (11) Chelated Protoheme Me2S0 1951 4 X 10 8 b 138 (18) Fe(anthracene-7/7)(Melm)(CO) CHC13 1966 -1 X 10 8 c 65 (19) Fe(anthracene-6/6)(Melm)(CO) C H C I 3 1975 6 X 10 5 65 (20) Fe(adamantane)(Melm)(CO) C H C I 3 1959 2 X 10 5 73 (25) FeSP-15(MeIm)(CO) CH 2C1 2 1945 -2 X 10 6 c 81 (24) FeSP-14(MeIm)(CO) CH 2C1 2 1939 2 X 1 0 5 81 (23) FeSP-13(MeIm)(CO) CH 2C1 2 1932 8 X 10 3 81 (56b)Fe(durene-7/7)(Melm)(CO) toluene 1974 -7 X 1 0 7 c (56b)Fe(durene-7/7)(l,2-Me 2Im)(CO) toluene 1972 3 X 10 6 (55b)Fe(durene-5/5)(Melm)(CO) toluene 1990 -9 X 1 0 7 c (55b)Fe(durene-5/5)(l,2-Me 2Im)(CO) toluene 1987 2 X 10 6 (54b)Fe(durene-4/4)(Melm)(CO) toluene 1988 7 X 10 6 (54b)Fe(durene-4/4)(l,2-Me 2Im)(C0) toluene 1985 2 X 10 5 Kru f o r chelated picket-fence quoted. value determined i n toluene. B - Dclm, as R-state base. - 217 -transfer can occur between Np and the bound CO, which, i n these extremely hindered systems, was considered to a r i s e with increased bending of the Fe-C-0 un i t from l i n e a r i t y 8 1 (section 2.5.7). 4.5.6 Dioxygen Binding (a) 0 2 a f f i n i t i e s For those heme systems with polar amide functions surrounding the s i x t h coordination s i t e , d i r e c t comparison of 0 2 a f f i n i t i e s i s considered to be of l i t t l e value since oxygen binding i s subject to contributions from both s t e r i c and e l e c t r o n i c e f f e c t s , c h a r a c t e r i s t i c to each system. In the durene heme s e r i e s , however, a hydrophobic 'cap' provides a r e l a t i v e l y non-polar d i s t a l environment, s i m i l a r to that of the solvent system, toluene, so that (lack of) p o l a r i t y e f f e c t s experienced by the bound dioxygen should be i d e n t i c a l f o r a l l three systems. V a r i a t i o n i n 0 2 a f f i n i t y within t h i s s e r i e s should therefore a r i s e l a r g e l y through s t e r i c and/or s o l v a t i o n e f f e c t s . Examination of °9 the K * values obtained (Table 4.5) reveals that f o r a l l three durene hemes, with e l e c t r o n i c a l l y i d e n t i c a l d i s t a l environments, 0 2 a f f i n i t i e s are e s s e n t i a l l y the same, within each of the R-and T-state systems, f o r B - Dclm and B - l,2-Me2Im, res p e c t i v e l y , This i s e n t i r e l y consistent with the complete absence of p o l a r i t y e f f e c t s . - 218 -(b) R/T state change Reduction in 0 2 affinity caused by an R- to T-state switch for each durene heme is similar in magnitude to that evidenced in the binding of C O (Table h . l ) . The only other heme series which allows comparison of K R / K J ratios is that of the pocket systems MedPoc and PocPiv°4. The durene hemes and these TPP derived complexes, both show similar R- to T-state affinity changes for dioxygen (Table 2.10). The kinetic consequence of this switch in the pocket systems, however, is different for 0 2 than for C O . While C O binding suffers almost equal and opposite changes in association and dissociation rates, the reduction in 0 2 affinity for the T-state system is primarily manifested in an increased off-rate with relatively l i t t l e change in on-rate. Since 0 2 binding is known to be subject to electronic effects which significantly alter dissociation rates, i t is difficult to determine whether this difference in relative C0/02 T-state binding is governed by electronic and/or proximal steric effects within the pocket systems. A Table 4.7 Comparison of R- and T-State Constants for CO and 0 2 Binding to Durene-Capped Hemes in Toluene at 20°C Compound Fe(durene-7/7)(DcIm)/(l,2-Me2Im) 7 - 3 20 20 Fe(durene-5/5)(DcIm)/(l,2-Me2Im) 10 - 3-4 - 30-40 30 Fe(durene-4/4)(DcIm)/(l,2-Me2Im) 4 - 7 - 6 - 24-40 20-30 or (Melm) 219 k i n e t i c comparison with the 'hydrophobic' durene hemes would be useful here i n evaluating the r e l a t i v e contribution of these e f f e c t s ; unfortunately, the lack of k i n e t i c data on 0 2 binding precludes a complete analysis. (c) M value An appreciation of the o v e r a l l d i f f e r e n t i a t i o n shown toward CO and 0 2 binding by a p a r t i c u l a r system may be gained from the r a t i o of equilibrium constants, K^/K 2, known as the M v a l u e 1 5 2 . P r i o r to any comparison of M values between the durene and simple 'open' hemes, however, i t should be noted that the measured 0 2 a f f i n i t i e s within open heme systems themselves vary; for instance there i s about a 5-fold difference i n M values between chelated TPP and the chelated proto- and mesohemes (Table 4.8). Traylor et a l . 4 6 and Lavalette and Momenteau 1 5 3 have determined on- and o f f - rate constants f o r 0 2 binding to the Im-chelated meso 4 6 (11) and TPP-heme 1 5 3 (53) systems, r e s p e c t i v e l y , using l a s e r photolysis of these hemes i n the presence of C0/02 mixtures. A s i g n i f i c a n t difference (6-fold) was found between the k L values determined independently for these two pyrr o l e - s u b s t i t u t e d and TPP hemes (Table 4.9). In contrast with t h i s are the k i n e t i c measurements for 0 2 binding to the Py-chelated deutero- and TPP systems, c a r r i e d out by Lavalette and Momenteau, 1 5 3 which afforded almost i d e n t i c a l o f f - r a t e values f o r both systems, i n d i c a t i n g that the e x i s t i n g differences for the Im-chelates r e s u l t not from differences i n t r i n s i c to the nature of the p y r r o l e - s u b s t i t u t e d and TPP-derived systems, but perhaps from the 220 -Table 4.8 Comparison of M Values (K c o/K 2 ) for Model Hemes Pyrrole-substituted Systems (11) Chelated protoheme (10) Chelated mesoheme In Toluene B=l,5-Dclm 2.4 x 10 4 1.2 x 10 4 In Toluene In o-Dichlorobenzene B=l,2-Me2Im B=DcIm (56b) Fe(durene-7/7)(B) 9 x 10' (55b) Fe(durene-5/5)(B) 7 x 10^ (54b) Fe(durene-4/4)(B) 5-7 x 10 2 9 x 10* 6 x 10 4 3-5 x 10 3 -1.6 x 10^ -1.0 x 10 4 -1.1 x 10 3 (18) Fe(anthracene-7/7)(B) (19) Fe(anthracene-6/6)(B) (20) Fe(Adamantane)(B) (26) Fe(pyridine-5/5)(B) 1.8 x 10 : 4.9 x 10 : 6.4 x 10: 14 (21) (22) (25) (24) (23) Fe-Cu-4 £ Fe-Cu-5 fe FeSP-15 FeSP-14 FeSP-13 & 3.1 x 10' 2.5 3.0 x 10 z x 10 2 80 46 TPP-Derived Systems (53) Chelated TPP °-(15b) Fe(C 2-cap)(B) (50) Fe(TTPPP)(B) (49) Fe-Im-Amide (30) Chelated Pf (14b) Fe(TPivP)(B) (29) Fe(TalPoc)(B) (28) Fe(MedPoc)(B) (27) Fe(PocPiv)(B) 5.9 x 10' 4.3 x 10 : 1.2 x 10 4 2.7 x 10" 5.5 x 10 2 2.7 x 10 2 2 x 10 4 5.6 x 10 4 4.3 x 10 J 3.5 x 10 3 4.8 x 10 2 2.2 x 10 2 1.4 x 10' £ B « Melm. - B «= THPIm. °- From r e f s . 64 and 153. 221 Table 4.9 K i n e t i c Constants for Dioxygen Binding to Durene and Open Heme Systems in Toluene at 20°C Compound k 2 (M _ 1 s" 1) -o • k 2 ( s ' 1 ) a K l (11) Im-Chelated protoheme 6.2 x 10 7 4 x 10 3 1 .5 x 10 4 (10) Im-Chelated mesoheme 8.4 x 10 7 4. .8 x 10 3 1 .7 x IO 4 Py-Chelated deuteroheme 2 x 10 8 7 x 10 4 2 .5 x IO 3 (53) Im-Chelated TPP 5 x 10 7 3 x 10 4 1 .7 x IO 3 Py-Chelated TPP 1 x 10 8 1 x 10 5 1 .1 x IO 3 (56b) Fe(durene-7/7)(Dclm) 9.8 x 10 6 (1 .5 x 10 4) 6 .6 x IO 2 — K i n e t i c determination I C O experimental methods X J . Unfortunately, no s a t i s f a c t o r y explanation can be provided at present for the difference i n k L values found between the Im-chelated meso or protohemes, r e l a t i v e to Im-TPP, which i n turn i s manifested as a 10-fold v a r i a t i o n i n o v e r a l l 0 2 a f f i n i t i e s (K ^) these respective systems (Table 4.9). For the durene-7/7 heme, where approximate dioxygen rate constants are a v a i l a b l e (Table 4.9), the reduction i n on-rate r e l a t i v e to the open chelated meso- and protoheme systems (6-8-fold) i s s i m i l a r i n magnitude to that seen for CO binding (-10-fold), where s t e r i c r e s t r i c t i o n imposed by the durene cap diminishes ligand access to the i r o n (see Table 4.3). This d i s t a l s t e r i c e f f e c t i s therefore exhibited toward both CO and 0 2 - 222 alike, with no significant discrimination shown for either diatomic within the 7/7-systera. The higher M value for the 7/7-heme, relative to the open, pyrrole - substituted chelated systems, results from a slight difference in dissociation rates for CO and 0 2 ( cf. Tables 4.3 and 4.8); in the light of the existing discrepancy between the open heme systems, as discussed above, however, discussion and over interpretation of this difference are minimized. The most interesting aspect of the relative C0/02 affinities shown within the durene heme series is the significantly lower M value of the 4/4-system relative to those of the larger-capped systems, despite similar 0 2 affinities. Unlike the pyrrole-substituted systems with distal amide functions, where a drop in M value can result from increased stabilization of the bound dioxygen via polarity effects, the differentiation between CO and 0 2 exhibited by the durene-4/4 heme, in the absence of dipolar interactions, must result largely from variation in steric and/or solvation factors, relative to those present in the 5/5 and 7/7-systems. Considering the latter effect first, on changing the solvent from toluene (*- 2.4) to o-dichlorobenzene ( t - 9.9) ca. a 4-7-fold drop in M value is observed for a l l three durene hemes (Table 4.8). This is in line with data from studies on other hemes75il°^t where higher 0 2 affinity in the more polar solvent system has been attributed to greater stabilization of the dipolar Fe-02 moiety relative to that of Fe-CO (section 2.7.4). Although data for direct comparison with an open system are not available, the reduction in M value indicates that solvent orientation is not restricted within the distal cavity of any durene heme. In contrast to this, the C2-capped heme showed very l i t t l e change 223 -i n C>2 a f f i n i t y (3-fold) between toluene and DMF solvent systems, despite a s u b s t a n t i a l change i n d i e l e c t r i c constant from 2.4 to 36.7, re s p e c t i v e l y (section 2 . 7 . 4 ) 7 8 a . This was a t t r i b u t e d to a r e s t r i c t e d d i s t a l environment formed within a cen t r a l cap anchored on a l l four sides of the porphyrin p e r i p h e r y 7 ^ 3 . Within the durene s e r i e s , however, the s i m i l a r r e l a t i v e change i n M value f o r a l l three hemes indicates that a l t e r a t i o n i n cap size does not r e s t r i c t solvent o r i e n t a t i o n , and more importantly, s o l v a t i o n e f f e c t s are e s s e n t i a l l y the same within the d i s t a l c a v i t i e s of a l l three systems. Therefore, r u l i n g out the p o s s i b i l i t y of v a r i a t i o n i n solvation, which may independently a l t e r CO/O2 a f f i n i t i e s between each system, the CO/O2 d i f f e r e n t i a t i o n exhibited by the hindered 4/4-complex r e l a t i v e to the larger-capped systems most l i k e l y a r i s e s from e f f e c t s that are s t e r i c i n nature; these appear to hinder the binding of CO without i n t e r f e r i n g appreciably with the binding of O2. This diminished a f f i n i t y toward CO i s a proximal e f f e c t r e f l e c t e d by a reduction i n on-as w e l l as an increase i n o f f - rate f o r CO (as discussed i n section 4.5.3). The durene-4/4 heme i s therefore a system which displays proximal s t e r i c d i s c r i m i n a t i o n against the binding of CO r e l a t i v e to that of O2. Since the magnitude of d i f f e r e n t i a t i o n i s p a r a l l e l e d i n the T-state system, unfavourable s t e r i c i n t e r a c t i o n between proximal base and porphyrin i s not responsible for t h i s discriminatory e f f e c t which must instead r e s u l t from the porphyrin plane doming inherent i n the 4/4-heme, and present to a s i m i l a r extent i n both R- and T-state systems. - 224 -I n t e r e s t i n g l y , the introduction of s k e l e t a l d i s t o r t i o n i n the 5/5-system appears to a f f e c t CO and 0 2 binding to a s i m i l a r extent - the M value i s not much d i f f e r e n t from that of the 7/7 heme; as the 'doming e f f e c t ' becomes more severe, however, the binding of CO i s hindered further, whereas that of 0 2 i s not diminished s i g n i f i c a n t l y . In view of t h i s trend, i t would be of i n t e r e s t to examine the r e l a t i v e C0/0 2 binding a f f i n i t i e s of a heme which possesses even more pronounced porphyrin plane d i s t o r t i o n than that found i n the 4/4-case, to determine whether t h i s d i f f e r e n t i a t i o n factor becomes further enhanced. - 225 4.5.7 Thermodynamic Considerations for CO and 0 2 Binding Traylor's group 7 5 and Collman's g r o u p 6 4 have independently suggested that for the binding of CO to hindered model hemes, the t r a n s i t i o n state and carbonylated complex both s u f f e r s t e r i c hindrance to a s i m i l a r degree. These interpretations were based on the grounds that reduction i n CO a f f i n i t y i s predominantly expressed i n decreased as s o c i a t i o n rates with l i t t l e change i n d i s s o c i a t i o n rate for encumbered systems r e l a t i v e to open hemes (section 2.5). Traylor et a l . 6 5 suggested further that a rapid pre-equilibrium e x i s t s between d i f f e r e n t conformational states for the five-coordinate complex, some of which r e s t r i c t access to the i r o n centre, such that k s t e r l c « K c o n^-, eq. (58). Since the major cont r i b u t i o n to the change i n free energy (<5AG° -B l^conf F e > ^ \ Fe •> " S h > <58> ^ s t e r i c where K c o - -k-C0 A G ° t e r ^ c ) f o r CO binding to a s t e r i c a l l y hindered heme ar i s e s from the extra free energy of a c t i v a t i o n $ A G ^ - A G g t e r j _ c ) required for formation of the t r a n s i t i o n state, with no further d e s t a b i l i z a t i o n i n the bound state ( 5AG ° - S A G * , see F i g . 4.31 and Table 4.10), the t r a n s i t i o n state - 226 -for CO binding i s considered to have s u b s t a n t i a l "product-like" character. reaction coordinate F i g . 4.31 Free energy b a r r i e r s f o r CO binding to five-coordinate encumbered and open heme systems Changes i n dioxygen a f f i n i t y f o r encumbered model complexes r e l a t i v e to open systems, on the other hand, are r e f l e c t e d by a decrease i n a s s o c i a t i o n as well as d i s s o c i a t i o n rates; t h i s i s c l e a r l y exemplified i n the Fe(PocPiv) and Fe(MedPoc) systems r e l a t i v e to the Table 4.10 L i g a t i o n Free Energy Changes f o r CO Binding to Encumbered Hemes Compared with Chelated Mesoheme Compound A G * a s s o c AG° OA G * a s s o c SAG 0 kcal/mol kcal/mol kcal/mol kcal/mol (10) Chelated mesoheme -9.4 -11.2 (18) Anthracene-7/7 -9.0 -10.8 0.4 0.4 (19) Anthracene-6/6 -6.0 - 7.7 3.4 3.5 (20) Adamantane -5.3 - 7.0 4.1 4.2 - 227 -picket-fence hemes (Table 2.5). The binding of 0 2 i s therefore considered to be influenced by factors which a f f e c t both the t r a n s i t i o n state (T.S.) and the ground state of the dioxygen complex, where the T.S. s u f f e r s greater d i s t o r t i o n than the "bound state", f o r the majority of encumbered systems studied. The severely d i s t o r t e d durene-4/4 complex, however, provides a system f o r which thermodynamic consequences d i f f e r e n t from those dis-cussed above apply f o r the binding of C O and 0 2. A comparison of the free energy changes f o r 0 2 binding to the 4/4- with the less d i s t o r t e d 5/5-system gives 5 A G ° - 0.3 kcal/mol, and 6 A G ° - 0 for the R- and T-state complexes, respectively, where 6 A G ° - -RTln[K° 2(4/4)/K° 2(5/5)] (from Table 4.5). An analogous comparison f o r C O r e s u l t s i n values of 5AG° - 1.7 (R-state) and 5 A G ° - 1.5 (T-state) (Table 4.11). The binding of C O to the 4/4-heme therefore s u f f e r s a les s favourable free energy Table 4.11 L i g a t i o n Free Energy Changes on CO Binding to the Durene-4/4 and -5/5 Hemes A G ° ^ G t s s o c A G d i s s o c (kcal/mol) (kcal/mol) (kcal/mol) Fe(durene-5/5)(Dclm) Fe(durene-4/4)(Dclm) Fe(durene-5/5)(l,2-Me 2Im) Fe(durene-4/4)(l,2-Me 2Im) 5AG i n parentheses -•10.6 -9.4 1.2 -8.9 -8.7 0.21 (1.7) (0.7) (-1.0) -8.4 -8.1 0.38 -6.9 -7.8 -0.84 (1.5) (0.3) (-1.2) • ( A G(durene-4/4) - A G(durene-5/5)) R-State T-State 228 change corresponding to ca. 1.4 kcal/mol, r e l a t i v e to 0 2 , on comparison with the less d i s t o r t e d 5/5-complex, for both R- and T-state systems. Examination of the r e l a t i v e values of S A G a s s o c and 8AG^^SSOC with 6 A G° o v e r a;Q for CO binding reveals that the major cont r i b u t i o n to t h i s less favourable free energy difference arises from increased de- s t a b i l i z a t i o n i n the bound CO complex (5AG(j^_SS0C) by 60% and 80% i n the R- and T-state systems, re s p e c t i v e l y (Table 4.11). From these comparisons with the 5/5-systems i t i s i n f e r r e d that, as a r e s u l t of severe heme d i s t o r t i o n i n the 4/4-system, the bound CO complex suffers greater d e s t a b i l i z a t i o n than the Fe-0 2 complex, presumably because of the d i f f e r e n t nature of the Fe-CO and Fe-0 2 bonds. In the case of dioxygen binding, heme d i s t o r t i o n i n the 4/4-system might be expected to impose greater r i d i g i t y on the Fe(durene-4/4)-(B)(0 2) complex, and thereby give r i s e to a larger o v e r a l l loss of t r a n s l a t i o n a l entropy between reacting species on 0 2 binding; t h i s would account for the more negative entropic contribution <As° ) observed r e l a t i v e to chelated protoheme (Table 4.12). For CO binding, however, the bound state s u f f e r s substantial d e s t a b i l i z a t i o n i n the 4/4-system r e l a t i v e to a less d i s t o r t e d heme. In t h i s case, the smaller entropy change (As 0 ) observed for both R- and T-state systems r e l a t i v e to 0 2 may well r e f l e c t a smaller o v e r a l l loss of t r a n s l a t i o n a l entropy as a r e s u l t of the d e s t a b i l i z i n g e f f e c t . Despite the even greater d e s t a b i l i z a t i o n i n the T-state system, compared with the R-state system, the reduced f l e x i b i l i t y inherent i n the T-state, with r e s t r i c t e d movement of the Fe atom etc., may demand a greater loss of t r a n s l a t i o n a l entropy (more negative A s ° ) for bond formation to occur on CO binding to t h i s system, r e l a t i v e to that for 229 Table 4.12 Thermodynamic Constants for CO and 0 2 Binding^ A 0 A ° A ° A 0 A H C O A S C O A H 0 2 A S 0 2 (kcal/mol) (eu)^ (kcal/mol) (eu)^ Hb -17.4 -13.6 to -27.7 to -15.5 -31.7 Mb -15.3 to -38.1 to -21.0 -56.1 (11) Chelated Protoheme0- -17.5 -34 -14.0 -35 (30) Chelated picket-fence -16.3 -40 (14b) Fe(TpivPP)(l,2-Me 2Im) -14.3 -42 (27) Fe(PocPiv)(l,2-Me 2Im) -13.9 -28 (9b) Fe(TPP)(l,2-Me 2Im) -12.8 -26.1 (15b) Fe(C 2-cap)(Melm) -10.5 -27.9 (15b) Fe(C 2-cap)(l,2-Me 2Im) - 9.7 -35.9 (54b) Fe(durene-4/4)(Dclm) - 9 -12 -14 -46 (54b) Fe(durene-4/4)(l,2-Me 2Im) -14 -32 -15 -52 — Solvent system i s toluene for a l l model systems with the exception of chelated protoheme. — Standard state 1 atm. — Aqueous suspension, pH 7. Error estimates for the durene-4/4 systems are given i n Appendix II I , K. the R-state 4/4-complex. For the TPP-derived picket-fence and pocket-systems 6 4, a s i g n i f i c a n t l y lower A s ° value for 0 2 binding r e l a t i v e to that for CO could be a r e f l e c t i o n of s t a b i l i z a t i o n of the bound 0 2 complex, i n this case, v i a dip o l a r i n t e r a c t i o n within the d i s t a l environment of these hemes; i n s u f f i c i e n t data preclude any further comparison. Notably, CO and 0 2 binding constants are very s i m i l a r for the pyrrole-substituted 2 3 0 f l a t open heme, chelated protoheme, i n aqueous suspension at pH 7 . 4.5.8 Evaluation of CO versus 0 2 Binding i n Model Systems (a) Pyrrole - substituted derivatives A l l pyrrole-substituted encumbered model hemes display a subs t a n t i a l d i s t a l s t e r i c e f f e c t toward the as s o c i a t i o n of diatomics such as CO and 0 2. Reduction i n a f f i n i t y f o r these gaseous ligands reaches a maximum and i s then enhanced to a le s s e r extent as s t e r i c hindrance i s increased further; t h i s i s i l l u s t r a t e d by the 'progressive reduction f a c t o r ' within a given s e r i e s (Table 4.13). D i f f e r e n t i a t i o n betwen CO and 0 2 binding (M value) may be determined by s t e r i c d i s c r i m i n a t i o n against CO and/or e l e c t r o n i c s t a b i l i z a t i o n of bound 0 2. With k i n e t i c constants for l i g a t i o n at hand, these factors may be separated into categories of asso c i a t i o n and d i s s o c i a t i o n rate changes, r e s p e c t i v e l y (Table 4.14). The s t e r i c discriminatory factor, a r i s i n g from reduction i n asso c i a t i o n rates r e l a t i v e to an open heme, i s seen to be small i n each case, except f o r the severely hindered FeSP-14 and FeSP-13 systems. S t a b i l i z a t i o n of the bound dioxygen, r e f l e c t e d by decreased 0 2 d i s s o c i a t i o n rates, however, i s co n s i s t e n t l y the largest contributory factor toward C0/0 2 d i f f e r e n t i a t i o n . A f t e r consideration of s o l v a t i o n and proximal e f f e c t s , which can undoubtedly a l t e r ligand d i s s o c i a t i o n rates, these planar p y r r o l e - s u b s t i t u t e d hemes (18-25) * With the exception of the planar adamantane heme for which the structure i s known, there i s no s t r u c t u r a l information on other pyrrole substituted systems to substantiate t h i s discussion further, however. Table 4.13 D i s t a l S t e r i c Effects Toward CO and 0 2 for Pyrrole-Substituted Systems Compound , C 0 (M* 1 s" 1) Reduction Progressive factor R.F. k 2 (M' 1 s' 1) Reduction factor Progressive R.F. (11) Chelated mesoheme 1.1 x 10y (18) Fe(anthracene-7/7)(Dclm) 6 x (19) Fe(anthracene-6/6)(Dclm) 3 x (20) Fe(adamantane)(Dclm) 9 x (22) FeCu-5(DcIm) 9 x (21) FeCu-4(DcIm) 2 x 10 b 10 4 10 3 10 4 10 4 (25) FeSP-15(THPIm) (24) FeSP-14(THPIm) (23) FeSP-13(MeIm) (56b) Fe(durene-7/7)(DcIm) 9.5 x (55b) Fe(durene-5/5)(DcIm) 1.1 x 10' (54b) Fe(durene-4/4)(Dclm) 2.7 x 10( 9 x 10 a 8 x 10 3 6 x 10 2 10-1 9 X 10? 1 1.8 1.8 6 X 10? 1.5 1.5 367 200 1 X 10 5 900 600 1200 3 1, .5 X 10 5 600 -122 122 1. .8 X 10 6 50 50 550 4 5. .2 X 10 5 173 3 122 122 1. .7 X 10 6 53 53 1400 11 3 X 10 5 300 6 18000 13 (2. .4 X 10 4) (3750) (13) 10 4 Unpublished data i n parentheses from Chang's systems 7 4, p r i v a t e l y communicated by T.G. Traylor. 2 3 2 Table A.14 0 2 Versus CO Binding f or Pyrrole-Substituted Model Hemes D i s t a l S t e r i c Discrimination against CO ^ El e c t r o n i c S t a b i l i z a t i o n of Oo fe (18) Fe(anthracene-7/7)(Dclm) 2 4 (19) Fe(anthracene-6/6)(Dclm) None 5 (20) Fe(adamantane)(Dclm) 3 -6 (26) Fe(pyridine-5/5)(Dclm) 3 60-70 (22) FeCu-5(DcIm) 3 45-50 (21) FeCu-4(DcIm) 4 25-30 (25) FeSP-15(DcIm) 3 16-19 (24) FeSP-14(DcIm) 6 35-40 (23) FeSP-13(MeIm) 6 35-40 ^ Determined from k ^/kP^, r e l a t i v e to chelated meso- or protoheme — Determined from k z r e l a t i v e to chelated meso- or protoheme provide a v a l i d series f o r comparison, whereby reduction i n k *• r e l a -t i v e to an open heme occurs with n e g l i g i b l e a l t e r a t i o n i n CO d i s s o c i a t i o n rate. The fa c t that change i n d i s s o c i a t i o n rates i s found almost e x c l u s i v e l y for dioxygen among these heme systems strongly indicates s t a b i l i z a t i o n of the bound dioxygen v i a dipolar i n t e r a c t i o n s , which are known to a f f e c t CO binding to a l e s s e r extent (section 2.7). One exception i s found i n the pyridine-5/5 system (26), where a 5-10-fold increase i n o f f - r a t e i s seen for CO; since 0 2 d i s s o c i a t i o n may 2 3 3 also be subject to s t e r i c d e s t a b i l i z a t i o n s i m i l a r to that found for CO - ° 9 i n t h i s system, the e f f e c t i v e decrease i n k , a r i s i n g from polar i n t e r a c t i o n s within the d i s t a l cavity, may i n fac t be even greater than 60-70-fold, r e l a t i v e to an open heme system. Therefore for the most part, studies with these pyrrole - substituted hemes strongly suggest that CO/O2 d i f f e r e n t i a t i o n i s determined by increased binding of 0 2 that r e s u l t s from dipolar e f f e c t s within the d i s t a l environment which s t a b i l i z e the dioxygen complex, thus lowering the rate of 0 2 d i s s o c i a t i o n from the heme. Although amide functions which increase,the p o l a r i t y of the d i s t a l pocket are present i n a l l the model hemes (18-25) studied, diverse k z values are evidenced among the d i f f e r e n t systems. These differences i n " ° 9 k ^ may well r e s u l t from the various conformational requirements cha-r a c t e r i s t i c of each 'cap', which can influence the d i r e c t i o n i n which a polar amide function points within the d i s t a l c a v i t y . Variations i n dioxygen d i s s o c i a t i o n rates may then be accounted f o r by changes i n proximity between an amide function and the bound dioxygen; within these systems incorporating two amide linkages (18-25), the extent of dipolar i n t e r a c t i o n s are found to a l t e r k from 5 to 50-fold. Such d i s t a l s i t e p o l a r i t y e f f e c t s are further substantiated i n the pyridine-5/5 cyclophane, 26, which incorporates a polar pyridine moiety i n addition to amide functions; dipolar interactions i n the d i s t a l environment of th i s system are enhanced to an even greater e x t e n t 7 5 as evidenced by the larger, 60-70-fold, decrease i n k z , and extremely low M value r e l a t i v e to other model systems (Table 4.8). - 234 -(b) TPP-Derived hemes The high association rates for both CO and 0 2 exhibited by chelated picket-fence (30) r e l a t i v e to chelated TPP (53) was at t r i b u t e d to- reduced s o l v a t i o n of the 'protected' d i s t a l c a v i t y i n the f i v e -coordinate state of the former complex 6 4 (Table 2.15, section 2.8). Inconsistent with t h i s suggestion, however, i s the CO a s s o c i a t i o n rate of the Fe(C 2-cap)(Melm) complex, 4.1 x 10 6 M"1 s" 1, which i s i d e n t i c a l to that f o r chelated TPP, 4.4 x 10 6 M"1 s " 1 i n toluene at 2 0 ° C 7 8 a . This capped heme was considered to have a protected d i s t a l cavity, r e s u l t i n g i n r e s t r i c t e d solvent o r i e n t a t i o n as Indicated by a very s l i g h t change i n 0 2 a f f i n i t y with a large change i n solvent p o l a r i t y 7 8 a (section 2.7.4). Furthermore, the "hanging-base" hemes, 45, 46, and 49, also display a high CO and 0 2 a s s o c i a t i o n rates r e l a t i v e to chelated TPP (see Table 2.12), although these systems incorporate only a single diagonal strap across the ' d i s t a l ' porphyrin face and therefore should experience solvent s t a b i l i z a t i o n s i m i l a r to that of an 'open' heme. (Proximal e f f e c t s r e s u l t i n g from favourable p o s i t i o n i n g of the Iron and attached coordinating base, may, however, be responsible f o r a higher rate of as s o c i a t i o n to these "hanging-base" systems). Such inconsistencies amongst the various TPP-derived hemes render i n t e r p r e t a t i o n d i f f i c u l t and general comparison les s useful between these systems, at the present time. Within the se r i e s of picket-fence and pocket complexes, those conformational e f f e c t s which are considered to influence dioxygen a s s o c i a t i o n and d i s s o c i a t i o n (see section 2.5.4) might also be expected to a f f e c t other diatomics such as CO to a s i m i l a r extent. The change i n 235 dioxygen d i s s o c i a t i o n rate with r e l a t i v e l y constant CO d i s s o c i a t i o n rate for both R- and T-state systems, suggests instead, that e l e c t r o n i c e f f e c t s are involved (section 2.7). The MedPoc and PocPiv systems c l e a r l y display a d i s t a l s t e r i c e f f e c t , roughly equal i n magnitude for both CO and 0 2. The primary k i n e t i c d i f f e r e n t i a t i o n between these two ligands l i e s i n decreased d i s s o c i a t i o n of 0 2 from the heme (Table 2.5). These progressively larger reductions i n k ' with decrease i n cap size for the two hemes may be taken as an i n d i c a t i o n of the importance of amide-hydrogen proximity i n r e l a t i o n to the bound dioxygen i n these systems also, as for the pyrrole-substituted hemes 6 5. In the PocPiv complex the r i g i d amide groups surrounding the small d i s t a l c a v i t y may be forced to orient i n a manner i n which dipolar i n t e r a c t i o n s with the Fe-0 2 moiety are enhanced r e l a t i v e to that i n the larger capped system, FeMedPoc. The rather high 0 2 d i s s o c i a t i o n rate f o r chelated picket-fence (only 10-fold lower than chelated TPP) i s su r p r i s i n g , considering the presence of four amide functions i n the d i s t a l environment of the former. The r i g i d amide linkages i n t h i s case may not be free to orient i n a manner i n which hydrogen-bonding interactions with Fe-0 2 are maximized. In contrast, a proton-n.m.r. study on the Fe-Amide-Im system", 49, indicated such an i n t e r a c t i o n to e x i s t i n solution; consistent with the above suggestion, dioxygen d i s s o c i a t i o n from t h i s heme with only two amides i s 5-fold lower than that from chelated picket-fence with four amides. Therefore, the large C0/0 2 d i f f e r e n t i a -t i o n revealed i n the pocket complexes Fe(MedPoc) and Fe(PocPiv) can lead also to an a l t e r n a t i v e conclusion from that provided i n section 2.5.4, - 236 -consistent with the concept of e l e c t r o n i c s t a b i l i z a t i o n of the bound dioxygen as opposed to s t e r i c d i s crimination against CO. (c) Durene Hemes The durene heme complexes with hydrophobic d i s t a l environments show d i f f e r e n t i a t i o n toward 0 2 binding to a much l e s s e r extent as CO ^9 r e f l e c t e d by large K u u/K r a t i o s , than model hemes with more polar bind-ing pockets (Table 4.8). The most s t r i k i n g comparison with respect to p o l a r i t y e f f e c t s i s found between the durene-5/5 and pyridine-5/5 hemes. Although the a s s o c i a t i o n rates of CO and 0 2 are dramatically d i f f e r e n t f o r the two systems (Table 2.4 and 4.3), presumably as a r e s u l t of the r i g i d amide linkages i n the pyridine-5/5 system that r e s t r i c t the d i s t a l pocket s i z e and hinder ligand coordination, t h e i r k 2 / k ^ r a t i o s are s i m i l a r : 17 and -10 f o r the pyridine- and durene-hemes, re s p e c t i v e l y (the k 2 value f o r the durene-5/5, taken as -1 x 10 8 M"1 s" 1, i s assumed to p a r a l l e l the k ^ value i n r e l a t i o n to the durene-7/7 heme, since CO and 0 2 a f f i n i t i e s f o r both these durene systems are the same). Further, the CO o f f - r a t e s of 0.24 s " 1 and 0.29 s " 1 f o r the pyridine- and durene-systerns, r e s p e c t i v e l y , are v i r t u a l l y i d e n t i c a l . The remarkable diffe r e n c e between t h e i r M values of 7 x 10 4 and 14 (for the durene and pyridine hemes, respectively, Table 4.8) therefore r e s u l t s from the much slower d i s s o c i a t i o n rate of 0 2 from the pyridine-5/5 system, that has a more polar d i s t a l environment. Although the rates of 0 2 and CO l i g a t i o n f o r the pyridine-5/5 model are considerably reduced compared to those f o r r e v e r s i b l e 0 2 (and CO) binding hemoproteins, the o v e r a l l - 237 -d i f f e r e n t i a t i o n f actor between the binding of these ligands, within the polar d i s t a l pocket of the model, i s comparable to that f o r protein systems that show very low M values (e.g. SW-Mb and Dd-Hb, Table 2.1). On the other hand, the association rates of CO and 0 2 to the durene-5/5 heme are i n the range exhibited by hemoprotein systems; the t o t a l lack of p o l a r i t y e f f e c t s , however, r e s u l t s i n an M value 10-fold higher than that for Gd-Hb, a hemoprotein that shows the poorest d i f f e r e n t i a t i o n toward the binding of 0 2. This substantiates the importance of e l e c t r o n i c e f f e c t s that serve to s t a b i l i z e the Fe-0 2 moiety i n re v e r s i b l e oxygen-carrying hemoproteins. The influence of severe porphyrin s k e l e t a l d i s t o r t i o n on CO and 0 2 a f f i n i t i e s has not been reported for any model heme to date. Studies with five-coordinate durene-4/4 hemes indicate that pronounced heme plane d i s t o r t i o n can discriminate against CO binding, r e l a t i v e to 0 2, predominantly v i a an increased d i s s o c i a t i o n rate of CO from the d i s t o r t e d heme complex; t h i s 'proximal' discriminatory e f f e c t i s p a r a l l e l e d i n both the R- and T-states systems. In contrast to the reduced a f f i n i t y f o r CO l i g a t i o n on the ' d i s t a l ' side of a five-coordinate durene-4/4 complex i s the enhanced coordination of t h i s l i g a n d to the unhindered side of a four-coordinate durene-4/4 heme, r e l a t i v e to the analogous comparison for a planar system: K c o (M"1) Kg (M-1) Durene-4/4 -4 x 10 5 4 x 10 6 £ Planar Heme 5 x 10 4 k 4 x 10 8 °-£ Dclm as base. ^ Fe(TPP). £ Chelated mesoheme. - 238 -This difference, presumably, i s a r e f l e c t i o n of the i r o n p o s i t i o n in r e l a t i o n to the porphyrin plane, and/or the extent of C-/7T overlap allowed between Fe and CO, as a r e s u l t of increased d i s t o r t i o n of the Fe-porphyrin system i n the durene-4/4 complex; ei t h e r , or both of these e f f e c t s may serve to enhance l i g a t i o n of CO to the unhindered side of a four-coordinate heme, and conversely, diminish a f f i n i t y for the ligand to the ' d i s t a l ' side of the five-coordinate system. 4.5.9 Evaluation of CO versus 0 2 Binding i n Hemoproteins Rate and equilibrium constants for the binding of CO and 0 2 to various oxygen carrying hemoproteins are l i s t e d i n Table 2.1. Assuming that d i s t a l side s t e r i c e f f e c t s are manifested predominantly as changes i n a s s o c i a t i o n rate, with l i t t l e change i n d i s s o c i a t i o n rate for a given ligand, the lower CO on-rate to Mb r e l a t i v e to that for Hb has been co n s i d e r e d 5 ^ > 6 8 to arise from d i s t a l s t e r i c i n t e r a c t i o n within the more crowded binding pocket of Mb 2 1. A d e t a i l e d evaluation of d i s t a l e f f e c t s f o r Mb, R-, and T-state Hb, c a r r i e d out by Olson et a l . 6 3 strongly indicates however, that the reduction i n CO a f f i n i t y displayed by Mb r e l a t i v e to R-state Hb derives from s t e r i c hindrance that i s proximal rather than d i s t a l i n o r i g i n . In t h i s study, the nature of the d i s t a l environment i n the three protein systems (Mb, R-, and T-state Hb) r e l a t i v e to chelated protoheme i n aqueous suspension, was probed using various i s o n i t r i l e s , d i f f e r i n g i n t h e i r respective s p a t i a l requirements on coordination to the Fe centre. I t was shown, f i r s t l y , that a l l three p r o t e i n a f f i n i t i e s e x h i b i t i d e n t i c a l dependencies with respect to ligand 239 -bulk, Indicative of a very s i m i l a r arrangement of residues surrounding t h e i r d i s t a l c a v i t i e s (Fig. 4.32). Secondly, the o v e r a l l s t e r i c b a r r i e r to l i g a t i o n i n Mb was i n between that of R- and T-state Hb, suggesting that the reduced a f f i n i t y of Mb toward CO and b u l k i e r ligands r e s u l t s predominantly from a proximal hindrance e f f e c t ; t h i s type of r e s t r a i n t i s considered to influence heavily the T-state of Hb, and give r i s e to a low ligand a f f i n i t y r e l a t i v e to that of R-state Hb, as discussed i n sect i o n 1.3. In Mb, the concept of proximal r e s t r a i n t i s borne out by c r y s t a l structure determinations, which show e c l i p s i n g between the plane of the proximal imidazole and N^-Fe-^ axis i n both deoxyMb and Mb02 complexes 2 1. In addition, the Fe i s displaced from the heme plane toward the proximal imidazole, presumably to r e l i e v e unfavourable non-bonded in t e r a c t i o n s between the imidazole and porphyrin plane on 0 2 binding. 1.0 - i . o ' — 1 — 1 — 1 — 1 — 1 — 1 I — 1 i t c o m e rvp n-b n-a rvh m e t-p t-b Fig. 4.32 6 A G ° dependencies on ligand bulk for Mb, R- and T-state Hb r e l a t i v e to chelated protoheme i n aqueous suspension at pH 7.0 Apart from SW-, AL-Mb and Ascaris Hb, the remaining hemoprotein systems i n Table 2.1 show higher ligand a s s o c i a t i o n rates r e l a t i v e to those for R-state Hb. This has been a t t r i b u t e d to the more 'open' bind-- 240 -ing pockets for some of these protein systems (e.g. Hb Zurich (/3)) 2 , 4^, where approach to the Fe centre i s more f a c i l e as a r e s u l t of less s t e r i c obstruction provided by d i s t a l residues close to the binding s i t e . Among such hemoproteins, Dd-Hb provides an extreme example 2 8. The ass o c i a t i o n rates of both CO and 0 2 to Dd-Hb approach those of the t h e o r e t i c a l d i f f u s i o n - c o n t r o l l e d rates f o r each l i g a n d 2 8 (with a l i m i t i n g r a t i o of k 2 / k ^ < - ) -2-3 expected on the s t a t i s t i c a l grounds that CO can coordinate only from one end, v i a the oxygen atom). The CO on-rate i n f a c t corresponds c l o s e l y to that f o r a four-coordinate model heme, i n which the Fe i s i n , or closer to, the porphyrin plane compared to the s i t u a t i o n i n the five-coordinate case (Table 4.15). As the Fe becomes displaced further away from the heme plane towards the proximal imidazole, as i n a five-coordinate system, the as s o c i a t i o n rate f o r CO diminishes ca. 10-fold r e l a t i v e to that f o r 0 2. Analogous to the studies c a r r i e d out on E r y ^ 7 (section 2.6.6), evidence concerning the Table 4.15 Comparison of CO and 0 2 Association Rates to Four- and Five-Coordinate Hemes 2 8'' 5 Compound k 2 (M"1 s' 1) kC0 (M'1 s" 1) Dd-Hb D i f f u s i o n - c o n t r o l l e d Four-coordinate model Five-coordinate model 3 x 10 8 7.5 x 10 2.6 x 10' 8 1 x 10 8 4 x 10 8 3 x 10 8 3.6 x 10 6 2 4 1 nature of the heme environment i n Dd-Hb, i s provided by the CO on-rate o o -pH p r o f i l e ^ , which indicates that the proximal imidazole remains coordinated to the Fe within the range of pH -4-9; therefore at neutral 9 D pH l i g a t i o n i s indeed to a five-coordinate Fe system . Further, proton nmr studies^ on the met-cyano form of Dd-Hb detected low l a b i l i t y of the proximal N^-H, compared to that for other hemoproteins such as Mb or LgHb, i n d i c a t i n g a less f l e x i b l e proximal environment i n Dd-Hb 2 8 - the Fe atom therefore being held f i r m l y i n place. The high a s s o c i a t i o n rates of CO and 0 2 to the deoxy form of Dd-Hb may therefore be r a t i o n a l i z e d r e a d i l y on consideration of the p o s i t i o n of the Fe, which i s presumably closer to the porphyrin plane i n t h i s p r o t e i n compared to that i n a five-coordinate i s o l a t e d subunit of Hb. A s i m i l a r explanation, with respect to Fe displacement from the porphyrin plane, was proposed to account for the high rate of CO and 0 2 a s s o c i a t i o n to deoxyEry^ 7. C r y s t a l structure d a t a 2 6 on t h i s p r o t e i n system show an ° ° ' 91 Fe-heme plane distance of -0.2 A compared to -0.4 A i n deoxyMb^ , consistent with the above suggestion. F i n a l l y , the higher CO asso c i a t i o n rate to HbZurich()3) r e l a t i v e to that for normal Hb has been suggested 4 6 to r e s u l t from quaternary s t r u c t u r a l differences, rather than from d i s t a l s t e r i c e f f e c t s alone. Proton nmr and k i n e t i c studies have indicated that T-state Hb Zurich behaves as though i t s (3 chains were i n the R - s t a t e 4 6 ' 1 5 4 . Also, differences i n the d i f f u s i o n of CO, r e l a t i v e to 0 2 5 2 a , through the protein matrix of the modified tetramer, complicates d i r e c t comparison with normal Hb, with respect to l i g a t i o n within the heme pocket. A l l the above considerations r e f l e c t the dominance of proximal e f f e c t s , which can modify the r e l a t i v e CO and 0 2 a f f i n i t i e s for various 242 hemoprotein systems. S t a t i c c r y s t a l s t r u c t u r a l data also indicate the existence of proximal d i f f e r e n t i a t i o n between CO and 0 2. In the dioxygen-bound systems, Ery0 2 Mb02 2 1 and the a-subunit of Hb 1 8, the Fe i s displaced toward the proximal imidazole, while within the CO bound analogs of EryCO 2 6 and HbCO 4 4, the Fe i s i n the porphyrin plane. In conclusion, model studies show that extremely hindered systems, such as FeSP-13 and FeSP-14, can discriminate against CO r e l a t i v e to 0 2 v i a d i s t a l s t e r i c e f f e c t s . The rates of ligand a s s o c i a t i o n to these models, however, are decreased s i g n i f i c a n t l y compared to those of hemoprotein systems. Model hemes that exhibit higher rates of ligand association, comparable to those of protein systems, however, do not show d i s t a l s t e r i c d i s c r i m i n a t i o n against CO, but instead, may d i f f e r e n t i a t e between CO and 0 2 a f f i n i t i e s through proximal s t e r i c e f f e c t s ; such systems are exemplified within the durene heme se r i e s . The most e f f e c t i v e d i f f e r e n t i a t i o n factor by f a r , that leads to the v a r i e d hemoprotein a f f i n i t i e s f o r CO and 0 2, however, i s the d i s s o c i a t i o n rate for dioxygen; while the change i n the o f f - r a t e f o r CO i s of the order of -90, k z values vary over a factor of 7 x 10 J within the hemoproteins. This i s e n t i r e l y consistent with conclusions drawn from model system studies. 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S c i . , U.S.A., 1975, 72, 1166. 144. D. Brault and M. Rougee, Biochemistry, 1974, 13, 4598. 145. B.B. Wayland, L.F. Mehne and J. Swartz, J . Am. Chem. S o c , 1978, 100, 2379. 146. (a) M. Gouterman i n "The Porphyrins", Vol. I l l , D. Dolphin, Ed., Academic Press, 1978, p. 1. (b) F. Adar i n "The Porphyrins", Vol. I l l , D. Dolphin, Ed., Academic Press, 1978, p. 167. 147. D. Lavalette, C. Tetreau and M. Momenteau, J . Am. Chem. S o c , 1979, 101, 5395. 148. J.B. Wittenberg, R.W. Noble, B.A. Wittenberg, E. Antonini, M. Brunori and J . Wyman, J . B i o l . Chem., 1967, 242, 626. 149. B.A. Wittenberg, E. Antonini, M. Brunori, R.W. Noble, J.B. Wittenberg and J. Wyman, Biochemistry, 1967, 6, 1970. 150. (a) M.W. Makinen, R.A. Houtchens and W.S. Caughey, Proc. Natl. Acad. S c i . U.S.A., 1979, 76, 6042. (b) W.J. Wallace, J.A. Volpe, J.C. Maxwell, W.S. Caughey and S. Charache, Biochem. Biophys. Res. Commun. 1976, 68, 1379. 151. R.D. Jones, J.R. Budge, P.E. E l l i s J r . , J.E. Linard, D. Sommerville and F. Basolo, J . Organomet. Chem., 1979, 181, 151. - 253 -152. G.B. Jameson and J.A. Ibers, Comments Inorg. Chem., 1983, 2, 97. 153. D. Lavalette and M. Momenteau, J.C.S. Perkin I I , 1982, 385. 154. V.S. Sharma, M.R. Schmidt and H.M. Ranney, J . B i o l . Chem., 1976, 251, 4267. - 254 -A P P E N D I C E S 255 -APPENDIX I Synthesis and Characterization of the Durene-Capped Porphyrins and Hemin Chloride Derivatives 1.1 General Methods Melting point determinations were obtained using a 6548-J17 Microscope equipped with a Thomas Model 40 Micro Hot Stage. Elemental analysis was performed by Mr. P. Borda of the Micro-an a l y t i c a l Laboratory, U.B.C. The *H-nmr spectra of a l l porphyrin intermediates were generally recorded at 100 MHz with a Varlan XL-100 Fourier-transform spectrometer. The spectra of the durene porphyrina were obtained at 400 MHz using a Bruker WH-400 spectrometer, i n a l l cases the chemical s h i f t s were recorded In the $(ppm) scale with tetramethylsllane (TMS, 5- 0) as an Internal standard. The l^C-runr Bpectra were generally run at 400 MHz on the Bruker WH-400 spectrometer using TMS ( 6 - 0) as an external standard. The chemical s h i f t s were obtained in the S (ppm) scale. Mass spectra were recorded on a Varlan MAT CH 4-B spectrometer or a Kratos/AEI MS-902 spectrometer. High resolution measurements were obtained on a Kratos/AEI MS-50 spectrometer. Electronic Absorption spectra In the uv and v i s i b l e region were recorded using a Cary 17 spectrometer. Thin layer chromatography ( t i c ) was carried out using precoated s i l i c a gel plates (Analtech-Unlplate, 250fl ) and the compounds were detected by uv l i g h t (254 nm). The mono-pyrroles 99 and 109 were synthesized following methods described i n d e t a i l by Wijesekera 1 0 9. Bis(chlororaethyl)durene (82) and a l l other reagents used were obtained commercially. Unless otherwise stated a l l solvents used were reagent grade. 1.2 Nomenclature of Intermediates The nomenclature used is based on the I U P A C numbering syst (shown below): The Intermediates have been labelled consistently as terminal bls-substltuted-2,3,5,6-tetramethylbenzene. For the dipyrromethanes tl methane bridge Is s i g n i f i e d by the label *(pyrrol-2-yl)methylpyrrole'. 1.3 Synthesis of Durene Diacid Chain Derivatives (a) l,4-Bls(2,2-dlearbox7Sthyl)-2.3,J .6-tetramethylbentene 8 4 Metallic sodium (25.70 g, 1.12 mol) was dissolved In anhydrous EtOH (1200 nL), under nitrogen. Dlethylnalonate (320.8 g, 2.0 mol) was s t i r r e d i n , followed by l,4-bls(chloromethyl)-2,3,5,6-tetraraethyl-benzene 82 (115.6 g, 0.5 mol), and the mixture refluxed for 0.5 h. A t i c analyals of the white suspension Indicated complete reaction of the st a r t i n g material to a single faster moving spot. Ethanol (800 mL) was then d i s t i l l e d off, and a solution of potassium hydroxide (400 g, 3.04 mol) i n water (800 mL) added. The remainder of the EtOH was d i s t i l l e d o f f and the mixture refluxed at 100°C for a further 1 h. The hot aqueous solution was next treated dropwlse with concentrated hydrochloric acid (500 mL) . The white s o l i d that precipitated when the solution became acidic was collected by f i l t r a t i o n and washed with water to give 174 g (95* yield) of 84. HJ. - 278.0-280.0°C. hml- calcd. for C 1 8H 2 20 8: C, 59.00; H, 6.05; Found: C. 58.95; H, 5.92. ^ • n n i r (5, DHS0-d6): 2.13 (a. 12H, benzene-CH 3), 3.13-3.22 ( m , 4 H , chain 1,1'-CH2), 3.22-3.33 (m, 2H, Cb(C0 2H) 2). Mass spectrum (m/e, r e l a t i v e Intensity): 278 (M-2C0 2 +. 72), 219 (100). 205 (54). (b) 1.4-Bls(2-carboxyeth7l)-2,3 ,5,6-tetramethylbenrene 85 l,4-Bls(2-carboxyethyl)-2,3,5,6-tetramethylbenzene 84 (172 g, 0.47 mol) was added slowly, i n small portions to a refluxing solution of DMF (600 mL). During the addition, vigorous evolution of carbon dioxide was observed. The resulting white suspension was refluxed for a further 0.5 h to ensure complete decarboxylation, and then poured slowly into an aqueous solution of 6 M hydrochloric acid (800 mL). The white diacid product 8 5 was collected by f l l t r s t l o n and washed with water, y i e l d 130 g (>99* y i e l d ) . M.p. - 284.0-286.0°C. Anal, calcd. for C 1 6 H 2 2 0 4 : C, 69.04; H, 7.97; Found: C, 68.48; H, 1H-ronr (5 , DMSO-d6): 2.13 («. 12H, benzene CH 3), 2.06-2.39 (ra. 4H, side chain 2,2'-CH2), 2.72-3.02 (a. 4H, chain l . l ' - C H j ) . Mass spectrum (m/e, relative Intensity): 278 (H 4, 80). (c) l,4-Bls(2-ethoxycarbonylethyl)-2,3,5.6-tetrainethylbenzene 86 A solution of 1,4-bls(2-ethoxycarbonylethyl)-2,3,5,6-tetramethyl-benzene 83 (128 g, 0.46 mol) In EtOH (600 mL) and concentrated sulphuric acid (25 mL) was refluxed for 3 h. The solution was cooled to y i e l d white, needle crystals, which were collected and washed with petroleum ether to give 123 g of product 86 (y i e l d 80%). ILfi. - 118.5-120.0°C. Anal, calcd. for C 2 0H 3 00 4: C, 71.82; H. 9.04; Found: C, 72.10; H, 9.11. 1H-ronr (6, CDCI3): 1.25 ( t . 6H, J - 7 Hz). -0CH2CH3), 2.18 (s, 12H, benzene CH 3), 2.28-2.50 (m, 4H, chain 2,2'-CH2). 2.90-3.12 (m. 4H, chain l . l ' - C H j ) . 4.14 (q. 4H, J - 7 Hz, -OCH2CH3). 1 3C-nmr (6, CDC13): 173.10 (C-0). 135.25 (durene 1-C and 4-C), 132.39 (durene 2-C. 3-C. 5-C, and 6-C), 60.41 (•0-CH2CH3). 34.21 (side chain 2-C), 26.15 (side chain 1-C), 16.30 (durene CH 3), 14.28 (0-CH 2£H 3). Mass spectrum (n/e, r e l a t i v e Intensity): 334 (M*. 86), 289 (16). 247 (100), 233 (38). (d) 1.4-Bis(3-hydroxypropyl)-2,3,5,6-tetramethylbenr.ene 1 <' 9 87 l,4-Bls(2-ethoxycarbonylethyl)-2,3,5,6-tetramethylbenzene 86 (122 g, 0.36 mol) was dissolved In dry THF (800 mL). under nitrogen. To this magnetically s t i r r e d solution, sodium borohydrlde (60.5 g, 1.6 mol) was added, followed by the rapid dropwise addition of boron t r l f l u o r l d e etherate (269 mL, 2.1 mol) from a pressure equalizing dropping funnel mounted with a calcium chloride drying tube. The temperature of the reaction solution was maintained below 50°C by the rate of the addition. The r e s u l t i n g thick suspension containing white boric acid by-product was s t i r r e d for a further 2 h. A t i c analysis of tha reaction mixture (in CH2C12> showed a single spot, with a lower Rf value than that of the st a r t i n g material. The excess diborane was destroyed by the dropvlse addition of acetic acid (150 mL), followed by water (500 mL) to dissolve the s o l i d boric acid by-product. The clear solution was then evaporated under reduced pressure to remove the THF, causing the d i o l 87 to separate out from the aqueous phase as a white s o l i d . The crude product was purif i e d by dissolving In a hot solution of EtOH (600 mL) and repreclpltatlng with water to give 81 g of pure d i o l 87, overall y i e l d 901. tLj;. - 158.0-159.5°C. Anal. Calcd. for C 1 6H 2 60 2: C, 76.75; H, 10.47; Found: C, 77.02; H. 10.64. 1H-ninr (5, CDC1 3): 1.50 (s, 2H, -OH), 1.58-1.94 (m, 4H, chain 2,2'-CH2), 2.26 (s, 12H, benzene CH3), 2.66-2.90 (m, 4H. chain 1,1'-CH2), 3.78 (t, 4H, -CH20H). 1 3C-nmr (5, CDCI3): 136.16 (durene 1-Cand4-C), 132.23 (durene 2-C, 3-C, 5-C, 6-C), 63.14 (CH20H), 32.82 (chain 1-C), 26.96 (chain 2-C), 16.38 (durene CH 3). Mass spectrum (o/e, re l a t i v e intensity): 250 (K 4, 87), 205 (94), 191 (47), 147 (100). (e) 1,4-Bis(3-bromopropyl)-2,3,5,6-tetraraethylbensene 88 l,4-Bis(3-hydroxypropyl)-2,3,5,6-tetramethylbenzene 87 (80 g, 0.32 mol) and 461 aqueous hydrobromic acid (240 ml) were heated at reflux under nitrogen. After 0.5 h, a t i c analysis Indicated complete con-version of the starting material into a single, faster moving spot. The emulsion was cooled, CH 2C1 2 (300 ml) added and the acid extracted out with water (200 ml) and saturated sodium bicarbonate (2 x 100 ml). The CH 2C1 2 solution was dried with anhydrous sodium sulphate and evaporated under reduced pressure after adding MeOH. The product 88 c r y s t a l l i z e d out as white needles, y i e l d 102.3 g (65%). HJI. - 113.0-114.0°C. AjjaJ,. Calcd. for C 1 6 H 2 4 B r 2 : C, 51.09; H, 6.43; Br, 42.48; Found: C, 50.95; H, 6.49; Br, 42.28. 1H-nmr (5, CDCI3): 1.64-2.10 (m, 4H, chain 2,2'-CH2), 2.17 (s, 12H, benzene CH3), 2.68-2.92 (m, 4H, chain 1,1 ,-CH 2). 3.45 ( t . 4H, J - 6.5 Hz, -CH 2Br). 13C-nmr (5. CDCI3): 135.39 (durene 1-C and 4-C), 132.33 (durene 2-C. 3-C, 5-C, «-C), 33.92 (£H 28r). 32.«» (chain 1-CH2). 29.37 (chain 2-£H 2). 16.44 (durene-C^) . Muss spectrum (m/e, relative Intensity): 374-376-378 (M*), 267-269 (30-30). 173 (15), 161 (26), 145 (27). (f) l,*-Bl«(3-cyanopropyl)-2.3,5,6-tetramethylbent«ne 89 l,4-Bls(3-bromopropyl)-2,3,5,6-tetramethylbenzene 88 (20.0 g, 0.05 mol) and potassium cyanide (13.0 g, 0.20 moL) were refluxed l n EtOH (250 mL), under nitrogen. After 4 h a t i c analysis showed only one spot, lower l n Rf rela t i v e to the starting material. The reaction mixture was cooled, and water (300 mL) added to cause complete c r y s t a l l i z a t i o n of the dlcyanlde 89. The product was then isolated by f i l t r a t i o n and thoroughly washed with water to ensure removal of any excess potassium cyanide, y i e l d 13.0 g (97%). HjJ. - 139.0-141.5°C. Anal. Calcd. for C 1 8H 2 4N 2: C, 80.55; H , 9.01; N, 10.44; Found: C, 80.46; H. 9,06; N, 10.15. 1 tbuml (6. CDC1 3): 1.57-2.05 (l. 4H, chain 2.2'-CH2), 2.29 (s, I2H. benzene CH3), 2.48 (t , 4H, J - 7 Hz, chsln 1,1'-CH2), 2.72-3.00 (m, 4H. chain -CH2CN). 13C-nmr (8, CDCI3): 135.07 (durene 1-C and 4-C), 132.39 (durene 2-C, 3-C, 5-C, 6-C), 119.57 (£N). 29.60 (chain 1-CH2), 25.37 (cnaln 2-£H 2), 17.33 (chain 3-£H 2), 16.44 (durene £ H 3 ) . Mass spectrum (m/e, rela t i v e Intensity): 268 (M*. 90), 214 (100), 200 (60). (g) l,4-Bls(3-carboxypropyl)-2.3.5,6-tetramethylbenzene oh 90 l,4-Bls(3-cyanopropyl)-2,3,5,6-tetramethylbenzene 89 (12.5 g, 0.047 mol), potassium hydroxide (21 g, 0.38 mol) and ethanol (250 mL) were heated at reflux, under nitrogen, for 5 h. After this time the reaction flask was flushed with nitrogen, using a Clalsen head adaptor, and the refiuxing solution checked, with moist litmus, to ensure that the evolution of ammonia gas had ceased. Water (200 mL) was then added and the EtOH d i s t i l l e d o f f u n t i l the reflux temperature reached 100°C. The resulting clear solution was refluxed for another 1 h, and then ON O cooled; careful a c i d i f i c a t i o n with concentrated hydrochloric acid yielded the white diacid product 90 which was collected and washed with water, y i e l d 14.1 g (98%). tUB- - 265.0-267.0°C. AMI. Calcd, for C l gH 2 60 6: C, 70.56; H, 8.55; Found: C, 70.8, H. 8.53. ' I LJUBI ( 6. 10% TFA/CDCI3): 1.60-2.10 (n, 4H, chain 2,2'-CH2), 2.32 (a. 12H, benzene CH3), 2.48-2.97 (n, 8H, chain 1,l',3,3'-CH 2). Mass spectrum (m/e, relative Intensity): 306 (M*, 40), 233 (100), 219 (30). (h) l.*-Bls(4.*-dlcarboxyt>utyl)-2.3.5,6-tetremethylbensene 1 0 9 91 l,4-Bls(3-broraopropyl)-2,3,5,6-t»tramethylbenzene 88 (101 g, 0.27 mol) was reacted to give 110 g of 91 ( y i e l d 95%) l n a preparation analogous to that described for 84 from bis(chlororaethyl)durene 82. ttjl. - 193.5-195.0°C. AnaJ,. Calcd. for C 2 2H3 0O g: C, 62.55; H, 7.16; Found: C, 62.32; H, 7.20. 1H-nmr ( 5 , DMS0-d6): 1.14-1.98 (m, 8H, chain 2-CH2, 3-CH2). 2.07 (s, 12H. benzene CH 3), 2.38-2.74 (m, 4H, chain 1-CH2), 3.25 (t. 2H, J - 7 Hz, CH(C0 2H) 2). Mass spectrum (m/e r e l a t i v e Intensity): 334 (M-2C02+, 20), 247 (100), 147 (70). (1) l,4-Bls(4-carboxybutyl)-2,3,5,6-tetramethylbenzene "V ^JyO>i 92 The d i a c i d 92 (86.8 g, 0.26 mol) was obtained l n quantitative y i e l d from 91 (109 g, 0.26 mol) in a manner analogous to that described for 85. M.P. 210-211.0°C. 1H-nmr (5 , DMSO-d6): 1.10-1.82 (m, 8H. chain 2-CH2 «nd 3-CH2), 2.13. 2.06-2.38 (a, m, 16H, durene CH3, chain 4-CH2), 2.42-2.76 (m, 4H, chain 1-CH2). Haas spectrum (m/e relative intensity): 334 (K*, 30), 247 (100), 233 (20), 147 (50). ( J ) 1.4-Bls(4-ethoxycsrbon7lbutyl)-2,3,3,6-tetramethylbenzene C M , 93 The e s t e r l f l c a t i o n of 92 (37.5 g. 0.11 mol) yielded 41.7 g of the bla ethylester 93 (951), following the method described for obtaining 86. ILB. - 55.0-56.0°C. Anal. Calcd. for C 2 4H3 80 4: C, 73.81; H, 9.81; Found: C, 73.89; H, 9.89. 1H-nmr (6 , CDCI3): 1.25 ( t , J - 7 Hz, 6H, -OCH2CH3), 1.35-1.95 (m, 8H, chain 2.2',3,3'-CH2), 2.20 (s, 12H, benzene CH3), 2.25-2.80 (m, 8H, chain 4,4',1.1,-CH2), 4.13 (q, J - 7.5 Hz, -OCH2CH3). 13C-nmr (5, CDCI3): 173.54 (C-0), 136.65 (durene 1-Cand4-C), 131.95 (durene 2-C. 3-C, 5-C, 6-C), 60.14 (-OCH2CH3), 34.24 (side chain 4-C), 30.36 (side chain 3-C), 29.37 (side chain 2-C), 25.49 (side chain 1-C), 16.28 (durene CH3), 14.23 (-0CH2CH3). Mass spectrum (m/e r e l a t i v e Intensity): 3.90 (M*. 40), 275 (50), 129 (100). (k) l , 4-Bls ( 5-hydroxypentyl ) - 2 , 3 , 5 , 6-tatramethylbenzene 1,4-Bis(4-ethoxycarbonylbuty1)- 2.3,5,6 -te tramethylbenzene 93 (41.0 g, 0.1 mol) was reduced via a diborone reduction, to give 32.1 g of 94 ( y i e l d 99%), analogous to the method described for obtaining 87 from 86. [LB.. - 109.5-111.0°C. Anal. Calcd. for C 2 0H 3 / i0 2: C. 78.38; H, 11.18; Found: C, 78.47; H. 11.40. JH-nmr (5, CDCI3): 1.35-1.80 (m, 14H, -OH, ch«ln 2,2'.3.3',4,4'- CHj), 2.27 (s, 12H, benzene CH3). 2.50-2.88 (m, 4H, chain 1,1'-CH2), 3.71 (t, 4H, -CH20H). 1 3C-nmr (5 , CDCI3): 136.71 (durene 1-C end 4-C), 131.90 (durene 2-C, 3-C, 5-C, 6-C), 62.85 (£H 20H), 32.66 (chain 1-C), 30.71 (chain 2-C), 29.73 (chain 3-C), 26.37 (chain 4-C), 16.34 (durene £ H 3 ) . Mass spectrum (m/e relative Intensity): 306 (M+, 80), 233 (100), 161 (20), 147 (30). (1) l,4-Bls(5-bromopentyl)-2 ,3,3,(-tatramathylbenzene Analogous to the preparation described for 68, 95 (38.3 g) was obtained from 94 (31.6 g, 0.1 mol) i n 86% y i e l d . M J I. - 100.0-102.0°C. Anal. Calcd. for C 2 0 H 3 2 B r 2 : C. 55.70; H, 7.46; Br, 36.97; Found: C, 55.82; H, 7.45; Br, 36.76. 1H-nmr (5, CDC13): 1.40-1.65 (m, 8H, chain 2,2',3,3'-CH2), 1.75-2.07 (m, 4H, chain 4,4'-CH2), 2.20 (s, 12H. benzene CH3), 2.50-2.85 (ra, 4H, chain, 1,1'-CH2), 3.43 (t , 4H, -QJ 2Br). 13C-nmr (5, CDCI3): 136.58 (durene 1-C and 4-C), 131.98 (durene 2-C. 3-C, 5-C, 6-C), 33.69 (CH 2Br), 32.71 (chain 1-CH2), 30.60 (chain 2-CH2), 29.07 (chain 3-£H 2), 28.79 (chain 4-£H 2), 16.37 (durene £ H 3 ) . Mass spectrum (m/e, r e l a t i v e i n t e n s i t y ) : 430-432-434 (K+, 55), 295-297 (100). (m) 1.4-Bla(6,6-dlcarboxylhexyl)-2,3,5,(-tetramethylbensene 96 In a preparation analogous to that described for 84, the tetra-acld 96 was obtained l n 96% y i e l d (39.8 g) from the dlbromlde 95 (37.3 g. 0.086 mol). M_j>. - 160.5-162.5°C (decomp.). H-nmr (6. 10% TFA-CDCI3): 1.35 (br s, 12H, chain 2,2 • , 3 . 3' ,4 ,4'-CH2) 1.58-1.93 (m. 4H, chain 5,5'-CH2), 2.12 (a, 12H, benzene CH3) , 2.33-2.73 (•, 4H, chain 1,1'-CH2). 2.97 ( t , 2H, J - 7 Hz, CH(COOH)2). Mass spectrum (m/e, relative Intensity): 390 (M-2C02+, 45), 275 (100). (n) l,4-Bls(6-carboxylhexyl)-2,3,5,6-tetramethylbenzene °** 97 The tetraacid 96 (39.3 g, 0.082 mol) vaa decarboxylated to give the diac i d 97 (31.6 g) In 991 y i e l d , by the method described in de t a i l for 85. SLE. - 147.0-149.0°C (decomp.). Anal . Calcd. for C 2 4H 3 80 4: C, 73.81; H, 9.81; Found: C, 73.77; H, 9.72. JH-nmr (6. 10» TFA-CDCI3); 1.35 (br s, 16H, chain 2,2',3,3',4,4',5,5'-CH 2), 2.13 (s, 12H, benzene CH 3), 2.20 (bs, 4H, chain 6,6'-CH2), 2.35-2.70 (m. 4H. chain, 1,1'-CH2). Mass spectrum (m/e relative intensity): 390 (M+, 30), 275 (30), 161 (60), 147 (50). 1.4 Synthesis of Durene Bis Pyrrole Derlvstives 1.4.1 The Bls-Pyrrole Dlketones 100 a, n - 4 c, n - 7 The durene d i a c i d chain derivative (90 or 97, 0.087 mol) and thlonyl chloride (40.6 g, 0.35 mol) were placed in a flask equipped with reflux condenser and calcium chloride drying tube. The mixture was heated on a steam bath u n t i l the vigorous evolution of gas subsided (-30 min). The excess thlonyl chloride was removed by evaporation under reduced pressure with CC1 4 (4 x 30 mL). The dark red residue of the bis acid chloride 98 was then taken up in CH 2C1 2 (30 mL) and transferred Into a flask containing 2-ethoxy-3,5-dlmethylpyrrole 99 (30.6 g, 0.175 mol), CH 2C1 2 (400 mL) and nltromethane (320 mL), under nitrogen. Anhydrous atannic chloride (91.2 g, 0.35 mol) was added rapidly, dropwlse, and the reaction mixture allowed to s t i r for 3 h at room temperature. The dark brown solution was then poured Into 0.1 M aqueous hydrochloric acid (600 mL) to quench the stannic chloride catalyst and s t i r r e d for -15 min causing p r e c i p i t a t i o n of the product. Yields of 70% and 66% were obtained for the 4/4-100a, and 7/7-diketone 100c derivatives, respectively. (In the case of the 7/7- derivative. a smaller volume of solvent, CH 2C1 2 (150 mL) and nltromethane (120 mL) could be used in the acylatlon step, because of the greater s o l u b i l i t y of the 7/7-intermediates.) ( i ) l,*-Bls(4-(5-ethoxycarbonyl-2,4-dlmethylpyrrol-3-yl)-4-oxobutyl]-2,3,5,6-tetramethylbenzene, 100* ( L i . - 246.0-248.0°C. Anal, calcd. for C3 6 H 4 gN 20 6: C, 71.50; H. 8.00; N, 4.63; Found: C, 71.26; H 7.91; N, 4.52. X H nmr (5, 10% TFA-CDCI3): 1.44 (t, 6H, J - 7.5 Hz, 0 -CH 2CU 3), 1.60-2.10 (m, 4H, chain 2,2'-CH2), 2.25 (s, 12H, benzene CH3), 2.55 (s, 6H, pyrrole 4-CH 3). 2.62 (s, 6H, pyrrole 2-CH3), 2.63-3.05 (n, 8H, chain 1,1',3,3'-CH2), 4.41 (q, 4H, J - 7 Hz), 0-CH2CH3), 9.65 (br a, 2H, NH). 1 3 C nmr (5, 10% TFA-CDCI3): 204.23 (chain C-O, 3,3'-C), 164.65 (ester C-O), 141.56 (pyrrole 2-C), 135.8 (durene 1,4-C), 132.25 (pyrrole 4-C, durene 2. 3, 5. 6-C), 122.8 (pyrrole 3-C). 118.24 (pyrrole 5-C), 62.07 (-OCH2CH3), 42.12 (chain C0£H 2), 30.00 (chain 1,1'-C), 25.64 (chain 2,2'-C), 16.27 (durene CH 3), 15.17 (pyrrole 2-CH3), 14.16 (-0CH2-CH3), 12.71 (pyrrole 4-CH3). Mass spectrum (m/e, relative intensity): 604 (K +, 20), 209 (100), 194 (95), 148 (95). (b) l,4-Bla[7-(5-ethoxycsrbonyl-2,4-dlmethylpyrrol-3-yl)-7-oxoheptyl]-2,3,5,6-tetramethylbenzene, 100c IUE. - 170.0-172.0°C. Axial, calcd. for C 4 2H 6 0N 20 6: C, 73.22; H, 8.78; N, 4.07; Found: C, 73.33; H, 8.00; N. 3.96. *H nmr (5 , 10% TFA-CDCI3): 1.41-1.59 (m, 15H, 0-CH2CU3, chain 2,2',3,3",4,4'-CH2), 1.64-1.82 (m, 4H, chain 5,5•-CH2), 2.25 (s, 12H, benzene CH3), 2.59 (s, 6H, pyrrole 4-CH3), 2.63 (s, 6H, pyrrole 2-CH3), 2.49-2.58 (m, 4H, chain 1.1'-CH2), 2.86 (t, 4H, chain 6,6'-CH2), 4.42 (q, 4H, J - 7 Hz, 0-CH.2CH3), 9.99 (bs, 2H, NH). • ro 1 3 C nmr (5, 10% TFA-CDCI3): 204.13 (chain C-O, 6,6',-C), 163.92 (ester C-O), 142.46 (pyrrole 2-C), 136.78 (durene 1, 4-C), 132.03 (pyrrole 4-C, durene 2,3,5,6-C). 122.50 (pyrrole 3-C), 118.37 (pyrrole 5-C), 62.28 (-0£H 2CH 3), 42.01 (chain C0CH2), 30.59, 29.81, 29.34 (chain 1,1' .2.2-.l.!' A.f-C), 25.81 (chain 5,5'-C), 16.48 (durene CH3) , 15.82 (pyrrole 2-CH3), 14.22 (-0CH 2£H 3), 13.12 (pyrrole 4-CH3). Mass spectrum (m/e, re l a t i v e Intensity): 688 (K*. 20), 209 (80), 194 (80), 148 (100). 1.4.2 The Bis Pyrrole Ethyl Esters 101 a, n - 4 c , n - 7 The dlketone (100a or c , 55.0 mmol) was suspended In THF (400 mL) and treated with sodium borohydrlde (6 g, 162 mnol) and boron t r l -fluorlde etherate (28 D L, 222 mnol) as described In the preparation of 87. During the addition the colour of the suspension darkened a l l g h t l y as the dlketone went into solution. The solution was s t i r r e d at RT for 1 h, when a t i c analysis indicated complete conversion of the starting material to a spot moving higher i n Rf value. The reaction mixture was then worked up In the usual way (described for 87), and the THF evaporated to precipitate the product 101. After the workup stage, a small amount of dark material remained undissolved. This brown impurity was f i l t e r e d away from the otherwiae clear aolutlon before attempts were made to precipitate the product. The bis pyrrole ethylester could be r e c r y s t a l l l z e d from a mixture of hot THF and HeOH to give 75» and 71% y i e l d of the 4/4-101a and 7/7-101c derivatives, respectively. (a) l,*-Blsl7-(5-«thox7carbonyl-2,*-dlmethylpyrrol-3-yl)butyl]-2,3,5,6-tetramethylbenzena, 101a H.p. - 225.0-227.0°C. Anal. calcd. for C 3 6H 52N 20 4: C. 74.96; H. 9.09; N. 4.86; Found. C, 73.83; H, 9.09; N, 4.62; Calcd. for 03^ 52^ 04 0.5H20: C, 73.56; H, 9.09; N, 4.76. 1H__nj5I (5, 10% TFA-CDCI3): 1.37 (t, 6H, J - 7 Hz, 0-CH2CH.3), 1.41-1.60 (m, 8H, chain 2,2',3,3'-CH2), 2.20 (s, 12H, benzene CH 3), 2.22 (s, 6H. pyrrole 2-CH3), 2.27 (s, 6H. pyrrole 4-CH3, 2.32-2.50 (m, 4H, chain 4,4'-CH2), 2.59-2.77 (m. 4H, chain 1,1'-CH2), 4.32 (q, 4H, J - 7 Hz, O-CU2CH3). 9-38 (br s, 2H, NH). Mass spectrum (m/e, r e l a t i v e intensity): 576 (M+, 40), 530 (50), 180 (95), 134 (100). 1 ro (b) l,4-Bls[7-(5-ethoxyearbonyl-2.4-dlmethylpyrrol-3-yl)heptyl)- tr. 2,3,5,6-tetramethylbenzene 101c n\£. - 127.0-129.0°C. Anal. Calcd. for C 4 2H 6 4N 20 4: C, 76.32; H, 9.76; N, 4.24; Found: C. 75.44; H, 9.84; N. 4.05; Calcd. for C 4 2H 6 4N 20 4 • 0. 5H20: C. 75.29; H, 9.78. N, 4.18. 1H_njsx (5, CDCI3): 1.34 ( t , 6H, J - 7.5 Hz, 0-CH2CH3), 1.26-1.47 (m, 20H, chain 2,2',3,3',4.4',5,5",6,6'-CH2), 2.19 (s, 18H, benzene CH3, pyrrole 2-CH3), 2.27 (s, 6H, pyrrole 4-CH3), 2.28-2.37 (m, 4H, chain 7,7'-CH2), 4.26 (q, 4H, 0-CH2CH3), 8.67 (br a. 2H. NH). Hass spectrum (m/e, re l a t i v e intensity): 660 (M*. 60), 6)4 (50). 180 (100), 134 (90). t 1.4.3 Th* Bl> Pyrrol* Benzyl Esters 102 a. n - 4 e, n - 7 The bis pyrrole ethylester (101a or e, 38 mmol) and r e d i s t i l l e d benzyl alcohol (300 nL or 100 mL, respectively) were heated under nitrogen. At the onset of reflux (205°C), a concentrated solution of sodium In benzyl alcohol was added. In 1 mL portions. During each addition, ethanol vapours were liberated and a aimultaneous drop in temperature was observed; the mixture was allowed to attain reflux each time before further addition of catalyst. After the addition of -3 mL of catalyst, no effervescence could be observed and the hot aolutlon was ca r e f u l l y poured Into MeOH (400 mL) with s u f f i c i e n t acetic acid (15 mL) to quench the catalyst. Water was added alowly to precipitate the product 102, which was f i l t e r e d and washed with 50% MeOH/water. The dlbenzylester could be obtained i n 98% and 90% y i e l d for the 4/4-102a and 7/7-102c derivatives, respectively. (a) 1,4-Bis[4-(5-benryloxycarbonyl)-2,4-dimethylpyrrol-3-yl)butyl]-2 , 3 , 5,6-tetramethylbenzene, 102a rL£. - 220.5-221.5°C. Anal. Calcd. for C 4 6H 5 6N 20 4: C, 78.82; H, 8.05; N. 4.00; Found: C, 78.32; H, 7.90; N, 3.92. lH nmr (6, 10% TFA-CDCI3): 1.47-1.69 (m, 8H, chain 2,2'.3,3'-CH2. 2.27 (s, 12H, durene CH3), 2.29 (s, 6H, pyrrole 2-CH 3), 2.32 (s, 6H, pyrrole 4-CH 3), 2.24-2.49 (ra, 4H, chain 4,4'-CH 2), 2.68-2.78 (m, 4H, chain 1,1'-CH 2), 5.37 (s, 4H, -OCH 2C 6H 5), 7.38-7.47 (m, 10H, C 6H 5). 1 1 3 C nmr (6, 10% TFA-CDC13): 164.32 (C - 0), 136.80 (durene 1-C and CT* 4-C), 135.62 (benzene 1-C), 133.63 (pyrrole 2-C), 132.07 (durene 2-C, -~J 3-C, 5-C, 6-C), 130.50 (pyrrole 4-C), 128.67, 128.43, 128.14 (benzene ' 2-C. 3-C, 4-C, 5-C, 6-C), 123.46 (pyrrole 3-C), 115.32 (pyrrole 5-C), 67.04 (-0-CH2CoH5), 31.23, 30.74, 29.75 (1,1',2.2',3,3'-C), 23.98 (chain, pyrrole termini, 4,4',-C), 16.33 (durene CH3), 11.58, 11.15 (pyrrole 2-CH3, 4-CH3). Mass spectrum (m/e, rela t i v e Intensity): 700 (M*. 40), 592 (30), 457 (55), 108 (50), 91 (100). (b) l ,4-Bls[*-(5-bent7loxycarbonyl)-2,*-dlmeth7lpyTrol-3-yl)heptyl]-2,3,5.6-tetramethylbenzene, 102c H.p. - 124.5-126.0°C. Anal. Calcd. for C 5 2H 6 8N 20 4: C, 79.55; H, 8.73; N, 3.57; Found: C, 79.45; H, 8.21; N, 3.43. 1H_nmi (6. CDC1 3): 1.24-1.53 (m, 20H. chain 2,2' ,3,3'.4,4', 5,5*,-6.6'-CH2), 2.17 (s. 6H. pyrrole 2-CH3), 2.20 (a, 12H. durene CH 3), 2.28 (s, 6H, pyrrole 4-CH3), 2.31-2.40 (m, 4H. chain 7.7'-CH2), 2.50-2.68 (m, 4H, chain 1,1'-CH2), 5.28 (a, 4H, -0CH 2C 6H 5), 7.29-7.45 (m, 10H, C 6H 5), 8.76 (bs, 2H, NH). 1 3 C nmr (5, CDC1 3): 161.30 (C-0), 136.90 (durene 1-C and 4-C). 136.87 (benzene 1-C). 131.87 (durene 2-C, 3-C, 5-C, 6-C), 129.72 (pyrrole 2-C), 128.49, 128.05, 127.95 (benzene 2-C, 3-C. 4-C. 5-C. 6-C), 127.70 (pyrrole 4-C), 122.60 (pyrrole 3-C), 116.52 (pyrrole 5-C), 65.35 (-0£H 2C 6H 5), 30.77, 30.13. 29.83, 29.34 (chain 1.1'.2,2'.3.3',4,4'.. 5,5' .6.6'-C), 24.07 (chain termini, 7,7'-C), 16.25 (durene CH 3), 11.50, 10.69 (pyrrole 2-CH3, 4-CH3). Mass spectrum (m/e re l a t i v e Intensity): 784 (M+, 70), 676 (70), 242 (80), 108 (100), 91 (100). 1.4.4 T h e Bis Formylpyrroles 106 a, n - 4 e, n - 7 (1) Debenzylstlon of 102: The dibenzylester 102 (a or c, 18 mraol) snd 10% palladium on charcoal (0.5 g) were s t i r r e d overnight under hydrogen (1 atmosphere) In f THF (400 mL) containing 5 drops of triethylamine. When the uptake of fo tT> hydrogen ceased, the catalyst was f i l t e r e d and the solution checked by 0 0 1 t i c for any unconverted s t a r t i n g msterlal. The solvent was then evaporated In" vacuo, leaving the bls(carboxy)pyrrole 103 as a white s o l i d . ( i i ) Decarboxylation of 103: The crude bis(carboxy)pyrrole 103 was refluxed ln DMF (200 mL) under nitrogen. A uv absorption spectrum showed a reduction of the band at 288 nm to Just a shoulder, within 2 h. No further change was observed. The solution containing the bis ot-free pyrrole product 104 was cooled ln ice and used d i r e c t l y in the next reaction. ( I l l ) Vlllsmeier forraylatlon of 104: To an lce-co61ed solution of DMF (21 mL) In dry CH^Clj (80 nL), phosphorus oxychloride (18.4 mL) was added dropwise and at l r r e d In. The Vlllsmeler reagent, thus prepared, was added dropwise rapidly to the c h i l l e d DMF aolutlon of the bis -free pyrrole 104 prepared above. Once the addition was complete, the aolutlon was s t i r r e d for a further 0.5 h to ensure completion of the reaction, and then poured onto crushed Ice (200 g). Solid sodium bicarbonate waa added carefully u n t i l the aolutlon became basic, and the CH2CI2 boiled o f f . Water (600 mL) was then added and the mixture heated at 75°C for 1 h. The grey-coloured s o l i d which separated out was f i l t e r e d and washed with water. Overall yields of 56% and 49% of crude 106a and c were obtained, respectively, from tha corresponding dlbenzylesters 102a and 102c. A p u r i f i e d sample of the bis formylpyrrole was prepared by deprotectlng the dlcyanovlnyl derivative 107 with aqueous potassium hydroxide in n-propanol. (a) l ,4-Bls(4-(5-formyl-2,*-dlmethylpyrrol-3-yl)butyl]-2,3,5,6-tetramethylbenzene, 106a " 253.0-254.0°C. Anil. Calcd. for C3 2H 4 4N 20 2: C, 78.65; H, 9.08; N, 5.73; Found: C, 78.36; M, 8.91; N, 5.70. lH nmr (5, CDCI3): 1.20-1.82 (m, 8H, chain 2 .2'. 3, 3'-CH2), 2.20 (s. 12H. benzene CHj), 2.23 (s, 6H, pyrrole 2-CH3), 2.26 (s, 6H, pyrrole 4-CH3), 2.28-2.80 (m, 8H, chain 1,1•,4,4•-CH2). 9.29 (br a, 2H, NH), 9.44 (a. 2H, HCO). Mass spectrum (m/e, r e l a t i v e Intensity): 488 (M*, 50), 460 (40), 445 (20). 136 (100). (b) 1.4-Bis[7-(5-formyl-2.4-dlmethylpyrrol-3-yl)heptyl]-2,3.S,6-tetramathylbenzene, 106c ILB. - 179.0-180.0°C. 1 Anal. Calcd. for C3 8H5 6N 20 2: C. 79.67, H, 9.85; N. 4.89; Found: C, o> 79.12; H, 10.00; N, 4.91. 1 1H_nmr. (5, CDC13): 1.41 (m, 20H, 2,2',3,3',4,4',5,5-,6,6'-CH2), 2.18 (a. 12H, benzene CH 3), 2.26 (s, 12H, pyrrole CH 3), 2.30-2.78 (m, 8H, side chain 1,1',7.T-CH2), 9.47 (s, 2H, HCO), 9.95 (br s, 2H, NH). Mass spectrum (m/e, r e l a t i v e Intensity): 572 (M*, 30), 544 (20), 395 (60), 347 (100). 1.4.5 The Bit Cyanovlnylpyrroles 107 «, n - 4 c, n - 7 The crude bis formylpyrrole 108 (a or c, -10 nmol), malononltrlle (1.7 g, 24 mnol) and cyclohexylamlne (1.5 mL), were refluxed l n toluene (300 mL), for 2 h. The toluene was evaporated in vacuo and MeOH added to precipitate the product. The crude yellow aolld thus obtained was pu r i f i e d by column chromatography using s i l i c a gel ( a c t i v i t y I, 150 g) and CH2CI2 as solvent. The pure lemon-yellow coloured dicyanovinyl derivative 107 eluted out f i r s t while a l l other impurities remained adsorbed on the column. Purification was carried out by dissolving the s o l i d i n hot CH2CI2. v i a Soxhlet extraction, and then pr e c i p i t a t i n g with HeOH. The overall y i e l d obtained from the dibenzyleater stage was 45% for 107« and 40% for 107c. (a) l,4-Bl»(*-I5-(2.2-dlcyanovlnyl)-2.4-dimethylpyrrol-3-yl]butyl)-2,3,3,6-tetraoethylbenzene. 107a M .P . - 280.0-283.0°C (decomp.). AnaJ,. Calcd. for C 3 8H 4 4N 6: C. 78.05; H. 7.58; N, 14.37; Found: C, 77.98; H, 7 . 6 6 ; N, 14.07. *H nmr (5, 10% TFA -CDCI3 ) : 1.39-1.61 (m, 8H , chain 2.2',3.3'-CH2). 2.18 (s, 6H . pyrrole 4 - C H 3 ), 2.20 (s, 12H, benzene CH 3), 2.37 (s, 6H, pyrrole 2- CH 3), 2.41-2.47 (m, 4 H , chain 4,4'-CH2), 2.62-2.68 (ra, 4H, chain 1,1'-CH2). 7.32 (s, 2H. C(H)-C(CN) 2). 9.47 (br s, 2H , NH). 1 3 C nmr (6, 10% TFA - C D C I 3 ) : 146.65 (pyrrole 2-C). 140.91 [C(H)-C(CN) 2). 139.69 (pyrrole 4-C). 136.64 (durene 1-C and 4-C), 132.14 (durene 2-C. 3- C, 5-C, 6-C), 127.25 (pyrrole 3-C), 125.35 (pyrrole 5-C), 116.86 (C N), 116.03 (C N), 56.89 ([C(H)-£(CN) 2]. 30.48 (chain 2,2'3.3'-C), 29.62 (chain durene termini 1,1'-C), 23.88 (chain, pyrrole termini 4,4'-C), 16.19 (durene CH 3), 12.32 (pyrrole 2-CH3), 9.56 (pyrrole 4 - C H 3 ) . Mass spectrum (m/e, r e l a t i v e intensity): 584 (H+, 50), 569 (50), 184 (100). (b) l,4-Blsl7-[5-(2.2-dlcyanovlnyl)-2.4-dlmathylpyTTol-3-yl)heptyl)-2,3.5,6-tetramethylbenzene, 107c tLp.. - 226.0-228.0°C. Anal- Calcd. for C 4 4H 5 6N 6: C, 79.00; H, 8.44; N. 12.56; Found: C, 78.83; H, 8.44; N, 12.51. 1H_rjmi (5. 10% TFA-CDC13): 1.23-1.48 (m. 20H. chain 2.2',3,3',4,4",-5,5',6,6'-CH2). 2.13 (a, 6H, pyrrole 4-CH3), 2.20 (a, 12H, benzene CH 3), 2.31 (a. 6H. pyrrole 2-CH3). 2.34-2.40 (m, 4H. chain 7.7,-CH2). 2.55-2.65 (B. 4H. chain 1.1'-CH2). 7.28 (a. 2H, C(U)-C(CN) 2), 9.39 (a. 2H. NH). 1 3C_noi (5. 10% TFA-CDCI3): 145.61 (pyrrole 2-C). 140.85 |C(H)-C(CN) 2). 139.12 (pyrrole 4-C), 136.97 (durene 1-C and 4-C). 132.02 (durene 2-C, 3-C, 5-C, 6-C), 127.23 (pyrrole 3-C), 125.12 (pyrrole 5-C). 117.09 (C N), 115.96 (C N), 57.92 <!C(H)-S(CN)2], 30.72 (chain durene termini 1, l'-C), 30.03, 29.81, 29.27 (chain, 2. 2', 3, 3'. 4, 4'. 5. 5', 6. 6'-C), 23.88 (chain pyrrole termini 7, 7'-C), 16.15 (durene CH 3), 12.34 (pyrrole 2-CH3), 9.54 (pyrrole 4-CH3). Hass spectrum (m/e, relative Intensity): 668 (K +, 60), 653 (20), 184 (100). 1.5 Synthesis of Durene Linked Dipyrromethane Dimers I.J.I Preparation of 110 (1) Monochlorlnatlon of the oc-methyl group In 107: The dlcyanovlnyl derivative 107 (a or c, 1.71 mmol) was dissolved in dry dichloroethane (300 mL), and treated dropwise with a solution of sulphuryl chloride (0.467 g, 3.44 mmol) i n dichloroethane (20 mL) at room temperature. The pale yellow aolutlon which turned orange during the addition, was allowed to s t i r for a further 0.5 h. The solvent was removed completely In vacuo, and the yellow residue of the corresponding ot-chloromethyl derivative 108 used d i r e c t l y in the next reaction without further p u r i f i c a t i o n . (11) Condensation of 108 with 109: The -chlororaethyl derivative 108 prepared above was suspended ln g l a c i a l acetic acid (350 ml.), and 2-ethoxycarbonyl-4-ethyl-3-methyl-pyrrole 109 (0.75 g, 2.5 mmol) ln acetic acid (5 mL) added. The mixture was warmed to 80°C under nitrogen. Within 1 h a l l of the starting material was dissolved. A t i c analysis indicated the product as a single yellow spot, coloured v i o l e t by bromine vapour. The acetic acid was evaporated down under reduced pressure to -20 ml, MeOH (100 mL) added and the solution allowed to stand overnight l n the refrigerator. The dipyrromethane dimer 110, c r y s t a l l i z i n g out as a dark yellow s o l i d , was f i l t e r e d and washed with MeOH to y i e l d 85% of product for both 4/4-a, and 7/7-c derivatives. (a) 1,4-Bis(A-I2-[(5-ethoxycarbonyl-3-ethyl-4-methylpyrrol-2-yl)-methyl]-5-(2.2-dlcy«novinyl)-4-methylpyrrol-3-yl]butyl)-2.3,5.6-tetramethylbenzene, 110a ILB. - 252.0-254.0°C. Anal. Calcd. for C5gH 7 0N 80 4: C, 73.86; H, 7.48; N, 11.88; Found: C, 73.56; H, 7.43; N, 11.65. XH nmr (8. 10% TFA-CDC13): 0.99 ( t , J - 7.5 Hz. 6H, 3'-CH2CH3). 1.38 (t, J - 7 Hz, 6H, -OCH2CH.3), 1.41-1.51 (m, 8H, chain 2,2',3,3'-CH2), 2.16 (s, 6H, pyrrole 4-CH3), 2.20 (s, 12H, benzene CH 3), 2.25 (s, 6H, pyrrole 4'-CH3), 2.31-2.44 (m, 8H, 4'-CH.2CH3, chain 4,4 •-CH2) . 2.53-2.65 (m, 4H, chain 1,1'-CH2), 4.00 (s, 4H, bridge CH 2), 4.33 (q, J - 7 Hz, pyrrole -OCH.2), 7.34 (s, 2H, C(HJ.-C(CN)2), 9.24 (bs, 2H, l'-NH), 9.50 (bs, 2H, 1-NH). 1 3 C nmr (8, 10% TFA-CDC1-,): 164.34 (C-0). 141.60 (£(H)-C(CN) 2) , 141.50 (pyrrole 2-C), 137.64 (pyrrole 4-C), 136.48 (durene 1, 4-C), 131.98 (durene 2,3,5.6-C), 129.37, 128.56. 125.79, 125.71 (pyrrole 3, 2', 3', 4'-C), 124.71 (pyrrole 5-C), 118.00 (pyrrole 5'-C), 116.63, 115.61 (£ N), 62.26 (0-£H 2CH 3), 61.64 (C(H)-£(CN) 2). 30.54 (chsln, durene termini, l . l ' - C ) , 24.02 (chain, pyrrole termini, 4,4'-C), 21.80 (chain 2,2'.-3,3'-C), 17.25 (pyrrole 3'-£H 2CH 3), 16.29 (durene CH 3), 15.17 (pyrrole 3'-CH 2£H 3), 14.18 (0-CH 2£H 3), 10.70 (pyrrole 4'-CH3), 9.62 (pyrrole 4-CH3). Mass spectrum (m/e re l a t i v e i n t e n s i t y ) : 942 (M 4). (b) 1,4-Bls(7-12-t(5-ethoxycarbonyl-3-ethyl-4-methylpyr^ol-2-7l)-meth7l)-5-(2,2-dlc7*novln7l)-4-meth7lp7rrol-3-7llhept7l)-2.3,5.6-tetramethylbenzene, 110c H j i . - 167.0-169.0°C. A n i l . Calcd. for C 6 4H 8 2N 80 4: C, 74.82; H, 8.05; N, 10.91; Found: C, 74.84; H. 8.18; N, 10.96. lH nmr (6. CDC13): 1.02 (t, J - 7.5 Hz, 6H, 3'-CH2CH3), 1.32 (t, J - 7 Hz, 6H, -0CH2CH.3). 1.21-1.48 (m, 20H. chain 2.2\3,3'.4,4',5,5\6,6'-CH 2), 2.13 («, 6H. pyrrole 4-CH3), 2.20 (a, 12H, benzene CH 3), 2.27 (a, 6H, pyrrole 4'-CH3), 2.38-2.48 (a, 8H, 4'-CH2CH3. chain 7,7 ,-CH 2). 2.56-2.65 (m, 4H, chain 1.1'-CH2), 3.92 (a, 4H, bridge CH 2), 4.23 (q, J - 7 Hz, 4H, 0-CU2CH3), 7.28 (», 2H, C(H)-C(CN) 2), 8.67 (br a. 2H, l'-NH), 9.13 (bs, 2H, 1-NH). 1 3 C nmr ( 6 , 10% TFA-CDC13): 164.31 (OO), 142.13 |£(H)-C(CN) 2), 141.46 (pyrrole 2-C), 138.06 (pyrrole 4-C), 136.88 (durene 1, 4-C), 131.92 (durene 2, 3, 5, 6-C), 129.47, 128.51, 126.18, 125.91 (pyrrole 1,2'.3'-C), 124.83 (pyrrol* 5-C), 118.02 (pyrrole 5'-C), 116.06, (C N), 61.54 IC(H)-C(CN 2)• 30.68 (chain, durene termini, 1, l'-C), 30.03 (4-C), 29.82, 29.45, 29.25 (chain 2,2',3,3',4, 4-,5,5',6,6'-C), 23.99, 23.82 (chain, pyrrole termini, 7,7'-C), 17.21 (pyrrole 3'-CH2CH3), 16.11 (durene CH 3), 14.98 (pyrrole 3'-CH2CH3), 14.02 (0-CH 2CH 3), 10.53 (pyrrole 4--CH3), 9.38 (pyrrole 4-CH3). 1.5.2 Hydrolyala of 110 0 0 111 a, n - 4 c . n - 7 The dipyrromethane dimer 110 (a or c, 1.06 mmol) was refluxed ro under nitrogen In a solution of n-propanol (60 ml) and 1.0 H aqueous ^ L O potassium hydroxide (100 ml). The reaction was followed by pe r i o d i c a l l y ( removing an aliquot of solution and recording the uv spectrum. Within 1.5 h a band at 407 nm, present in the starting material, had completely disappeared Indicating the removal of the dicyanovlnyl protecting group. The band at 275 nm and a shoulder at 315 nm remained, and became more intense. At thla stage, the n-propanol was boiled o f f under nitrogen, and the aqueous brown emulsion refluxed for a further 3 h to ensure complete hydrolysis of the pyrrole ester function. The solution was then cooled under nitrogen and a c i d i f i e d with g l a c i a l acetic acid. The resulting gelatinous brown s o l i d was f i l t e r e d and washed with water to give quantitative y i e l d of the corresponding dipyrromethane dimer 111a or e. ( • ) 1 , 4 - B i s ( 4 - [ 2 - | (5-earboxy -3-ethyl -4-aethylpyrrol-2-yl)methyl] - 5 -formyl - 4-Bethylpyrrol - 3-yl]butyl ) - 2 , 3 , 5 , 6-tetramethylbenzene, 111» M.p. - 215.0-217.0°C (decomp.). lti nmr ( . DHS0-d6): 0.71-0.92 (m, 6H, 3'-CH2CH3). 1.29-1.56 (m, 8H, chain 2,2',3,3'-CH2). 2.03-2.74 (m, br, 36H, durene CH3, pyrrole 2-, 4-CH3, chain 1.1'.4,4'-CH2, 3'-CH2CH3), 3.93 (a, br. 4H. bridge CH 2). 9.54 (a, 2H, HCO), 11.12 (ba, 2H, 1-NH), 11.57 (br a, 2H, l'-NH). Mass spectrum (m/e, relative Intensity): 702 (M-2C02+, 30), 673 (40), 94 (100). ( b ) 1 . 4 - B l e ( 7 - [ 2 - [ ( 5 - c a r b o x T - 3 - e t h y l - 4 - m e t h y l p y r r o l - 2 - y l ) m e t h y l ) - 5 -formyl - 4-methylpyrrol - 3-ylJheptyl) - 2 , 3 , 3 , 6-tetramethylbentene, 111c ILfi. - 140.0-142.0°C (decomp.). *H nmr (8, DMSO-dj): 0.67-1.50 (m, br, 26H, 3'-CH2CH.3, chain 2,2' ,3,3',4,4',5,5',6,6'-CH2), 1.72-2.59 (m, br, 36H, benzene CH3, pyrrole 2-, 4-CH3, chain 1,1',7,7'-CH2, 3'-CH2CH3), 3.78 (br, 4H, bridge CH 2). 9.41 (s, 2H, HCO), 10.94 ( b s , 2H, 1-NH), 11.48 (br a, 2H. l'-NH). Mass s p e c t r u m ( n / e , relative Intensity): 786 (M-2C02+, 10). 1 . 6 Cyclization to the Durene Capped Porphyrins (1) Decarboxylation of 111: The dipyrromethane derivative 111 (a or c, 1.01 mmol) was refluxed ln DMF (200 mL) under nitrogen. A uv spectrum of the reaction solution showed a considerable decrease ln the intensity of the band at 275 nm rel a t i v e to that at 315 TUB. This was taken as an indication of the extent of decarboxylation. After 2.5 h no further change in the spectrum occurred. The aolution was cooled and the DMF evaporated down under reduced pressure to 10 mL. Because of the low s o l u b i l i t y of the compound 111 l n CH 2C1 2, THF (450 mL) was added to the dipyrromethane dimer in DMF and this solution was then used d i r e c t l y in the c y c l i z a t i o n step. (11) Intramolecular "2+2'' coupling: The c y c l i z a t i o n was carried out under high d i l u t i o n conditions, in the dark. The solution of the dipyrromethane dimer prepared above was added elowly, by Beans of a ayringe pump, to a aolutlon of CH2CI2 (1000 mL). containing toluene-p-sulphonlc acid (8.0 g) dissolved i n HeOH (5 mL). Ideally, 20 mL of the dimer 112 • or c was added over a period of 7 h. Once the addition was complete (ca. 6-7 d), the reddish-violet solution wss concentrated down (150 mL) and extracted with saturated sodium bicarbonate aolutlon (3 x 50 mL) to remove the acid catalyst. The solvent waa evaporated and the residue chromatographed as described below. (a) 7,l7-Dlethyl-2,B,12.1B-tatramethyl-3,13t2,3,5.6-tetramethyl-phenylene-1,4-bis(tetamethylene)]porphyrin, 54a Chromatographic p u r i f i c a t i o n : The crude porphyrin waa f i r s t chromatographed on s i l i c a gel (120 g) using CH2CI2 as solvent. Two faint bands which eluted down f i r s t were discarded. The aolvent waa then changed to 1% MeOH/CH^C^ and the porphyrin eluted down with aome brown Impurities. Tha p a r t i a l l y purified porphyrin was then chromatographed on alumina ( a c t i v i t y I II, 35 g), using toluene as solvent. Some fast moving impurities eluted down I n i t i a l l y with a change of solvent to CH2CI2. the porphyrin eluted down cleanly, leaving a l l other Impurities adsorbed at the origin. C r y s t a l l i z a t i o n from a solution of Ch^C^/MeOH afforded 0.10 g of purified porphyrin 54a. Typically, yields varied between 11-16% depending on the success of the dipyrromethane hydrolysis to the intermediate 111a. ILE. - 272.0-273.0°C. Mol. wt. Calcd. for C 4 6H 5 f tN 4: 664.4509; Found by high resolution mass spectrometry: 664.4495. Anal. Calcd. for C 4 6H 5 6N 4: C, 83.09; H, 8.49; N, 8.42; Found: C, 81.06; H, 8.20; N, 8.20. Calcd. for C 4 6 H 5 6 N 4 H 2 0 : C, 80.89; H, 8.56; N. 8.20. *H nmr (6, CDCI3): 9.83 (B, 2H, methlne protons 10-H and 20-H), 9.61 (a, 2H, methlne protons 5-H and 15-H), 4.06 (m, 4H, -CH 2CH 3), 3.82 (m. ' ts> 2H, chain protons), 3.62 (m, 8H, two CH3 and 2 chain protons), 3.32 (s, -^j C n 6H, two CH3), 1.88 (t , J - 7.5 Hz. 6H, -CH 2CU 3), 1.71 (m, 2H, chain , protons), 1.43 (m, 4H, chain protons), 1.30 (m, 2H, chain protons), 0.29 (s, 6H, durene CHj), 0.21 (s, 6H. durene CH 3), -1.51 (m, 2H, chain protons), -1.72 (m, 2H, chain protons). -3.54 (br s, 2H, N-H). 1 3 C nmr (6. 10% TFA-CDCI3): 146.54, 144.87, 143.00. 140.94, 140.31. 139.57. 139.42, 138.62 (16C, - and -py r r o l l c carbona), 135.69 (2C, durene 1,4-C), 129.49, 129.17 (4C, durene 2,3,5,6-C), 99.93, 98.84 (4C, meso carbona 5,10,15.20-C), 28.66, 28.00, 27.55, 25.20 (BC, chain carbons), 20.07 (2C, CH2CH3), 16.38 (2C, CH 2CH 3), 14.46 (4C, durene CH 3), 12.00, 11.59 (4C, 2,8,12,18-CH3). V i s i b l e spectrum (CH 2C1 2): Xmax (nm), 400.7 502.0 538.0 571.5 626.0 log t . 5.22 4.04 4.07 3.76 3.64 (b) 7.17-Dlethyl-2.8,12.18-tetramethyl-3,13-[2,3.5,6-t«tramethyl-phenylene-1 ,4-bla(heptamethylene)]porphyrln, 5 6 a Chromatographic p u r i f i c a t i o n : The crude porphyrin was chromatographed on alumina ( a c t i v i t y III, 30 g) using 25% hexane/toluene as solvent. Some fast moving brown impurities eluted down i n i t i a l l y . The aolvent was then gradually changed to 20% CH 2Cl 2/toluene and the porphyrin eluted down cleanly leaving a l l other Impurities adsorbed at the or i g i n . Recrystalllzatlon from a solution of CH2Cl2/Me0H afforded 0.088 g of the durene-7/7 porphyrin. The yields obtained varied between 9-13% depending on the purity of the dipyrromethane dimer 111c. ttji. - 314.5-315.5°C. Mol. wt. Calcd. for C^Hgglfy: 748.5444; Found by high resolution msss spectrometry: 748.5446. Anal. Calcd. for C 5 2H 6 8N 4: C, 83.37; H, 9.15; N, 7.48; Found: C, 82.58; H, 9.34; N. 7.30. Calcd. for C 5 2H 6 8N 40.5H 20: C, 82.38; H, 9.17; N. 7.39. 1H nmr (S, CDC1 3): 10.04 (a, 2H, methlne protons 10-H and 20-H), 10.02 (a. 2H, methlne protons 5-H and 15-H), 4.41 (m, 2H, chain protons), 4.16 (m, 2H, chain protons), 3.97 (m, 4H, -C{J2CH3). 3.60 (s, 6H, two CH3), 3.57 (s, 6H, two CH3), 2.31 (m, 2H, chain protons), 2.05 (m, 2H, chain protons), 1.84 ( t . J - 7.5 Hz, 6H, -CH2CH.3), 1.57 (m, 2H, chain protons), 1.38 (m, 2H, chain protons), 1.30-0.95 (m, 12H, chain protons), -0.01 to -0.16 (a, 4H, chain protons), -0.20 (s, 12H, durene CH 3), -3.71 (br a, 2H, NH). l 3 C nmr ( 5 . 10% TFA-CDCI3): 145.45, 143.45, 142.68, 142.46, 141.65, 140.85, 139.71, 138.78 (16C, - and -pyrrolic carbons), 135.52 (2C, durene 1.4-C), 130.60, (4C, durene 2,3,5,6-C), 99.28, (4C, meso carbons 5,10,15,20-C), 31.19, 29.85. 29.26, 28.44, 28.37. 27.76, 26.82, (14C, chain carbons), 20.15 (2C, CH 2CH 3), 16.43 (2C, CH2CH3), 15.20 (4C. durene CH 3), 12.19, 11.66 (4C, 2,8.12,18-CH3). V i s i b l e spectrum (CH 2C1 2): Xmax (nm), 397.0 495.5 528.5 565.0 618.5 log € , 5.16 4.12 3.98 3.81 3.72 Z.7 Synthesis of th« Durene-Hemln Chloride Complexes F e 1 1 1 ( S 4 b ) C l n - 4 F e 1 1 1 ( 5 5 b ) C l n - 5 F e 1 1 1 ( 5 « b ) C l n - 7 The free base durene porphyrin ( 5 4 a , 5 J a , or 5 6 a , 0.075 mmol), dissolved i n THF (30 mL), waa added rapidly, dropwise, to a s t i r r e d solution of ferrous chloride (0.75 mmol) In MeOH (30 mL), under nitrogen atmosphere. The mixture was refluxed for 1 h, after which time a uv-vlslble spectrum lndicsted complete conversion to the hemin chloride. The solution wss then evaporated down i n vacuo, the realdue taken up in CH2CI2, washed with water and chromatographed on alumina ( a c t i v i t y V, 30 g) using CH2CI2 a a solvent. The green fL-oxo dimer, elutlng down from the column, was shaken with dilute aqueous hydrochloric acid (0.2 H) to eff e c t converalon back to the hemin chloride. The CH2CI2 aolutlon was evaporated i n vacuo and hexane added to c r y s t a l l i z e the hemin chloride, ( y i e l d -85%). ( a ) Durene-4/4 hemin chloride. F e ( 5 4 b ) C l : 6naJ.. Calcd. for FeC 4 6H 5 4N 4Cl: C, 73.25; H, 7.22; N, 7.43; Cl, 4.70; Found: C. 73.02; H, 7.35; N, 7.19; C l . 4.56 Mol. Ut, Calcd for FeC 4 6H 5 4N 4Cl: 753.3386; Found by high resolution mass spectrometry: 753.3415. Vi s i b l e spectrum (toluene): Xmax (nm), 370.0 400.0(sh) SOO.O(sh) 527.0 629.0 log e . 4.86 4.80 3.92 3.95 3.69 (b) Durene -5/5 hemin chloride, Fe(55b)Cl: Anal. Calcd. for F e C 4 8 H 5 8 N 4 C l : C, 73.69; H, 7.47; N. 7.16; C l . 4.53; ^ Found: C, 73.37; H, 7.60; N, 6.96; C l , 4.33. 1 Mol. Wt. Calcd for FeC 4 8 H 5 8 N 4 C l : 781.3699; Found by high resolution mass spectrometry: 781.3716. V i s i b l e spectrum (toluene): Xmax (nm), 372.0 400.0(sh) 503.0 530.0 630.0 log t , 4.88 4.80 3.93 3.96 3.71 (c) thirene-7/7 hemin chloride. Fe(3«b)Cl: AxuU. Calcd. for FeC 5 2H 6 6N 4Cl: C. 74.69; H. 7.93; N, 6.68; CI, 4.23; Found: C, 74.10; H, 8.00; N, 6.50; CI, 4.36. Mol. Wt. Calcd for FeC 5 2H g 6N 4Cl: 837.4321; Found by high resolution mass spectrometry: 837.4340. Vi s i b l e soectrun (toluene): Xmax (nm), 374.0 400.0(ah) 505.0 532.0 631.0 log t . 4.94 4.82 3.94 3.98 3.72 ro CO - 279 -APPENDIX II Spectral Data for the Iron(II) Durene-Capped Systems - 280 -Fe(durene-7/7) log e , 384.0 4.91 409.0 4.96 527.0 3.99 561.0 4.09 Fe(durene-5/5) ^max ( n n i) • log e , 386.0 4.91 411.0 4.96 527.0 3.99 561.0 4.09 Fe(durene-4/4) vmax ( m ) • log C , 389.0 4.91 412.0 4.96 527.0 3.99 561.0 4.09 Fe(durene-7/7) (B) B - l,2-Me2Im: B - Dclm *max ( n m) > ^max ( n m) • log e, 420 (br) 415 (br) 5.08 548 (br) 548 (br) 4.04 Fe(durene-5/5) (B) B - l,2-Me2Im: B - Dclm : Amax ( n n l)-A max (nm), log e. 420 (br) 418 (br) 5.08 550 (br) 550 (br) 4.04 Fe(durene-4/4) (B) B - l,2-Me2Im B - Dclm B - Melm Amax (nm>-Amax <™>. ^max ( n m) • log 6 , 423 (br) 420 (br) 419 (br) 5.08 560 (br) 560 (br) 560 (br) 4.04 281 -Fe(durene-7/7)(Dclm)(Melm) X m a x (nm), 414.0 518.0 547.0 log €, 5.32 4.18 4.43 Fe(durene-7/7)(Dclm)(TMIC) X B a x (run), 418.0 520.0 550.0 log 6, 5.43 4.12 4.16 Fe(durene-7/7)(Dclm)(t-BuNC) X m a x (run), 421.0 520.0 550.0 log 6, 5.33 4.12 4.16 Fe(durene-5/5)(Dclm)(RNC) RNC - TMIC or t-BuNC X m a x (nm), 422.0 522.0 553.0 log e , 5.19 4.06 4.24 Fe(durene-7/7)(TMIC)2 X m a x (nm), 420.0 530 (br) log € , 5.60 4.10 Fe(durene-5/5)(TMIC) 2 X Bax ( ™ ) . 424.0 520.0 546.0 log 6 , 5.49 3.96 4.04 - 282 -Fe(durene-4/4)(Dclm)(n-BuNC) X m a x (nm), 424.0 525.0 558.0 log €, 5.13 4.02 4.31 Fe(durene-4/4)(TMIC) X m a x (nm), 399.0 510 (br) 541.0 log € , 5.23 3.67 4.04 Fe(durene-4/4)(C0) X m a x (nm), 395 (sh) 403.0 553 (br) log C, 5.05 5.21 4.10 Fe(durene-5/5)(CO) X m a x (nm), 383 (sh) 402.0 525.0 555.0 log 6, 4.67 5.38 4.04 4.12 F e ( d u r e n e - 7 / 7 ) (B) (CO) B - l,2-Me2Im: X m a x (n™)• 390 (sh) 409.0 529.0 B - Dclm Xmax (nm) , 390 (sh) 411.0 529.0 log €, 4.60 5.44 4.06 Fe(durene-5/5) (B) (CO) B - l,2-Me2Im: Xmax ( n m) • 393 (sh) 413.0 543 (br) log e, 4.60 5.39 4.07 B - Dclm : Xmax ^ n m) • 395 (sh) 415.0 530 (br) logC , 4.60 5.41 4.02 555.0 555.0 4.02 550 (br) 4.03 - 283 -Fe(durene-4/4) (B) (CO) B - l,2-Me2Ini: X m a x (nm), 395 (sh) 415.0 log 6, 4.60 5.33 : X B - Dclm, or Melm m a x (nm), 395 (sh) 418.0 log 6 , 4.60 5.36 544.0 4.12.. 520 (br) 549.0 4.08 Fe(durene-7/7)(Dclm)(02) log e , 407 5.07 535.0 3.98 565.0 4.04 Fe(durene-5/5)(DcIm)(02) log € , 408 5.03 540 (sh) 4.05 566 (br) Fe(durene-4/4)(B)(02) ;max ( n m ) -log e, 409.0 5.00 550 (sh) 566 (br) 4.06 - 284 -APPENDIX III Raw Data for the Binding of Imidazoles, Isocyanides, CO and 0 2 to the Durene-Capped Hemes The data were processed by least-squares analysis i n a l l cases. Values of the equilibrium and k i n e t i c constants, determined at two d i f f e r e n t wavelengths i n the 460-350 nm region, were found to be consistent to within — 15% error. Maximum and minimum values of the Van't Hoff pl o t s f or the Fe(durene-4/4)(B) systems (see appendix IIIK) afforded error estimates of ±15% and ± 25% for the A H 0 and A s ° thermodynamic constants, r e s p e c t i v e l y . o o o o o o «> C* *> i> m U» U> ^ M <0 O M 00 O K> OB o o o o o o v i >J - » J 17< CTi 0> w i* H oo y H N t o *o m *o o o o o o o o b b b o b b vl (71 y U M O •> OB N« cn o o o o o o o W M M M h* O U H >0 O W CD »> a*, r-o a> - J o o o o o o o o o o o o r— fs» u i *> m cn «0 Ul »-* Ki W >-0 OOOOO© O O h- i — M v j O H ^ M H w >o O f O vO U> Kl «-•>-» O O OMJ) CO l-t IT> M CO ffi sj H si vfl 4> O H- « «*0 O o o o o o o U i Mv) O Wl O O vl C- Ui U (Ji CD NJ vO CO CD > U> PO I-* O O PO *> -J r-« OB) Ui Ul £• CD O vO o o o m o s t o o o o o o > > > i s a 1 3 - a 5 3 o n uia w u u w w O O O O O O O U» Ul Ul *> *> *» si t> SJ m OI w O M rv U v) K) o o o o o o Ui U» U» CJ* ON (Ji 91 N J iO O H U N i N | O £ T \ V j Ul o o o o o o r-l H. H- M b O V3 N J ^ M « ui ^ vO O {• \fl O O O O O O O O O O O O 00 Nj ON ±> U> K-1 Q> OI U N j 91 CO O O O o o o O M y i co o «• «> si fs> *— si r\» < O O O O O O o o o o o •— C" Ul Cl 00 vO H SJ M yi H is) o f- O O O o 00 U « o> £< M vO N I oa ON ui c* *> O O ui 4> C-fN) i— o o o o O I s O u i u p* ON cn co so cn CO M vO O H J> • • O o o o ro »-» • . . . N l W O H U OI M N I O N J ^ H CO Ui Ui • • O O O O W H • • • • H H I O M N | M Ul H W O CO •> en - J ON U> U i U> W •> P* U l U l U i U l i> 4> - 5 8 3 -(•) Fe (durene-5/5) • Dclm ^max " 420 nm *o " • 0.474 .850 A„-A log [Dclm A A 0-A A-A » log ( ) IDcIm) A-A„ A-A«, 10" 5 M 0.531 0.057 0. 319 0.179 -0.748 9.48 -4.02 0.552 0.078 0. 298 0.262 -0.582 15.80 -3.80 0. 59? 0.123 0. 253 0.486 -0.313 25.30 -3.60 0.621 0.147 0 229 0.642 -0.193 34.80 -3.46 0.657 0.183 0. .193 0.948 -0.023 44.30 -3.35 0.669 0.195 0 .181 1.077 0.032 53.80 -3.27 Slope - 1.08, av • KDcIm - 2.0 x 10 3 M"1 at \>ax 420 and 387 nm (3) Fe(durene-5/5) + l,2-«e2Im X „ „ - 420 nm A. - 0.480 0.870 A-Ao A_-A A-A0 A^-A A A 0 log ( ) A « A Il,2-Me2Iml log |l,2-Me2Ira) 10 -5 0.612 0.670 0.702 0.726 0.753 0.132 0.190 0.222 0.246 0.273 0.258 0.200 0.168 0.144 0.117 0.512 0.950 1.320 1.708 2.333 -0.291 -0.022 0.121 0.233 0.368 7.48 15.00 22.50 33.70 52.40 -4.13 -3.83 -3.65 -3.47 -3.28 Slop. - 0.9, av. K l f 2 . M e 2 i „ - 5.8 x 10 3 M"1 at X o a x 420 and 387 nm (6) Fe(durene-7/7) + Dclm ^max ~ 4 2 0 ™ A„ - 0.470 A„ - 0.707 A 0 A A 0-A A A 0 A A "A, log ( ) (Dclm) log [Deli A-A„ 10 " M 0.542 0.072 0 .165 0.436 -0.360 1 .38 -3.86 0.573 0.103 0 .134 0.769 -0.114 2 .30 -3.64 0.598 0.128 0 . 109 1.174 0.070 3 .45 -3.46 0.620 0.150 0. .087 1.724 0.237 4 .83 -3.32 0.640 0.170 0. .067 2.540 0.404 6 .67 -3.17 0.657 0.187 0 .050 3.740 0.573 8 .97 -3.05 Slope - 1.1, av. KDclm - 3.3 x 10 3 M-1 a t * m „ 420 and 384 nm (7) Fe(durene-7/7) + l,2-Me2Im ^max " 420 nm A„ - 0.560 A„ - 0.928 A-A 0 A-AG A A A 0 A«-A log ( ) [1.2 •Me2Im] log [1,2-1 A„-A A„-A 10 5 M 0.620 0.058 0.308 0.188 -0.725 1 .45 -4.84 0.652 0.090 0.276 0.326 -0.487 2 89 -4.54 0.693 0.131 0.235 0.557 -0.254 5 .06 -4.30 0.715 0.153 0.213 0.718 -0.144 7 .23 -4.14 0.737 0.175 0.191 0.916 -0.038 10 .10 -3.99 0.753 0.191 0.175 1.091 0.038 13 .70 -3.86 Slope - 0.80, av • K1.2-He 2 Im " 1 04 x IO 4 M'1 420 and 384 nm (8) Fe(OEP) + 1, >2-Me2Im ^max ~ 384 run A , - 0.513 A „ - 0.352 A 0 - A A D - A A A D - A A A » log ( ) [l,2-Me2Im) log [1,2-1 A - A B A - A „ IO" 5 M 0.477 0.036 0 125 0.288 -0.541 2.69 -4.57 0.445 0.066 0 093 0.731 -0.136 5.38 -4.27 0.428 0.085 0. 076 1.112 0.049 8.07 -4.09 0.409 0.104 0. .057 1.825 0.261 13.50 -3.87 0.400 0.113 0. .048 2.350 0.372 21.60 -3.67 0.388 0.125 0 ,036 3.470 0.541 35.10 -3.45 Slope - 0.95. av • K l , 2 - H e 2 l n - 2 x 10* M 1 et XB 1 1 ) t 418 and 384 nm (9) Fe(durene-7/7)(Dclm) + Helm \ oax - 412 nm 0.610 A . - 1. .106 A - A Q A - A 0 A A< A> log ( ) (Helm) log [Meli A „ - A A ^ - A 10" 5 M 0. 656 0. 046 0.450 0.102 -0.990 2.58 -4.59 0. .710 0. 100 0.396 0.253 -0.598 6.02 -4.22 0. .750 0. .140 0.356 0.393 -0.405 9.46 -4.02 0. 787 0. 177 0.319 0.555 -0.256 12.90 •3.89 0. .813 0. 203 0.293 0.693 -0.159 16.30 -3.79 0. 845 0. 235 0.261 0.904 -0.044 20.60 -3.69 0. .869 0. 259 0.237 1.093 0.039 25.80 -3.59 Slope - 1.04. I i o n l t r l l e A f f i n i t y Conatant Determination, K K In toluene, at 20°C. (Dclm) -0.3 M TMIC, mv - 195.24 t-BuNC. m - 83.13. d - 0.735 g/mL (1) Fe(durene-5/5) (Dcln) + t-BuNC [heme) -3 x 10" 6 M AAo 422 nn A.A A 0 AA,, 0.430 A. - 0.557 A-A,, log ( ) I'BuNC] A.-A A„-A 10' 5 M log ['BuNC) 0 .450 0 .020 0. .107 0. 187 -0, 728 0 .95 -4 .02 0 ,470 0. .040 0. 087 0. 460 -0. ,337 2. ,38 -3 .62 0. .489 0. .059 0. ,068 0. 868 -0. ,062 4 .28 -3 .37 0 ,504 0. .073 0. .053 1. 396 0. ,145 6 .66 -3 .18 0 .516 0 .086 0. ,041 2. ,098 0. .322 9. ,51 -3 .02 0 .525 0 .095 0, .032 2 .970 0. .473 13, .30 -2 .88 Slope - 1.06, t-BuNC i 1 • t • v- KDcIm - 2.1 X 10 3 M'1, f i n a l I'BuNC) -1 x 10" 2 M (2) Fe(durene-7/7) (Dclm) + t-BuNC [hene] -2 x 10" 6 M ^nax " 420 mi Ao " 0.311 A«, - 0.645 A A 0 A-A0 log I'BuNC) A A\> A.-A log ( ) I'BuNC) A„-A v» 10" 5 M 0. .448 0.137 0.197 0.695 -0.158 0.282 -5.55 0. 514 0.203 0.131 1.550 0.190 0.564 -5.25 0, ,543 0.232 0.102 2.275 0.357 0.846 -5.07 0. 565 0.254 0.080 3.175 0.502 1.310 -4.88 0. ,582 0.271 0.063 4.302 0.634 1.960 -4.71 t - BuNC e , /. Slope - 0.94, av. K D c i „ - 2.62 x 10 5 M"1, f i n a l ^BuNC] -2 x 10' a M (3) Fe(durene-5/5) (Dclm) (CO) • TMIC (heme] -2.6 x IO" 6 M, [CO] - 2.4 x 10' 4 M, [Dclm] -1 K ^max _ 4 1 5 A D - 0.650 A,, - 0.240 Ao-A A O - A log ( ) (TMIC) log [TMIC] -4 A-A„ 10 0.602 0.480 0.362 0.133 -0.877 2.77 -3.56 0.563 0.087 0.323 0.269 -0.570 4.84 -3.32 0.530 0.120 0.290 0.414 -0.383 6.92 -3.16 0.493 0.157 0.253 0.621 -0.207 9.00 -3.04 0.471 0.179 0.231 0.775 -0.111 11.10 -2.95 0.450 0.200 0.210 0.952 -0.021 13.50 -2.87 0.431 0.219 0.191 1.147 0.059 16.90 -2.77 Slope - 1.11, KCO.TMIC . 6.99 x 10 2 M"l, K ™ „ - *l°cla-[CO] KCO.TMIC - 6.4 x 10 6 M 1 (4) Fe(durene-7/7) (Dclm) (CO) + TMIC [heme) -3 x 10"* M, [CO] - 2.4 x 10" 4 M. [Dclm] -1 M A- B„, - 418 nm A D - 0.350 - 0.846 A Q A A 0-A A D-A A-A. log ( ) [TMIC) log [TMIC] A-A„ A-A«, IO" 4 M 0. ,693 0. .343 0, .153 2, .242 0, .351 2. 57 -4 .59 0. ,721 0. .371 0, .125 2, ,968 0. .472 3. 88 -4 .41 0. .758 0. .408 0, .088 4. .636 0. .666 5. .17 -4 .29 0. ,784 0. ,434 0, ,062 7, ,000 0. .845 6, ,77 -4 .17 TMIC Slope - 1.18. K C 0 ' ™ 1 C - 7.17 x 10 4 M"1, K D c I n - 8.3 x 10 8 M"1 o o o o o o o 51 o> o> w u< u* 3 ! W O ^  u J O U w fr fr u» *-» O 0* 0 0 0 0 0 0 0 CD U< H OB OD U ** f U> H O t * O O O O O O O O H H H IO fO W vj fO * W J< M CO U> Nl K 9> > fr iv» O O O M M OA O M Ul M H M O O 9) <J 0> LC O S3 H C U O O O O O O O ONU M O H N Ul fs> fr O <-J O fr N I i»r> cfN C7^  CO (T» vO > I > VldT fr N> •— W >-> fr ^ A fr S> O ON UA UV Lt> r—• C*» O C»» O M fr fr O O O O O O O PO O o o o o fr H OS i" w N H O "-J fr * — o> >o u u >o 0 O 1 O (-» -a —• 3 1 3 " n f l a » 1 — S —• >-4 r t t o "0 M IX c ™ O 3 O O O O O O •O OJ CB Nl sj * (71 « U< CO O H N) Ul NJ M O 4 O O O O O O m m fr fr fr u> W H CD (7i M CD O M CO M vj K) O O O O O O U> fr fr U* PO H fr O U VI si fr Nj fr fr M M O O O O O O M UJ U fr U> ON Ca u i 4) cn u i u> ca u i co co o M I O O O O O O O O O O O O O K H M M N U st -O H fr P S J M O fr U l O > 1 0 »-»»-»>-• o o o vj N O Nl fr N CO U» Ul Nl y i >o u i fr co H *-»»-• o o o o r o O N I y i w H N H CO CO CD >fl O O U l U l SO fr 0 »-• M m o o — 1-1 ui Ps) O C*> fr 1-1 cn so N I O O O O O O O O H M fr N! CO O O s-i 1-* »-* Nl U l U l w o U l > 8, > NJ IS) >-• C O O" W Co Ul fr W vTi ^ Ln r-» a 3 fr U) M M H 1 — ijj fr N J o Ul H O « >0 0D o co CD fr o 0J a 3 O O O O O O ^ 0 b o o b o o • N*mfri**POr-» - »i U O i » U l fr fr 1 O O O O O O O O O O O O U K) M H M o Ul vO M W O Cn fr U* O 0  m O - 682 -t 4 Fe(durene-*/4) (1.2-Me2Im) • CO (heme) -3 x 10"* M, |l,2-Me2Im] -0.5 M (1) At 4°C, P c 0 ln bulb - 2.23 torr. P T o t a i - 742.8 torr 416 nm *o - 0.380 A„ - 0. 810 A-AQ A-A„ Change l n pCO log P c o A A- Ao A«,-A log ( ) mono, reading A,p-A A„-A (mm) (torr) 0. .505 0. 125 0.305 0.410 -0.387 24.2 0.073 -1.139 0. .572 0. 192 0.238 0.807 -0.093 39.6 0.119 -0.925 0 .603 0 223 0.207 1.077 0.032 50.6 0.152 -0 . 8 1 8 0 .637 0 257 0.173 1.486 0.172 66.6 0 . 2 0 0 - 0 . 6 9 9 0 .668 0 . 2 8 8 0.142 2.028 0.307 87.6 0.263 -0 . 5 8 0 0 .699 0 .319 0.111 2.874 0.458 110.6 0.332 -0.479 CO Slope - 1.17, p l / 2 " 0 1 4 5 torr (1) At 12°C, P c o ln bulb - 2.08 torr, Ptotal _ 7 2 8 2 t o r r \ n a ) t - 416 nm A Q - 0.340 A^ - 0.717 A A „ A-A 0 Change in pCO A A- A o A.-A log (- ) mono reading log Pc o Aa-A V * (mm) (torr) 0. .411 0. .071 0.306 0.232 -0.634 27.8 0.079 -1.10 0. .461 0. .121 0.256 0.473 -0.325 51.4 0.147 -0.833 0 .510 0. .170 0.207 0.821 -0.086 75.8 0.217 -0.665 0 .550 0. .210 0.167 1.257 0.100 109.8 0.314 -0.504 0 .582 0. .242 0.135 1.793 0.253 153.8 0.439 -0.357 0 .605 0 .265 0.112 2.366 0.374 204.2 0.583 -0.234 0 .632 0 .292 0.085 3.435 0.536 275.6 0.787 -0.104 Slope - 1.17. P j y 2 - 0.270 torr ( i l l ) At 17°C, P 1 / 2 - 0.41 torr (iv) At 20.5°C, P c o ln bulb - 2.52 torr, P T o t s i - 740.6 torr ^max ~ 4 1 6 A„ - 0.487 - 1.030 A-A„ A„-A A.-A A A 0 log ( ) A..-A Change ln mono. reading (mm) PC0 (torr) log ?<> 0. 587 0. 100 0. .443 0. .226 -0. 646 52 .6 0 .179 -0 .747 0. 635 0. 148 0. 395 0 .375 -0. 426 85 .2 0 .290 -0 .538 0. 695 0. 208 0. .335 0 .621 -0. 207 130 .4 0 .444 -0 . 3 5 3 0. 739 0. 252 0. .291 0 .866 -0. 062 173. .6 0. 591 -0. 2 2 9 0. 780 0. .293 0 250 1. .172 0. 069 216. 4 0. 736 -0. 133 0. 808 0. 321 0. 222 1. .446 0. 160 264. 4 0. 900 -0. 046 0. 838 0. 351 0. 192 1. .830 0. 262 334. 4 1. 140 0. 056 CO Slope - 1.1, ?i/2 - 0.662 torr CO *1 2-Me2Im -1-5 x 10 5 M 1 r o v O o (v) At 29°C, P c o in bulb - 3.98 torr. P T o t a l _ 7 3 0 8 t o r r X B M - 416 nm A Q - 0.533 A„ - 1.130 A..-A A-A„ A«,-A Change l n mono. reading (mm) pCO (torr) log P1 CO 0. .678 0. .145 0. 452 0 .321 -0 .494 63 .6 0. .347 -0 460 0. 739 0. 206 0. 391 0 .527 -0, .278 102 .0 0. 555 -0. 256 0. 783 0. 250 0. 347 0. .720 -0. 142 143 .0 0. .778 -0. 109 0. 822 0. 289 0. 308 0 .938 -0. 028 187 .4 1. .020 0 0 0 8 0. 858 0. 325 0. 272 1. .195 0. 077 246 .2 1 .339 0 127 0. 901 0. 368 0. 229 1. .607 0. 206 317. .6 1 .728 0. 238 0. 930 0. 397 0. 200 1. .985 0. .298 388 .0 2. 111 0. 324 CO Slope - 0.99, Pjy 2 - 1.08 torr ( 5 ) »e(durene-5/J) (Dcln) (t-BuHC) • CO [hemel -3 x I O - 6 M. (Dclm) - 0.5 M, (t-BuNC) - 0.032 M P c o l n bulb - 5.10 torr, P T o t a i - 744.8 torr X n a x - 413 nm A„ - 0.268 A., - 0.800 A-Ao *-A„ Change l n A-AQ A^-A log ( ) mono, reading P C 0 log P C 0 A„-A A„-A (mi • ) (torr) 0. 621 0 .352 0 .179 1.97 0 .295 20 .0 0.137 -0 .863 0. 670 0. .402 0. .130 3.09 0 490 30 .0 0.205 -0 687 0. .694 0. .426 0, ,106 4.02 0. .604 36 .4 0.249 -0. 603 0. 706 0. .438 0. ,094 4.66 0. .668 43. .0 0.294 -0. 531 0. 730 0. .462 0. .070 6.60 0. .820 59. .4 0.407 -0. ,391 CO Slope - 1.1, P 1 / 2 - 0.074 torr. vt'" u"C,C0 - 1.35 x 10 6 I T 1 KDcI» " K O ^ . C 0 • Rt-BuNC (t-BuNC) - 8.6 x ID 7 M'1 («) Pe(durene-7/7) (Dclm) (Melm) + CO (heme) -3 x IO" 6 M, (Dclml - 0.5 M. (Melm) - 0.0572 M P c 0 l n bulb - 1.51 torr, Piotal - 7*9 0 torr X „ a x - 409 nm A„ - 0.540 A„ - 0.762 Change ln 3. readl (mm) (torr) mono, ing P c 0 log P c 0 A A-*o 0. 600 0. .060 0. .636 0. ,096 0. .660 0. ,120 0. .689 0 .149 0 .710 0 .170 Slope - 1.2, A-A0 A.-A *«,-* 0. .162 0.370 0 .126 0.762 0 .102 1.176 0 .073 2.040 0 .052 3.270 A-A c log ( ) A..-A -0.431 -0.118 0.071 0.310 0.514 82.6 145.2 225.2 332.2 457.6 2.7 x 10 5 M"1 0. 167 -0. 779 0. 293 -0. 534 0. 454 -0. 343 0. .670 -0. 174 0. .923 -0. 035 p l / 2 " 0 V t o r r - K M e I m - c o -KDcIm ~ K H e l"' C 0 • K M e I n [Melm] - 6.64 x 10 7 M - 1 (7) Fe(durene -4 /4 ) (Melm) (CO), a t 20°C (heme) -3 x I O - 6 M, [Melm| - 0.5 M P c o l n bulb - 0.034 torr, P T o t a i - 748.0 torr ^max - 418 nm *o - 0.515 A . " 1 .056 A -*o A-A„ Change l n pCO A A-A„ log ( ) mono, reading log P1 A -A A B-A (mm) (torr) 0 .571 0.056 0.485 0, .115 -0.938 50.0 0.0023 -2.64 0 .612 0.097 0.444 0. 218 -0.661 90.8 0.0041 -2.38 0. .669 0.154 0.387 0. 398 -0.400 142.8 0.0065 -2.19 0. 709 0.194 0.347 0. 559 -0.253 185.6 0.0084 -2.07 0. .747 0.232 0.309 0. 751 -0.124 244.2 0.0111 -1.95 0. 786 0.271 0.270 1. 004 0.002 311.0 0.0141 -1.85 0. 820 0.305 0.236 1. 292 0.111 415.0 0.0189 -1.72 CO CO Slope - 1.2, P 1 / 2 - 0.0145 torr, f^tim - 6.9 x 10 6 M"1 ro o («) »e(durane-4/4) (Dcla) + CO (hemel -3 x IO" 6 H. [Dclm] -0.5 M (1) At *°C, P c o l n bulb - 0.035 torr, Ptotal " 739.2 torr ^max " 418 nm *o - 0.472 A. - 0 .955 A-A„ A-A 0 Change l n A A-A„ A.-A log ( ) mono, reading pCO log P1 A.-A A„-A (mm) (torr) 0. .607 0.135 0.348 0.388 -0.411 84.5 0.004 -2.40 0, .645 0.173 0.310 0.558 -0.253 126.7 0.006 -2.22 0 682 0.210 0.273 0.769 -0.114 169.0 0.008 -2.10 0. .720 0.248 0.235 1.055 0.023 253.4 0.012 -1.92 0. .767 0.295 0.188 1.569 0.196 316.8 0.015 -1.82 CO >co Slope - 1.01, Pj/2 " 0.010 torr (11) At 11°C, P c o l n bulb - 0.088 torr, P T o t a l - 7 4 7 2 t o r r \nax " 418 nm Ao - 0. 453 A „ - 0. .954 A A 0 A-A„ Change l n pCO A A-A„ A.-A log ( ) mono, reading log P1 A„-A A.-A (mm) (torr) 0. 645 0.192 0.309 0.621 -0.207 86.6 0.010 -1.99 0 668 0.215 0.286 0.752 -0.124 107.8 0.013 -1.90 0, ,711 0.258 0.243 1.062 0.026' 146.2 0.017 -1.76 0. .749 0.296 0.205 1.444 0.160 197.8 0.023 -1.63 0. 787 0.334 0.167 2.000 0.301 256.4 0.030 -1.52 0, 821 0.368 0.133 2.770 0.442 327.8 0.039 -1,41 CO Slope - 1.1, 1 p l / 2 - 0.016 torr ,co ( U l ) At 20°C, P c o ln bulb - 0.212 torr, P T o t a l - 744.2 torr ^max ~ 418 nm Ao - 0.462 A„ - 0 .980 A-AQ A-Ac Change l n pCO A A A 0 A.A log ( ) mono, reading log P' A„-A A.-A (mm) (torr) 0 .637 0.175 0.343 0.510 -0.292 40.8 0.012 -1.94 0 .687 0.225 0.293 0.768 -0.115 61.2 0.017 -1.76 0. .730 0.268 0.250 1.072 0.030 85.8 0.024 -1.61 0. ,766 0.304 0.214 1.421 0.152 116.4 0.033 -1.48 0. .800 0.338 0.180 1.878 0.274 157.0 0.045 -1.35 0. .835 0.373 0.145 2.572 0.410 208.2 0.059 -1.23 CO CO Slop e - 1.0, Pl/2 " 0.023 tor r . K D c I o - 4.3 x 10 6 M"1 , C 0 ro (lv) At 31.9°C, P c o In bulb - 0.245 torr, P T o t a l - 748.4 torr ^max " 418 n n Ao - 0. 450 A . " 0. .968 A A 0 A-A0 Change in pCO A A A „ A.-A log ; ( ) mono, reading log P' A«-A A..-A (mm) (torr) 0. 593 0.143 0.375 0.381 -0.419 40.2 0.013 -1.88 0. .622 0.172 0.346 0.497 -0.304 57.6 0.019 -1.72 0, .660 0.210 0.308 0.682 -0.166 87.6 0.029 -1.54 0. .704 0.254 0.264 0.962 -0.017 127.6 0.042 -1.38 0. .735 0.285 0.233 1.223 0.087 175.8 0.058 -1.24 0. 766 0.316 0.202 1.564 0.194 250.0 0.082 -1.09 0. 788 0.338 0.180 1.878 0.274 327.8 0.107 -0.97 CO Slope - 0.8, P 1 / 2 - 0.046 torr , C 0 D CO Association Rata Constant Determination, k' In toluene, at 20°C, [hemel -3 x IO" 6 M (1) Fe(durene-7/7) (Dclm) (CO), [Dclm] - 0.1 M Tonometer volume «• 347.4 ml, P Q - 753.5 torr ^max 6 1 5 ™ ^max 4 2 5 ™ Vol. CO P c 0 (torr) k ^ a " 1 ) * 1 ° 3 ^ b a ^ ' 1 ) * 1 0 ' added (mL) 15.0 32.20 0.649 0.629 35.0 75.13 1.48 60.0 128.79 2.59 2.64 85.0 182.45 4.36 110.0 236.11 5.10 4.94 135.0 289.77 6.51 5.53 Slope - k c 0 (412 nm) - 25.2 t o r r ' V 1 , k c 0 (425 nm) - 21.4 t o r r ' V 1 ave. k c 0 - 2.33 x 10 6 M _ 1 a - 1 , [Dclm] -0.1 M Dclm k c 0 - 9.5 x 10 5 M"1 s " 1 . [Dclm] -1.0 H Dclm (2) Fa(duren«-5/5) (Dclm) (CO), [Dclm] - 0.1 M Tonometer volume - 347.4 ml, P 0 - 755.4 torr X M X 415 nm X n „ 425 nm Vol. CO P c o (torr) kobsC"'1) x 1 0 * k o b s ( a - l ) * 1 0 * added (mL) 20.0 43.05 0.450 0.471 40.0 86.10 0.904 0.987 60.0 129.15 0.133 0.151 80.0 172.20 0.179 0.191 100.0 215.25 0.209 0.232 120.0 258.30 0.253 0.296 Slope - k c o (415 nm) - 95.7 t o r r ' V 1 , k c 0 (425 nm) - 111.8 t o r r ' V 1 k c o - 1.1 x 10 7 M"1 a " 1 Dclm (3) Fa(durana-4/4) (Dclm) (CO), Tonometer volume - 339.6 ml. [Dclm] - 0.1 H fa - 749.2 torr ^max 4 1 ' nm X Vol. CO pCO added (mL) max 428 50.0° io o i n ^ 100.0 2 1 8 . 3 6 0.355 150.0 327.54 o l o o ? ' 7 ° 2 s l o p , - k c o ( 4 1 8 „ „ , . 2 6 6 t o r r . V 1 k c o ( 4 2 8 n ) _ 3 3 3 t o r r . v l k n ° T " 3 0 * 1 0 6 M"1 o 1 Dclm (4) Pe(durena-4/4) (Malm) (CO). [Melm] - 0.1 M Tonometer volume - 347.4 ml, P Q _ 752.8 torr ^max *1 ? ™> X max 425 d"ded Zy P C 0 W - - » > x 10* k o b s ( . - l , x «0.0 n i l 0.453 80 0 , 7 , , ?- 5 7* 0.689 2U.I 0 7 4 6 Slope - kC0 ( 4 1 7 „„, . 4 2 , t o r r - l 8 - l k C 0 ( „ 5 n ) _ M > 8 ~ * 9 * 1 ° S M-l S - l Me In (5) Fe(durene-7/7) (l,2-Ha 2In) (CO), (1.2-H« 2Is] -1 M ^max " *10 ™ ^max ~ 4 2 0 ™> P c 0 (torr) ^ . ( a ' 1 ) x 10 2 ^ . ( e ' 1 ) x 10 2 109 1.43 1.44 218 2.91 327 4.52 4.12 436 5.34 5.67 545 7.05 Slope - k c o (410 nn) - 1.26 t o r r ' V 1 , k c 0 (420 na) - 1.28 t o r r ' V kl,2-Ne 2Ia " 1-3 " 1 ° 5 " ' (6) Fa(durene-3/5) (1.2-He2Im) (CO), (1.2-Me2Im) -1.6 H ^nax " 4 1 3 ™ ^max " 4 2 3 nm P c o (torr) kob«<*'l> x 10 3 k ^ C ' 1 ) " l o 3 109 1.23 1.29 218 2.34 327 3.02 3.78 436 4.50 5.12 545 5.68 6.49 654 6.38 7.46 Slope - k c o (413 nm) - 9.76 t o r r ' V 1 , k c 0 (423 nm) - 11.5 t o r r " 1 * ki?2-Me2Im - 1 1 x 10* H"1 o'1 (7) F*(dur*n*-4/4) (l,2-H* 2Im) (CO), (l,2-Me2Im) -2.0 H ^max - 4 i 6 ™ ^max ~ 4 2 6 ™ P c o (torr) k o b s ( e ' l ) * 1 ° 2 k o b 9 ( a ' l ) x 10 2 43.6 2.87 2.68 98.1 5.94 5.92 185.3 1.12 1.16 272.5 1.71 1.74 359.7 2.08 2.28 446.9 2.79 2.84 534.1 3.23 3 43 Slope - k c o (416 nm) - 6.05 t o r r ' V l , k c 0 (426 nn) - 6.45 t o r r ' V l kl,2-Me 2l« ~ « • « x 105 M-l a " l (8) In CH 2C1 2, Fe(durene-7/7) (Dclm) (CO). (Dclm) - 0.8 M ^max - 4 1 ° ™ ^max ~ 4 2 5 ™ P c o (torr) k o b , ( a - l ) x 10 3 k a b,(»'l> x 10 3 83.4 1.08 1.11 110.0 1.43 1.53 221.0 2.62 ' 2.61 333.0 4.09 4.53 444.0 5.53 5.81 555.0 7.49 7.51 Slope - k c 0 (410 nm) - 13.2 t o r r ' V 1 , k c 0 (425 nm) - 13.5 t o r r ' V l CO , , , kl,2-Me2Im " 1 1 x 10* N'l a ' 1 (9) In CH 2C1 2, Fe(durena-4/4) (Melm) (CO), [Melm) - 2.1 M Slope - k c o (415 nm) - 19.8 t o r r ' V 1 , k c 0 (425 nm) - 25.4 t o r r ' V l k c o - 1.9 x 106 M'l a ' l Dclm E . k c 0 Determination Oslng Varlad [Dclm] (1) Fe(durene-7/7) (Dclm) (CO) (Heme| -3 x 10' 6 H Two separata experlmenta ware carried out: (a) I n i t i a l (Dclm] - 0.91 H In 4.8 mL aolutlon, waa gradually dilut e d to a tot a l volume of 48.3 mL to give (Dclm) - 0.09 M. (b) To an I n i t i a l (Dclm] - 0.0045 H, waa added aliquots of a Dclm aolutlon of 0.509 H, to give a f i n a l (Dclm] - 0.087 In a to t a l solution volume of 3.82 mL. In both cases 80 mL of CO was added Into the tonometer at the start of the t i t r a t i o n , and P c o corrected for each successive aliquot of toluene added into the tonometer. Values of k O D S/(C0] obtained are plotted for each [Dclm]. at for Fe(Por)(DcIm)(C0) and Fe(Por)(Dclm) apecles. 412 nm 425 nm |DcIm] M k c o (M-1 a" 1) x 10 s k c 0 (M'1 a" 1) x 101 0. 91 9 .89 9, .20 0. 81 9 ,71 9 ,43 0. 63 9 .94 10, .20 0. .43 10. .70 11 .40 0. 27 11. .70 13, .20 0. .16 14. .50 14. .80 0. .090 17. .00 18, .80 0. 087 17 .50 19 .20 0. 063 19, .70 21 .20 0. 049 22 .80 25 .60 0. 025 30, .20 35, .20 0. .013 39 .90 48 .20 0. 0099 47. .10 55, .40 0. 0072 53 .60 62. .10 0. 0045 68. .40 77. ,40 (2) r*(durene-3/3)(DcIm)(C0) 415 nm 425 nm (Dclm] M k c 0 (M"1 s" 1) x 10 7 k c o (IV 1 s" 1) x 0 .94 1 .09 1 .02 0 .83 0 .988 1 .04 0, .64 0, ,956 1 .05 0 .44 0 .941 1 .03 0, .27 0. .904 0, .988 0, .16 0, ,953 1 .04 0, .098 0 ,890 1 .14 0. .086 0. ,890 1, .08 0, .072 0, .913 1 .20 0. 048 0. ,955 1, .30 0, .022 1, ,03 1 .73 0. 010 1. 09 2. 73 0. ,0072 1. 05 2. 94 0. 0056 1. 04 3. 47 0. 0041 0. 975 3. 97 (3) Fa(durena-4/4)(DeIm)(C0) 418 nm 428 nm [Dclm] H kC0 ( M-1 x 1 Q 6 kC0 ( M-1 „-l, , 1.12 2.70 2.94 0.98 2.68 2.97 0.74 2.54 2.82 0.50 2.54 2.66 0.30 2.49 2.58 0.17 2.23 2.59 0.091 2.27 2.73 0.072 2.35 3.22 0.048 2.30 4.24 0.025 2.21 6.23 0.010 1.788 .10.30 0.0078 1.73 12.20 0.0059 1.60 13.60 0.0040 1.38 17.90 t CO Dleaoclatlon R*t« Constant Determination,k _ c o (1) Fe(duren*-4/4)(1.2-Me2Im)(C0). U.2-He2Im] -1 M [Heme] 1 X IO" 6 M Tonometer volume - 347.4 mL, aolutlon volume - 3.95 mL P c - .756.5 torr Vol. CO [CO] x I O - 5 H [CO] x 10" 5 H kobsU' 1) added (mL) before flashing after flashing 416 nm 0.4 0.872 0.972 10.3 0.6 1.31 1.41 14.2 0.8 1.74 1.84 15.8 1.0 2.18 2.28 17.8 1.2 2.62 2.72 21.0 1.6 3.49 3.59 26.7 -CO 1 [CO] after flashing vs. k o b , -> y- intercept - kj^-MejIm - 4.8 s - 1 (2) F*(dur*ns-7/7)(l,2-Me 2Im)(C0) • TMIC; Cling Stopped Flow Apparatus After mixing: [Heme) -3 x 10" 6 M. [1,2-MejIm] - 1.0 M free |C0) -1 x 10'* M, [TMIC] - 1 x 10" 3 M *msx 4 1 1 ™ In (A-Aa) time (sees) slope — -0.05 s" 0. .647 6 24 0. .554 7. .93 0 405 10. 11 0. .315 12. 02 0. .131 16. 12 0 .163 22. .10 kl,2-Me 2Im " 0 0 5 »" (3) Fe(durena -5/5)(l,2-He 2Im)(C0) + TMIC; Using Stoppad Flow Apparatus After mixing: (Heme) -3 x IO" 6 M, |l,2-Me2Im) - 1.0 M free' (CO) -1 x IO' 4 M. [TMIC] - 1 x 10 - 3 M Xmax 4 1 1 n n In (A-AJ time (seca) 0.351 1.41 0.157 1.31 slope - -1.1 s" 1 -0.030 1.50 -0.248 1.69 -0.545 1.95 c o • ° - 9 4 2 2 4 1 k l i 2 . M I n - l . l -1.661 2.88 2 (4) Fa(0EP)(l,2-Me 2Im)(C0) + TMIC; Using Stopped Flow Apparatus After mixing: [Heme) -3 x 1 0 - 6 M. [l,2-Me2Im] - 0.5 M free (CO) -5 x 10' 5 M. [TMIC] - 5 x IO" 4 M ^max 4 0 9 ™ Xmax 4 1 5 ™" In (A-A,,) time (aecs) In (A^-A) time (sees) 1. 105 1 .17 -0. .317 1 .20 1 030 I .37 -0. 553 I. 63 0. 880 1 .63 -0. 711 1. 89 0. 742 1. .86 -0 823 2 .08 0. 599 2 15 -1 .070 2 .54 0. 322 2 54 -1. 347 3. 02 0. 020 3. 06 -1 826 4 08 0. 274 3 .51 --2. .064 4 .55 Slope at 409 nm - -0.6 s - 1 . at 415 nm - -0.52 a - 1 kl,2-Me 2lm " 0 5 6 • Fe(dur*ne-5/5)(DcIm)(CO) + TMIC; Cling Stopped Flow Apparatus After mixing: (Heme] -3 x 10"* M. (Dclm] - 0.3 M free' (CO] -1 x 1 0 - 5 M, (TMIC] - 1 x 10 -* M X n a x 413 nm In (A^-A) 1.34 1.25 1.13 1.02 0.87 0.68 0.400 0.039 -0.371 time (sees) 2.22 2.57 3.02 Slop* a tX , . , 413 na - -0.32 a" 1 , at X,,,, 422 nm - -0.27 a - 1 -CO . ( 6 ) Fe(durene-7/7)(DcIm)(C0) + TMIC; Using Cary spectrometer After mixing: (Heme] -2 x 1 0 - 6 M, (Dclm) - 1.0 H free (CO) -1 x IO" 5 M. (TMIC) - 5 x IO" 5 K In (A-A,,,), Xj,,, 413 nm time (sees) -1.55 0 -1.65 4 slope - -0.021 s - 1 -1.74 8 -1.83 12 -1.95 18 - C O -2.12 26 kl,2-Me,Ia " - ° 0 2 »' -2.31 36 1 -2.65 51 -3.02 71 ro ^ 1 C CO Binding to Fe(durane -4 /4) . " c o Datennlnatlon In Toluene at 20°C (1) Fe(durene-4/4)(C0) + Malta [Heme) ~3 x IO" 6 M, P c 0 -1 ata *a.x " " 3 na *o " 0.660 A„ - 0. 117 A„-A A 0-A A A„-A A-A» log ( ) [Mela] log [Meli A-A,, A-A„ 10 - 5 M 0.521 0.139 0. .404 0.344 -0.463 0.532 -5.27 0.437 0.223 0. .320 0.697 -0. 157 1.06 -4.97 0.362 0.298 0 .245 1.22 0. .085 1.59 -4.80 0.319 0.341 0 .202 1.69 0. .227 2.12 -4.67 0.283 0.377 0 .166 2.26 0, .356 2.65 -4.58 0.260 0.400 0 .143 2.80 0. 447 3.18 -4.50 0.235 0.425 0 .118 3.60 0. .557 3.89 -4.41 Slope — 1.' 18. K - 7.37 x 10' ' M'l. •^ CO - 5.3 x 10 5 M" 1 (2) Fe (durene -4/4) (CO) + Dclm *max " 3 9 3 nn *o " 0.808 A„ - 0. 248 A„-A A--A A A„-A A -\p log ( ) [Dcla] log [Dc: A-A. AA,, IO' 5 M 0.771 0.037 0 .523 0.071 -1. 15 0.217 -5.66 0.725 0.083 0 .477 0.174 -0. 759 0.507 -5.29 0.673 0.135 0 .425 0.318 -0. 498 0.869 -5.06 0.625 0.183 0 .377 0.485 -0. 314 1.30 -4.89 0.588 0.220 0 .340 0.647 -0. 1B9 1.73 -4.76 0.543 0.265 0 .295 0.898 -0. 047 2.24 -4.65 0.511 0.297 0 .263 1.13 0.053 2.82 -4.55 Slope - i . : I. K - 3 .97 x 10 4 M-l. 6.5 x 10 5 M'l (3) Fe(durene-4/4)(C0) + l,2-Ma 2Ia *max " 393 na *o " 0.742 A«, - 0.215 A„-A A c-A A A„-A A-Ao log ( ) [l,2-Me 2In] log [1,2-A * . A-Agj 10" 5 M 0.561 0.181 0. .346 0.523 -2.80 0.362 -4.44 0.502 0.240 0. .287 0.836 -0.078 0.724 -4.14 0.459 0.283 0. .244 1.16 0.064 1.09 -3.96 0.424 0.318 0. 209 1.52 0.182 1.45 -3.84 0.397 0.345 0. .182 1.90 0.278 1.81 -3.74 0.369 0.373 0. 154 2.42 0.384 2.29 -3.64 0.348 0.394 0. 133 2.96 0.472 2.89 -3.54 Slope - 0.85. K - 1.18 x 10 4 M-- 1. K c o - » - 5 X 10 4 M'l ave. Kco - 4.3 x 1° " ro vo oo o o o o o o Ui Ul C> U> »4 M w CN o m o c* O Ul Ul O Ul Ul o o o o o o UJMrJHMO H Ni W 4 U> u) O W M O U< Ui o o o © o o H M K ) M U U WMOUIHUI O M « O N H H P-* r- o O O M OI H Nl t N Nl O U U M S© CO * * rO i> (-• > *— to > • C L * « M 3 « r Ul X *» \ ax r-> *• l °, O ch o ro X a ro • 3 + Dc o ro i o o o o o ON Cf* N t N t OB C CD ul N l U Ul CO O v3 Kl OOOOO M N H P O Nl W 4 C CO UIWOHH OOOOO S) M u y C* r-* m so c * o U O I CO Nl N | r- O O O O M lO C H SO O U J O SO O ON CD ON S0 > o > > 4* *> r-« N^ I — O I NV a O O O o o o U M O K W Ul Ui O Ui ro ON CP* co ON ui so ro o> > 8 . IdT > (V rO OOOOO > o > I 3 -1 ON ON O O O ON O O ON o o so Co N I ui ui r-* Co *> i> ro u> m so ON C* ro *> o o o o o o U» i_j £» j> r> f> ON CO O PO Ui CO O O O r-« Ui O O O O O O O ON ON OI Nl Ni CO H Ul CO Ul Nl N Ui o so o ro CD O O O O O O r- I- r- r-» O O co ON •> ro so ON CO CO CO Nl U l CO O O O O o o O ro j> ON so ro Ul Ul Ul C* CO Ul C y a * I O O O O O O M M M H K O vO Ul h— N J Ul CO U> CO sO CO Oi o o o o o o o h-> ro ro ui ui •> sO fO ON O *> O ro Ni ON *o so ui r- r- r- O O O CD ui O N t £• u> ro ON ui N I N I O Ui ON *> *> o ui > I— r- O O O O W r* OO Ul U p* ro ui ro co so so ON N t U I o O CO > O O o o o o N H O H U Ul C*l U ) H H IS) H r* Ul Ul H 00 Oi > o o o o o o I-* O O N J *> N t CO Ul CO Ul O O * • ON *> N l SO C* I o U M H M O r-» Ul O ON Ui cn co d O J ON Nt O *> O ON O ON B o i 9 w CB a r -3 I O Nl O O ON o e 9 i M H M ifl Ul M CN sO *> • N1 CO Ul CO ON Ui O *> co i- O o o o o 0 « N I U I U M ON Nl SO sO Ul Ul CD N J Ul «0 C* H- O O O J > Ul I—» sO N J i> U» O ON CO Nl ON CN Ui CD - 663 -- 300 -• f t i n sC O C* sD O (N CO CM t£ <T vfi IN I u3 ffv »N d iA O 0> vO O vj N CO r-t O o o o o • o . - i o m o < • O O - 7 O M N >} CO H tS M C ^ IN d o o d d o H O e o i N O CO ^ r-l CO J rH CO r i n n r \ N CN H O O O O O O O C co o vo co i n f i i n f » CN o f t r » O f t O O H H H N N O O O O O O O O CO ( N O 1*1 ^ M l A H CO lA H co i n N M C <c * m O O O O O O O N -3 O tn i n CM i n t o co IN i n H O ^ i n N o> H r i O O O O O rt rt I v i o « N o n o * a m i f l n w n H n vo o vo «j i n H H CN n I 3 H o 4 N co m O D M n N H <C O o * i n « * > N o H rt O O O O O o O s t lO IN N i n (N o o M N o i n N r l CM CM <T CO Cs* O O O O O O O 0> ffi H O O i N 9> <j t-i O r - r"i O so f l 1*1 f l N CN CM H O O O O O O O m i n m <j m r«- i n W C C O H J N H O O O rt rt rt CM O O O O O O O O O H O C i N O i CO iTl s i N <t o <o vC <£ i n i n i n O O O O O O O ^ n i f l s c c o N Oi i n c i co H N n m N co o O O O O O <-H H IN n i n s o O \ C st O (N oo W vC <N lA fs CO H r t tN s t v3 l O l O O 4 O u i CN ps co rs csi i s rt P i s i iC Oi •} O rt CN < H O « r i s i co i n o> O sO O O*1 *n co <n rt O rt r i ^ b o o o o b Nl O o> cy> O co r s «o •<T so O i n csi o O O rt rt CN) fl • < t i n CN i n i n o M» <t CN O*» r s vo rt rt rt o O O O O O O O O o f t o o m co o CM m r - » oo O rt rt rt rt rt O O O O O O i n f t I <N CN \ O rt B CM rt O CN t> • X <-N CM CM ac rt lO W I •s O rt > „ 9 (I Ns « I <0| < < PS CM CO CO CM rt f s n e o r t i f l p i - j r t c - m J (N H O N O O O O O O O \ A CM CM CM CO co r s H IO co m n o> s t rt CM co i n r s o nO O O O O O rt rt M? CM Oi l O f l o i n f l f l CM CM CM rt rt O O O O O O O CM NT <N N? CM CM CM s t eo H 4 S r l m O O rt rt rt CM CM O O O O O O O rt I CM CM \ O -* oi o fN o o «n rtOMAPlrt Nt s* f * f l f l f l O O O O O O O CO O CO o o o CM t** i n rt o i i n rt f s so so so i n «n m O O O O O O O (Iv) At -J1°C, X n a x - 424 nm A^ , - 0.707 A. - 0.332 P ln bulb - 12.4 torr, PjOTAL " 754.4 torr *o-A Ao-A Change l n 0 2 A A o * A-A. log ( ) mono, reading P A-A. A A . (mm) (torr: 0, .654 0.053 0 .322 0.165 -0.784 60.0 0.986 0. 629 0.078 0. ,297 0.263 -0.581 103.6 1.70 0. .600 0.107 0, .268 0.400 -0.399 165.8 2.73 0. 570 0.137 0. ,238 0.576 -0.240 247.0 4.06 0 .547 0.160 0 .215 0.744 -0.128 351.4 5.78 0 .520 0.1B7 0 .188 0.995 -0.002 473.0 7.77 0. .500 0.207 0. .168 1.232 0.091 597.0 9.81 °2 Slope - 0.9. P j / 2 - 7.82 torr (v) At -*6°C. X n a x - 424 nm A„ - 0.880 A. - 0.375 P0 2 l n bulb - 7.2 torr, PxoTAL " 7 4 5 2 t o r r A 0-A AQ-A Change ln 0 2 A Ag-A A-A. log ( ) mono, reading P A-A. A-A. (mm) (torr) 0 .790 0 .090 0. ,415 0. 217 -0. .664 22 ,2 0. 213 0 .750 0. .130 0. .375 0. .347 -0 .460 37. 2 0, ,358 0 .700 0, .180 0, ,325 0. 554 -0 .257 60. .0 0, ,577 0 .650 0. 230 0. 275 0. 836 -0 .078 91. .4 0. 878 0. .610 0. 270 0. 235 1. 149 0. .060 131. .2 1. 261 0, ,570 0. 310 0. 195 1. 590 0. .201 181. 8 1. ,747 0. 535 0. 345 0. 160 2. 160 0. ,334 260. ,2 2. 500 o 2 Slope - 0.9, Pjyj - 1.08 torr log P -0.006 0.231 0.435 0.609 0.762 0.891 0.992 0, 0 2 Aaaoclatlon Rate Constant Determination, k * Fe(durene-7/7)(Dclm)(CO) under C0/02 mixtures *max 4 0 5 °2 0 2 P (torr) k o b s (a - l ) x 10 4 133 .0 5 .73 Slope - 118.0 165 .0 6 .29 208. .1 7. .00 °2 k-DcIm - 9.8 x 272, 5 7. 78 444. 2 9. 50 r - l ."I o o 2 log P -0.671 -0.447 -0.239 -0.056 0.101 0.242 0.398 J Kinetic Determination of K * In Toluene at 20°C (1) Fe(durene-7/7)(DeIoi)(CO)/(02) [Heme] -3 x 10 6 M. (Dclm] -0.1 K P c o added - 85.76 torr, k c o|C0] - 1. 50 x 10 3 a" 1 P (torr) kobs<»' -1) x 10 3 kCO[CO]Aobs 412 nm 425 mi 412 nm 425 nm 4.29 1.39 1.08 15.01 1.33 1.44 1.13 1.10 36.45 1.17 1.24 1.28 1.28 68.61 0.977 1.02 1.54 1.56 100.77 0.810 0.883 1.85 1.80 132.93 0.753 0.776 1.99 2.05 165.09 0.653 0.674 2.30 2.36 207.97 0.615 0.563 2.44 2.82 S l o p e a v e - 6.6 x 10- 3 t o r r " 1 a" 1, °2 KDclm " 5 5 * 10 2 H 1 (2) F«(<hir«n«-3/5)<DcIm)(CO)/(Oj) [Heme] -3 x 10 6 M, (Dclm) -0.1 H P c o added - 43.07 torr. k c o|CO) - 4.10 x 10 3 a" 1 0 2 P (torr) koba(«' 412 nm - 1) x 10 3 425 na i k c o 412 nm |CO)/k„ b s 425 ni 10.77 32.30 53.83 75.36 96.89 161.48 3.51 2.93 2.53 2.24 1.96 1.48 4.16 3.39 2.86 2.33 2.04 1.44 1.14 1.37 1.58 1.79 2.05 2.71 1.18 1.44 1.71 2.10 2.40 2.40 0 2 K (415 nm) - 8.7 x 10 2 M'1. °2 K (425 nu) - 1 .2 x 10 3 H - 1 (3) Fe(durena-V*)<DcIm)(CO)/(0 2) [Heme] -3 x 10* M, (Dclm] -0.9 H P c o added - 43.90 torr, k c o[CO] - 1.10 x 10 3 a" 1 °2 ' kobi(»" 1) * 1 ° 3 k c o ( C O ] A o b , 418 nm 428 nm 418 nm 428 nm 10.97 32.91 54.85 76.79 109.70 153.5B 197.46 1.01 0.994 0.858 0.819 0.707 0.627 0.534 1.34 1.05 0.922 0.814 0.698 0.585 0.523 1.089 1.107 1.282 1. 343 1.556 1.754 2.060 1.082 1. 381 1.573 1.781 2.077 2.479 2.772 °2 02 K (418 nm) - 4.4 x 10 2 M"1, K (428 nm) - 7.5 x 10 2 M 1 °2 « v e - KDcIm - « x 10 2 M-1 (4) Fe(durene-4/4)(MeIm)(C0)/(0 2) (Heme] -3 x 10 s M, (Melm) -1.0 M P c o added - 42.88 torr, k c 0(CO] - 1.75 x 10 3 a ' 1 °2 p ("") "ob .C" 1 ) « 1 ° 3 k C ° [ C 0 ] A o b 9 417 nn 425 nn 417 nm 425 nm 21 .44 1. .45 1. .95 1 .207 1 .210 53 .60 1. .08 1. .39 1 .620 1 .698 85 .76 0, .912 1. .10 1 919 2 .145 117 .92 0. 766 0. 878 2 .285 2 .688 150. .08 0. 622 0. 765 2 .814 3 .085 182. .24 0. 534 0. 635 3 .277 3 .717 °2 0 2 K (417 nm) - 1.06 x 10 3 M"1. K (425 nm) - 1.28 x 10 3 H 1 °2 - 303 -<n ic to o o fN f~- CT1 c\ j O O O H N N *Ti st *3 <1 *3 -sT CO SO v? O CO sO eo t*» "3 s i N H CN <t tn so \ c-i r s o co <—> n r s m f s O O O rt rt vO r s m m <T CO N O* N O O rt rt CN J rt SO /-s o r- rt r» K C I o CO M m vo sS t N O rt csi *n rt CM r-i <t •s €N I o K B •a *sl B a <-s CM ««< O *» u ac IB CM rt O e M * CM * X O CM K rt MO CO *-\ o m -N rt i n K I ^ • 1 !•< • O o o * J p « 6 w *> o CM *0 X o o O so w o m ci tji rt O O Oi d w r i H H O O Oi eo r s so m O W N W s t rt CM n <f »n K Van't Hoff Plot* for CO and Oj Binding to Fe(durene-4/4) (B) Systems (1) Fe(durene-4/4)(DeIm) + CO K ( t o r r - 1 ) ln K + l n 760 T (°C) 1/T (°K) x 10" 3 100.0 62.5 43.5 21.7 11.24 10.77 10.40 9.71 5,5 11.0 20.0 31.9 3.59 3.52 3.41 3.28 Slope Y-lntercept - A H ° / R . 6 . 7 X 1 0 3 A H ° - -9 ± 1 kcal/mol A s" / R.. 5.8 A S ° - -12 ± 3 eu (2) Fe(durene-4/4)(l,2-Me 2Im) + CO K ( t o r r " 1 ) l n K + l n 760 T (°C) 1/T (°K) x 10 - 3 0.93 1.5 2.4 3.7 6.9 6.56 7.04 7.51 7.94 8.56 29.0 20.5 17.0 12.0 4.0 3.31 3.41 3.45 3.51 3.61 Slope - - ^ H ° / R - 6.9 x 1 0 3 A H ° - - 1 4 ± 2 kcal/mol Y-lntercept As' / R - - 1 6 . 3 As° - -32 ± 8 eu (3) Fe(durene-4/4)(Delm) + 0 2 K ( t o r r ' 1 ) l n K + l n 760 T (°C) !/T (°K) x a(0.0072 0.0219 0.0578 0.172 0.279 1.70 2.81 3.78 4.87 5.35 20.0 6.0 - 3.5 -13.0 -17.5 3.41] 3.58 3.71 3.85 3.91 Slope Y-Intercept AH° / R - 7.3 x 10 J A H ° - - 1 4 ± 2 kcal/mol As" / R - - 2 3 . 4 As° - - 4 6 ± 11 eu (4) Fe(durene-4/4)(1.2-Me 2lm) + 0 2 K ( t o r r - 1 ) l n K + l n 760 T (°C) 1/T (°K) x fi[4.1xl0"4 0.00785 0.0193 0.0595 0.128 0.926 -1.17 1.78 2.68 3.81 4.57 6.55 20.0 - 2.5 -13.5 -23.0 -31.0 -46.0 3.41) 3.70 3.85 4.00 4.13 4.42 Slope - - A H ° / R - 7.5 X 10 3 A H ° - -15 ± 2 kcal/mol Y-lntercept - ^ S ° / R - -26.2 As° - -52 ± 13 K i n e t i c a l l y determined at 20.0°C 

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