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Syntheses of derivatized capped iron(II) porphyrin complexes and their interaction with CO and O₂ Tang, Hang 1990

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SYNTHESES OF DERJVATIZED CAPPED IRONfJI) PORPHYRIN COMPLEXES AND THEIR INTERACTION WITH CO AND O, by Hang Tang B. Sc., Peking University, P.R. China, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY LN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1990 © Hang Tang, 1990 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 Vancouver, Canada Date Oct. H, iff0 DE-6 (2/88) Abstract The affinity of hemoglobin for CO relative to 0 2 is decreased compared to simple iron-porphyrin systems mainly due to electronic effects. Hydrogen-bond formation with the His E7 residue of hemoglobin has been revealed to stabilize the bound dioxygen. This thesis describes the development of a series porphyrin models, benzene-4/4 (49a), benzene-5/5 (49b), amidobenzene-4/4 (49c) and amidobenzene-5/5 (49d) capped porphyrins, which serve as simple models to imitate the hydrogen bonding in hemoglobin. The chain linked bis pyrroles 38 were synthesized by Friedel-Crafts acylation of the benzene and nitrobenzene diacid chain derivatives with two equivalents of the (5-unsubstituted pyrrole 36, followed by diborane reduction of the ketonic groups. After the nitro functions were transformed to the acetamides, modification of the ethyl ester functions of 38a, 38b and 39 via the benzylesters 40, the carboxypyrroles 41 and the a-unsubstituted pyrroles 42 afforded the important bis formyl pyrroles 43. The cyanoacrylate protected formyl pyrrole derivatives 44 were subjected to monochlorination at the a-methyl groups followed by condensation with two equivalents of the cc-unsubstituted pyrrole 46 to give dipyrromethane dimers 47. Strong aqueous alkali caused saponification of the two ester groups and deprotection of the formyl functions to give 48. The a-formyl-a'-unsubstituted dipyrromethane dimer, resulting from the thermal decarboxylation of 48, was cyclized to produce the porphyrin 49 in acidic medium. The interactions of CO and 0 2 with the heme derivatives, Fen(porphyrin)(DcIm), have been studied. The CO binding constant of the amide substituted heme is two fold ii larger than that of the non-amide substituted heme. In contrast, the low temperature (-45°C) 02 binding constants of the hemes have shown a 6-8 fold increase from nonamide to amide substituted hemes, probably because of a hydrogen bonding between the amide function and bound dioxygen similar to the hydrogen bonding in hemoglobin. ( C H 2 ) n 49a n=4, R=H n=5. R=H n=4. R=NHC0CH 3 n=5, R=NHCOCH 3 w ( C H 2 ) n - ^ - ( C H 2 ) < A XL, H H 38 X = C 0 2 E t 39 X = C 0 2 E t 40 X = C 0 2 C H 2 P h 41 X = C 0 2 H 42 X=H 43 X=CH0 44 X = C ( H ) = C ( C N ) C 0 2 C H 3 n=4, R=H n=5, R=H n=4, R=N0 2 (38 ) or NHCOCH 3 (39 , 40, 41, 42, 43, 44) n=5, R=N0 2 (38 ) or NHC0CH 3 ( 39 , 40, 41, 42, 43, 44) Y H H X ( C H 2 ) n ^ > ( C H 2 ) n 47 C(H)=C(CN)C0 2 CH;j 48 CHO o n=4, R=H b n=5, R=H c n=4, R=NHC0CH 3 d n=5, R=NHC0CH 3 Y C 0 2 E t C 0 2 H iii Table of Contents Page Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Abbreviations xiii Acknowledgements xv Chapter 1 Introduction 1 1.1.1 The porphyrin macrocycle 1 1.1.2 Iron spin state and geometry 2 1.2 The structure and functions of hemoglobin 3 1.2.1 Hemoglobin structure and cooperativity 3 1.2.2 Role of the distal residues 10 1.3 The nature of 0 2 and bound 0 2 12 1.4 Carbon monoxide binding in hemoproteins 15 1.5 Studies of hemoprotein distal electronic effects with model compounds 16 1.5.1 Requirements for the model compounds 17 1.5.2 Model compounds 19 1.6 Brief overview of porphyrin synthesis 27 iv Chapter 2 Results and discussion of the porphyrin syntheses 32 2.1 Synthetic objective 32 2.2 Synthetic plan 34 2.3 Benzene and nitrobenzene diacid chain derivatives 38 2.4 Chain linked bis pyrroles 49 2.5 Dipyrromethane dimers and porphyrins 61 2.6 Electronic absorption spectra of benzene and amidobenzene capped porphyrins 70 2.7 Proton nmr spectral assignments of benzene and amidobenzene capped porphyrins 78 Chapter 3 Experimental for the porphyrin syntheses 94 3.1 General methods 94 3.2 Nomenclature of porphyrins and their intermediates 95 3.3 Syntheses of benzene and nitrobenzene diacid chain derivatives 96 3.4 Syntheses of benzene, nitrobenzene and amidobenzene bis pyrrole derivatives 114 3.4.1 The bis pyrrole diketones 114 3.4.2 The bis pyrrole ethyl esters 119 3.4.3 The amide bis pyrrole ethyl esters 123 3.4.4 The bis pyrrole benzyl esters 126 3.4.5 The bis formylpyrroles 131 3.4.6 The bis cyanoacrylates 136 3.5 Syntheses of benzene and amidobenzene linked dipyrromethane dimers 141 3.5.1 Preparation of 47 141 v 3.5.2 Hydrolysis of 47 147 3.6 Cyclization to the capped porphyrins 151 3.7 Syntheses of the benzene and amidobenzene-hemin chloride complexes 158 Chapter 4 Interaction of the capped hemes with CO and 0 2 161 4.1 Materials and apparatus 161 4.1.1 General 161 4.1.2 Thermostatting equipment 161 4.1.3 Electronic absorption spectra 162 4.2 Solution preparation 162 4.3 Mathematical analyses 166 4.4 Results 169 4.4.1 Carbon monoxide binding to the five-coordinate hemes: K c o values 169 4.4.2 Dioxygen binding to the five-coordinate hemes, K 0 2 174 4.5 Discussion 178 4.5.1 Carbon monoxide binding: K 0 0 178 4.5.2 Thermodynamic considerations for CO-binding 183 4.5.3 Dioxygen binding 184 References 188 Appendices 196 Appendix I Spectral data for the iron(LT) capped systems 196 Appendix II Raw data for the binding of CO and 0 2 to the capped hemes 198 vi A CO affinity constant (tetennination, K00 in toluene at 20°C 198 B CO affinity constant determination, K00, in toluene at varied temperatures 201 C Van't Hoff plots for CO binding to Fe(benzene-4/4)(DcIm) and Fe(amidobenzene-4/4)(DcIm) systems 205 D 0 2 affinity constant determination, K m , in toluene at -45°C 206 vii List of Tables Table Page 1.1 Properties of the dioxygen moiety 14 1.2 CO & 0 2 binding constants of 4 & 5 in toluene at 20-22°C 23 1.3 Thermodynamic properties for 0 2 binding to cobalt porphyrins 25 1.4 Kinetic rate parameters & equilibrium constants for the binding of CO & 0 2 with basket-handle porphyrins 26 2.1 Comparison of electronic absorption spectral data of the porphyrins in CH2C12 73 4.1 Equilibrium constants for CO binding to the capped hemes compared to those for chelated open hemes in toluene at 20°C 175 4.2 Pm values for dioxygen binding to the capped hemes in toluene 182 4.3 Thermodynamic constants for CO binding 184 viii List of Figures Figure Page 1.1 d-Orbital populations for five- and six-coordinate (d6)Fe(LT) 3 1.2 Tertiary structure surrounding the heme prosthetic group of hemoglobin 4 1.3 Saturation curves for dioxygen binding to hemoglobin and myoglobin 5 1.4 Changes of quaternary structure of hemoglobin on transition from T to R 8 1.5 Proximal environment of deoxy-hemoglobin 8 1.6 Protein residues that comprise the proximal and distal environments of the heme 9 1.7 The environment of the bound 0 2 in hemoglobin, possible hydrogen bonding with the His E7 11 1.8 Molecular orbital diagrams for 0 2 and CO 13 1.9 Formation of a dipyrromethane from pyrrole precursors 28 1.10 Formation of dipyfromethenes from pyrrole precursors 29 1.11 Synthesis of porphyrins from dipyrromethanes 30 1.12 Synthesis of porphyrins from dipyrromethenes 30 2.1 A retrosynthetic analysis of the strapped porphyrin 35 2.2(a) Synthetic scheme for the chain derivatives 40 2.2(b) Synthetic scheme for the chain derivatives 41 2.3 NMR spectra at 7.0-8.0 ppm range 47 2.4 Possible mechanism for the formation of the ketone by-product 49 ix 2.5 Syntheses of chain linked bis pyrrolic intermediates 51 2.6 The alternative procedure for the syntheses of the porphyrins 56 2.7 Scheme for the synthesis of the a-free pyrrole 55 2.8 Transformation of dipyrromethane dimers and cyclization to the porphyrins 63 2.9 Resonance structures of pyrrylcarbinyl cations 62 2.10 Formation of a porphyrin via the acid-catalyzed cyclization of dipyrromethanes 69 2.11 Trends in visible spectra for various pyrrole-substituted porphyrins 72 2.12 UV-visible spectrum of the capped benzene-4/4 porphyrin 49a 74 2.13 UV-visible spectrum of the capped benzene-5/5 porphyrin 49b 75 2.14 UV-visible spectrum of the capped amidobenzene-4/4 porphyrin 49c 76 2.15 UV-visible spectrum of the capped amidobenzene-5/5 porphyrin 49d 77 2.16 'H-nmr spectrum of the benzene-4/4 capped porphyrin. 49a 79 2.17 'H-nmr spectrum of the benzene-5/5 capped porphyrin 49b 80 2.18 'H-nmr spectrum of the amidobenzene-4/4 capped porphyrin 49c 81 2.19 'H-nmr spectrum of the amidobenzene-5/5 capped porphyrin 49d 82 2.20 Etioporphyrin II 78 2.21 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin 49a with simultaneous irradiation at 8 4.07 and 8 4.00 85 2.22 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin 49a with simultaneous irradiation at 8 3.86 and 8 1.88 86 2.23 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin 49a with simultaneous irradiation at 8 1.44 and 8 1.24 87 x 2.24 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin 49a with simultaneous irradiation at 8 0.88 and 8 0.13 88 2.25 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin 49a with simultaneous irradiation at 8 0.00 89 2.26 The first-order assignment for the chain methylene protons of the 5/5-benzene capped porphyrin (49b) 92 2.27 The first-order assignment for the chain methylene protons of the 5/5-amidobenzene capped porphyrin (49d) 92 4.1 Thermostatted cell-holder for temperatures above 4°C 163 4.2 6 cm Path-length quartz cell 164 4.3(A) Apparatus used for reducing Fem(Por) to Fen(Por) using aqueous dithionite 165 4.3(B) Tonometer for measuring ligand binding constants of five-coordinate hemes, Fen(Por)(DcIm) 165 4.4 Vacuum line set-up used for preparing and admitting CO/Ar mixtures into a tonometer 170 4.5 Spectral trends for Fen(benzene-4/4), Fen(benzene-4/4)(DcIm) and Fen(benzene-4/4)(DcIm)(CO) systems in toluene 172 4.6 Isosbestic spectral changes for: Fen(Por)(DcIm) + CO Fen(Por)(DcIm)(CO) 173 4.7 Hill plot for Fen(benzene-4/4)(DcIm) + CO ^ s Fen(benzene-4/4)(DcIm)(CO) at 20°C 174 4.8 Van't Hoff plot for: Fen(benzene-4/4)(DcIm) + CO ^ Fen(benzene-4/4)(DcIm)(CO) 176 4.9 Van't Hoff plot for: Fen(amidobenzene-4/4)(DcIm) + CO v xi FeD(amidobenzene-4/4)(DcIm)(CO) 176 4.10 Spectral trends for Fen(benzene-5/5)(DcIm), Fen(benzene-5/5)(DcIm)(02) and pv-oxo-dimer of the Fen(benzene-5/5)(DcIm) systems in toluene 179 4.11 Isosbestic spectral changes for: Fen(Por)(DcIm) + 0 2 v. -Fe^ PbrXDcImJCOj) at -45°C in toluene 180 4.12 Hill plot for Fen(benzene-4/4)(DcIm) + 0 2 N Fen(benzene-4/4)(DcIm)(02) at -45°C in toluene 181 4.13(A) "Basket-shaped" conformation of the distorted durene-5/5 and -4/4 systems 182 4.13(B) "Squashed" conformation of the benzene-5/5 system 182 xii List of Abbreviations arm atmosphere B base Dclm 1,5-dicyclohexylimidazole diglyme dimethylether of diethylene glycol DMF N,N-dimethylformamide DMSO dimethyl sulfoxide DPG 2,3-diphosphoglycerate ESR electron spin resonance FAB fast atom bombardment Hb hemoglobin Hb02 oxyhemoglobin His histidine 'H-nmr proton nuclear magnetic resonance LHP inositol hexaphosphate KB dissociation rate constant k™ association rate constant L ligand: OO or 0 2 Mb myoglobin Mb02 oxymyoglobin Melm 1-methylimidazole MO molecular orbital xiii M. P. melting point pyrrole nitrogens Por porphyrin t-bu tert-butyl TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMS tetramethylsilane TPP meso-tetraphenylporphyrin TpivPP tetra(a,a,a,a-o-pivalamidophenyl)porphyrin Val valine Abbreviations in UV/visible and nmr assignments br broad rise X,(nm) wavelength in nanometers bs broad singlet A absorbance d doublet e extinction coefficient m multiplet sh shoulder q quartet s singlet t triplet xiv Acknowledgements I wish to express my sincere gratitude to Professor David Dolphin for his guidance and encouragement throughout this work. I would like to extend special thanks to Dr. Tilak Wijesekera for his many invaluable suggestions and discussions during the synthetic work, and for his assistance with the preparation of this thesis. Thanks are due to Professor Brian R. James for helpful discussions on binding studies. Finally, I would like to take this opportunity to thank all the members of Professor Dolphin's research group, over the years, for making my stay at U. B. C. a pleasant one. xv Chapter 1 Introduction 1.1.1 The porphyrin macrocycle1 All porphyrins are derived from porphin (1) by substitution of peripheral H atoms with side chains. In the Fischer system of nomenclature, the peripheral positions are COOH numbered 1 - 8 and the methine (meso) positions are designated a, p\ y and 8. Various substitution patterns at the periphery give rise to several naturally occurring porphyrins. The iron complex of protoporphyrin-LX (2) is the prosthetic group of hemoglobin, myoglobin, peroxidases, oxidases and many cytochromes.2 The heterocyclic ring of a porphyrin contains 24 atoms forming a highly aromatic system with 18 conjugated % electrons. Unlike smaller aromatics, however, highly distorted porphyrin systems do exist3 Skeletal distortion occurs in two ways: ruffling and doming. Doming of a porphyrin occurs when all of four pyrrole nitrogens are out of the porphyrin plane on the same side. Ruffling of a porphyrin results when opposite 1 pyrrole nitrogens are out of the porphyrin plane on the same side and adjacent pyrrole nitrogens are out of the plane on the opposite sides. These deformations tend to mimmize angular bond strain at a, p\ y and 8 meso positions. However, there is little loss in aromatic stabilization since individual pyrrole rings tend to remain planar. The two protons attached to the nitrogens in a porphyrin are readily ionizable resulting in the porphyrin dianion. These dianions are very stable tetradentate ligands towards a variety of metal ions. Due to the high degree of electron delocalization, both porphyrins and met^oporphyrins are highly colored compounds with characteristic spectra in the UV/visible range. 1.1.2 Iron spin state and geometry4 The neutral four-coordinate Fe(H)-porphyrin moiety can coordinate a variety of ligands in the remaining two axial positions. The ligands include species bearing lone pair electrons for donation to the metal center. The ferrous ion contains six d-electrons which can populate the orbitals as shown in Fig. 1.1. It can exhibit three spin states: S = 0, low spin; S = 1, intermediate spin; and S = 2, high spin state. The spin state and stereochemistry of the iron(LI) center is controlled almost entirely by the nature and number of axial ligands. The coordination of strong field ligands such as imidazole, CO and 0 2 leads to low spin six-coordinate hemes. Weaker field ligands, typically anionic ones such as chloride and azide, lead to five-coordinate high spin derivatives. The d-orbital populations for the five and six-coordinate states seen in hemoproteins are shown in Fig. 1.1. When ferrous heme is coordinated by only one imidazole, it is high spin and out of plane towards the imidazole yielding a roughly square pyramidal bonding geometry. When coordinated by two imidazoles or an imidazole and CO, the iron is low spin and located much closer to or within the 2 porphyrin plane to form an octahedral type complex. 2 2 2 1_ _1_ xz ,yz xy 5 * x y , x z , Coordinat ion n u m b e r : F ive 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 Fig. 1.1 d-orbital populations for five and six-coordinate (d6)Fe(LI) 1.2 The structure and functions of hemoglobin 1.2.1 Hemoglobin structure and cooperativity Hemoglobins are essential to the life of all vertebrates; they also occur in some invertebrates and in the root nodules of leguminous plants. The main task of hemoglobin is to convey oxygen from the lungs to the tissues and facilitate the return of carbon dioxide from the tissues back to the lungs. Another hemoprotein in the muscle, monomelic myoglobin, combines with the oxygen released by hemoglobin, stores it and transports it to mitochondria, where the oxygen generates chemical energy by the "combustion" of glucose to carbon dioxide and water. Hemoglobin is a tetramer of molecular weight 64,500 and consists of four polypeptide chains; two a chains each containing 141 amino acid residues and two p4 chains each containing 146 amino acid residues. Each chain has a prosthetic heme 3 group (the active site), iron(LT) protoporphyrin LX. The a and P chains (subunits) have different sequences of amino acids but fold up to form similar three-dimensional structures. Each a subunit is linked to a P unit to give two sets of dimers, aipi and cx2P2. These dimers are in turn held together so as to form cxip2 and a2pi contacts which make up the overall quaternary structure of the tetramer.5 Fig. 1.2 Tertiary structure surrounding the heme prosthetic group of hemoglobin subunits5 The hemes in Hb are held separately in protein pockets formed by several helical and non helical segments (Fig. 1.2). Helices E and F constitute the "distal" and "proximal" sides of the heme, respectively. The iron of the heme is 5-coordinated to the four porphyrin nitrogens (ISL^J and N £ of the proximal histidine F8 which is the only covalent linkage between the heme and the protein. The porphyrin is in van der Waals contact with another histidine on the distal side (E7) and also makes contact 4 with 18 other ajmno acid side chains, most of which are nonpolar. The oxygen transport property of hemoglobin is dependent upon the ability of its ferrous iron binding 0 2 reversibly instead of being irreversibly oxidized. Hemoglobin binds oxygen cooperatively5 with a free energy of cooperativity of 3.5 kcal/mol under physiological conditions, this corresponds to the rise in 0 2 affinity between zero and full oxygen saturation being about 500-fold.6 The cooperative binding of dioxygen by hemoglobin is manifested by its sigmoidal equilibrium curve7 (Fig. 1.3). It ensures uptake and release of dioxygen over Fig. 1.3 Saturation curves for dioxygen binding to hemoglobin and myoglobin the comparatively narrow range of partial 0 2 pressures that distinguish the lungs (Po2 -100 mm Hg) from the tissues (POj -30-40 mm Hg). The oxygen equilibrium curve of single chain hemoglobin, such as the one found in myoglobin, is hyperbolic.7 The oxygen affinity of hemoglobin rises with increasing oxygen binding so that a sigmoidal oxygen equilibrium curve results. Initially, hemoglobin is reluctant to take up 5 oxygen, but its affinity increases with oxygen uptake. At arterial oxygen pressure, both myoglobin and hemoglobin are nearly saturated. But at venous pressure, myoglobin gives up only about 10 percent of its oxygen, whereas hemoglobin releases roughly half. At any partial pressure of 02, myoglobin has a higher 0 2 affinity than hemoglobin, which allows oxygen to be transferred from the blood to the muscle. Studies on ligation to myoglobin7 indicate a first order dependence on oxygen K02 Mb Mb + 0 2 s N Mb02 concentration, where the equilibrium constant is defined by: [MbOJ K0 2 _ Mb -[Mb][OJ The sigmoidal curve of hemoglobin7 bearing four active sites indicates a greater than first order dependence on oxygen concentration, Kai Hb (Hb)a + n02 v > (HbCU With the affinity constant defined by: [HbOJ,, V-02 _ ^Hb -tHb]B[OJ" Hill's equation fits the experiment best for a Hill's constant n -2.7. 6 The oxygen affinity of hemoglobin is also modulated by protons, chloride ions, 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (MP), known collectively as the heterotropic ligands.8,9 Increasing the concentration of any one of the heterotropic ligands shifts the oxygen equilibrium curve of the hemoglobin to the right, towards lower oxygen affinity, and a more sigmoidal oxygen equilibrium curve results. The cooperative effects of hemoglobin are due to an equilibrium between two alternative quaternary structures of the tetramer, the deoxy or T and the oxy or R structure. Owing to the ingenious and laborious work of Kendrew et al.10 and Perutz,5 we now have clear structural pictures of hemoglobin and myoglobin. Crystallographic analyses of deoxy- and oxyhemoglobin have revealed differences in their structures and therefore made it possible to investigate the mechanism of cooperativity. The arrangement of the subunits (quaternary structure) and the conformation of the subunits (tertiary structure) of R and T states have been found to be different. The quaternary T—»R transition consists of a rotation of the dimer a2|32 relative to cel01 by 12 - 15° and a translation of one dimer relative to the other by 0.8 A 1 1 (Fig. 1.4). In deoxyhemoglobin,12 the iron is high-spin ferrous (S = 2) and five-coordinate. The iron atoms of the a and |3 hemes are displaced from the mean planes of resulting in the doming of the porphyrins towards the proximal histidine F8. The imidazole ring of the proximal histidine F8 is in an asymmetric position with respect to the porphyrin nitrogens of the heme (Fig. 1.5).11,12 Quantitative energy calculations13 have indicated that the steric repulsion between the asymmetric axial imidazole and the porphyrin ring causes the doming of Fe towards the proximal imidazole. Studies by crystallography14,15 have revealed that the tight packing of the heme and the intersubunit contacts inhibit the iron from assuming symmetrical R-like coordination and the heme from becoming planar. However, there is little strain on the heme in deoxyhemoglobin. 7 Fig. 1.4 Changes of quaternary structure of hemoglobin on transition from T (full lines) to R (broken lines)**0 p Fig. 1.5 Proximal environment of deoxy-hemoglobin, the distances are measured in A 6 8 Fig. 1.6 Protein residues that comprise the proximal and distal environments of the heme13<,) On the distal side of the (3 subunit, valine E l l , histidine E7, and phenylalanine CD4 residues are in close proximity to the heme, and occupy the space that would be required for a sixth ligand to coordinate (Fig. 1.6). In the relaxed (R) oxyhemoglobin,16 the iron atoms become low-spin (S = 0) and six-coordinate. The heme group appears to be nearly planar in the a subunit but ruffled in the (3 subunit, and the iron atom has moved towards the porphyrin plane. As a result the proximal histidine comes closer to the porphyrin plane in oxy- than in deoxyhemoglobin. The position of His F8 relative to the heme becomes more symmetric, the geometry of which reduces steric repulsions between the imidazole and porphyrin nitrogens.11 On the distal side of the P subunit, the Val E l l side chain 9 whose presence hinders ligand binding in deoxyhemoglobin has shifted away. 1.2.2 Role of the distal residues Free ferrous porphyrins are rapidly oxidized by oxygen, and their affinity for oxygen is several thousand times less than for carbon monoxide. Globin keeps iron from being oxidized, which is necessary because only ferrous iron combines reversibly with oxygen, and also discriminates in favor of oxygen against carbon monoxide. Stabilization of the bound dioxygen moiety via hydrogen-bonding with the His E7 residue was first suggested in view of the electronic picture of an end-on coordinated geometry in the distal pocket by Pauling.17 Infrared18 and resonance Raman19 appear to support this hypothesis indirectly. Kinetic studies20 of the 02, CO and isocyanide binding properties of a variety of vertebrate and invertebrate heme proteins have indicated that the distal histidine is capable of forming a hydrogen bond with bound dioxygen molecules. The strength of this hydrogen bond was estimated to be 2 and 1 kcal/mol for mammalian myoglobin and hemoglobin, respectively. ESR spectra21"23 of some myoglobins and hemoglobins have also provided more direct evidence for such H-bonding. The first direct evidence of the H-bonding was provided by a neutron diffraction study of oxymyoglobin that yielded the N—02 distance of 2.97 A 2 4 (Fig. 1.7). This was followed by X-ray analysis of oxyhemoglobin which gave the N—02 distances of 2.7 and 3.2-3.4 A for the a and P subunits, respectively.16,25 Therefore, in the a subunits of hemoglobin, NE of the distal histidine forms a hydrogen bond with the terminal oxygen atom and stabilizes 0 2 bound to the heme iron. In the P subunit, the distal histidine Ne-H is further away and equidistant from both O atoms suggesting a weaker interaction with either or both O atoms. The absence of such H-bonding to the 10 Fig. 1.7 The environment of the bound 0 2 in hemoglobin, possible hydrogen bonding with the His E7 oxygen in CO of carbonmonoxyhemoglobin is consistent with electroneutrality of the Fe-C-0 unit.26 Olson et al?1 have replaced the distal histidines in myoglobin and hemoglobin by glycine, which opened an access to the heme pocket. It left the oxygen affinity and kinetic constants of the P-subunits unchanged, but diminished the oxygen affinity of the cx-subunits 8-fold. The reduction in the oxygen affinity was brought about by a 60-fold increase in the dissociation (off) rate that more than compensates the 10-fold increase in associate (on) rate due to the opening of the heme pocket. Thus, it has confirmed further that in both myoglobin and a hemoglobin, His E7 stabilizes bound 0 2 by H-bonding. In synthetic model compounds which offer no steric hindrance to the ligands, oxygen binds with an Fe-O-O angle of 120°, while CO lies on the heme axis perpendicular to the heme plane. The heme pockets of myoglobin and hemoglobin seem to be tailored to accommodate the bent oxygen and force the CO off the heme axis, which might have accounted for their low CO affinity. However, X-ray crystallographic study of human carbonmonoxyhemoglobin at 2.2 - 2.3 A resolution28 11 has shown a very small inclination, but the porphyrins are ruffled. X-ray analysis29 of a synthetic "hindered pocket" iron porphyrin having a lower CO affinity than that of the unhindered "picket fence" iron porphyrin shows similar geometry. Fe-C-O is slighdy but detectably bent and tilted off the axis normal to the porphyrin plane. This modest distortion of the carbonyl unit is accompanied by considerable ruffling of the porphyrin periphery. It may be concluded from these experimental data that in both the "hindered pocket" porphyrin and in hemoglobin, the strain energy responsible for the low CO affinity is from a combination of structural changes involving both ligand distortions and overall porphyrin skeletal deformations. Springer et al.30 have studied the protection of the heme iron from oxidation by replacing the distal histidine in sperm-whale myoglobin by ten different amino acid residues. The replacements reduced the oxygen affinity and accelerated autoxidation. Usually oxidation occurs in the fraction of molecules which are deoxygenated. The faster autoxidation is therefore partly attributed to the decreased oxygen affinity. Furthermore, Perutz31 has suggested that the distal histidine protects the ferrous heme iron by acting as a proton trap since autoxidation is catalysed by protons. At neutral pH, the distal histidine is protonated only at N 5 which faces the solvent (Fig. 1.7). Any proton entering the heme pocket of deoxymyoglobin would be bound by NE, and simultaneously N 8 would release its proton to the solvent. When the histidine side-chain swings out of the heme pocket, the protons would interchange, restoring the previous state. 1.3 The nature of 0 2 and bound 0 2 Molecular oxygen is a paramagnetic molecule with a formal bond order of two. 12 It has a triplet ground state with two unpaired electrons. This is explained readily by simple molecular orbital theory (Fig. 1.8A). The MO description for molecular dioxygen shows vacancies for the addition of a single electron in both of the antibonding 2p %\ orbitals. The addition of one or two electrons to a neutral dioxygen molecule results in the formation of the superoxide (02) and peroxide (O )^ anions respectively, leaving superoxide with a bond order of 1.5 and peroxide O-O link a bond order of one. The reduction in bond order is associated with an increase in 0-0 bond length and a decrease in the stretching frequency, tVo, all of which are useful guides to the assessment of the properties of 02-metal complexes (Table 1.1). The nature of iron-oxygen bonding in hemoglobin has been the subject of much discussion. There have been three major proposals on the electronic nature of the 02-a molecule molecule Fig. 1.8 Molecular orbital diagrams for 0 2 (A) and CO (B) 13 Table 1.1 Properties of the dioxygen moiety S p e c i e s Bond Compound 0-0 - Bond P(0-0) Order D i s t a n c e E n ergy cm (A) ( k c a l / m o l ) 0 2 + 2.5 0 2 A s F 6 1.123 1-49.4 1858 0 2 2 0 2 1.207 117.2 1554.7 02- 1.5 K0 2 1.28 — 1145 0 2 2" 1 Na 20 2 1.49 48.8 842 metal moiety. Pauling's model17 is as follows: approach of dioxygen to the Fe center leads to spin pairing of the electrons in the TC* orbitals, following their loss of degeneracy. The Fe-02 bond then involves o donation from dioxygen into empty d/ orbital of Fe, which is balanced by d-TC back donation from Fe into the antibonding molecular orbitals of 02. This model postulates low-spin Fe(II) with neutral dioxygen, which must be in a singlet state to account for the overall diamagnetism of the system.32 In contrast, Weiss33 has put forward a scheme in which electron transfers to dioxygen from iron has taken place, giving Fe3+(02")- The diamagnetism then results from strong antiferromagnetic interaction between low-spin iron(UI) and superoxide ion. Infrared1800 and flash photolysis34 have shown support for this Fe(HI)-superoxide species. X-ray fluorescence has also pointed to the presence of unpaired electron density on the iron atom.35 Another molecular orbital calculation favors a model based on the electronic structure of ozone.36 It suggests that the similarity of the infrared 0-0 stretching 14 frequencies of Mb02 and superoxide anion, used as evidence for the Weiss' model, may be coincidental, and there is littie electron transfer from Fe to 02. In this model, low spin Fe(II) is considered to exist in an excited state while dioxygen retains a triplet configuration. Excitation of one electron from the d^ into cL? orbital of Fe(LI), pointing towards the triplet dioxygen, leads to a o bond, while the interaction between the remaining electron in the d^ orbital and TC-system of the 0 2 forrriing a three center, four electron TC bond. This model is consistent with a diamagnetic Fe-02. The facile formation and dissociation of the Fe-02 bond is easily rationalized in this model since the 0 2 always retains its triplet ground state character in the system. The high resolution 170-nmr spectrum of the Fe02 linkage of a dioxygen-imidazole-iron(d6) porphyrin complex37 has given a spectrum pattern similar to ozone.38 The crystal structures of MbOj39 and Hb0216 have shown that the Fe-O-0 has a bent, end-on geometry, as predicted by Pauling.17 The angle of Fe-O-0 is 115° in oxymyoglobin, 153° in a oxyhemoglobin and 159° in the P oxyhemoglobin. The oxygen molecule forms a hydrogen bond with N£ of His E7 in the a, but either none or a weak one in the P subunit 1.4 Carbon monoxide binding in hemoproteins Carbon monoxide with a bond order of three (Fig. 1.8B) binds with a ferrous heme with much higher affinity than oxygen. The bonding of CO to ferrous ion can be regarded as follows:40 overlap of a filled carbon o-type orbital on the metal atom is enhanced by TC back donation from Fe to CO, which is maximized at an angle of 180°. Thus, the resultant Fe-C-0 unit is linear and with near electrical neutrality. Whereas CO is bound to ferrous ion and simple porphyrin Fe(U) in a linear 15 manner,41 the crystal structure determination has indicated that the CO lies off the heme axis in both a and (3 subunits of carbonmonoxyhemoglobin and myoglobin.26,42, 4 3 This has implied the distal residues as the source of the distortion. 1.5 Studies of hemoprotein distal electronic effects with model compounds Although the reversible binding of 0 2 and CO to heme proteins occurs at the iron atom, the steric and electronic controls provided by the protein play major roles. The crystal structure determinations of various hemoglobins (oxy or deoxy) and myoglobins (oxy or deoxy) have shown that the most important changes on ligand binding involve the changes in the immediate vicinity of the heme (Section 1.2). In the aspect of distal side effects, the steric obstruction may serve to lower the affinity for the sixth ligand (e.g. CO),42 and hydrogen bonding24 between dioxygen and the distal residue may electronically stabilize the 02-complex. On the proximal side, non-bonded globin constrains can prevent the required movement of the iron into the porphyrin ring on ligand binding.13"15 In addition, electronic interactions (hydrophobic or polar) between the porphyrin ring and protein residues in close proximity could alter electron density of the heme moiety.44 Heme proteins have complex structures and their dynamic equilibrium studies are extremely complicated due to the influence of both tertiary and quaternary changes on ligand binding. Theoretical studies of CO and 0 2 binding to myoglobin45 have indicated that approach of a ligand to the heme is controlled by successive barriers, in addition to the most inner barrier of Fe-ligand bond formation. In order to evaluate the influence of the immediate protein environment (heme pocket) on the ligand binding, it is necessary to construct models with the minimum of added molecular structure necessary 16 to mimic the active site behavior of a hemoprotein. These synthetic iron-containing oxygen carriers are more readily characterized than the hemoproteins. A more accurate X-ray crystal structure can be obtained than for the much larger, more complicated hemoprotein. By designing different model compounds, it is possible to investigate separately the effects of the heme environment on heme binding reactivity.46 1.5.1 Requirements for the model compounds46 A synthetic model compound contains an active heme site in which a ferrous atom is bound to give a complex which is then able to combine reversibly with dioxygen. But, difficulty arises with obtaining porphinatoiron-dioxygen complexes. Simple ferrous porphyrins, in the absence of large excess of base B, undergo rapid and irreversible oxidation by molecular oxygen to yield the (i-oxo dimer: Fen(Por)(B)2 % Fen(Por)(B) + B Fen(Por)(B) + 0 2 v ^ Fe(Por)(B)(02) Fe(Por)(B)(02) + Fen(Por)(B) k s (B)(Por)Fera-02-Fem(Por)(B) rapid (B)(Por)Fe-02-Fe(Por)(B) > 2FeIV(Por)(B)(02-) rapid FeIV(Por)(B)(02-) + Fen(Por)(B) > (Por)Fem-0-Fem(Por) + 2B Autoxidation proceeds via formation of the monomelic iron-dioxygen adduct Fe(Por)(B)(02), followed by formation of a |i-peroxo-dimer, (B)(Por)Fe-02-Fe(Por)(B). Then, experimental data indicate that this peroxo-bridged complex decomposes via the formation of a presumed ferryl, Fe(IV)(Por)(B)(02") intermediate to give the p>oxo 17 dimer.46,47 This reaction does not occur in hemoproteins because the polypeptide chain surrounding the heme prevents the close approach of two hemes and the subsequent oxidation. Another major difficulty encountered is that the simple ferrous porphyrins in solution, in the presence of strongly coordinating N-donor ligands, tend to form a six-coordinate complex: K K Fen(Por) + B v ' s Fen(Por)(B) + B ^ ' » Fen(Por)(B)2 Often in such systems K 2 > K 1 } making Fen(Por)(B)2 the dominant species in solution. This can be largely understood in terms of the ligand field stabilization energy that results from the spin state change in going from 5-coordinate high spin to 6-coqrdinate low spin. In order to overcome these difficulties, three strategies have been devised: (1) steric - in this case the dimerization and 6-coordination of the base are inhibited; (2) low temperature - to slow down the reactions leading to dimerization; (3) rigid surfaces - iron-porphyrins in this case are attached to the surface (polymer) in such a way as to prevent dimerization and six-coordination. Several hemoglobins and myoglobins are known to undergo autoxidation reactions promoted by anions. This autoxidation is known to proceed in the presence of protons.48 Formation of superoxide ion (or the hydroperoxyl radical H0 2) has been implicated in this oxidation. In a recent 170-nmr study49 of a "basket handle" porphinatoiron dioxygen adduct, H 2 1 7 0 and H 2 1 7 0 2 species have been detected in the autoxidation process, strongly supporting the implication of superoxide (or the hydroperoxyl radical H0 2) in the reaction. The hydrophobic environment for the heme center in hemoglobin and myoglobin is very important to prevent this kind of 18 autoxidation. Therefore, experiments with model systems of heme oxygen carriers are generally conducted in nonaqueous media. 1.5.2 Model compounds Continued efforts to overcome the problems associated with synthetic oxygen carriers have led to the development of various elaborate model hemes. These model compounds with different structures have provided much information about the effects of the protein environment on ligand binding. The mechanism of ligand binding by heme proteins has been established based partly on model studies. This section mainly deals with the model studies relevant to distal electronic effects - hydrogen bonding between bound dioxygen and His E7 residue. A detailed discussion about the distal steric effect of the hemoproteins is available.50 CO and 0 2 binding experiments of heme proteins and synthetic analogues have indicated the importance of the local polarity at the binding site to the differential binding of CO and 0 2. Direct evidence such as from neutron diffraction and X-ray analysis studies (Section 1.2.2) of oxymyoglobin and oxyhemoglobin has left little doubt that heme-bound dioxygen has a tendency to form a hydrogen bond with His E7. However, quantitative estimations of the energetic contribution of this interaction to the overall stability of the metal-oxygen complex still depend on the model binding studies. By using a cobalt salicylidenimine complex, Drago et al.51 observed a 400 fold increase in oxygen affinity when trifluoroethanol was added to a CH2C12 solution, suggesting intermolecular hydrogen bonding. More sophisticated porphyrin models53, 5 4 1 5 7 1 5 8 , 6 0 with amide substitution have exhibited intramolecular hydrogen bonding quite similar to natural heme proteins, however, in no cases has such hydrogen bonding been confirmed by X-ray structural analysis. 19 (i) Picket-Fence systems Synthetic analogues of dioxygen-carrying complexes with amide substituents in the distal protected pocket have the potential to form hydrogen bonds between the NH and bound dioxygen. One of them, the "picket fence" porphyrin: tetra(a,cc,a,a-o-pivalarmdophenyl)porphinatoiron (3)s was the first synthetic model capable of binding dioxygen reversibly at room temperature. This compound has steric bulkiness 3 TpivPP a M=2H b M=Fe constructed with the pivalamido groups on one side of the porphyrin plane and leaves the other side unencumbered. An imidazole ligand is allowed to coordinate to the unhindered side of the porphinatoiron, while the other side remains as a pocket for oxygen-binding. Moreover, the fence discourages dimerization to a p>oxo dimer. Since this compound has polar amide substituents in the distal pocket, much work has been carried out to investigate the possible interactions between coordinated dioxygen and carbon monoxide with the amides. Single crystal X-ray analysis data53,54 have shown a suitable geometry for such hydrogen-bonding. The terminal oxygen atom and the N-H group point towards each other. The shorter N(H)-0 separations of 3.88 20 and 4.19 A in the picket fence Cycomplex can be compared to typical values of 2.94 A for primary amides55 and 2.85 A for secondary amides56 where strong hydrogen-bonding interactions exist. The separations in the model Cyadduct are -1 A longer, suggesting a weaker hydrogen bond. Calculation54 has suggested significant enthalpic stabilization due to this hydrogen bond. ESR studies22 of mono-ortho and para acetamido derivatives of cobalt(II)TPP [(o-NHCOCH3)TPPCo and (p-NHCOCH3)TPPCo], in the presence of N-methylimidazole and molecular oxygen, have provided information concerning the dynamics of bound oxygen: when hydrogen bonding is possible (from the N-H of the ortho acetamide), the ESR spectra of the dioxygen adduct, like oxymyoglobin, are not motionally averaged as they are for the para acetamide. The motion of the (p-NHCOCH3)TPPCo as shown in the averaging of the ESR signals in solution originates from the rotation of the dioxygen moiety about the Co-0 bond in the absence of a hydrogen bond. (ii) Substituted "chelated" porphyrins Chang and co-workers57 have made a model system consisting of a series of 5-coordinate heme compounds (4) differing principally in the dipole moment of the R R 4a R=H R'=t-Bu / oo 4b R=CH2OMe R'=H HN R'=H R'=H R'=H R'=H R'=H 21 phenyl substituents above the 0 2 binding site. Binding studies on these complexes revealed that the polar groups in near proximity to the binding site of the CO or 02, dramatically affect for 0 2 but not for CO (CO binding kinetics appear to be only affected by steric interactions). From Table 1.2, it is apparent that compound 4e with a secondary amide substituent has a much stronger 02-affinity than the other hemes. Its 0 2 dissociation rate is much slower than for the other hemes due to H-bonding. The other two compounds 5a and 5 b have a polar acetamido group either on the same or the opposite side of the OJCO binding site. The binding data suggested that enhanced 0 2 affinity of 5b relative to 5a must be due to a favorable interaction of the Fe-OO and amide dipoles. It was concluded that this enhanced affinity is derived from a non-bonding dipole-dipole interaction and not from direct hydrogen bonding (too distant for effective H-bonding compared with a typical peptide H-bonding distance of (2.8 - 3 A). 5a R=H R'=NHCOCH3 5b R=NHCOCH3 R'=H Co11 1-naphthyl porphyrins (6)58 substituted with amido, carboxy and hydroxyl 6a 6b 6c 6d R=H R=C02H R=CONH2 R=CH2OH 22 Table 1.2 CO & 0 2 binding constants of 4 & 5 in toluene at 20 - 22°C 5 7 k01 PQ2 r 1/2 kco pco r 1/2 Compound M V s-1 torr M V s1 torr 4a 4.7xl07 15,500 33 2.5x10s 0.14 0.0057 4b 2.6xl07 9,900 38 l.lxlO 6 0.028 0.0075 4c 2.3xl07 2,900 13 1.2x10s 0.090 0.0075 4d 2.2xl07 11,000 48 1.5x10s 0.13 0.0086 4e 1.3xl07 300 2.3 1.3x10s 0.042 0.0032 4f 1.4xl07 4,750 34 0.59x10s 0.048 0.0081 4g 1.3xl07 9,300 72 0.47x10s 0.053 0.011 5a 3xl07 38,000 126 2.3x10s 0.12 0.0054 5b 2.6xl07 4,100 16 1.6x10s 0.072 0.0045 groups at the 8-naphthyl positions display significant enhancements in 0 2 affinity which correlates well with H-bond strength (Table 1.3). The data clearly demonstrate that the presence of a protic group near the dioxygen binding site drastically increases the Co-0 2 formation constant, achieving a free energy gain between 3 kcal/mol for 6b and 1.1 kcal/mol for 6d, with reference to 6a in DMF at -42°C. The enthalpy change for these intramolecular H-bonded dioxygen complex formations also increases significantly. Compared to literature thermodynamic parameters of hydrogen-bonding,59 the large gain in enthalpy for the models is attributed to the intramolecular H-bond. The large negative entropy is also consistent with the loss of rotational degrees of freedom of the metal-bound 0 2 due to the hydrogen bonding. (iii) "Basket Handle" models Momenteau et a/.60 were the first to show the dramatic differences in oxygen binding to a heme equipped with amido groups versus a heme with less polar ether groups on their distal side. These "basket handle" porphyrins contain one strapping group with a pyridine as the axial ligand of the parent porphinatoiron and the other strapping group consists of a simple alkyl chain bridging over the oxygen-binding site. The distal cavity size in both hemes is considered to be very similar with only slight differences in shape owing to the more rigid amide linkages. Two of these "basket handle" porphyrins, 7a and 7b, include the long aliphatic C 1 2 chain which maybe expected to introduce little, if any, steric interference. The CO affinities of these 5-coordinate systems are nearly identical with k*00 and k"00 are simultaneously decreased by a factor of 2. K 0 2 is ten times larger for compound 7b than for compound 7a (Table 1.4). The change of K 0 2 is found to result exclusively from a ten fold reduction of the oxygen dissociation rate in compound 7b. Thus the presence of the NH (amide) 24 Table 1.3 Thermodynamic properties for O z binding to cobalt porphyrins58 Compound Solvent T(°C) Pifl(torr) AH(kcal/mol) AS(e.u.)* 6a DMF -42 21 -11+0.4 -52+2 -30 70 6b DMF -42 0.028 -30 0.73 -22+1 -88+5 0 49 6c DMF -42 0.30 -30 4.0 -16+2 -70+7 0 91 6d DMF -42 2.00 -30 4.9 -13+1 -56+4 0 100 CoMb(sperm whale) 25 57 -13.3 -53 CoMb(horse) 25 57 -11.3 -46 CoHba-SH 15 24.3 -13.9 -54.4 CoHbP-SH 15 24.5 -16.5 -63.8 * The entropy units are J-mol'K"1 group in 7b strongly increases the mtrinsic stability of the oxy derivative. Proton nmr61 spectroscopy has indicated that the bridged chain pulls the amide protons towards the center of the porphyrin as shown by the high field shift (up to 3 ppm) of the corresponding resonances in the Zn(LT) or in the carbon monoxide iron(II) complexes 7a R=(CH2)12 7b R=CO(CH2)10CO relative to aromatic amides. Ring current shift calculations have estimated that the distance between one of the amide nitrogen atom and the terminal O atom in a bent configuration may be close to 3 A, consistent with an intramolecular hydrogen bond. Table 1.4 Kinetic rate parameters & equilibrium constants for the binding of CO & 0 2 with basket-handle porphyrins (Solvent: toluene; temperature: 20°C)60 Heme 7a 7b 10-7xkco (MV1) 6.8 3.5 lOhk™ (s1) 69 30.4 Kr'xK 0 0 (M1) 9.9 11.5 KTxk02 (MV) 30 36 lObdc"02 (s-1) 40 5 lO-'xK02 (M'1) 7.5 70 26 All of the above model studies have provided evidence that the polar functions in the heme distal side can increase the 0 2 affinity greatly, this is possibly due to the hydrogen bonding stabilization of 02-complex. 1.6 Brief overview of porphyrin synthesis There have been several reviews62'65 dealing with porphyrin synthesis. This section gives only a brief summary of the topic. The porphyrin macrocycle can be constructed in essentially three different ways. They are: (i) "one-pot" condensation of monopyrroles6200 (ii) "2+2" addition of dipyrrolic precursors620'' (iii) intramolecular cyclization of linear tetrapyrroles62(c)'65 The first method has very limited synthetic value since it is useful only for the synthesis of symmetrically substituted porphyrins and some "meso" substituted porphyrins. The synthesis of the well-known meso-tetraphenylporphyrin (TPP)66 is an example of this method. The "2+2" coupling of dipyrrolic intermediates can provide porphyrins with varied peripheral substitution of certain symmetries. In order to obtained a single porphyrin product, one of the two dipyrrolic intermediates should be symmetrical. The most useful dipyrrolic intermediates are dipyrromethanes and dipyrromethenes. A dipyrromethane is prepared by the condensation of an cx-free pyrrole with a pyrrylcarbinyl cation 9 (Fig. 1.9), derived from the precursor 8, in an acidic medium. The porphyrin formation from dipyrromethanes involves the acid-catalyzed coupling of 5,5'-diformyldipyrromethane with 5,5'-ch^ nsubstirateddipyrromethane (or self-27 condensation of 5'-vmsubstimted-5-formyldipyrromethane) to produce a porphodimethene intermediate which is further oxidized to the porphyrin. etc. Fig. 1.9 Formation of a dipyrromethane from pyrrole precursors Dipyrromethenes are relatively strong bases and are best handled in their more stable protonated forms, the dipyrromethenium salts. A general synthesis of dipyrromethenes involves the condensation of an a-formylpyrrole with an a-free pyrrole in the presence of strong acid (Fig. 1.10). There are also some specialized syntheses of dipyrromethenes. Solvolysis of a variety of monopyrroles in a hot solution of hychobromic acid and formic acid leads to dipyrromethenes of head-to-head symmetry. In the presence of bromine, a-methyl-a'-unsubstitutedpyrroles condense in a head-to-tail fashion, giving a niixture of dipyrromethenes. Porphyrins can be made from dipyrromethenes in acidic media. Intramolecular cyclization of a linear tetrapyrrole is a very useful way to make porphyrins with unsymmetrically arranged |i-substituents. It involves the coupling of the individual pyrrole in an unambiguous manner, utilizing stable intermediates leading to a linear tetrapyrrolic compound, which in the final step is cyclized in a head-to-tail 28 Fig. 1.10 Formation of dipyrromethenes from pyrrole precursors fashion to the required porphyrin. Biladiene-ac 10 (Fig. 1.11) is the most common used tetrapyrrolic precursor in porphyrin synthesis. It can be synthesized from either dipyrromethanes or dipyrromethenes. The acid-catalyzed condensation of two equivalents of 2-formyl-5-methylpyrrole with dipyrromethane-5,5'-dicarboxylic acid, or alternatively of 5-unsubstirated-2-methylpyrrole with 5,5'-diformyldipyrromethane gives the 1,19-dimethylbiladiene-ac which can be cyclized in the presence of cupric salts to give the Cu complex of the porphyrin (Fig. 1.11). Instead of condensing with two equivalents of the same monopyrrole, a stepwise condensation of dipyrromethane with different mono pyrroles leads to a completely unsymmetrical porphyrin. Coupling of two dipyrromethenes, 5'-bromo-5-bromomethyldipyrromethene and 5'-unsubstituted-5-methyldipyrromethene gives l-bromo-19-methylbiladiene-ac (Fig. 1.12). This 29 Fig. 1.11 Synthesis of porphyrins from dipyrromethanes Fig. 1.12 Synthesis of porphyrins from dipyrromethenes 30 tetrapyrrolic precursor can be cyclized to the corresponding porphyrin by simply heating in o-dichlorobenzene or leaving in DMSO/pyridine at room temperature for several days. In a recent report,65 Wijesekera and Dolphin have described the development of various monopyrroles and dipyrromethenes. These dipyrromethenes make it possible to construct very useful biladienes which lead to various porphyrins. 31 Chapter 2 Results and discussion of the porphyrin syntheses 2.1 Synthetic objective Differentiation of CO and 0 2 binding in model systems of oxygen binding heme proteins may arise from electronic and/or steric effects (Section 1.2.2). On the distal side of the heme, steric interplay with amino acids can influence the orientation of bound dioxygen or induce tilting of carbon monoxide. Alternatively, electronic interactions may stabilize or destabilize the bound moiety. There has been a simple reference series developed in this group, the durene capped hemes, 11, 12 and 13, where the influence of distal steric effects on CO and 0 2 binding has been examined in 11, Durene-4,4, n=4 12, Durene-5,5, n=5 13, Durene-7,7, n=7 a, M=2H b, M=Fe the absence of stabilizing electronic interactions.30 The study of CO and 0 2 binding within the completely hydrophobic distal cavity of the durene capped porphyrin series has revealed the relative contribution of steric effects, both proximal and distal, for R-and T- state heme systems. 32 Introduction of a polar factor to such heme model systems may help to understand the contribution of electronic effects to the discriminatory binding of CO and 0 2. Neutron diffraction data24 have indicated that a strong hydrogen bond exists between the terminal oxygen and the distal histidine E7 residue in the binding pocket of Mb0 2 and a subunit of Hb02, an interaction that is thought to stabilize the bound 02. An amide substituted benzene capped porphyrin model may well help in understanding the nature of this hydrogen bond if the conformational requirement, i.e., the direction in which the polar amide function points within the distal cavity, for such an interaction is satisfied. A hydrogen bond with bound 0 2 then would be realized and thus increase the stability of the 02-complex. The cap sizes chosen in this study were 4/4 and 5/5 methylene groups because from the previous study of the durene analogues,50 it was known that the CO and 0 2 binding constants of the 7/7 methylene system were very similar to those of the 5/5 methylene system. Therefore, one may not be able to see a significant difference in these two hemes. Synthesizing the 4/4 and 5/5 methylene chain amide substituted benzene capped porphyrins 49c and 49d was considered to be sufficient to investigate the electronic effects of the binding properties of this system. For the purpose of comparison, the benzene capped porphyrins 49a and 49b were also synthesized and studied. R a, Benzene-4,4, n=4, R=H c, Amidobenzene-4,4, n=4, R=NHCOCH3 b, Benzene-5,5, n=5, R=H 49, M=2H, d, Amidobenzene-5,5, n=5, R=NHCOCH3 50, M=Fe 33 2.2 Synthetic plan The synthetic strategy for the hydrocarbon chain strapped porphyrins and the durene capped porphyrins were developed in this group by Wijesekera63 for the purposes of distorting the porphyrin mean plane, and making a myoglobin model with a fully hydrophobic distal cavity, respectively. The "retrosynthetic" analysis of the target molecule via dipyrromethane dimers has previously been described63 (Fig. 2.1). The first disconnection of the target molecule 14 leads to the most crucial intermediate, 15, of the entire synthetic scheme. With the peripheral substituents chosen as Ri=R4, R2=Rs and R3=R6, the double disconnection leads to a symmetric dipyrrolic intermediate already carrying the chain. The reverse of this disconnection is a simple acid catalyzed reaction of an a-unsubstitutedpyrrole with an a-formylpyrrole to produce a methene link. In order to prepare this bis a-formylpyrrolic derivative, the hydrocarbon and the durene chains are made first and are then attached to two f3-free pyrroles by two simultaneous Friedel-Crafts acylation reactions. Reduction of the carbonyl group followed by modification of the ethyl ester groups at positions 5 to formyl groups gives a derivative which is ready to be transformed to dipyrromethane dimers via coupling of a second pyrrole unit. However, before doing so, the formyl functions are protected as dicyanovinyl groups. The dipyrromethane dimers, after deprotection and decarboxylation are subjected to acid-catalysed intramolecular 2+2 coupling to give the desired porphyrins. It was anticipated that this strategy could be used to construct the amide substituted benzene-capped porphyrins. Since the two porphyrins 49a and 49b without amide groups on the benzene cap are quite similar to the durene-capped porphyrins, no special problems were anticipated. However, for the porphyrins with the amide 34 35 functionality, it was necessary to consider the best stage to introduce the sensitive functional group, since although the hydrocarbon strap of other porphyrins survived the entire reaction sequence, the amide was not expected to. Therefore, it was considered appropriate to introduce the polar functionality to the molecules as late as possible, and utilize the previous developed synthetic route to the greatest extent. Generally, an aromatic amide can be obtained by reducing the corresponding nitro compound. This raised the question as to how and when the nitro group should be introduced. Of course, if the nitro group could be introduced into the benzene-capped porphyrins, it would save time and avoid possible complications. However, because the porphyrin ring system itself is highly aromatic, there was no reason to expect that nitration would occur only on the appended benzene ring but not on the porphyrin periphery. Classical nitration procedures require the use of acidic reagents, and the resulting N,N'-diprotonated porphyrins are less nucleophilic, a feature that would have shown some advantage in this strategy. Protonation of a porphyrin with the acidic nitration medium would deactivate the porphyrin periphery and therefore nitration of the benzene ring be preferred. Thus, the porphyrins with benzene cap, 49a and 49b, were made and nitration was examined. The benzene capped porphyrin 5/5 49b was stirred with a mixture of acetic acid, sulphuric acid and nitric acid at room temperature for two hours. The brown-black solid obtained after work-up showed a characteristic UV/visible spectrum of a porphyrin. However, the mass spectrum showed only dinitrated and trinitrated compounds and no mononitrated product or unreacted starting material. Even by lowering the reaction temperature to 0°C, no mononitrated product was obtained. Since the meso positions of the porphyrin were unsubstituted, they should be ideal places for electrophilic attack of the nitrating agent, nitronium ion, NOj. The deactivating factor was not sufficient to prevent the nitration of the porphyrin 36 periphery. There have been examples of porphyrin peripheral nitration even under very mild conditions.67 This led us to the conclusion that the nitro functionality had to be introduced before the formation of the benzene-strapped porphyrin. The aromatic nitro group is one of the most active towards catalytic hydrogenation and it might not be expected to survive such conditions which were necessary for some steps in the synthesis. Therefore, nitration was postponed until after protection of the a-formyl function, by conversion to a dicyanovinyl derivative, because this compound was relatively stable, and no further catalytic hydrogenation was required beyond this stage. The dicyanovinyl derivative prepared during the synthesis of the benzene-capped porphyrin 4/4 (49a) was used for nitration studies. The lemon yellow l,4-bis{4-[5-(2,2-dicyanovinyl)-2,4-dimethylpyrrol-3-yl]butyl}benzene was stirred with an excess of nitronium tetrafluoroborate, a reagent which efficiently nitrates aromatics at low temperature. The common solvent for this reaction, sulfolane, was used because of its ability to solubilize both inorganic salts and organic compounds. The resulting reddish-brown solution was allowed to stir for one hour and the mixture was diluted with water and methylene chloride. The disappearance of the characteristic lemon yellow color of the starting material implied that the starting material had been destroyed. NMR and mass spectra of the isolated material confirmed the conclusion. It suggested that the pyrrole nucleus, sensitive to oxidizing agents, had been attacked by strong oxidizing species of the nitrating agent. Therefore, a simple pyrrole, 2-ethoxy carbonyl-3,4,5-trimethylpyrrole, was examined with nitronium tetrafluoroborate in methylene chloride at -80°C. Even at such a low temperature, the pyrrole was destroyed. It was concluded that nitration could only be achieved before the pyrroles were attached to the chain. Nitration was then investigated at several stages: dicyanide 26, dibromide 25, 37 diester 23 and diester 28. Except for the dibromide 25, the other three compounds could be nitrated. Since the diester 28 is the step just before the coupling of the pyrroles, it was decided that nitration should be carried out at this stage. However, problems arose from this early stage nitration. In the synthetic sequence, transbenzylation and debenzylation were necessary to make a clean, high yield cc-free pyrrolic derivative. The nitro function is very sensitive to the catalytic hydrogenolysis conditions of debenzylation. Possible discrimination of such reaction conditions towards the reduction of a nitro group relative to debenzylation was examined. A THF solution of nitrotoluene and 2-benzyloxycarbonyl-4-ethyl-3,5-dimethylpyrrole (1:1 ratio) was stirred under hydrogen in a hydrogenator in the presence of Pd/C catalyst. Within 10 min, more than half of the total material had been reduced. It indicated that no discrimination existed. It was therefore decided that the catalytic debenzylation step should be eliminated by saponifying the ethyl ester 38 directly instead of the indirect transbenzylation-debenzylation sequence. 2.3 Benzene and nitrobenzene diacid chain derivatives The synthetic scheme for the chain derivatives is shown in Fig. 2.2. The commercially available terephthaldehyde 20 was reacted with malonic acid by Knoevenagel reaction68 to form the diacid 21. Pyridine and piperidine were used as the catalysts. An excess of malonic acid (>2:1) was used to obtain a high yield. The accompanying decarboxylation was observed during the condensation of the aldehyde with malonic acid (evolution of carbon dioxide). The double bonds of compound 21 were then subjected to reduction. Two methods were tried, both being successful. The diacid 21 was first esterified to its 38 ethyl ester, and then reduced by hydrogenation using 10% Pd/C as the catalyst. The product was very clean and obtained in almost quantitative yield. But, with such a large quantity of the starting material (around 0.5 mol), a large volume of hydrogen (22.4 L) had to be absorbed to accomplish the reduction. The reduction took a long time (2 days) to consume the hydrogen and fresh catalyst had to be used several times during the reaction. Therefore, an easier and faster method was sought. The diacid 21 was reduced directly by Raney Nickel-Aluminium alloy69 using a low pressure catalytic hydrogenation. To the warm aqueous KOH solution of the diacid 21, small portions of Raney Nickel-Aluminium alloy were added. The hydrogen liberated from the reaction of aluminium with strongly basic solution was adsorbed on the surface of the newly formed Raney nickel catalyst to perform the reduction. The product obtained by this method was very pure and the reaction was faster. In order to increase the chain length by two carbons, the next step was to transform the acid group to some active functionality for further extension of the chain. The reduction of the acid to an alcohol was chosen. The commonly used reagent for this purpose is diborane. Even though carboxylic acids are generally more active towards diborane reduction than their ester derivatives, it was impossible to reduce compound 22 completely using diborane because of its low solubility in THF; only partial reduction could be achieved. Therefore, this reduction was carried out on the diester 23 which is significantly more soluble in the same solvent. The esterification was first attempted in toluene and ethanol using a Dean-Stark apparatus. After being refluxed with p-toluene-sulphonic acid for two days, much of the solid diacid was remaining. Therefore the ester had to be prepared via the acid chloride, though it was not as clean a reaction as direct esterification. The dark brown acid chloride prepared by the reaction of 22 with thionyl chloride was used directly 39 CHO CHj(COOH), pyridine/piperidine CHO toluene 20 C00H Raney-Ni KOH C00H 1) S0CI2 2) EtOH/Et jN O HOOC HOOC COOH COOH 29 DMF H00C> 'COOH 30 1) CH2(C00CHjCH3)2 NoOEt 2) Aq.KOH (T} 3) HCI CH2Br KCN EtOH HBr 1)Aq.KC)H H 0 0 C CN 2 ) H + Fig. 2.2(a) Synthetic scheme for the chain derivatives N0 2 C O O C 2 H 5 1)Aq.K0H 2)H* N02 COOC2H5 1 1)Aq.K0H 2>H* N02 HOOO COOH Fig. 2.2(b) Synthetic scheme for the chain derivatives without purification. The crude diester 23 was washed with methanol until the filtrate was colorless, to give a white crystalline solid of analytical purity in 84% yield. The diester 23 was then subjected to diborane reduction. To a THF solution of 23 and sodium borohydride, boron trifluoride etherate was added dropwise, and the reaction mixture was allowed to stir for four hours, before being quenched by the addition of acetic acid. Crystallization of the product was attempted and failed, which made purification by chromatography necessary. As the product bore two hydroxyl groups, it was very polar and did not move well on silica gel. Thus, the product 24 could only be collected as an impure brown liquid and this caused problems in the next few reactions. In order to avoid this, diborane was produced externally instead. In this case, diborane was conveniently prepared by the. reaction of boron trifluoride etherate with sodium borohydride in dimethylether of diethylene glycol (diglyme). It was then distilled into the THF solution containing the starting material 23 by a small stream of nitrogen. The reduction took place smoothly and after work-up, the white slurry was dried in a vacuum desiccator giving an analytically pure product The next step was to convert the alcoholic groups in 24 to good leaving groups for further extension of the carbon chain. The. readily prepared dibromide 25 was chosen for this purpose. The preparation of the diacid 27 required the extension of the methylene side chain by only one carbon. This was easily achieved by the preparation of the nitrile 26 from the dibromide 25 using potassium cyanide. The nitrile 26 was readily hydrolysed in basic medium to give the corresponding pure diacid 27 in a yield greater than 90%. To extend the methylene side chain by two carbons, a malonate synthesis was employed. The reaction was carried out in anhydrous ethanol with sodium ethoxide as the base. A common difficulty encountered in the alkylation of active CH2 compounds 42 is the formation of unwanted dialkylated products. Since the reaction was carried out in ethanol, during the alkylation of diethyl sodiomalonate, the monoalkyl derivative initially formed is in equilibrium with its anion as indicated below: RCH(C0 2C 2H 5) 2 + C H C C O , ^ ) , R C C C O ^ ) , + OT2(COAH5)2 QHjOH R C H C C O , ^ ) , + C J H J O " However, dialkylation does not take place to any appreciable extent because ethanol is sufficiently acidic to reduce the concentration of the anion of the monoalkyl derivative to a low value.70 Nevertheless, to avoid possible dialkylation, an excess of diethyl malonate (4:1) was used. After 30 min reflux, some white solid came out of the solution which was believed to be sodium bromide. With 2 hours reflux, TLC indicated that no starting material remained. The tetraester was not isolated, but saponified in situ directly to the tetracid 29. Usually such tetracarboxylic acids are unstable and lose C0 2 spontaneously on heating to produce the dicarboxylic acid. However, effervescence upon addition of concentrated hydrochloric acid was mistakenly thought to be the indication of decarboxylation. Characterization of the acid prepared above indicated that no decarboxylation had occurred. Instead, effervescence originated from malonic acid which was produced by saponification of the excess diethyl malonate. Decarboxylation of tetracid 29 was then effected in DMF to give the white analytically pure product 30. With the diacids 27 and 30 in hand, the next consideration was to introduce a nitro function onto the benzene moiety. Although aromatic nitration has been studied extensively and there are many nitration methods available, our starting material was 43 such that the strong nitration conditions may affect other parts of the molecule. Direct nitration of the diacid 27 was tried first, but it proved not to be a good method. It was very difficult to determine if the reaction was complete and separation of the product from the unreacted starting material and the other impurities was difficult. Therefore, it was considered better to nitrate the ester of the diacid and obtain the nitro-diacid later by saponification. Since there are a wide range of nitrating agents available,71 .nitration of these diesters seemed to be easy at the beginning. Firstly, the most common nitrating reagent, a mixture of concentrated nitric acid and sulphuric acid, was examined. To a mixture of concentrated nitric acid and sulphuric acid at room temperature, the diester 28 was added, and upon warming the mixture to 85°C for 20 min, some solid precipitated out. Work-up gave decomposed materials. The powerful oxidizing components in the solution might have been too violent at high temperature for the active benzylic positions. Thus, the use of a nitronium salt, nitronium tetrafluoroborate,72 was examined. It is one of the most powerful nitrating agents and is preferred for nitrating aromatic compounds which are susceptible to acid-catalyzed hydrolysis. It also gives high yields at low temperatures73 which can prevent oxidation of the substrates. The common solvents for this nitration are aprotic solvents such as sulfolane. However, sulfolane has a high boiling point (285°C) and is miscible with water and organic solvents. It is therefore difficult to separate the solvent and the product after reaction. Moreover, crystallization of the product might not be possible because the nitroester product might be a liquid. Fortunately, methylene chloride can dissolve nitronium tetrafluoroborate at a low concentration. A simple analogue of the starting material, toluene, was examined first with this solvent. At -80°C, toluene could be nitrated with nitronium tetrafluoroborate in methylene chloride without complication. 44 Therefore, nitration was tried with the diester 28 at different temperatures. The starting material in methylene chloride was stirred with nitronium tetrafluoroborate (1.1 equivalent) in an acetone-dry ice bath at -65°C for six hours, the nmr spectrum indicated that no nitration occurred and the clean starting material recovered. The temperature was raised gradually to -45°C, -30°C, -20°C and then 0°C. At this temperature, some reaction took place and a mixture of the starting material and the product resulted. The nmr spectrum of this material in the range of 7.0-8.0 ppm exhibited some new peaks at 7.32, 7.40 and 7.76 ppm in addition to the strong starting peak at 7.03 ppm. Raising the reaction temperature to 25°C gave more product but still a significant amount of the starting material remained. The starting material and the product had very similar Rf values on TLC so that it was not easy to separate them. To overcome this obstacle, nitration had to be driven to completion. At the same time, testing of trifluoroacetic acid with sodium nitrite for nitration74 was underway. At 0°C, a mixture of the starting material and the product was obtained. At 25°C, TLC analysis showed that there was more product than at 0°C, but more by-product with a slightly lower Rf value and more impurities at origin. In order to avoid decomposition of the compound and push the reaction towards the product, a lower temperature (0°) and more sodium nitrite were found to be useful. However, scale-up of the reaction caused problems. With 10 g of the starting material, more decomposition and more by-product resulted, and only a 40% yield of the product obtained. Therefore, the reaction was finally carried out in several small batches (5 g of the starting material). Because of the solubility problem of nitronium tetrafluoroborate in methylene chloride, the trifluoroacetic acid-sodium nitrite method was eventually employed in the synthesis. TLC of the reaction mixture after work-up showed three very close spots: the 45 fastest moving one was the unreacted starting material, the middle one was believed to be the product and the one with the lowest R, value, bright yellow in color, was a major by-product Since the starting material, the product and the by-product were quite close on a TLC plate, it was very difficult to separate them. To purify the product, a column chromatographic separation had to be performed. Firstly, methylene chloride alone was used to elute out any possible unreacted starting material. Then, 1% methanol/methylene chloride was used to elute two bands, the faster moving one was the product and the other the by-product. Characterization of the product was mainly achieved by proton nmr and mass spectrometry. Since a nitro group exerts inductive, resonance and magnetic anisotropy effects on the benzene moiety, it causes a deshielding effect on the benzene protons. The usual chemical shifts of the protons of nitro benzene with respect to benzene are: 8 ^ = 0.92, 8m M = 0.25, 8^ = 0.38 ppm downfield. The proton nmr [Fig. 2.3(b)] obtained from the nitrobenzene derivative 33 was in agreement with such a resonance pattern. The doublet at 7.28 ppm was assigned to the proton meta to N0 2. The doublet at 7.35 ppm was due to the proton para to N0 2 and the most downfield singlet at 7.72 ppm due to the proton ortho to N0 2. The proton nmr in the range 1-4 ppm also reflected the asymmetry caused by nitration, e.g. the triplet from the benzylic position, now split into two triplets. In the mass spectrum, the parent peak was not seen but a parent-45 corresponding to the loss of OCHjCHa was observed. But, it was also the mass for the parent peak of the starting material. This was checked by high resolution mass spectrometry. For the nitrodiester 32, the peak at mass 306 was examined. From calculation, the starting material should have the mass of 306.1831 and the product minus OCFf2CH3 of 306.1342. The high resolution mass spectrum showed that the 306 peak (306.1352) was from the product instead of the starting material. This characterization was further 46 confirmed by elemental analysis. f 8 1 i i i • r JL 7 V -A ' I I I I I | I I 7 a b c Fig. 2.3 NMR Spectra at 7.0-8.0 ppm range: (a) starting material; (b) nitro-product; (c) by-product The bright yellow by-product was separated and fully characterized. Its proton nmr spectrum showed two doublets in the 7-8 ppm range each accounting for two protons [Fig. 2.3(c)]. This implied that something must have happened to the side chain instead of the benzene ring itself. The peaks assigned to the benzylic positions accounted for only two protons and the others were unchanged. The peaks from the 2,2'-positions had split into two sets. Therefore, only one benzylic position had been affected causing the asymmetry. The mass spectrum gave a parent peak amounting to the parent(the starting material)+0-2H which suggested that one of the benzylic positions had been oxidized to carbonyl function during the reaction. The nmr spectra comparison of this material to the known compound (4'-ethylacetophenone)75 revealed a similarity. Thus the structure below was proposed for the by-product: 47 0 H 6 C 2 O O C ^ The structure was in agreement with the proton nmr and mass spectra of the by-product and was further confirmed by high resolution mass spectrometry and carbon-13 nmr. The carbon-13 nmr spectrum showed three typical carbonyl signals at 172, 173 and 197 ppm which were from two carbonyls of the chain terminal ester and a benzylic carbonyl. The conclusion was manifested by the mechanism of the reaction proposed by Uemura et al.16 The following reactions were involved in the reaction solution: NaN02 + CF3COOH HN0 2 + CF3COONa (1) 2HN02 = F = ^ N A + H 2 0 (2) HN0 2 + H + v. NO + + H 2 0 (3) N 2 0 3 v NO + N0 2 (4) N 2 0 3 s NO + + N02" (5) 2N02 ^ % N 2 0 4 + H + ^ N N0 2 + + HN0 2 (6) The nitronium ion (N02) for aromatic nitration could be formed by reaction (6). For the formation of aromatic ketones from alkylbenzenes with labile hydrogens (benzylic hydrogens), hydrogen abstraction by radical species (NO or N02) to give a benzylic radical is possible. This radical forms a hydroperoxide and the alkoxyl radical breaks down to form a ketone, or dimerizes to dialkyl oxide77 (Fig. 2.4). It is interesting to note, however, that an appreciable amount of ketone was obtained even at low temperature (0°C, gave 30% of the total material) in our case. The two nitrodiesters were saponified in aqueous KOH-ethanol solution under 48 - © - C H -0 I •0 - H -0 o- 0 0 H Fig. 2.4 Possible mechanism for the formation of the ketone by-product nitrogen. The mass spectrum did not show the parent peak but a very strong parent-18. High resolution mass spectrometry indicated that it originated from the loss of one molecule of water to give, presumably the intramolecular anhydride. Thus, with the desired diacids 27, 30, 34 and 35 in hand, the pyrrole coupling reactions were studied. 2.4 Chain linked bis pyrroles The first step towards dipyrromethane dimers was to convert the diacid chain to the chain linked dipyrrolic intermediate by a Friedel-Crafts reaction (Fig. 2.5). It was successfully achieved by reacting two moles of the {3 unsubstituted pyrrole with the diacids 27, 30, 34 and 35.™ There were two choices for the appropriate P-free pyrroles: 2-ethoxycarbonyl-3,5-dimethylpyrrole or 2-benzyloxycarbonyl-3,5-dimethyl 49 pyrrole. The latter appeared to be better because it would not only save a step, but would also bypass the high temperature transbenzylation. However, acylation with 2-benzyloxycarbonyl-3,5-dimethylpyrrole was examined and the benzylester pyrrole was found to be sensitive to the acylation conditions. By changing reaction conditions such as temperature, quantity of the catalyst used and solvent proportions, the reaction gave some product heavily contaminated by decomposed material. TLC of the reaction mixture showed a small spot with a lower R, value and another faint spot with higher R f value. These materials were postulated as being the half-acylation product and a very small amount of the product. Because of the insolubility of both starting material and product, it was impossible to purify and characterize them. The ethyl ester analogue 36 was therefore used in the reaction. The coupling, carried out in two stages, involves the initial formation of the acid chloride from the diacid, which is subsequently acylated in the presence of stannic chloride, in a mixture of methylene chloride and nitromethane. Four equivalents of stannic chloride were used in the reaction to give reasonable yields (60-80%) compared to 60-70% for the durene analogues.50, 6 3 It should be noted that the nitro compounds gave higher yields than the compounds without the nitro function. The next step was the reduction of the keto functions to methylene groups.79 Initially, diborane produced in situ (Section 2.3) was used. For the two diketones without nitro substituents, 37a and 37b, the reaction proceeded smoothly without any complication. But, for the nitro compounds 37c and 37d, problems arose and the yields were low (-57%). The products obtained by this method were so contaminated that they were brown colored. With this kind of method producing diborane in situ, too many species might exist in the reaction medium: NaBH4, BF 3, BjHg and B 2 0 3 etc. It is generally believed that sodium borohydride alone does not affect nitro groups; only 50 l H , / P d - C 44c n-4. R-NHCOCHs 44d n-5. R-NHCOCHs Fig. 2.5 Syntheses of chain linked bis pyrrolic intermediates 51 the combination of sodium borohydride with other catalysts such as palladized charcoal80 can affect nitro functionality. However, there are some examples showing that the nitro groups of aromatic compounds can be reduced by sodium borohydride alone.81,82 In the latter case, the reaction has been considered to involve a 1,4 addition-ehmination mechanism: Since our starting material had many active sites, side reactions may have taken place and the possible attack on the nitro function by NaBH4 may be one of them. In order to make the reaction cleaner, the borane-THF complex was used. The starting material was suspended in anhydrous THF, and addition of 1 M BH3-THF complex caused the material to go into solution. Once the reaction was complete, it was quenched by adding methanol. A white, clean product was crystallized in high yield (-80%) from the concentrated solution upon addition of methanol. Since the reaction went well with the commercially available BH 3-THF complex, the reduction of diketones 37a-d was finally completed with this reagent. With the methylene chain of the desired length in the capping group, the next consideration in the synthetic plan was to change the cc-ethyl ester to the synthetically useful a-formyl derivative. This included the removal of the ethyl ester functionality, thermal decarboxylation of the resulting diacid to give the a-free pyrrole, and transformation to the a-formyl derivative by a Yilsmeier reaction.83 The direct saponification in strongly basic medium gave gelatinous solid products in low yields. In addition, the reaction went very slowly, requiring prolonged reflux with the risk of 52 subsequent decarboxylation to the air-sensitive a-free pyrrole which occurred in the synthesis of the durene analogue.50,63 Therefore, hydrogenolysis of the corresponding benzylester derivatives was chosen as the preferred route.63 The transesterification, performed at high temperature, was a modification84 of the procedure developed by Hayes et al" Benzyl alcohol and the starting material were brought to reflux under a stream of nitrogen and a solution of sodium in benzyl alcohol was added in 1 mL portions. During each addition, vigorous evolution of ethanol and a simultaneous drop in temperature were observed. When no more ethanol was evolved, the hot solution was poured into a stirred mixture of acetic acid and methanol, the product was crystallized out by adding water. Because prolonged reflux would result in decomposition of the starting material and the product, the reaction time was minimized by immediately adding the catalyst once reflux occurred and quenching the mixture within a few minutes after the final addition of the catalyst. The solid product was filtered off as soon as possible because the decomposed materials in the solution tended to color the product pink; this colored material could only be removed by chromatography. This transesterification method was suitable for diethylester 38a and 38b. It gave a clean reaction and the yield was high. But, for the two diethylesters with the nitro group, the reaction did not give any product. Reflux of the starting material in benzyl alcohol did not cause any problems; the solution was clear yellow at reflux. Upon addition of sodium benzyloxide catalyst, however, the solution turned dark red immediately without effervescence of ethanol. After work-up, no product could be crystallized out and a deep red solution remained. Evaporation of the solvents gave a reddish black residue which could not be dissolved in any solvent nor be scraped off from the reaction flask. By introduction of a nitro function on the benzene moiety, the 53 property of the compound towards strongly basic catalysts at high temperatures had changed dramatically so that the compound decomposed. This may be attributed to the increasing acidity of the molecule due to the nitro functionality. If the acidity of the benzylic protons increased after introduction of the nitro group, the benzylic protons may be able to dissociate from the starting material. A literature survey suggested this possibility. The pKa of benzyl alcohol in DMSO at 25°C 8 6 is 21.30 and the pKa of toluene in DMSO is ~42.87 This means that at room temperature, toluene is a much weaker acid than benzyl alcohol, and it is not possible for the benzylic protons of toluene to react with the base, sodium benzyl alkoxide. After introduction of a nitro group, the pKa of 4-nitrotoluene in DMSO decreases to 22.5.86 This value is comparable to that of benzyl alcohol. Even though the pKa of 2-nitrotoluene could not be found in the literature, one might expect that it should be lower than that of 4-nitrotoluene, considering inductive and conjugative effects. Therefore, in the reaction medium, some of the protons may dissociate and destruction of the starting material be initiated. To avoid the transesterification step, a direct saponification of the ethyl ester in aqueous base had to be considered even though it did not give a clean product with similar compounds.63 If the diacid could be obtained by this method, hydrogenolysis could be avoided. This seemed to be a better route for the synthesis. Saponification was investigated in a refluxing KOH solution of water and n-propanol. The amount and ratio of the materials (solvents, base) used in the reaction and reaction time were varied, but no product was obtained and the starting material was always destroyed. In most cases, some solid remained undissolved. As indicated by the appearance of the reddish colored material, decarboxylation might have taken placed under these strongly basic, high temperature conditions giving an air-sensitive a-free pyrrole derivative. 54 Because transesterification and saponification did not afford any product for the N0 2 substituted compounds, an alternative procedure was proposed (Fig. 2.6, p. 56). Instead of transforming the 5,5'-positions of the bis pyrrole derivatives to the a-formyl function, a 5-formyl simple pyrrole could be used in the coupling to the dipyrromethane dimer. This required the 2,2'-positions of bis pyrrolic derivatives to be unsubstituted, for coupling with the a-formylpyrrole. The resulting product of this route is the same as the dipyrromethane from the original synthetic plan. Transformation of a simple pyrrole to an a-free pyrrole is a well developed procedure63 (Fig. 2.7), which could be adopted in our bis pyrrolic derivative. The reaction was investigated on a nmr scale. Fig. 2.7 Scheme for the synthesis of the a-free pyrrole The nitroethylester 38d (Fig. 2.5, p.51) dissolved in CDC13 in a nmr tube was treated with three equivalents of S02C12 and the reaction monitored by nmr. The reaction proceeded very slowly. After two hours, nmr indicated that a large amount of the starting material still remained, but, peaks corresponding to the ethyl groups of the ethyl ester and the chain methylenes had changed. Some new peaks around 5-6 ppm and 1 ppm appeared. In addition, the pyrrole NH had split into two groups of peaks. As the reaction continued the nmr spectrum became more complicated. Finally, the 2,2'-CH3 signal disappeared completely but the resulting compound was definitely not the desired a-trichlorinated derivative. Chlorination had taken place at other parts of the molecule. Therefore, this route was also not suitable. Because the final purpose of this work was to make porphyrins with amide caps, 55 Fig. 2.6 The alternative procedure for the syntheses of the porphyrin it was then decided that the nitro group should be transformed to amide at an early stage. The aromatic nitro function is one of the most active towards catalytic hydrogenation. It is generally presumed that nitroso compounds and hydroxyl amines are intermediates in the reduction. Both of these types of compounds give amines when treated with reducing agents: f0i NO NOH NHj In our reaction, the starting material dissolved in THF was stirred with 10% Pd/C, acetic anhydride and sodium acetate under hydrogen. The amine produced was transformed to the amide in situ. Now the amide substituted compounds were ready to undergo transesterification. To avoid the high temperature of boiling benzyl alcohol (205°C) which might cause decompose of the compounds, transesterification at 100°C and 12 mm Hg was employed as described by Kenner and coworkers.88 Nitrogen had to be passed through the benzyl alcohol to exclude air before heating. Sodium was added followed by the starting material and the reaction vessel was evacuated so that any ethanol produced was pumped away to drive the reaction to the product side. After 5 hours of heating on a steam bath, most of the starting material had decomposed to a red colored material with only little product formed. The reaction time was reduced to 30 min which was found to be suitable for the reaction to go to completion. The solution was cooled, acidified by acetic acid and benzyl alcohol distilled off at 0.1 mm Hg in a boiling water bath. The impurities and the remaining benzyl alcohol were washed away with ethyl acetate and the white product was obtained in 60-70% yield. The next consideration in the synthesis was hydrogenolytic debenzylation. The 57 reaction went smoothly at room temperature and 1 atm hydrogen. In the cases of the amide substituted benzylesters 40c and 40d, uptake of hydrogen was very slow at the beginning, but speeded up on addition of fresh catalyst. Removal of the catalyst by filtration, and evaporation of the solvent, produced a white solid. The next major concern in the synthesis was to introduce a formyl function at the 2-positions of the pyrroles. The diacid was first decarboxylated by heating in DMF and formylation was carried out in situ. The diacid derivatives 41a-d were directly used in the decarboxylation without purification. Decarboxylation was monitored by UV/visible spectrometry. The 282 nm band of the starting material was reduced to a shoulder after two hours refluxing. Continuous refluxing, however, did not remove the shoulder completely. The solution containing the a-free pyrrole was cooled in an ice bath under nitrogen for the next reaction. Introduction of a formyl group into the chain linked bis pyrrole was achieved by a Vilsmeier reaction,83,6200 whereby the a-free pyrrole in the decarboxylation solvent was treated with either phosphorus, oxychloride-DMF or benzoyl chloride-DMF.89 The reactive intermediate (a) has been suggested for the phosphorus oxychloride reaction and the similar species (b) for the benzoyl chloride type reaction. The reaction is known to proceed via an iminium salt (c). The iminium salt was subsequently T H , C ^ = c / o J C , T f H , C ^ = < 0 C P h Y r i i ^ H H , C CH> a b c hydrolysed by using a weak aqueous base, sodium bicarbonate, to give the a-formylpyrrole.63 In the course of this work, it was found that formylation of the two compounds 58 without an amide substituent on the benzene moiety could be effected by phosphorus oxychloride. But for the two amide substituted analogues, phosphorus oxychloride gave little product. After addition of the Vilsmeier reagent to the amide substituted cx-free pyrrole, the solution turned reddish purple and hydrolysis gave some intractable tars mixed with the product. In fact, modification of the Vilsmeier formylation procedure by substitution of benzoyl chloride for phosphorus oxychloride89 is better for acid sensitive compounds. Eventually, formylation of the two amide substituted compounds was effected using benzoyl chloride-DMF as the Vilsmeier reagent. In all cases, the iminium salts were not isolated but hydrolysed directly in aqueous base. The next stage in the synthetic scheme was to attach a second pyrrole to each end of the chain linked intermediates to give the desired dipyrromethane dimers. This required the use of sulphuryl chloride for cx-methyl oxidation and acidic conditions for coupling. Protection of the formyl function was therefore essential. The most useful of these protecting groups was first used by Fischer and Hofelmann90 and later by Woodward91 in the synthesis of chlorophyll. These are the cyanovinyl derivatives, prepared by a Knoevenagel condensation of a formyl pyrrole with malononitrile or a cyanoacetate ester, under basic conditions.92 These compounds are usually crystalline, less soluble than the aldehyde counterparts and move well on silica gel, allowing the use of the Knoevenagel reagent to scavenge the crude aldehyde and then purify it by chromatography. Due to the extended conjugation, the products have absorption maxima in the 390-410 nm range, resulting in yellow to orange colors, and fluorescence under UV light. However, strongly basic conditions, which are required for regenerating the aldehyde, limit the usefulness of these protecting groups to pyrroles bearing no ester groups. This was not a matter of concern in this work since this synthesis was 59 designated63 to make the best use of the strongly basic conditions. When the protecting group was removed after formation of the dipyrromethane dimer, the use of alkali would not only regenerate the aldehyde function but also saponify the two ester functions and produce intermediate 48. The choice of which protecting group should be used depends on the nature of the compounds. Malononitrile gives the less soluble derivative, and its use should be avoided in systems of inherently low solubility. But the corresponding dipyrromethane dimer can be crystallized cleanly giving a pure product The solubility of cyanoacrylates are much higher than dicyanovinyls and they can be deprotected more readily. But, in principle, Z and E isomers (four of them could result in this case, one with the two bulky ester groups being opposite to the pyrrole nucleus should predominate) could exist. Crystallization of the corresponding dipyrromethane dimers was impossible due to the existence of these isomers. During the course of this synthesis, the two protecting groups were investigated in detail. Both reactions to form dicyanovinyl and cyanoacrylate derivatives proceeded smoothly in refluxing toluene with cyclohexylarnine as catalyst. The reaction solvent had been chosen to prevent premature crystallization of the monoprotected aldehyde.63 The reaction was monitored by TLC where the monoreacted derivative moved faster than the starting material, whereas, the diprotected derivative moved well ahead of both. It was found that within two hours, all of the starting material would be converted to N C D I C Y A N O V I N Y L C Y A N O A C R Y L A T E 60 the unprotected derivative. The advantage of the dicyanovinyl derivatives was ease of purification on a silica gel column, which was essentially a filtration process using methylene chloride as the eluting solvent. Column chromatography of cyanoacrylate derivatives was more complicated, ethyl acetate or methanol in methylene chloride had to be used to move the product. Both dicyanovinyl and cyanoacrylate derivatives could be crystallized out giving lemon yellow, analytically pure products. Both protected chain linked dipyrrolic derivatives were examined for pyrrole coupling reaction to make dipyrromethane dimers 47. In the case of the cyanoacrylates, the reaction went well without any complication, but just as expected, the product 47 could not be crystallized. After the solvent was evaporated under vacuum, column chromatography was necessary to purify the product. In some cases, a second column of alumina or the use of the chromatotron with a silica gel rotor was necessary since impurities were very difficult to be removed. The purification of the dicyanovinyl analogue was relatively easy and the product could be crystallized. But a large volume of reaction solvent, methylene chloride, was needed since the solubility of the starting material was very low, especially in the case of the amide substituted dipyrromethanes. Changing of the solvent to 1,2-dichloroethane did not improve the solubility much. 40 mg of the starting material needed more than 200 mL of 1,2-dichloroethane to dissolve it completely. Therefore, a compromise had to be made and the cyanoacrylate protecting group was employed eventually in the synthesis. 2.5 Dipyrromethane dimers and porphyrins With the dialdehyde protected, the next major concern was to couple the second pyrrole to make the synthetically useful intermediates: dipyrromethane dimers 47 which 61 could be then further transformed into porphyrin 49 (Fig. 2.8, p.63). This was achieved by coupling two a-free pyrrole nuclei to the chain linked derivatives of pyrrylcarbinyl cations. Pyrrylcarbinyl cations are highly stabilized by delocalization of the positive charge throughout the electron-rich pyrrole nucleus (Fig. 2.9), and as a result they are etc. Fig. 2.9 Resonance structures of pyrrylcarbinyl cations formed readily from a variety of precursors (51) by an SN1 mechanism. Of the three commonly used pyrrylcarbinyl cation sources (bromide, chloride and acetoxy), = -Br, -CI, -OCH 3, -OH, -OCOCH3, -NR3, -NHR2 etc. chloromethylpyrrole was chosen to be the source of pyrrylcarbinyl cation in this synthesis because of its stability. It is known that the ct-methyl position can be selectively chlorinated over a p-methyl group when using sulphuryl chloride,62(b) but multichlorination at the cx-methyl is possible. Usually this is controlled by the use of low temperatures and calculated amount of reagent. In addition, the presence of an electron withdrawing cyanoacrylate group deactivates the ring towards dichlorination. Since in the starting material, there were two positions to be chlorinated, two equivalents of the chlorination reagent must be used. Compounds 44 contain a benzene moiety which is likely to undergo chlorination 62 R 0 49b n=5, R=H 49c n - 4 , R -NHCOCH, 49d n -5 . R=NHCOCH 3 Fig. 2.8 Transformation of dipyrromethane dimers and cyclization to the porphyrins 63 as well under the reaction conditions. From the detailed discussion63 of the mechanism of the pyrrole side chain chlorination, it is known that both electrophone93"95 and free radical98 mechanisms are possible. Therefore, any attempt to chlorinate the pyrrolic side chain might chlorinate the benzene moiety. Wijesekera63 has shown that exclusive pyrrole side chain chlorination takes place in the presence of a hexaalkyl substituted benzene during the reaction with sulphuryl chloride. It was expected that with compounds 44a and 44b there should not be any problem with this reaction. But, in the cases of amide substituted 44c and 44d, the electron donating amide might make the benzene ring active enough towards chlorination. Since a pyrrolic nucleus is known to be more reactive towards electrophilic attack, it was hoped that this reaction center could compete favorably with the amide benzene center and lead to only pyrrole chlorination. In fact, treatment of the starting material in dry methylene chloride with slightly more than two equivalents of sulphuryl chloride led to exclusive a-chlorination at the pyrrole nucleus and a high yield of dipyrromethane dimer 47 was isolated. No benzene chlorinated material was observed. The a-chloromethyl derivative was isolated by evaporating the solvent under vacuum. The yellow powdery product was used for coupling with the a-free pyrrole 46, without further purification. A variety of conditions has been discussed for the coupling of pyrrole monoesters.620* 6 3 In our synthesis, the coupling in acetic acid according to the procedure developed by Wijesekera63 was adopted with success. The bis-a-chloro methylpyrrole derivative was suspended in glacial acetic acid together with the 2-ethoxycarbonyl-4-ethyl-3-methylpyrrole (46) and the mixture was warmed in a water bath. In order to prevent any oxidation of the a-free pyrrole at high temperature, a nitrogen atmosphere was provided for the reaction mixture. When the temperature 64 reached 70°C, the chloromethyl compound began to dissolve resulting in a transparent red solution. An aliquot was taken out for TLC analysis. There were two major spots existed which could be assigned to the product and the half coupled compound. After the mixture being heated at this temperature for 30 min, a TLC analysis exhibited only a single yellow spot with some impurities at the origin. This yellow spot could be colored reddish violet by bromine vapour which is characteristic for dipyrromethanes due to the ready oxidizability at the methane bridge.97 Since no other spot on TLC plate was colored by bromine vapour, the two oc-chloromethyl positions in a single starting material molecule had been attached to cc-free pyrrole and no half coupling compound remained. Because of the possible isomers (up to four), crystallization of the product was impossible. The solvent was therefore removed completely by evaporating under vacuum, and the product was purified by chromatography. In all cases, silica gel was used. The product did not move on the column with methylene chloride alone, and either methanol or ethyl acetate had to be added to facilitate its elution. Even after column chromatography, the compounds 47a and 47b were still not pure, and separation in a chromatotron followed by an alumina column had to be performed to obtain an analytically pure sample. In spite of the difficulty of purification, the cyanoacrylate protecting group was still preferred because of the high yield (> 80%). The final stage of the synthesis was to convert the dipyrromethane dimers to porphyrins. The first step here was to remove the cyanoacrylate protecting groups and the ethyl esters simultaneously. Since an amide is not as susceptible as an ester towards hydrolysis, it would be possible to hydrolyse the ester function and leave the amide unattacked by choosing proper reaction conditions. The reaction was therefore examined on a small scale. Dipyrromethane dimer 47c was refluxed in aqueous n-65 propanoVpotassium hydroxide (10 equivalents) under nitrogen. Although cleavage of the cyanoacrylate function can be followed by UV/visible spectroscopy, complete formation of the acid from the ethyl ester was only guaranteed by prolonged reflux in a strongly basic medium. Prolonged refluxing might lead to decarboxylation of the acid function and possible hydrolysis of the amide function. After 2 hours at reflux, the reaction mixture was cooled, neutralized and the gelatinous precipitate isolated by filtration. The mass spectrum (FAB) gave the parent peak. The 'H-nmr spectrum of this material indicated that no ester group remained and the other peaks, which were very complicated, could not be assigned. The product was carried forward. The oc-acid function was decarboxylated in boiling DMF to give the ex-free derivative. This solution diluted with methylene chloride was added slowly, to the reaction flask containing the acid catalyst, by syringe pump. Work-up afforded the porphyrin in extremely low yield (5%). During the addition of the a-free derivative, precipitation of some black tar was observed and the porphyrin obtained appeared to decompose. It was possible that the amide function might have partially hydrolysed during the deprotection step giving an unstable amine derivative. To avoid the problem, the material after hydrolysis was subjected to acetylation, by stirring with acetic anhydride for 20 min. Decarboxylation seemed to be unsuccessful; the band at 280 nm in the UV/visible changed, but, the system didn't behave like the previous reaction. In fact, no porphyrin was obtained after the cyclization reaction. When the amine was converted to the amide, the acid function was also affected by acetic anhydride, giving a mixed anhydride which could not be decarboxylated and therefore gave no porphyrin product. The above results suggested that hydrolysis should be carried out under more controlled conditions. Therefore, various conditions, not only the concentration of the 66 base, but also its ratio to the reactants, were examined. With just enough base and time to deprotect the cyanoacrylate group, a large amount of ester function still existed. By a gradual increase of the amount of the base used, the amount of unsaponified compound decreased. Finally, a 30:1 molar ratio of base:starting material and a base concentration of 1.5 M in water and watenn-propanol of 2:1 were found to be optimal. The reaction was carried out under a nitrogen atmosphere in order to prevent the oxidation of any decarboxylated pyrrole derivative which was highly sensitive to the air. The reaction was followed by UV/visible spectroscopy. The starting material exhibited a strong band at 388 nm (due to the cyanoacrylate function), and a band of medium intensity at 280 nm. As reflux continued, the band at 388 nm decreased in intensity (also moving to lower wavelength) with a simultaneous increase in intensity of the band at lower wavelength and a growth of a new absorption at 324 nm (due to pyrrole aldehyde). The reaction appeared to have gone to completion within three hours. n-Propanol was boiled off and the mixture was allowed to cool to room temperature under nitrogen, at which time partial precipitation of some brown solid was observed. The solution was filtered off and the solid left on the filter paper was dissolved by adding water. The resulting clear solution was acidified and the product was collected by filtration. The yield of this reaction was high (over 80%) and the product was quite pure. Its mass and proton nmr spectra were in agreement with its proposed structure. However, a small peak at around 6 ppm was noted in the nmr spectrum, which indicated some decarboxylation during reflux. The product was not quite pure for this reason and an elemental analysis was therefore not obtained. As discussed by Wijesekera,63 decarboxylation can be carried out thermally under mildly acidic, neutral or weakly basic conditions. In practice the acidic decarboxylation approach gives considerably low yields of final porphyrin and should be avoided.98 The 67 low yield might be due to prernature condensation of the a-free and a-formyl functions which leads to intermolecular coupling. Decarboxylation of compounds 48a-d was carried out in refluxing DMF under nitrogen. The reaction was monitored by UV/visible spectra again. In two hours, the absorption band at 280 nm (due to the carboxypyrrole group) was reduced to a shoulder and the solution was cooled and DMF evaporated under vacuo to give a small final reaction volume. Methylene chloride was added and the solution was extracted with water to remove the last trace amount of DMF. Even though precipitation of some black material during cyclization in the previous reaction had been attributed to decomposition of the amine derivative, it might also be caused by the low solubility of the compound in methylene chloride since this series of compounds had an inherently lower solubility than the durene systems. Therefore, methylene chloride was substituted for by THF to improve solubility. The methylene chloride solution containing the a-free derivative was dried over sodium sulphate, filtered and evaporated down to about 10 mL and then diluted with distilled THF to 200 mL and stored in a refrigerator for the subsequent cyclization procedure. The dipyrromethane dimers bearing a-free and a-formyl functions were thus ready for the intramolecular cyclization to form the porphyrins via a b-bilene to porphodimethene (Fig. 2.10). The strain introduced by imposition of a short strap has less adverse effect on the more flexible porphodimethene, which is a non planar system, than on a porphyrin, and so that the initial cyclization step should proceed smoothly. Once formed, the porphodimethene undergoes rapid autoxidation where the net gain in resonance stabilization energy is the driving force for porphyrin formation. Furthermore the bilene-b can accommodate the diagonal strap, which also allows for correct orientation of the remaining a-formyl and a-free functions to undergo a second intramolecular coupling to the porphodimethene.63 The reaction of an a-free with an a -68 formyl group must occur in an intramolecular head-to-tail fashion. Intermolecular reaction between functions on different molecules will only cause polymerization without porphyrin formation. Therefore the acid-catalysed condensation must be carried out under high dilution in order to mmimize intermolecular coupling. Since the acid-catalysed rearrangement of dipyrromethanes is a well known H H H Por phodimethtne Porphyrin Fig. 2.10 Formation of a porphyrin via the acid-catalyzed cyclization of dipyrromethanes 69 phenomenon," the acid used in the condensation has to be carefully selected. Toluene-p-sulphonic acid, first used by Kenner's group100 and later used successfully by Wijesekera63 in the synthesis of the durene-capped porphyrins, was employed in our work. The THF solution of the decarboxylated 48 was added extremely slowly to the acid catalyst using a syringe-pump. This is a device that is used to add a solution at a constant rate, by means of a hypodermic syringe. At the slowest speed of operation, the pump emptied a 50 mL syringe in approximately 9 hours. Two separate syringes were used to add the solution to two flasks, each containing toluene-p-sulphonic acid in methanol and methylene chloride. Two stages of chromatography purification were needed to purify the porphyrins. Since the porphyrins were not easily eluted on silica gel, methanol was necessary to facilitate separation, resulting in the elution of some impurities. Chromatography on an alumina column was therefore used to remove these impurities and obtain pure 49a and 49b. In the cases of the amide substituted porphyrins 49c and 49d, the alumina column was not efficient, and thus chromatotron purification was needed. The hemin chloride complexes were synthesized from the porphyrins and ferrous chloride. 2.6 Electronic absorption spectra of benzene and amidobenzene capped porphyrins The electronic spectra of a typical porphyrin consists of an intense Soret band and four weak bands in the visible range. The Soret band, often recorded at around 400 nm with a molar extinction coefficient around 400,000, is characteristic of the macrocyclic conjugation of porphyrins. The two hydrogens in the center of the free-70 base poiphyrin break the conjugated ring symmetry, and give rise to two possible tautomeric forms.101 It is generally considered that the Soret band is due to one n-n' transition which is similar for both tautomers, and that the four bands at longer wavelengths (I, II, III, IV in the order of increasing energy) arise in pairs, two from each tautomer, from a second n-n' transition.101 The intensities of the four satellite visible bands are correlated to the nature of the side chains on porphyrins.10100 The etio spectrum [Fig. 2.11(a)] is found in all porphyrins carrying alkyl side chains at six or more peripheral positions. The spectral pattern for the band intensities is rV>ILI>II>I. If a single carbonyl (aldehyde, ketone), a carboxylic acid, ester or an acrylic acid side chain is present, band HI becomes more intense than band IV, affording a rhodo-spectrum. These strongly electron-withdrawing groups, which also cause shift of absorption to longer wavelengths, are termed "rhodofying" groups. Two rhodofying groups on adjacent 'pyrrole' subunits cancel each other's rhodofying effect and an etio-spectrum results. But, two rhodofying groups on diagonally opposite rings enhance each other's effect and an oxorhodo spectrum is obtained [Fig. 2.11(c), HI>n>IV>I]. Finally, if four or more peripheral positions are unsubstituted, the spectrum is of the phyllo-type. Upon protonation of the free-base porphyrin, a dication results with a proton attached to each central nitrogen. Since the symmetry changes to D4h, the four bands in the visible range collapse to two, the oc-band and fi-band. The Soret band in the absorption of dicationic porphyrins is displaced to longer wavelength compared to that in neutral species. Insertion of a metal at the porphyrin core also results in similar changes in spectra. Since the porphyrins synthesised here have only alkyl substituents on the porphyrin periphery, one might expect the etio-type absorption spectra. This was 71 2.11 Trends in visible spectra for various pyrrole-substituted porphyrins 72 indeed the case of the 5/5-methylene chain capped porphyrins. But, there has been evidence63 showing that distortion of the porphyrin skeleton also leads to a change in intensities of the four visible bands. Our porphyrins also showed similar trends. As the chain length decreases from a 5/5-methylene chain (Figs. 2.13, 2.15) to a 4/4-chain (Figs 2.12, 2.14), the spectra changed from an etio-type to a typical rhodo-type with in>IV>n>I. This change in intensities is due to distortion of the porphyrin macrocycle as the chain becomes shorter.63 Table 2.1 Comparison of electronic absorption spectral data of the porphyrins in CH2CI2 K*x nm (log e) Porphyrin Soret Band IV Band III Band II Band I Benzene-4,4 49a 404 506 542 572 626 (5.26) (4.08) (4.10) (3.91) (3.60) Benzene-5,5 49b 400 498 534 568 622 (5.10) (3.90) (3.77) (3.46) (3.13) Amidobenzene-4,4 49c 404 506 542 572 624 (5.23) (4.07) (4.09) (3.89) (3.60) Amidobenzene-5,5 49d 400 500 536 568 622 (5.26) (4.20) (4.13) (3.96) (3.82) 73 -u . uoa - . 1 , , , , , , , , , 1 , , , , 1 , , — - , , , 300 400 500 600 700 W A V E L E N G T H Fig. 2.12 UV-visible spectrum of the benzene-4/4 capped porphyrin 49a in C H 2 Q 2 Fig. 2.13 UV-visible spectrum of the benzene-5/5 capped porphyrin 49b in CH2C12 Fig. 2.14 UV-visible spectrum of the amidobenzene-4/4 capped porphyrin 49c in C H 2 Q 2 Fig. 2.15 UV-visible spectrum of the amidobenzene-5/5 capped porphyrin 49d in C H 2 d 2 2.7 Proton nmr spectral assignments of benzene and amidobenzene capped porphyrins The ^-nmr spectra (Figs. 2.16 - 2.19) of the porphyrins were recorded at 400 MHz in deuterated methylene chloride. In order to minimize the variation of chemical shifts caused by aggregation of the porphyrin,102 concentrations of the solutions were kept low and consistent (in all cases, concentrations of the porphyrins were -0.01 M). The alkyl substitution pattern surrounding the porphyrin periphery of the system is essentially identical to that of etioporphyrin U (Fig. 2.20), except that the QHj groups at positions 3 and 13 are replaced by a methylene chain linking of the cap. As for several other similar strapped and capped porphyrins that have been synthesised, the simple 'H-nmr spectrum of etio II porphyrin is used as a suitable reference.50,63'64 Fig. 2.20 Etioporphyrin II The diamagnetic ring current of the highly delocalized porphyrin macrocycle causes protons associated with the periphery to experience a deshielding effect, and therefore these protons resonate further downfield from TMS. For etio n, the peripheral groups' resonances appear as four distinct groups of signals: the protons from all four equivalent methyl groups resonate at 8 3.62, the ethyl groups as a triplet and a quartet at 8 1.87 and 8 4.11 respectively, and the four methine protons (5, 10, 15, 78 Fig. 2.16 'H-nmr spectrum of the benzene-4/4 capped porphyrin 49a in CD2C12 1 4 P P M Fig. 2.17 'H-nmr spectrum of the benzene-5/5 capped pcqphyrin 49b in CD2C12 20) occur as a singlet at 8 10.11. The inner N-H protons appear upfield of TMS at approximately 8 -3.78 due to extensive shielding by the porphyrin ring current.103 Introduction of a strap diagonally across one face of the porphyrin is equivalent to joining the ethyl groups at 3 and 13 positions of etio II and leads to asymmetry in the molecule. The asymmetry results in a splitting of the methyl and methine proton signals. It has been pointed out that the splitting of the methyl signals depends on the length of the strap.63 The splitting of the methyl proton resonances for the long chain 5/5 system is smaller (0.13 ppm) than that of the shorter chain 4/4 system (0.19 ppm). As the chain length is decreased, both signals corresponding to the methyls are shifted upfield, because the distortion of rings A and C from the porphyrin plane63 , 6 4 towards the tight connecting strap. Such distortion affects the aromaticity of the porphyrin macrocycle and results in diminished deshielding for the methyl protons, thereby giving rise to an upfield shift. Within these shifts, the higher field signals shift more than low field ones, corresponding to methyl groups at the 2 and 12 positions. A similar trend is found in the methine proton resonances. As the chain length decreases from 5/5 to 4/4, both meso signals are shifted upfield, with the peak at higher field being shifted more. Irradiation of the multiplet due to the 7 and 17 methylene protons (at 4.07 ppm in the case of 4/4-benzeneporphyrin) causes an enhancement in the intensity of the higher field meso-proton resonance, indicating that this signal corresponds to the protons at the 5 and 15 positions. It is these protons that are closer, in terms of carbon-carbon bonds, to the chain linked 3 and 13 ring positions. Distortion caused by a strap linking positions 3 and 13 is therefore expected to affect the 5 and 15 meso protons more than the 10 and 20 protons, thus giving rise to the increasing separation between their respective resonances.50,63, ( A A reversal of this trend is displayed for the N-H protons in a downfield shift 83 relative to etio U. The shorter chain compounds have shifted more. Situated within the porphyrin core, these N-H protons experience extensive shielding from the porphyrin ring current that causes them to resonate at high field (8 -3.78 ppm for etio II). A disruption of the electron delocalization, caused by increasing distortion of the macrocycle from planarity, results in a dirninished shielding effect and thus a gradual shift downfield.50-63,64 A striking feature of the 'H-nmr spectra is the large upfield shifts observed for some of the proton resonances of the methylene straps which have previously been observed for various strapped porphyrin derivatives.50, 6 3 , 6 4 1 1 0 4 Those protons positioned above the porphyrin macrocycle experience a strong shielding effect from the diamagnetic ring current, and thus have upfield shifts (relative to that of the free chain derivative). The protons associated with carbon atoms attached to the porphyrin periphery, however, experience the deshielding effect of the porphyrin ring current and thus resonate further downfield relative to those above the porphyrin macrocycle. A series of double resonance experiments were carried out on the 4/4-benzene capped porphyrin in order to obtain a first-order assignment of the peaks arising from the chain methylene protons. The sixteen chain methylene protons appear as seven distinct multiplets, each consisting of two protons except the one at 0.13 ppm which consisted of four protons [Fig. 2.21(a)]. Figs. 2.21-2.25 illustrate the spectra obtained by a series of double resonance experiments carried out on the methylene chain protons of the 4/4-benzene capped porphyrin 49a. The spectra were recorded in the same range and on the same expansion in order to give a direct comparison with the undecoupled spectrum. The seven resonances due to the chain methylene protons and the two signals from the peripheral ethyl groups have been labelled A to I, starting from the one at the lowest 84 u LA Fig. 2.21 PPM 0 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin (49a) (a) Normal spectrum (b) Simultaneous irradiation at 8 4.07 (c) Simultaneous irradiation at 8 4.00 85 IL Fig. 2.22 PPM Partial 'H-nmr spectra of the benzene-4/4 capped rx>rphyrin (49a) (a) Normal spectrum (b) Simultaneous irradiation at 5 3.86 (c) Simultaneous irradiation at 5 1.88 86 PPM Fig. 2.23 Partial 'H-nmr spectra of the benzene-4/4 capped r^hyrin (49a) (a) Normal spectrum (b) Simultaneous irradiation at 8 1.44 (c) Simultaneous irradiation at 8 1.24 87 A J U L Fig. 2.24 PPM 0 Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin (49a) (a) Normal spectrum (b) Simultaneous irradiation at 5 0.88 (c) Simultaneous irradiation at 5 0.13 88 b A_A1 I A -JL E F A . M 5 0 Fig. 2.25 PPM Partial 'H-nmr spectra of the benzene-4/4 capped porphyrin (49a) (a) Normal spectrum (b) Simultaneous irradiation at 8 0.00 field. Among the two lowest field resonances A and B, the one at lower field is a quartet which contains four protons from the two CHj groups of the ethyl substituents. The one at higher field is a multiplet consisting of two protons assigned to the two protons at the chain termini (C-4 and C-40. Being attached to the periphery of the porphyrin ring, these protons experience the largest deshielding effect due to the delocalized 7t-electron system of the macrocycle. B at higher field corresponds to one proton each from C-4 and C-4' of the chain, 89 and not two protons of the same carbon. This is identical with other systems.63 Irradiation of the resonance A (Fig. 2.21b) resulted in the triplet at 1.87 ppm becoming a singlet while the other resonances remained unchanged. Irradiation at B (Fig. 2.21c) resulted in a significant change in the fine structure of the resonance C. In addition, this irradiation caused a change at resonance E and sharpening of the lines at resonance F, indicating that the protons at E and F are coupled to those at B. However, G, H and I are unaffected. Therefore, both E and F should consist of one proton each from C-3 and C-3' of the chain and C, of one proton each from C-4 and C-4'. This assignment was further confirmed by irradiation at resonance C (Fig. 2.22b) which affected only B, E and F. Irradiation at D (Fig. 2.22c) caused the quartet of A to turn into a singlet while the other resonances remained unaffected. This again confirmed that the signal A consisted of four protons from the ethyl groups. Fig. 2.23b illustrates the result of decoupling of the protons at resonance E. As expected, changes were observed at B, C, F and I. However, the peak H was left unchanged, strongly suggesting that I and not H contains the two protons from C-2 and C-2'. Although G did not show any significant change, this was tentatively assigned to the other two protons of C-2 and C-2'. Irradiation at F (Fig. 2.23c) provided confirmation for the earlier assignment that G was due to the two protons of C-2 and C-2'. Decoupling at G (Fig. 2.24b) caused changes in E (C-3, C-3'), F (C-3, C-3'), H and I (C-2, C-2'). From the previous decoupling, G had been assigned to the two C-2, C-2' protons, thus suggesting that H corresponds to the four protons of C-l and C-l'. Irradiation at I (Fig. 2.24c) changed E, F, G and H, confrrrning that I is due to C-2 and C-2' not C-l and C-l'. Finally, irradiation at H (Fig. 2.25b) affected G and I but not 90 E, F, indicating that it is from C-1 and C-1'. Thus, the first order assignment of the protons at the different chain carbon atoms could be made as shown below: 1 1 1 1—: 1 1 5 4 3 2 1 0 PPM Similar decoupling experiments were carried out with the 5/5-benzene and 5/5-amidobenzene porphyrins. The assignments based on the analysis of the spectra are shown in Figs. 2.26 and 2.27. In the case of the 5/5-benzene capped porphyrin, there is an overlap of signals from C-3,3' and C-2,2' or C-1,1' at resonance H. Irradiation at I, J or K did not affect E and G (assigned to 0-4,40. Therefore, none of these is due to C-3,3', suggesting that H consists of 4 protons from C-3,3' and two protons from either C-2,2' or C-1,1'. However, assignments for L J and K were not possible since irradiation of any one of them caused changes in the other two and H. The only thing that can be decided is that they contain six protons from C-2,2' and C-1,1'. Another interesting feature observed in the spectra of the 5/5-benzene and 5/5-amidobenzene porphyrins is the existence of the methylene protons of the ethyl group as two complex multiplets rather than a simple quartet expected for the CH 2 protons of 91 an ethyl group as in etio porphyrin TX This inequivalence in nonmetallated systems has been interpreted in terms of the intrinsic asymmetry in the porphyrin molecule.63'105 -CH2CH3 C-5 C-5' k A B C D PPM 0 -1 Fig. 2.26 The first-order assignment for the chain methylene protons of the 5/5-benzene capped porphyrin 49b ~~r 2 -7--1 PPM Fig. 2.27 The first order assignment for the chain methylene protons of the 5/5-amidobenzene capped porphyrin 49d 92 The resonances from the methylene chain protons of the 4/4-amidobenzene capped porphyrin are either overlapped or very close, so that a first order assignment of the peaks arising from these protons through decoupling is precluded. The resonances corresponding to the benzene protons in the porphyrins have been shifted upfield. The signals of the 4/4 and 5/5-benzene capped porphyrins have been shifted from -7 ppm, for the normal aromatic protons, to 2.40 ppm for the 4/4 and 2.69 ppm for the 5/5 porphyrins respectively. The upfield shifts of the signals are easily explained by a strong shielding effect from the diamagnetic ring current due to the protons' positions above the porphyrin macrocycle. But, the reason for the 4/4 analogue having a larger upfield shift than that of the 5/5 is not so obvious. From the UV/visible spectrum of the 4/4-system, it is known that the porphyrin plane is distorted. The distortion would lower the aromaticity of the porphyrin system which should result in a reduced upfield shift. One possible reason for this reversal might possibly be the closer positioning of the benzene moiety in the benzene-4/4 capped porphyrin than that of the 5/5 analogue, to the porphyrin plane. The protons on the 4/4-benzene moiety would therefore experience a stronger shielding effect. 93 Chapter 3 Experimental for the porphyrin syntheses 3.1 General methods Melting point determinations Melting points were obtained using a Bristoline 6548-J17 microscope equipped with a Thomas Model 40 Micro Hot Stage, and are uncorrected. Elemental analysis Elemental analysis was performed by Mr. P. Borda of the Microanalytical Laboratory, U.B.C. Nuclear magnetic resonance spectroscopy The proton nmr spectra were recorded either at 300 MHz with a Varian XL-300 Fourier-transform spectrometer or at 400 MHz using a Bruker WH-400 spectrometer. In all cases the chemical shifts were recorded on the 8 (ppm) scale with tetramethylsilane (TMS, 8 = 0 ppm) as an internal or external standard. Mass spectrometry Mass spectra were recorded on a Varian Mat CH 4-B spectrometer or a Kratos/AEI MS-902 spectrometer. High resolution measurements were obtained on a 94 Kratos/AEI MS-50 spectrometer. Electronic spectroscopy A HP 8452A Diode Array Spectrophotometer was used to obtain UV/visible spectra. Chromatography Column chromatography was performed using silica gel obtained from Merck (70-230 mesh, activity I) and alumina from Fisher (neutral, 80-200 mesh, activity I). Thin layer chromatography (TLC) was performed using precoated silica gel plates (Analtech-Uniplate, 250 u.) and compounds were detected by UV light (254 nm). The chromatotron was a Harrison Research chromatotron apparatus with self-made silica gel rotors (Silica Gel PF-254 with CaSCyl/2H20 type 60 TLC, Merck) and alumina rotors (Alumina GF-254 type E for TLC, Merck and Adsorbsil-Plus-2P, Applied Science). Mixtures of solvents used in chromatography are expressed as volume/volume ratios. Starting materials The mono-pyrroles 36 and 46 were synthesized following methods described in detail by Wijesekera.63 Terephthaldehyde 20 and all other reagents used were obtained commercially. Unless otherwise stated all solvents used were reagents grade. 3.2 Nomenclature of porphyrins and their intermediates The nomenclature of the porphyrins is based on the IUPAC numbering system as shown below: 95 11 10 13 12 All mtermediates have been labelled consistendy as terminally bis-substituted-benzene, 2-nitrobenzene or 2-acetamidobenzene with the chain attached to position 3 of the pyrrole ring. For nitro- and amide-substituted compounds, the chain positions were labelled as 1, 2 and 1', 2' etc. as shown below: R For dipyrromethanes the methane bridge is signified by the label '(pyrrole-2-yl)methylpyrrole'. The pyrrole nuclei of dipyrromethanes were numbered in such a way as to assign 2 and 2' to the methane bridged a positions: 3 . 3' 3.3 Syntheses of benzene and nitrobenzene diacid chain derivatives 1.4-Bis(2-carboxvethenyl)benzene (21) 96 COOH COOH 21 Malonic acid (125 g, 1.20 mol) was placed in a 2L-round-bottom flask containing pyridine (200 mL) and dissolved by warming and shaking on a steam bath. Terephthaldehyde 20 (70 g, 0.52 mol), toluene (200 mL) and piperidine (18 mL) were added. The flask, fitted with a reflux condenser, was moved to an oil bath and heated gradually to 80°C. The reaction mixture was maintained at this temperature overnight During this time, a white solid precipitated and carbon dioxide was released. After being cooled, the reaction mixture was poured into a large beaker containing a mixture of concentrated hydrochloric acid (250 mL) and water (250 mL). The white solid was filtered and washed with water till neutral, yield 108 g (95%). M.P. = > 300°C (reported10600 > 300°C). Mol.wt. Calcd. for C 1 2 H 1 0 O 4 : 218.0579; Found, by high resolution mass spectrometry: 218.0581. Anal. Calcd. for C ^ C v C, 66.05; H, 4.62; Found: C, 65.78; H, 4.72. 'H-nmr (80 MHz, 5, DMSO-dg): 6.60(d, 2H, J=16Hz, side chain 2-CH), 7.60(d, 2H, J= 17Hz, side chain 1-CH), 7.73(s, 4H, benzene-H), 12.44(bs, 2H, -COOH). 97 Mass spectrum [m/e(relative intensity)]: 218(M+, 100), 173(91), 155(32), 147(38), 127(56). 1.4-Bis(2-carboxvethvnbenzene (22) COOH COOH 22 Potassium hydroxide (50 g) was dissolved in water (800 mL) in a 2L-wide-mouth-Erlenmeyer flask, followed by l,4-bis(2-carboxyethenyl)benzene (21) (103.5 g, 0.47 mol) and the rnixture was heated on a stirrer-hot plate until all of the material dissolved. To the warm solution was added Raney nickel-aluminum alloy (40 g) in small portions, over a period of 45-60 min to avoid too much frothing (if any excessive frothing occurred, a few drops of octyl alcohol was added). During addition of the alloy, the temperature of the rnixture was kept around 80°C where it was held for an additional 3 h with stirring. The hot solution was then filtered using a steam-warmed buchner funnel, and the nickel residue was disposed of by pouring it into a mixture of nitric acid (50 mL) and water (100 mL). The filtrate was cooled and acidified by adding concentrated hydrochloric acid. The resulting mixture was allowed to stand overnight and the white solid was filtered off and washed with water. The solid was dried in air for a few days to give 98.5 g product (93.5%). 98 M.P. = 231.0 - 233.0°C (reported106™ 223 - 224°C). Mol.wt. Calcd. for C u H 1 4 0 4 : 222.0892; Found: by high resolution mass spectrometry: 222.0894. Anal. Calcd. for C u H 1 4 0 4 : C, 64.85; H, 6.35; Found: C, 64.95; H, 6.52. lH-nmr (80 MHz, 8, DMSO-ds): 2.05-2.78(m, 8H, side chain l-CH, and 2-CKy, 7.14(s, 4H, benzene-H), 12.09(bs, 2H, -COOH). Mass spectrum [m/e(relative intensity)]: 222(M+, 17), 204(6), 176(22), 162(38), 117(100), 91(34). 1.4-Bis(2-ethoxvcarbonylethvl)benzene (23) COOC2H5 COOC2HS 23 l,4-Bis(2-carboxyethyl)benzene (22) (96.4 g, 0.43 mol) was placed in a lL-round-bottom flask and thionyl chloride (158 mL, 2.17 mol) was added. The mixture was refluxed on a steam bath with a calcium chloride drying tube on the top of the condenser. After 3 h, all of the diacid had reacted to give a yellow crystalline diacid 99 chloride in a brown reaction solution. The excess SOCl2 was driven off by evaporating, in vacuo, and chased with carbon tetrachloride (2x50 mL). Dry ethanol (150 mL) was added to dissolve the diacid chloride, with heat being generated by the partial formation of the ester. A 1:1 mixture (226 mL) of triethylamine and dry ethanol was added dropwise through a pressure equalizing funnel while the reaction flask was cooled in an ice bath. Once the addition was complete, the pale yellow crystalline diester started falling out of solution and the solution was stirred at room temperature overnight. Acetic acid was added to acidify the reaction mixture. The pale yellow solid was filtered off and washed thoroughly with a 1:1 mixture of methanol and water to remove acetic acid. The product was dried in air, yield 101.6 g (84%). M.P. = 70.3 - 72.0°C (reported106*0 69°C). Mol.wt. Calcd: for C^H^O^ 278.1518; Found: by high resolution mass spectrometry: 278.1518. Anal. Calcd. for C^H^A: C, 69.04; H, 7.97; Found: C, 69.14; H, 7.89. 'H-nmr (300 MHz, 8, CDC13): 1.23(t, 6H, J=9Hz, - P O L O U ; 2.60(t, 4H, J=6Hz, 2-CH2); 2.92(t, 4H, J=6Hz, 1-CHJ; 4.13(q, 4H, J=6Hz, -OCROHU); 7.1 l(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 278(M\ 24), 233(12), 204(62), 190(29), 159(22), 130(69), 117(100). 100 l,4-Bis(3-hvdroxvTjropvl')benzene (24) CH 2OH r CH 2OH 24 Sodium borohydride (41.1 g, 1.09 mol) was dissolved in diglyme (400 mL), under nitrogen, in an ice-cooled lL-3-neck-round-bottom flask. The gas oudet of this flask was connected to a second ice-cooled lL-3-neck-round-bottom flask which contained l,4-bis(2-ethoxycarbonylethyl)benzene (23) (75.5 g, 0.27 mol) dissolved in anhydrous THF (400 mL). To the first magnetically stirred solution, boron trifluoride etherate (182.5 mL, 1.45 mol) was added slowly by dropping funnel. The B2U^ produced was introduced by N 2 to the second reaction flask. After addition of BF 3, the ice baths were removed and the reaction solution was stirred for a further 30 min. A TLC analysis of the reaction rnixture showed a single spot, with a lower R, value than that of the starting material indicating a complete reaction. The excess diborane was destroyed with methanol and the reaction solution was filtered to remove some brown materials. The solvent was evaporated under reduced pressure and the remaining white compound was taken into CILClj (50 mL) and washed with water (3x50 mL). After removal of CHjClj, a white slurry was obtained which was dried on the vacuum line to give 46.36 g of the product (88%). M.P. = 49.5 - 51.0°C (reported10i(d) 55°C). 101 Mol.wt. Calcd. for C l 2Hi 80 2: 194.1307; Found, by high resolution mass spectrometry: 194.1307. Anal. Calcd. for C ^ O , : C, 74.19; H, 9.34; Found: C, 73.70; H, 9.32. 'H-nmr (300 MHz, 5, CDC13): 1.61(s, 2H, -OH); 1.83-1.93(m, 4H, side chain 2-OLJ; 2.68(t, 4H, J=7.5Hz, side chain 1-CILJ; 3.67(t, 4H, J=6Hz, -CTLOH); 7.14(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 194(M+, 50), 176(18), 158(27), 148(17), 131(100), 117(89), 104(59), 91(75). 1.4-Bis(3-bromopropyl)benzene (25) l,4-Bis(3-hydroxypropyl)benzene (24) (46.4 g, 0.24 mol) and 48% aqueous hydrobromic acid (200 mL) were heated at reflux under nitrogen. After 4 h, a TLC analysis indicated complete conversion of the starting material to a compound which moved faster on TLC plate. The emulsion was cooled to room temperature, methylene CH 2 Br CH 2 Br 25 102 chloride (100 mL) added and the acid extracted with water (3x50 mL) and saturated aqueous sodium bicarbonate (3x50 mL). The CrLClj solution was extracted again with 0.1 M KOH aqueous solution (2x100 mL) to remove any trace amount of bromine and washed with water (2x100 mL). The resulting solution was dried with anhydrous sodium sulphate, filtered and evaporated under reduced pressure to give a light yellow liquid product (72.6 g, 92.8%). Mol.wt. Calcd. for C ^ B ^ : 317.9618-319.9598-321.9578; Found, by high resolution mass spectrometry: 317.9626-319.9611-321.9606. Anal. Calcd. for C^H^Br,: C, 45.03; H, 5.03; Found: C, 45.43; H, 5.19. 'H-nmr (300 MHz, 5, CDC13): 2.25-2.34(m, 4H, side chain 2-CH2), 2.89(t, 4H, J=7.5Hz, side chain 1-CH2), 3.54(t, 4H, J=6Hz, -CH,Br). 7.28(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 318-320-322(M+, 28-60-28), 211-213(100-99), 172(17), 158(12), 131(28). 1,4-Bis(3-cvanopropyl)benzene (26) l,4-Bis(3-bromopropyl)benzene (25) (34.3 g, 0.107 mol) and potassium cyanide CN 26 103 (28 g, 0.43 mol) were refluxed in dry EtOH (500 mL) under nitrogen. After 7 h a TLC analysis showed only one spot, lower in relative to the starting material. Water (1 L) was added to the reaction mixture while it was still warm to dissolve excess KCN. The solution was cooled with continuous stirring. The reaction mixture containing the yellow oil separated from the aqueous phase was extracted with CH2C12 (150 mL) and washed with water thoroughly. Methylene chloride was evaporated off under reduced pressure to give a quantitative yield of the product (22.6 g, 100%). The solution containing KCN was treated with excess 5% sodium hypochlorite solution for 24 h and then poured into the drain in a fumehood. M.P. = 54.3 - 56.0°C. Mol.wt. Calcd. for C M H 1 6 N 2 : 212.1314; Found, by high resolution mass spectrometry: 212.1313. Anal. Calcd. for C 1 4 H 1 6 N 2 : C, 79.21; H, 7.60; N, 13.20; Found: C, 79.27; H, 7.65; N, 13.00. 'H-nmr (300 MHz, 8, CDC13): 2.03-2.13(m, 4H, side chain 2-CH2), 2.44(t, 4H, J=7.5Hz, side chain 1-CH2), 2.87(t, 4H, J=7.5Hz, side chain -CI^CN), 7.50(s,4H,benzene-H). Mass spectrum [m/e(relative intensity)]: 212(M+, 12), 185(61), 172(46), 158(42), 131(65), 117(100). 104 1 ^ -BisO-carboxvpropyDbenzene (27) H 0 0 C iQL^ C O O H 27 l,4-Bis(3-cyanopropyl)benzene (26) (23.8 g, 0.112 mol), potassium hydroxide (63 g, 1.12 mol) and ethanol (600 mL) were heated at reflux, under nitrogen, for 8 h. After this time the reaction flask was flushed with nitrogen, using a Claisen head adaptor, and the refluxing solution checked, with moist litmus, to ensure that the evolution of ammonia gas had ceased. Water (400 mL) was then added andv the EtOH distilled off until the reflux temperature reached 100°C. The resulting clear solution was refluxed for another hour, and cooled; careful acidification with concentrated hydrochloric acid afforded the white diacid product 27 which was filtered and washed with water, yield 26 g (93%). M.P. = 170.0 - 172.0°C (reported10** 176 - 177°C). Mol.wt. Calcd. for C 1 4 H 1 8 0 4 : 250.1205; Found, by high resolution mass spectrometry: 250.1207. Anal. Calcd. for C l 4 H l g 0 4 : C, 67.18; H, 7.25; Found: C, 67.45; H, 7.35. 'H-nmr (300 MHz, 5, 10% TFA/CDCI3): 1.96-2.06(m, 4H, side chain 2-CH2), 2.48(t, 4H, J=7.5Hz, side chain 3-CH,), 2.69(t, 4H, J=9Hz, side chain l-CH,), 7.14(s, 4H, benzene-H). 105 Mass spectrum [m/e(relative intensity)]: 250(M+, 21), 232(10), 214(30), 190(32), 130(67), 117(100). 1.4-Bis(3-ethoxvcarbonvlpropyl)benzene (28) l,4-Bis(3-carboxypropyl)benzene (27) (19.3 g, 0.077 mol), ethanol (80 mL), toluene (120 mL) and p-toluene-sulphonic acid (0.5 g) were placed in a 250 mL Erlenmeyer flask fitted with a Dean-Stark trap surmounted by a reflux condenser. The solution was refluxed on a stirrer-hot plate for 4 days. During this period, the azeotrope collected in the trap was removed and more toluene and ethanol were added to the reaction flask. After the reaction mixture was cooled, the solvents were removed under reduced pressure. The remaining yellow oil was taken into CHjClj (100 mL) and extracted with saturated aqueous sodium bicarbonate (3x50 mL) and water (3x50 mL). The product was purified by silica gel (300 g) on a column with CHjClj as the eluent to give 20.8 g of a colourless oil as product, yield 88%. Mol.wt. Calcd. for QgH^Cv 306.1831; Found, by high resolution mass spectrometry: 306.1840. Anal. Calcd. for C 1 8 H 2 6 0 4 : C, 70.56; H, 8.55; Found: C, 70.49; H, 8.60. 28 106 'H-nrnr (300 MHz, 5, CDC13): 1.26(t, 6H, J=9Hz, -OCILCH3), 1.88-1.98(m, 4H, side chain 2.32(t, 4H, J=10.5Hz, side chain 3-CHj), 2.62(t, 4H, J=7.5Hz, side chain l-CHj), 4.12(q, 4H, J=10.5Hz, -OC^CH^, 7.10(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 306(M+, 11), 260(13), 232(12), 215(58), 204(26), 173(96), 130(100), 117(92). 1.4-Bis(4.4-dicarboxvbutvl')benzene (29) Freshly cut metallic sodium (5.7 g, 0.248 mol) was dissolved in anhydrous ethanol (200 mL), under nitrogen. Diethyl malonate (71.8 g, 0.448 mol) was added with stirring, followed by l,4-bis(3-bromopropyl)benzene (25) (35.8 g, 0.112 mol) in anhydrous ethanol (100 mL), and the mixture refluxed for 2 h. A TLC analysis of the suspension indicated complete reaction of the starting material to a compound with a lower R,. Ethanol (-100 mL) was then distilled off and a solution of potassium hydroxide (75 g, 1.34 mol) in water (180 mL) added. The remaining ethanol was distilled off and the mixture refluxed at 100°C for a further 2 h. The hot aqueous solution was next treated dropwise with concentrated hydrochloric acid (100 mL). The white solid that precipitated when the solution became acidic was collected and washed with water to give 36.5 g (89%) of 29. 29 107 M.P. = 140.0 - 141.5°C. Anal. Calcd. for C ^ C v C, 59.01; H, 6.05; Found: C, 59.30; H, 6.24. 'H-nmr (300 MHz, 8, 10%TFA/CDC13): 1.75-1.85(m, 4H, side chain 3-CH2), 2.02-2.12 (m, 4H, side chain 2-OLJ, 2.70(t, 4H, J=6Hz, side chain l-CILJ, 3.60[t, 2H, J=6Hz, CH(COOH)J, 7.15(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 278[(M-2C02)+, 8], 260(27), 242(100), 191(39), 145(51), 131(68). 1,4-Bis(4-carboxvbutyl)benzene (30) l,4-Bis(4,4-dicarboxybutyl)benzene (29) (36 g, 0.098 mol) was added slowly, in small portions to a refluxing solution of DMF (250 mL) in a lL-Erlenmeyer flask. During addition, vigorous evolution of carbon dioxide was observed. The resulting white suspension was refluxed for a further 2 h to ensure complete decarboxylation, and then poured slowly into an aqueous solution of 6 M hydrochloric acid (300 mL). The white diacid product 30 was collected by filtration and washed with water, yield 26.3 g H 0 0 O COOH 30 (96.5%). 108 M.P. = 149.0 - 151.0°C. Mol.wt. Calcd. for CigH^Cv 278.1518; Found, by high resolution mass spectrometry: 278.1519. Anal. Calcd. for C ^ O , : C, 69.04; H, 7.97; Found: C, 69.51; H, 7.82. 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 1.69-1.79(m, 8H, side chain 2- and 3-CH2), 2.50(t, 4H, J=6Hz, side chain 4-OLJ, 2.65(t, 4H, J=7.5Hz, side chain 1-CH2), 7.13(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 278(M+, 11), 260(26), 242(100), 191(33), 145(67), 131(92). l,4-Bis(4-ethoxvcarbonylbutvl)benzene (31) In a preparation analogous to that described for 28, the diethylester 31 was obtained in 99% yield (15.8 g) from the diacid 30 (13.3 g, 0.048 mol). Mol.wt. Calcd. for C o^H^C :^ 334.2144; Found, by high resolution mass spectrometry: 31 334.2143. 109 Anal. Calcd. for C ^ C v C, 71.82; H, 9.04; Found: C, 71.56; H, 9.00. 'H-nmr (300 MHz, 8, CDC13): 1.25(t, 6H, J=9Hz, -OCt^CH,), 1.61-1.70(m, 8H, side chain 2-, 3-0^), 2.32(t, 4H, J=6Hz, side chain 4-CHJ, 2.60(t, 4H, J=6Hz, side chain l-CHj), 4.11(q, 4H, J=6Hz, -OQLOL.), 7.08(s, 4H, benzene-H). Mass spectrum [m/e(relative intensity)]: 334(M+, 13), 288(31), 242(100), 214(62), 186(65), 117(92). l,4-Bis(3-ethoxvcarbonylpropvl)-2-nitrobenzene (32) To a stirred ice cooled 500mL-Erlenmeyer flask, fitted with a CaS04 drying tube, were added l,4-bis(3-ethoxycarbonylpropyl)benzene (28) (5.13 g, 0.0168 mol) and trifluoroacetic acid (50 mL), followed by sodium nitrite (2.33 g, 0.0336 mol). The resulting dark brown rnixture was stirred in an ice bath until no brown gas came out of the solution (-24 h). The yellow solution was then poured onto crushed ice (50 g) and methylene chloride (50 mL) was added. The organic layer was extracted with saturated sodium bicarbonate (3x50 mL) and water (3x50 mL). The impure brown oil was purified using silica gel (50 g) in a column with 1% MeOH/CILClj as the eluent to give the yellow liquid product 4.3 g (73%). 32 110 Mol.wt. Calcd. for C ^ H ^ N A 351.1682; Found, by high resolution mass spectrometry: 351.1671. Anal. Calcd. for C ^ N A : C, 61.53; H, 7.17; N, 3.99; Found: C, 61.26; H, 7.20; N, 3.89. 'H-nmr (300 MHz, 8, CDC13): 1.30(t, 6H, J=12Hz, -OCILCH3), 1.97-2.07(m, 4H, side chain 2, 2'-CH2), 2.34-2.43(m, 4H, side chain 3, S'-CHj), 2.74(t, 2H, J=7.5Hz, side chain l'-CH,), 2.92(t, 2H, J=6Hz, side chain 1-CHj), 4.17(q, 4H, J=6Hz, -OCFLCHj), 7.32(d, IH, J=9Hz, m-N02-benzene-H), 7.40(d, IH, J=9Hz, p-NCybenzene-H), 7.76(s, IH, o-N02-benzene-H). Mass spectrum [m/e(relative intensity)]: 351(M\ very weak), 306[(M-OC2H5)+, 13], 276(29), 269(100), 242(23), 232(43), 172(47). 1.4-Bis(4-ethoxvcarbonvlbutvl)-2-nitrobenzene (33) l,4-Bis(4-ethoxycarbonylbutyl)-2-nitrobenzene (33) was synthesized in 65.8% yield using the method described for compound 32. 33 111 Anal. Calcd. for C a j H ^ A : C, 63.31; H, 7.70; N, 3.69; Found: C, 63.12; H, 7.63; N, 3.86. 'H-nmr (300 MHz, 8, CDC13): 1.21(t, 6H, J=7.5Hz, -OCH.CH,), 1.60-1.70(m, 8H, side chain 2, 2'-, 3, 3'-CH2), 2.28-2.3 l(m, 4H, side chain 4, 4'-CH2), 2.64(m, 2H, side chain l'-CH,), 2.81(t, 2H, J=7.5Hz, side chain l-CH,), 4.08(q, 4H, J=7.5Hz, -OQLCHj), 7.28(d, IH, J=11.3Hz, m-N02-benzene-H), 7.35(d, IH, J=7.5Hz, p-N02-benzene-H), 7.72(s, IH, o-N02-benzene-H). Mass spectrum [m/e(relative intensity)]: 349(40), 334[(M-OC2H5)+, 100], 288(43), 274(71), 256(73), 246(98). l,4-Bis(3-carboxvpropvl)-2-nitrobenzene (34) l,4-Bis(3-ethoxycarbonylpropyl)-2-nitrobenzene (32) (7.6 g, 0.022 mol) was dissolved in potassium hydroxide (7.5 g) solution of water (75 mL) and ethanol (225 mL) and refluxed under nitrogen. After 2 h, the condenser was removed and EtOH was distilled off until the temperature reached 100°C. The reaction mixture was cooled in an ice bath and acidified by adding concentrated hydrochloric acid. The off-white solid was collected by filtration and washed with water to give 5.97 g (92%) product. 34 112 M.P. = 157.0 - 159.0°C. Anal. Calcd. for C M H l 7 N,0 6 : C, 56.95; H, 5.80; N, 4.47; Found: C, 57.08; H, 5.82; N, 4.55. 'H-nmr (300 MHz, 8, 10%TFA/CDC13): 1.91-2.09(m, 4H, side chain 2, 2'-CH2), 2.48-2.58(m, 4H, side chain 3, S'-CH,), 2.76(t, 2H, J=9Hz, side chain l'-CH,), 2.95(t, 2H, J=9Hz, side chain l-CH,), 7.31(d, IH, J=9Hz, m-benzene-N02-H), 7.41 (d, IH, J=7.5Hz, p-benzene-N02-H), 7.96(s, IH, o-benzene-N02-H). Mass spectrum [m/e(relative intensity)]: 277[(M-H20)+, 2], 250(24), 232(19), 214(37), 190(48), 117(100). 1.4-Bis(4-carboxvbutvl)-2-nitrobenzene (35) l,4-Bis(4-carboxybutyl)-2-nitrobenzene (35) was prepared according to the method described for compound 34 in a yield of 81%. 35 M.P. = 114.0 - 116.0°C. 113 Anal. Calcd. for C H ^ N A : C, 59.43; H, 6.55; N, 4.33; Found: C, 58.93; H, 6.50; N, 4.07. 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 2.47-2.53(m, 4H, side chain 3, y-CHJ, 1.83-1.93(m, 8H, side chain 2, 2'-, 4, 4'-CH2), 2.68-2.74(m, 2H, side chain l'-CHj), 2.90(t, 2H, J=7.5Hz, side chain 1-CH2), 7.27(d, IH, J=6Hz, m-N02-benzene-H), 7.37(d, IH, J=6Hz, p-N02-benzene-H), 7.76(s, IH, o-N02-benzene-H). Mass spectrum [m/e(relative intensity)]: 305[(M-H2O)+, 13], 293(34), 270(22), 260(19), 246(100), 228(48), 218(79), 206(79). 3.4 Syntheses of benzene, nitrobenzene and amidobenzene bis pyrrole derivatives 3.4.1 The bis pyrrole diketones ( C H 2 L r C I (CH 2 ) n - 1 / / \> 37a, n=4, R=H 37b, n=5, R=H 37c, n=4, R=N02 37d, n=5, R=N02 (a) 1.4-Bisr4-(5-ethoxvcarbonvl-2,4-dimethvlpvrrol-3-yl)-4-oxobutvl1 benzene (37a) 114 l,4-Bis(3-carboxypropyl)benzene (27) (10 g, 0.04 mol) and thionyl chloride (19 g, 0.16 mol) were placed in a flask equipped with a reflux condenser and calcium chloride drying tube. The mixture was heated on a steam bath until all of solid went into solution (-30 min). The excess thionyl chloride was removed by evaporation under reduced pressure and chased with CCL, (4x25 mL). The dark red residue of the bis acid chloride was then taken up in distilled CHjClj (30 mL) and transferred into a flask containing 2-emoxy-3,5-dimethylpyrrole (36) (16.7 g, 0.1 mol), distilled CH2C12 (140 mL) and nitromethane (150 mL), under nitrogen. Anhydrous stannic chloride (41.7 g, 0.16 mol) was added rapidly and the reaction mixture was stirred overnight at room temperature. The dark brown solution was then poured into 0.1 M aqueous hydrochloric acid (100 mL) and stirred for -15 min resulting in precipitation of the product. The yellow sohd was filtered and washed with water, CH2C12 and methanol to give a creamy yellow product. The second crop was collected from the filtrate by evaporating all of the solvents and thoroughly washed with methanol. Overall yield was 12.5 g (57%). An analytical sample was recrystallized by adding methanol to a concentrated solution of 37a in CH2C12 and minimum trifluoroacetic acid. M.P. = 185.0 - 186.5°C. Mol.wt. Calcd. for QjIL^NA: 548.2886; Found, by high resolution mass spectrometry: 548.2894. Anal. Calcd. for C^rL^NA: C, 70.05; H, 7.35; N, 5.11; Found: C, 70.00; H, 7.35; N, 5.24. 115 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 1.48(t, 6H, J=6Hz, -OCH.CH,), 1.99-2.09(m, 4H, side chain 2-CH2), 2, 53(s, 6H, pyrrole 4-CH3), 2.57(s, 6H, pyrrole 2-CH3), 2.74(t, 4H, J=6Hz, side chain 1-CH2), 2.90(t, 4H, J=6Hz, side chain 3-CH,), 4.45(q, 4H, J= 6.2Hz, -OCHjCH,), 7.15(s, 4H, benzene-H), 10.25(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 548(M\ 4), 340(17), 209(100), 194(66), 148(74). (b) 1.4-Bisr5-(5-ethoxvcarbonvl-2.4-dimethvlpvrrol-3-vl)-5-oxopentvl1benzene (37b) l,4-Bis[5-(5-emoxycarbonyl-2,4-dimethylpyrrol-3-yl)-5-oxopentyl]benzene ( 3 7 b ) was synthesised in the manner described for 3 7 a from the diacid 30. The product was isolated in two crops to give an overall yield of 70%. M.P. = 175.0 - 177.0°C. Mol.wt. Calcd. for C^H^NjCv 576.3199; Found, by high resolution mass spectrometry: 576.3205. Anal. Calcd. for C ^ H ^ N A : C, 70.81; H, 7.69; N, 4.86; Found: C, 70.90; H, 7.65; N, 4.97. 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 1.47(t, 6H, J=5.6Hz, -OCHjCty, 1.75(b, 8H, side chain 2-, 3-CHj), 2.56(s, 6H, pyrrole 4-CH3), 2.59(s, 6H, pyrrole 2-CH3), 2.61-116 2.68(m, 4H, side chain 1-Cry, 2.86-2.91(m, 4H, side chain 4-CH^, 4.45(q, 4H, J=7.5Hz, -OCJiCHs), 7.1 l(s, 4H, benzene-H), 10.16(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 576(M+, 9), 530(2), 409(4), 363(4), 209(37), 194(67), 148(100). (c)1.4-Bisr4-(5-ethoxvcarbonvl-2,4-dimethvlpvrrol-3-vl)-4-oxobutyll-2-nitrobenzene (37c) l,4-Bis[4-(5-emoxycarbonyl-2,4-dimemylpyrrol-3-yl)-4-oxobutyl]-2-nitrobe 37c was obtained according to the method for 37a with a yield of 82%. M.P. = 181.5 - 182.5°C. Mol.wt. Calcd. for C^H^NjO,: 593.2723; Found, by high resolution mass spectrometry: 593.2747. Anal. Calcd. for C ^ H ^ A : C, 64.74; H, 6.62; N, 7.08; Found: C, 64.72; H, 6.52; N, 6.89. 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 1.40(t, 6H, J=7.5Hz, -OOLCFL,), 1.96-2.06(m, 4H, side chain 2, 2'-CH2), 2.48-2.54(m, 12H, pyrrole 2-, 4-CH3), 2.73(t, 2H, J=5.6Hz, side chain l'-CH,), 2.83-2.96(m, 6H, side chain 1-, 3, S'-CHJ, 4.39(q, 4H, J=7.5Hz, -OQLCH,), 7.29(d, IH, J=7.5Hz, m-N02-benzene-H), 7.31(d, IH, J=7.5Hz, p-NQ2-117 benzene-H), 7.74(s, 1H, o-N02-benzene-H), 10.21 (bs, 2H, NH).. Mass spectrum [m/e(relative intensity)]: 593(M+, 3), 576(4), 209(45), 194(78), 148(100). (d)l,4-Bisr5-(5-emoxycarbonvl-2,4-Q^ l,4-Bis[5-(5-ethoxycarbonyl-2,4-dimethylpyrrol-3-yl)-5-oxopentyl]-2-nitrobenzene (37d) was obtained in a yield of 74% from the diacid 35 according to the method described for 37a. M.P. = 193.5 - 194.5°C. Mol.wt. Calcd. for C^H^NjO,: 621.3050; Found, by high resolution mass spectrometry: 621.3035. Anal. Calcd. for C ^ N j O g : C, 65.68; H, 6.97; N, 6.76; Found: C, 64.53; H, 6.88; N, 6.24; Calcd. for C^Ho^CyCHaOH: C, 64.30; H, 7.25; N, 6.43. 'H-nmr (300 MHz, 8, 10% TFA/CDC13): 1.43(t, 6H, J=9.2Hz, -OOLOL.), 1.71-1.82(b, 8H, side chain 2, 2'-, 3, S'-CH,), 2.57-2.62(m, 12H, pyrrole 2-, 4-CH3), 2.68-2.75(b, 2H, side chain l'-CH2), 2.86-2.95(m, 6H, side chain 1-, 4, 4'-CH2), 4.42(q, 4H, J=7.7Hz, -OCFLCHj), 7.27(d, IH, J=9Hz, m-N02-benzene-H), 7.38(d, lH, J=9Hz, p-N02-benzene-H), 7.75(s, IH, o-N02-benzene-H), 10.23(bs, 2H, NH). 118 Mass spectrum [m/e(relative intensity)]: 621(M+, 2), 591(8), 209(10), 194(74), 148(100). 3.4.2 The bis pyrrole ethyl esters R (a) 1.4-Bisr4-(5-ethoxvcarbonyl-2.4-chrnethvlpvrrol-3-vl)butvl1benzene (38a) The diketone 37a (14 g, 0.0255 mol) was suspended in anhydrous THF (150 mL) in a 250 mL-two-neck-round-bottom flask which was kept closed with rubber septa; one needle was inserted into each septum allowing a very small flow of N 2. A I M borane-THF complex (10 mL) was injected in the solution and the resulting mixture was stirred for 1 h, during which time all of the white suspension had gone into solution. TLC analysis of an aliquot, quenched by acetic acid, indicated the reaction had not gone to completion. Another 10 mL of BH3-THF solution was added and the mixture was stirred for another hour. TLC analysis, which showed a single spot with a higher Rf value than that of the starting material, indicated a complete reaction. The excess BHj was destroyed by slowly adding methanol (20 mL) and the 38a, n=4, R=H 38b, n=5, R=H 38c, n=4, R=NQ2 38d, n=5, R=N02 119 solvents were evaporated off under reduced pressure and methanol was added to this concentrated solution to crystallize the white product, yield 10.6 g (80%). M.P. = 162.0 - 163.0°C. Mol.wt. Calcd. for Q j H ^ N A : 520.3301; Found, by high resolution mass spectrometry: 520.3307. Anal. Calcd. for C^fL^NA: C, 73.81; H, 8.52; N, 5.38; Found: C, 73.69; H, 8.52; N, 5.45. 'H-nmr (300 MHz, 8, CDC13): 1.35(t, 6H, J=7.5Hz, -OC^CFy , 1.44-1.51(m, 4H, side chain 3-CH2), 1.58-1.68(m, 4H, side chain 2-CH2), 2.18(s, 6H, pyrrole 2-CH3), 2.26(s, 6H, pyrrole 4-CH3), 2.38(t, 4H, J=7.5Hz, side chain 4-CH2), 2.59(t, 4H, J=9.4Hz, side chain 1-CH2), 4.30(q, 4H, J=7.5Hz, -OCTLCH,), 7.08(s, 4H, benzene-H), 8.70(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 520(M+, 19), 474(23), 428(13), 206(100), 180(71), 134(94). (b) 1.4-Bisf5-(5-ethoxvcarbonvl-2.4-dimethvlpvrrol-3-vl)pentvl1benzene (38b) The bis pyrrole ethylester 38b was synthesised by the method described in detail for 38a, yield 84%. 120 M.P. = 181.5 - 182.5°C. Mol.wt. Calcd. for C^H^NA: 548.3614; Found, by high resolution mass spectrometry: 548.3612. Anal. Calcd. for C ^ R ^ C v C, 74.42; H, 8.82; N, 5.10; Found: C, 72.00; H, 8.72; N, 4.92; Calcd. for C3 4H4 8N204-CH3OH: C, 72.38; H, 9.02; N, 4.82. 'H-nmr (300 MHz, 8, CDC13): 1.37, 1.34-1.490, m, 14H, - O C H J C H J , side chain 3-, 4-CHj), 1.58-1.68(m, 4H, side chain 2-CH,), 2.20(s, 6H, pyrrole 2-CH3), 2.28(s, 6H, pyrrole 4-CH3), 2.30-2.38(m, 4H, side chain 5-CH2), 2.58(t, 4H, J=7.5Hz, side chain 1-OLJ, 4.31(q, 4H, J=7.5Hz, -OCH^CH,), 7.08(s, 4H, benzene-H), 8.66(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 548(M+, 17), 502(27), 180(90), 134(100). (c) l,4-Bisr4-(5-ethoxvcarbonvl-2,4-dimethvlpvrrol-3-vl)butyl1-2-nitrobenzene (38c) The diketone 37c was converted to the ethylester 38c by the method described for 38a, yield 83%. MP. = 137.0 - 138.5°C. Mol.wt. Calcd. for Q j H ^ N A : 565.3152; Found, by high resolution mass spectrometry: 121 565.3153. Anal. Calcd. for Q H ^ N A : C, 67.94; H, 7.66; N, 7.43; Found: C, 67.43; H, 7.61; N, 7.62. 'H-nmr (300 MHz, 5, CDC13): 1.33(t, 6H, J=7.5Hz, -OCHjQL,), 1.42-1.53(m, 4H, side chain 3, S'-CHj), 1.58-1.66(m, 4H, side chain 2, 2'-CH2), 2.15(s, 6H, pyrrole 2-CH3), 2.23(s, 6H, pyrrole 4-CH3), 2.35(t, 4H, J=7.5Hz, side chain 4, 4'-CH2), 2.63(t, 2H, J=9.4Hz, side chain l'-OLJ, 2.83(t, 2H, J=7.5Hz, side chain 1-CH2), 4.27(q, 4H, J=6.3Hz, -OCHjCHj), 7.19(d, IH, J=7.5Hz, m-N02-benzene-H), 7.26(d, IH, J=7.5Hz, p-N02-benzene-H), 7.65(s, IH, o-NCybenzene-H), 8.54(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 565(M+, 3), 535(5), 519(3), 428(6), 206(12), 180(60), 134(100). (d) l,4-Bisr5-(5-emoxvcarbonvl-2.4-dimethvlpvrrol-3-vl)pentvll-2-nitrobenzene (38d) The product 38d was obtained in 79% yield using the method described for 38a. M.P. = 137.5 - 138.0°C. Mol.wt. Calcd. for C ^ H ^ N A 593.3465; Found, by high resolution mass spectrometry: 593.3466. 122 Anal. Calcd. for C^H^NA: C, 68.78; H, 7.98; N, 7.08; Found: C, 68.99; H, 7.98; N.6.83. 'H-nmr (300 MHz, 8, CDC13): 1.35, 1.32-1.50(t, m, 14H, -OOLOL., side chain 3, 3'-, 4, 4'-CH2), 1.58-1.67(m, 4H, side chain 2, 2'-CH2), 2.19(s, 6H, pyrrole 2-CH3), 2.26(s, 6H, pyrrole 4-CH3), 2.34-2.38(m, 4H, side chain 5, S'-CHj), 2.63(t, 2H, J=7.5Hz, side chain l'-CHj), 2.83(t, 2H, J=7.5Hz, side chain l-CHj), 4.29(q, 4H, J=7.5Hz, -OQLCH,), 7.20(d, IH, J=8Hz, m-NCybenzene-H), 7.29(d, IH, J=9Hz, p-NCybenzene-H), 7.67(s, IH, o-N02-benzene-H), 8.80(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 593(M+, 7), 563(9), 547(5), 456(7), 206(7), 180(61), 134(100). 3.4.3 The amide bis pyrrole ethyl esters N H C O C H 3 39a, n=4 39b, n=5 (c) 1.4-Bisr4-(5-ethoxvcarbonvl-2.4-dimemvlpvrrol-3-vl)butvl]-2-acetarmd (39c) 123 l,4-Bis[4-(5-emoxycarbonyl-2,4-mmemylpyrrol-3-y (38c) (7.3 g, 12.9 mmol) was dissolved in THF (200 mL), followed by 10% Pd/C (0.7 g), acetic anhydride (10 mL) and sodium acetate (0.5 g). The reaction flask was then connected to a hydrogenation apparatus and the solution was stirred under an Hj atmosphere (1 atm) overnight. TLC analysis indicated that only part of the starting material had reacted. The catalyst was filtered off and fresh 10% Pd/C catalyst (0.7 g) was added and the reaction flask connected to the hydrogenator again. The mixture was stirred under an Hj atmosphere overnight, when TLC analysis indicated a complete reaction. The catalyst was filtered off and the white solid was crystallized out by concentrating the solution, in the presence of hexane, yield 90% (6.7 g). M.P. = 215.5 - 216.5°C. Mol.wt. Calcd. for C^H^NjOj: 577.3516; Found, by high resolution mass spectrometry: 577.3514. Anal. Calcd. for C ^ N A : C, 70.68; H, 8.20; N, 7.27; Found: C, 70.44; H, 8.39; N, 7.07. 'H-nmr (400 MHz, 8, CDCLJ: 1.27(t, 6H, J=8Hz, -OCHjCH,), 1.35-1.58(m, 8H, side chain 2, 2'-, 3, S'-OLJ), 2.10(s, 9H, pyrrole 2-CR,, NHCOCB,), 2.18(s, 6H, pyrrole 4-CH3), 2.28-2.34(m, 4H, side chain 4, ^ - O L J , 2.44-2.54(m, 4H, side chain 1, l'-CH,), 4.22(q, 4H, J=6.7Hz, -OQL.CH,), 6.83-6.87(s, d, 2H, NHCOCH 3, m-NHCOCH3-benzene-H), 7.00(d, IH, J=8Hz, p-NHCOCH3-benzene-H), 7.48(s, IH, o-NHCOCH3-benzene-H), 8.49(b, 2H, NH). 124 Mass spectrum [m/e(relative intensity)]: 577(M+, 15), 531(18), 485(14), 206(16), 180(46), 134(100). (d)l,4-Bisr5-(5-emoxvcarbonvl-2.4-dimemvfo (39d) The product was obtained in the same way described for 39c, yield 96%. M R = 167.0 - 169.0°C. Mol.wt. Calcd. for QgH^NA: 605.3829; Found, by high resolution mass spectrometry: 605.3821. Anal. Calcd. for C ^ N A : C, 71.37; H, 8.48; N, 6.94; Found: C, 71.39; H, 8.68; N, 6.84. 'H-nmr (400 MHz, 8, CDC13): 1.33-1.48(t, m, 14H, -OCHjCFL, side chain 3, 3'-, 4, 4'-CH2), 1.55-1.65(m, 4H, side chain 2, 2'-CH2), 2.19(s, 9H, pyrrole 2-CH3, N H C O C H 3 ) , 2.26(s, 6H, pyrrole 4-CH3), 2.34-2.38(m, 4H, side chain 5, S'-CHJ, 2.50-2.60(m, 4H, side chain 1, l'-CH,), 4.30(q, 4H, J=6.7Hz, -OQj^CH,), 6.95(b, 2H, NHCOCH 3, m-NHCOCH3-benzene-H), 7.08(d, IH, J=8Hz, p->fHCOCH3-benzene-H), 7.56(s, IH, o-NHCOCH3-benzene-H), 8.58(b, 2H, NH). Mass spectrum [m/e(relative intensity)]: 605(M+, 26), 587(47), 559(44), 541(14), 513(18), 180(61), 134(100). 125 3.4.4 The bis pyrrole benzyl esters R ( C H 2 ) n - ^ - ( C H 2 ) n H 6 40a, n=4, R=H 40b, n=5, R=H 40c, n=4, R=NHCOCH3 40d, n=5, R=NHCOCH3 (a) l,4-Bisr4-(5-benzvloxvcarbonvl-2.4-m^ethvlpvrrol-3-vl)butvllbenzene (40a) l,4-Bis[4-(5-emoxycarbonyl-2,4-mmemylpyiTol-3-yl)butyl]benze^ (38a) (7.8 g, 15 mmol) and distilled benzyl alcohol (100 mL) 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 vapor was liberated and a simultaneous drop in temperature was observed; the mixture was allowed to attain reflux each time before further addition of the catalyst. After addition of 3 mL of the catalyst, no effervescence could be observed and the hot solution was carefully poured into methanol (150 mL) with sufficient acetic acid (10 mL) to quench the catalyst. Water was added slowly to precipitate the product, which was filtered and washed with 50% MeOH/water. The dibenzylester was obtained in 86% yield (8.3 g). M.P. = 147.0 - 149.0°C. 126 Mol.wt. Calcd. for QjFL^NjCv 644.3614; Found, by high resolution mass spectrometry: 644.3610. Anal. Calcd. for C«H,iN a0 4: C, 78.23; H, 7.50; N, 4.34; Found: C, 78.04; H, 7.59; N, 4.38. 'H-nmr (400 MHz, 5, CDC13): 1.42-1.49(m, 4H, side chain 3-CH^, 1.57-1.66(m, 4H, side chain 2-CHj), 2.16(s, 6H, pyrrole 2-CH3), 2.27(s, 6H, pyrrole 4-CH3), 2.37(t, 4H, J=8Hz, side chain 4-CH2), 2.58(t, 4H, J=8Hz, side chain l-CH^, 5.29(s, 4H, -OCHjCA). 7.07(s, 4H, benzene-H), 7.31-7.43(m, 10H, -OCH.C^) , 8.54(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 644(M+, 8), 536(4), 509(7), 401(10), 268(23), 108(31), 91(100). (b) l,4-Bis[5-(5-benzvloxvcarbonvl-2.4-dimethvlpvrrol-3-vl)pentvl]benzene (40b) The dibenzylester 40b was obtained with the method described for 40a. The yield was 92%. MP. = 131.0 - 133.0°C. Mol.wt. Calcd. for CUHJJNJCV 672.3927; Found, by high resolution mass spectrometry: 672.3923. 127 Anal. Calcd. for C ^ N A : C, 78.54; H, 7.79; N, 4.16; Found: C, 78.30; H, 7.95; N, 4.14. ^-nmr (400 MHz, 8, CDC13): 1.33-1.48(m, 8H, side chain 3-, 4-CH2), 1.57-1.66(m, 4H, side chain 2-CHj), 2.16(s, 6H, pyrrole 2-CH3), 2.27(s, 6H, pyrrole 4-CH3), 2.31-2.37(m, 4H, side chain 5-CHi), 2.55(t, 4H, J=8Hz, side chain l-CH^), 5.29(s, 4H, -OCHjC^), 7.07(s, 4H, benzene-H), 7.30-7.43(m, 10H, -OCH 2CJL), 8.70(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 672(M+, 12), 564(8), 537(10), 493(9), 429(14), 242(12), 108(56), 91(100). (c)l,4-Bisr4-(5-benzvloxvcarbonyl-2.4-m^emvlpvn-ol-3-vl)butvll-2-acetarrudobenzene(40c) l,4-Bis[4-(5-ethoxycarbonyl-2,4-dimethylpyrrol-3-yl)butyl]-2-acetamidobenzene (39c) (1 g, 1.73 mmol) was placed in distilled benzyl alcohol (10 rnL) and a small piece of metallic sodium (-0.05 g) was added. The resulting mixture was heated at 100°C under 12 mm Hg pressure controlled by a pumptable. After 30 min of heating, all of the starting material had dissolved resulting in a brownish yellow solution which was cooled to room temperature while still on the vacuum line. Acetic acid was slowly added to the solution under nitrogen to neutralize the reaction mixture. When some pink color appeared, wet pH paper showed the solution was neutral and dry ice was added. Benzyl alcohol was distilled off under 0.1 mm Hg at 70 - 100°C. The slightly 128 pink solid was then taken into methylene chloride (30 mL) and washed with water (3x30 mL) to remove salts. After evaporating the methylene chloride, the sohd was taken into ethyl acetate (10 mL), warmed, shaken in a supersonic apparatus and cooled in a refrigerator at 4°C. A white solid was filtered off and washed with cold ethyl acetate to give 0.91 g product (75%). M.P. = 187.0 - 189.0°C. Mol.wt. Calcd. for C^H^NjOj: 701.3829; Found, by high resolution mass spectrometry: 701.3834. Anal. Calcd. for C4 4Hs 1N 3O s: C, 75.29; H, 7.32; N, 5.99: Found: C, 75.13; H, 7.50; N, 5.78. 'H-nmr (400 MHz, 5, CDC13): 1.42-1.65(m, 8H, side chain 2, 2'-, 3, S'-CHj), 2.16(s, 9H, pyrrole 2-CH3, NHCOCHj), 2.27(s, 6H, pyrrole 4-CH3), 2.35-2.4l(m, 4H, side chain 4, 4'-CHa), 2.52-2.61(m, 4H, side chain 1, l'-CHJ, 5.29(s, 4H, -OQLCsHj), 6.86(b,lH, NHCOCH3), 6.92(bd, IH, m-NHCOCH3-benzene-H), 7.06(bd, IH, p-NHCOCH3-benzene-H), 7.32-7.45(m, 10H, -OCHjQHj), 7.56(b, IH, o-NHCOCH3-benzene-H), 8.53(b, 2H, NH). Mass spectrum [m/e(relative intensity)]: 701(M+, 3), 593(3), 458(9), 108(69), 91(100). 129 (d) l,4-Bisr5-(5-benzvloxvcarponvl (40d) The diethylester 39d (1.3 g, 2.15 mmol) was transesterified in 10 mL benzyl alcohol as described for the analogue 40c. The solution was neutralized by adding dry ice only and worked up as for 40a. The yield was 59% (0.92 g). M.P. = 164.0 - 166.0°C. Mol.wt. Calcd. for CJrljjNjOj: 729.4142; Found, by high resolution mass spectrometry: 729.4140. Anal. Calcd. for C ^ N j O , : C, 75.69; H , 7.59; N, 5.76; Found: C , 75.38; H , 7.55; N, 5.56. 'H-nmr (400 MHz, 8, CDC13): 1.33-1.49(m, 8H, side chain 3, 3'-, 4, 4'-CH2), 1.56-1.65(m, 4H, side chain 2, 2'-CH2), 2.17(s, 9H, pyrrole 2-CH3, NHCOCHj), 2.28(s, 6H, pyrrole 4-CH3), 2.33-2.38(m, 4H, side chain 5, 5'-CH2), 2.50-2.60(m, 4H, side chain 1, l'-CH,), 5.30(s, 4H, - O C H J O H J ) , 6.93(b, 2H, NHCOCH3, m-NHCOCH3-benzene-H), 7.07(b, IH, p-NHCOCH3-benzene-H), 7.32-7.44(m, 10H, -OCTLCJi), 7.56(b, IH, o-NHCOCH3-benzene-H), 8.60(b, 2H, NH). Mass spectrum [m/e(relative intensity)]: 729(M+, 7), 685(6), 621(10), 595(13), 486(17), 460(9), 108(100), 91(86). 130 3.4.5 The bis formyl pyrroles R 43a, n=4, R=H 43b, n=5, R=H 43c, n=4, R=NHCOCH3 43d, n=5, R=NHCOCH3  (a) l,4-Bisr4-(5-formvl-2,4-dimethvlDvrrol-3-vl)butvllbenzene (43a) (i) Debenzylation of 40a: The dibenzylester 40a (5 g, 9.62 mmol) and 10% Pd/C (0.5 g) were stirred under hydrogen (1 arm) in THF (150 mL) overnight. When the uptake of hydrogen ceased, the catalyst was filtered and the solution checked by TLC for any unconverted starting material. The solvents were then evaporated in vacuo, leaving the bis(carboxy)pyrrole 41a as a white solid. (ii) Decarboxylation of 41a: The crude bis(carboxy)pyrrole 41a was refluxed in DMF (100 mL) under nitrogen. The UV absorption spectrum showed a reduction of the band at 282 nm to just a shoulder, within 2 h. No further change was then observed. The solution containing the bis a-free pyrrole 42a was cooled in an ice bath and used direcdy in the next reaction. (iii) Vilsmeier formylation of 42a: 131 To an ice cooled solution of DMF (11 mL) in dry CH2C12 (40 mL), phosphorus oxychloride (9.8 mL) was added dropwise. The Vilsmeier reagent, thus prepared, was added rapidly to the chilled DMF solution of the bis a-free pyrrole 42a prepared above. Once addition was complete, the solution was stirred for a further 30 min to ensure completion of the reaction, and then poured onto crushed ice (100 g). Solid sodium bicarbonate was added carefully until the solution became basic, and CH2C12 was then boiled off. Water (300 mL) was added and the rnixture was heated at ~75°C for 1 h. The grey colored solid which crystallized out was filtered and washed with water. An overall yield of 85% of crude 43a was obtained from the dibenzylester 40a. An analytically pure sample of the bis formylpyrrole was prepared by deprotecting the cyanoacrylate derivative 44a with aqueous potassium hydroxide in n-propanol. M.P. = 217.0 - 219.0°C. Mol.wt. Calcd. for C^HjgNA: 432.2777; Found, by high resolution mass spectrometry: 432.2783. Anal. Calcd. for C^H^NA: C, 77.74; H, 8.39; N, 6.48; Found: C, 77.57; H, 8.28; N, 6.32. 'H-nmr (300 MHz, 8, CDC13): 1.40-1.5 l(m, 4H, side chain 3-CH2), 1.58-1.68(m, 4H, side chain 2-CH^, 2.21(s, 6H, pyrrole 2-CH3), 2.25(s, 6H, pyrrole 4-CH3), 2.38(t, 4H, J=7.5Hz, side chain 4-Ctij), 2.60(t, 4H, J=7.5Hz, side chain 1-CH2), 7.07(s, 4H, benzene-H), 9.47(s, 4H, NH, HCO). 132 Mass spectrum [m/e(relative intensity)]: 432(M\ 44), 404(34), 389(18), 296(10), 136(100), 108(58). (b) 1,4-Bisf5-(5-fonnvl-2,4-m^emvlpvrrol-3-vl)pentvl1benzene (43b) This compound was prepared by the same procedure as that employed in the preparation of dialdehyde 43a. The crude bis formylpyrrole was obtained in 88% yield from the dibenzylester 40b. M.P. = 152.0 - 154.0°C. Mol.wt. Calcd. for C^H^r^C^: 460.3090; Found, by high resolution mass spectrometry: 460.3081. Anal. Calcd. for C J O H J ^ C Y C, 78.22; H, 8.75; N, 6.08; Found: C, 77.90; H, 8.85; N, 6.13. 'H-nmr (400 MHz, 8, CDC13): 1.32-1.40(m, 4H, side chain 3-OLJ, 1.43-1.5l(m, 4H, side chain 4-CH2), 1.57-1.65(m, 4H, side chain 2-CHJ, 2.22(s, 6H, pyrrole 2-CH3), 2.25(s, 6H, pyrrole 4-CH3), 2.37(t, 4H, J=8Hz, side chain 5-CH2), 2.57(t, 4H, J=8Hz, side chain 1-OLJ, 7.06(s, 4H, benzene-H), 9.18(bs, 2H, NH), 9.48(s, 2H, HCO). Mass spectrum [m/e(relative intensity)]: 460(M+, 15), 432(17), 150(14), 136(100), 133 108(78). (c) l,4-Bisr4-(5-formvl-2.4-(i^ (43c) The dibenzylester 40c (3.6 g, 5.14 mmol) was debenzylated and decarboxylated according to the procedure described for 40a and 41a. The ice cooled bis a-free pyrrole 42c was treated dropwise with the Vilsmeier reagent, which was made by adding benzoyl chloride (5.4 mL) to a chilled mixture of DMF (5.8 mL) and dry CH2C12 (21 mL). Then, the resulting mixture was treated same way as described for 42a. After being heated at 70°C for 1 h, the product was extracted with CH2C12 to give an impure greyish black.43c. An analytical sample was prepared by deprotecting the cyanoacrylate derivative 44c with aqueous potassium hydroxide in n-propanol. M.P. = 206.5 - 208.5°C. Mol.wt. Calcd. for Cjo j^^ Oa: 489.2991; Found, by high resolution mass spectrometry: 489.2995. Anal. Calcd. for CjoH^aO,: C, 73.59; H, 8.03; N, 8.58; Found; C, 73.23; H, 8.23; N, 8.55. 'H-nmr (400 MHz, 8, CDC13): 1.44-1.67(m, 8H, side chain 2, 2'-, 3, S'-CHj), 2.18(s, 134 3H, N H C O C H 3 ) , 2.20(s, 6H, pyrrole 2-CH3), 2.24(s, 6H, pyrrole 4-CH3), 2.36-2.4l(m, 4H, side chain 4, 4'-CH2), 2.53-2.62(m, 4H, side chain 1, l '-CH 2), 6.88-6.93(b, 2H, NHCOCH 3, m-NHCOCHj-benzene-H), 7.05-7.07(b, IH, p-NHCOCH3-benzene-H), 7.55 (bs, IH, o-NHCOCH3-benzene-H), 8.87(bs, 2H, NH), 9.48, 9.52(s, s, 2H, HCO). Mass spectrum [m/e(relative intensity)]: 4.89(M+, 19), 461(23), 446(11), 169(40), 136(92), 108(100). (d) 1.4-Bisr5-(5-formvl-2.4-dimethvlpvrrol-3-vl)pentvl1-2-acetamidobenzene (43d) The product 43d was obtained according to the procedure employed in the synthesis of 43c. M.P. = 143.0 - 144.0°C. Mol.wt. Calcd. for Q j H ^ N A : 517.3304; Found, by high resolution mass spectrometry: 517.3303. 'H-nmr (400 MHz, 8, CDC13): 1.31-1.65(m, 12H, side chain 2, 2'-, 3, 3'-, 4, 4'-CH2), 2.19(s, 3H, NHCOCH,), 2.20(s, 6H, pyrrole 2-CH3), 2.25(s, 6H, pyrrole 4-CH3), 2.34-2.38(m, 4H, side chain 5, S'-CH,), 2.50-2.58(m, 4H, side chain 1, l'-CH2), 6.91, 7.02-7.09, 7.56(b, m, b, 4H, NHCOCH 3, benzene-H), 8.98(bs, 2H, NH), 9.48(b, 2H, HCO). Mass spectrum [m/e(relative intensity)]: 517(M+, 20), 498(8), 489(23), 474(10), 150(18), 135 136(81), 122(27), 108(100). 3.4.6 The bis cyanoacrylates R 44a, n=4, R=H 44a, n=5, R=H 44a, n=4, R=NHCOCH3 44a, n=5, R=NHCOCH3 (a) 1.4-Bis f 4-f 5-(2-cvano-2-memoxvcarbonvlvinvl)-2.4-d^emvlpyrrol-3-vl1butvl) benzene (44a) The crude l,4-bis[4-(5-formyl-2,4-dimethylpyrrol-3-yl)butyl]benzene (43a) (1 g, 2.31 mmol) was taken up in toluene (50 mL) with methyl cyanoacetate (0.56 g, 5.65 rnmol) and cyclohexylamine (0.5 mL). The rnixture was heated at reflux on a stirrer-hot plate under nitrogen. The reaction was monitored by TLC and was found to be complete in 2 h. The solvent was evaporated, in vacuo, and the residue was taken to dryness on a vacuum line. The crude product was dissolved in CHjCL, and chromatographed on silica gel (50 136 g). The solvent polarity was gradually increased, 0.5% ethyl acetate/CH2Cl2 and then 1% ethyl acetate/CHjClj, to facilitate elution without causing the impurities to move. The bright yellow product was crystallized from the eluates by concentrating, in the presence of methanol, to yield 1.06 g (76.9%). The solid thus obtained was analytically pure. M.P. = 198.3 - 200.3°C. Mol.wt. Calcd. for C^H^NA: 594.3206; Found, by high resolution mass spectrometry: 594.3207. Anal. Calcd. for C A N A : C, 72.70; H, 7.12; N, 9.42; Found: C, 72.87; H, 7.27; N , 9.32. 'H-nmr (400 MHz, 8, CDC13): 1.43-1.50(m, 4H, side chain 3-CH,), 1.58-1.66(m, 4H, side chain 2-CHj), 2.15(s, 6H, pyrrole 4-CH3), 2.28, 2.29(s, s, 6H, pyrrole 2-CH3), 2.38-2.43(m, 4H, side chain A-CR^, 2.60(t, 4H, J=8Hz, side chain l-CH^, 3.86(s, 6H, -OCH3), 7.07(s, 4H, benzene-H), 7.20, 7.91[s, s, 2H, C(H)=C(CN)COOCH3], 9.48(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]: 594(M\ 49), 562(14), 530(26), 217(68), 185(100). 137 (b) 1.4-Bis(5-r5-(2-cvano-2-methoxvcarbonvlvinvl)-2,4-dimethvlpvrrol-3-vllpentvl)  benzene (44b) The bright yellow product 44b was synthesized according to the method described for 44a in a yield of 81.8%. M.P. = 168.5 - 170.0°C. Mol.wt. Calcd. for QgH^NA^ 622.3519; Found, by high resolution mass spectrometry: 622.3531. Anal. Calcd. for C ^ N A : C, 73.28; H, 7.44; N, 9.00; Found: C, 73.32; H, 7.41; N, 8.98. 'H-nmr (400 MHz, 6, CDC13): 1.32-1.41(m, 4H, side chain 3-CH,), 1.41-1.50(m, 4H, side chain 4-CH2), 1.59-1.67(m, 4H, side chain 2-CHj), 2.15, 2.17(s, s, 6H, pyrrole 4-CH3), 2.28, 2.30(s, s, 6H, pyrrole 2-CH3), 2.37(t, 4H, J=8Hz, side chain 5-CH2), 2.58(t, 4H, J=8Hz, side chain l-CH^), 3.86(s, 6H, -OCH3), 7.07(s, 4H, benzene-H), 7.20, 7.92[s, s, 2H, C(H)=C(CN)COOCH3], 9.49(bs, 2H, NH). Mass spectrum [m/e(relative intensity)]:_622(M+, 28), 590(7), 558(8), 217(54), 185(77). (c) 1.4-Bis(4-r5-(2-cvano-2-methoxvcarbonvlvinvl)-2.4-dimethvlpyrrol-3-vl1butvn-2- acetamidobenzene (44c) 138 l,4-Bis[4-(5-benzyloxycarbo^ (40c) (3.5 g, 5.0 mmol) was initially converted to the bis formylpyrrole 43c in the manner described previously. The crude 43c was then taken up in toluene (100 mL) containing methyl cyanoacetate (1.78 g, 18 mmol) and cyclohexylamine (1 mL), and the mixture refluxed under nitrogen for 2 h to reach completion. The residue obtained after evaporating the solvent was chromatographed on silica gel (100 g, activity I) with 1% MeOH/CH2Cl2 as the eluent The bright yellow powdery product was crystallized by concentrating the solution in the presence of methanol, yield 1.76 g (54.1% overall from the bis benzylester.40c). M.P. = 228.0 - 230.0°C. Mol.wt. Calcd. for QgtL.jNjOj: 651.3421; Found, by high resolution mass spectrometry: 651.3440. Anal. Calcd. for C ^ N A : C, 70.02; H, 6.96; N, 10.74; Found: C, 69.71; H, 6.88; N, 10.58. 'H-nmr (400 MHz, 8, CDC13): 1.43-1.66(m, 8H, side chain 2, 2'-, 3, 3'-CHa), 2.15(s, 6H, pyrrole 4-CH3), 2.17(s, 3H, NHCOCH,), 2.27, 2.30(s, s, 6H, pyrrole 2-CH3), 2.37-2.42(m, 4H, side chain 4, 4'-CH2), 2.52-2.61(m, 4H, side chain 1, l'-CH2), 3.85(s, 6H, -OCH3), 6.89-6.93(m, 2H, NHCOCH 3, m-NHCOCH3-benzene-H), 7.06(b, IH, p-NHCOCH3-benzene-H), 7.20, 7.54(bd, IH, o-NHCOCH3-benzene-H), 7.90, 7.92[s, s, 2H, CQi l^CNJCOOCHJ, 9.49(bs, 2H, NH). 139 Mass spectrum [m/e(relative intensity)]: 651(M+, 54), 619(12), 587(14), 245(25), 217(85), 185(100). (d) 1.4-Bis f 5-r5-(2-cvano-2-methoxvcarbonvlvinvl)-2.4-dimethvlpvrrol-3-vl1pentyl) -2- acetamidobenzene (44d) The bright yellow product was obtained in a yield of 53.4% from the bis benzyl ester 40d using the method described for 44c. M.P. = 184.0 - 186.0°C. Mol.wt. Calcd. for Q ^ A N ^ : 679.3733; Found, by high resolution mass spectrometry: 679.3734. Anal. Calcd. for C ^ O j N , : C, 70.67; H, 7.26; N, 10.30; Found: C, 70.51; H, 7.30; N , 10.17. 'H-nmr (400 MHz, 8, CDC13): 1.33-1.50(m, 8H, side chain 3, 3'-, 4, 4'-CH2), 1.57-1.67(m, 4H, side chain 2, 2'-CH2), 2.16(s, 6H, pyrrole 4-CH3), 2.20(s, 3H, NHCOCHj), 2.29, 2.31(s, s, 6H, pyrrole 2-CH3), 2.35-2.40(m, 4H, side chain 5, 5'-CH2), 2.52-2.60(m, 4H, side chain 1, l'-CH,), 3.87(s, 6H, -OCH3), 6.93(b, 2H, NHCOCH 3, m-NHCOCH3-benzene-H), 7.07(b, IH, p-NHCOCH3-benzene-H), 7.21, 7.58(b, IH, o-NHCOCH3-benzene-H), 7.91, 7.93[s, s, 2H, C(H)=C(CN)COOCH3], 9.49(bs, 2H, NH). 140 Mass spectrum [m/e(relative intensity)]: 679(M+, 53), 647(10), 615(12), 273(8), 217(83), 185(100). 3.5 Syntheses of benzene and amidobenzene linked dipyrromethane dimers 3.5.1 Preparation of 47 0 0 47a, n=4, R=H 47b, n=5, R=H 47c, n=4, R=NHCOCH3 47d, n=5, R=NHCOCH3  (a) 1.4-Bis(4-r2-((5-ethoxvcarbonvl-3-ethvl-4-methvlpvrrol-2-yl)methvl)-5-(2-cvano-2- methoxvcarbonvlvinvl)-4-methvlpvrrol-3-vnbutyl} benzene (47a) (i) Monochlorination of the a-methyl group in 44a: 141 l,4-Bis{4-[5-(2-cyano-2-methoxycarbonylvinyl)-2,4-dimethylpyrrol-3-y benzene (44a) (0.44 g, 0.74 mmol) in dry methylene chloride (50 mL) was treated, dropwise, with a solution of sulfuryl chloride (0.22 g, 1.63 mmol) in methylene chloride (10 mL) over a period of 10 min at room temperature. The pale yellow solution which turned orange during addition was stirred for a further 0.5 h. The solvent was then removed completely in vacuo, and the yellow residue of the corresponding cx-chloromethyl derivative 45a was used directly in the next reaction without purification, (ii) Condensation of 45a with 46: The a-chloromethyl derivative 45a, prepared above, was suspended in glacial acetic acid (50 mL) together with 2-ethoxycarbonyl-4-ethyl-3-methylpyrrole 46 (0.35 g, 1.9 mmol) and heated under nitrogen. When the temperature reached 70°C, all of the starting material went into solution resulting a clear dark red solution. After the reaction mixture was heated for a further 30 rnin, TLC analysis of the reaction mixture indicated the product as a single yellow spot, colored violet by bromine vapor. The mixture was evaporated to dryness in vacuo and the residue taken up in methylene chloride (50 mL). The remaining acid was removed by extraction with saturated aqueous sodium bicarbonate (3x50 mL) and water (3x50 mL). The dried methylene chloride solution was then chromatographed on silica gel (50 g). The excess a-free pyrrole starting material 46 was eluted with methylene chloride and the product was eluted with 5%-10% ethyl acetate/methylene chloride. Crystallization of the product was tried in several solvents system and all failed. The product was eventually isolated as a non-crystallisable glass, by evaporating the solvents, yield 0.65 g (92%). An analytically pure sample was obtained using a chromatotron (silica gel, 1 mm plate, 0.5% methanol/methylene chloride as the eluent). 142 M.P. = 244°C (decomp.). Mol.wt. Calcd. for CjgHjgNgOg: 952.5099; Found, by high resolution mass spectrometry: 952.5090. Anal. Calcd. for C ^ N A : c, 70.56; H, 7.19; N, 8.82; Found: C, 69.83; H, 7.36; N, 8.36; Calcd. for C ^ N A ' C H s O H : C, 69.49; H, 7.37; N, 8.53. ^-nmr (400 MHz, 8, CDC13): 1.01 (t, 6H, J=8Hz, S'-OLOL,), 1.31, 1.29-1.40(t, m, 10H, -OCHjCHj, side chain 3-CH2), 1.55-1.64(m, 4H, side chain 2-CHj), 2.10, 2.11, 2.12(s, s, s, 6H, pyrrole 4-CH3), 2.29(s, 6H, pyrrole 4'-CH3), 2.33-2.43(m, 8H, 3'-CHjCH,, side chain 4-CHJ, 2.53-2.61 (m, 4H, side chain l-CHJ, 3.78, 3.83(s, s, 6H, -OCH 3 isomers), 3.87-4.20(m, 4H, bridge-CH,), 4.24(q, 4H, J=8Hz, -OCH^CH^, 7.06(s, 4H, benzene-H), 7.20, 7.84, 7.85[s, s, s, 2H, C(H)=C(CN)COOCH3 isomers], 9.00(b, 2H, l'-NH), 9.37(b, 2H, 1-NH). Mass spectrum [m/e(relative intensity)]: 952(M+, 3), 906(15), 860(20), 698(29), 666(20). 0V) 1.4-Bis(5-r2-((5-ethoxvcarbonvl-3-ethvl-4-methvlpvrrol-2-yl)methvl)-5-(2-cvano-2- methoxvcarbonvlvinvlV 4-methvlpvrrol-3-vllpentvl 1 benzene ( 4 7 b ) This compound was prepared by the same procedure as that employed in the preparation of compound 4 7 a . With 0.51 g (0.82 mmol) of the starting material 44b, a yield of 85.8% was obtained. The product was purified by silica gel (50 g) first, then 143 by alumina (30 g) and was found to be analytically pure. M.P. = 96.0 - 98.0°C. Mol.wt. Calcd. for C^H^NA^ 980.5412; Found, by high resolution mass spectrometry: 980.5394. Anal. Calcd. for C^HaNA* C, 71.00; H, 7.40; N, 8.56; Found: C, 68.88; H, 7.43; N, 8.07; Calcd. for C 5 8 H 7 2 N A " 1/201,0,: C, 68.64; H, 7.19; N, 8.21. Mass spectrum [m/e(relative intensity)]: 980(M+, very weak), 934[(M-CH3CH2OH)+, very weak], 374(41), 343(41). (c) l,4-Bis(4-r-2((5-ethoxvcarbonvl-3-ethvl-4-methvlpvrrol-2-vl)methyl)-5-(2-cvano-2- methoxvcarbonylvinyl)-4-methvlpvrrol-3-vl] butyl 1 -2-acetamidobenzene (47c) Compound 44c (0.42 g, 0.65 mmol), in dry methylene chloride (50 mL) was chlorinated using a solution of sulfuryl chloride (0.19 g, 1.4 mmol) in methylene chloride (10 mL) as described for 47a. The bis a-chloromethyl derivative was then reacted with 2-ethoxycarbonyl-4-ethyl-3-methylpyrrole (46) (0.3 g, 1.66 mmol) in acetic acid (50 mL) to give the dipyrromethane dimer 47c. The crude product was purified by silica gel (30 g, activity I), 1% MeOH/CH2Cl2 as the eluent, yield 0.58 g (89%). 144 M.P.= 132.0 - 134.0°C. Mol.wt. Calcd. for CjgH7 1N709: 1009.5313; Found, by high resolution mass spectrometry: 1009.5289. Anal. Calcd. for C a H 7 1 N 7 Cy C, 68.96; H , 7.08; N, 9.71; Found: C, 68.58; H, 7.10; N, 9.18. 'H-nmr (400 MHz, 8, CDC13): 0.96-1.03(m, 6H, 3'-CH2CH3), 1.26-1.35(m, 6H, -OCHjCHj), 1.35-1.50(m, 4H, side chain 3, 3'-CH2), 1.50-1.68(m, 4H, side chain 2, 2'-CH2), 2.11(s, 6H, pyrrole 4-CH3), 2.16(s, 3H, NHCOCH,), 2.27(s, 6H, pyrrole 4'-CH3), 2.34-2.43(m, 8H, S'-CFLCH,, side chain 4, 4'-CH2), 2.48-2.63(m, 4H, side chain 1,1'-CH2), 3.71, 3.76, 3.78, 3.82(s, s, s, s, 6H, -OCH, isomers), 3.86-3.92(m, 4H, bridge-CH2), 4.20-4.30(m, 4H, -OCH^CH,), 5.63-5.68(bd, IH, NHCOCH3), 6.92(bd, IH, m-NHCOCH3-benzene-H), 7.05(bd, IH, p-NHCOCH3-benzene-H), 7.43(b, IH, o-NHCOCH3-benzene-H), 7.17-7.20, 7.84, 7.86[b, s, s, 2H, C(H)=C(CN)C02CH3], 8.92, 9.25(b, b, 2H, l'-NH isomers), 9.36, 9.40(b, b, 2H, 1-NH isomers). Mass spectrum [m/e(relative intensity)]: 1009(M+, very weak), 963[(M-CH3CH2OH)+, 2], 784(6). (d) lt4-Bis(5-r2-((5-ethoxvcarbonvl-3-ethvl-4-methvlpyrrol-2-vl)methvl)-5-(2-cvano-2- methoxvcarbonvlvinvl)-4-methvlpvrrol-3-vl1pentvll-2-acetamidobenzene (47d) 145 The product 47d was synthesised using the same procedure as described for 47c, yield 94%. M.P. = 99 - 101°C. Mol.wt. Calcd. For C ^ H ^ N A : 1037.5626; Found, by high resolution mass spectrometry: 1037.5535. Anal. Calcd. For C ^ N A : C, 69.41; H, 7.28; N, 9.44; Found: C, 68.94; H, 7.32; N, 9.07. 'H-nmr (400 MHz, 6, CDC13): 0.98-1.05(m, 6H, V-CH^), 1.29-1.43(t, m, 14H, -OCHjCR,, side chain 3, 3'-CH2, 4, 4'-CH2), 1.52-1.63(m, 4H, side chain 2, 2'-CH2), 2.14, 2.16(s, s, 6H, pyrrole 4-CH3), 2.20(s, 3H, NHCOCH,), 2.27, 2.29, 2.30(s, s, s, 6H, pyrrole 4'-CH3), 2.34-2.45(m, 8H, S'-Q^CH,, side chain 5, 5'-CH2), 2.49-2.57(m, 4H, side chain 1, l'-CH2), 3.78-3.85(m, 6H, -OCH 3 isomers), 3.91(s, 4H, bridge-CH2), 4.21-4.30(m, 4H, -OCH^CH,), 6.05-6.13(bd, IH, NHCOCH3), 6.91(bd, IH, m-NHCOCH3-benzene-H), 7.05(bd, IH, p-NHCOCH3-benzene-H), 7.14, 7.54(b, IH, o-NHCOCH3-benzene-H), 7.23, 7.89, 7.92[b, s, s, 2H, C(H)=C(CN)COOCH3 isomers], 8.78, 8.94(b, b, 2H, l'-NH isomers), 9.37, 9.40(b, b, 2H, 1-NH isomers). Mass spectrum [m/e(relative intensity)]: 1037(M+, very weak), 991[(M-CH3CH2OH), 2]. 146 3.5.2 Hydrolysis of 47 0 . 0 48a, n=4, R=H 48b, n=5, R=H 48c, n=4, R=NHCOCH3 48d, n=5, R=NHCOCH3 (a) 1.4-Bis f 4-r2-((5-carboxv-3-emvl-4-memvlpvrrol-2-vl)methyl)-5-form 3-vllbutvl)benzene (48a) In a 25 mL-round-bottom flask fitted with a Claisen adaptor, a nitrogen inlet and a reflux condenser, were placed l,4-bis{4-[2-((5-emoxycarbonyl-3-ethyl-4-methylpyrrol-2-yl)memyl)-5(2-cyancH2-memoxycarbonylvmyl)-4-memylpyrro }benzene (47a) (0.46 g, 0.48 mmol), KOH (0.81 g, 14.5 mmol) and water (9.6 mL). The magnetically stirred suspension was heated to reflux and n-propanol (4.8 mL) was added. The yellow solid starting material went into solution immediately resulting in a pale brown solution. Refluxing was continued and every half an hour an aliquot was removed, diluted with water and its UV/visible spectrum was recorded. This spectrum was compared to that of the starting material which showed a strong absorption at 388 nm 147 and a moderately intense absorption at 280 nm. During the reaction, the peak at 388 nm diminished with a simultaneous increase in the intensity of the peak at lower wavelength and a new peak appeared at 324 nm. In 3 h, the peak at 388 nm disappeared completely, indicating complete removal of the cyanoacrylate protecting groups. The lower wavelength bands (now at 268 nm and 322 nm) were very intense. The condenser was removed at this time to evaporate off n-propanol until the temperature reached 100°C. The solution was cooled to room temperature, while under nitrogen, when the product partially separated out as a brown slimy material. The solution was filtered under suction and the brown material was dissolved by adding water while on the filter paper. The clear solution was then cooled in ice and acidified with glacial acetic acid. The brown powder was collected by filtration and washed with water. The final product was dried in a vacuum desiccator over KOH for 2 days, yield 0.33 g (93%). M.P. = 179.0 - 181.0°C. 'H-nmr (300 MHz, 8, DMSO-dJ: 0.90(m, 6H, 3'-CH,CH,), 1.04-1.60(0, 8H, side chain 2-, 2.12(s, 12H, pyrrole 4, 4'-CH3), 2.20-2.36(m, 8H, side chain 4-CH2, 3'-CHjCHs), 2.40-2.56(m, 4H, side chain 1-CH2), 3.80(s, 4H, bridge-CH^, 7.00(s, 4H, benzene-H), 9.44(s, 2H, HCO), 11.06-11.48(m, 4H, pyrrole NH). Mass spectrum [m/e(relative intensity)]: 734(M+, from FAB), 646[(M-2COO)+, 3], 617(3), 508(5), 169(11), 122(42), 109(58), 94(100). 148 (b) 1.4-Bis{5-r2-((5-carboxv-3-eAvl-4-m  3-yllpentyl) benzene (48b) A brown powdery product was obtained by the same method described for compound 48a in a yield of 86%. M.P. = 250.0 - 252.0°C. 1H1nmr (300 MHz, 8, DMSO-cL.): 0.90(t, 6H, S'-OLCR,), 1.04-1.36(m, 8H, side chain 3-, 4-CHj), 1.36-1.58(b, 4H, side chain 2-OL.), 2.14(s, 12H, pyrrole 4, 4'-CH3), 2.22-2.30 (m, 4H, side chain 5-CH,), 2.30-2.48(m, 8H, side chain 1-CH2, y-CU.CH,), 3.80(s, 4H, bridge-CH2), 7.00(s, 4H, benzene-H), 9.44, 9.52(s, s, 2H, HCO), 11.04-11.60(m, 4H, pyrrole NH). Mass spectrum [m/e(relative intensity)]: 761[(M-H)+, from FAB], 674[(M-2COO)+, 2], 645(3), 536(3), 524(7), 122(30), 109(56), 94(100). (c) 1,4-Bis f 4-r2-((5-carboxv-3-emvl-4-methvlpvrrol-2-vl)methvl)-5-formvl-4-methvlpvrrol- 3-vHbutvl 1 -2-acetamidobenzene (48c) The procedure used for the synthesis of this compound was the same as that employed in the preparation of 48a. The quantities of the reagent and solvents used for 0.33 g (0.33 mmol) starting material 47c were, potassium hydroxide (0.55 g), water (6.5 mL) and n-propanol (3.2 mL). The brown powdery product was obtained in a yield of 89% (0.23 g). 149 M.P. = 165.0 - 167.0°C. 'H-nmr (300 MHz, 8, DMSO-cLJ: 0.94(m, 6H, S'-OLCHj), 1.12-1.64(m, b, 8H, side chain 2, 2'-, 3, S'-OLJ, 2.02(s, 3H, NHCOQi) , 2.18(s, 12H, pyrrole 4, 4'-CH3), 2.24-2.40(m, 8H, side chain 4, 4'-CH2, S'-CIL.CHj), 2.40-2.62(m, 4H, side chain 1, l'-CH2), 3.80(s, 4H, bridge-CHj), 6.88-7.20(m, 3H, benzene-H), 9.24, 9.50, 9.58(d, s, s, 2H, HCO), 11.10-11.50(m, 4H, pyrrole NH). Mass spectrum [m/e(relative intensity)]: 791(M+, from FAB), 703[(M-2COO)+, 4], 674(4), 568(8), 122(24), 109(48), 94(100). (d) 1.4-Bis (5-r2-((5-carboxv-3-emvl-4-memvlpvrrol-2-vl)methvl)-5-formvl-4-rnethvlpvrrol- 3-vllpentvl I-2-acetamidobenzene (48d) The product was synthesised by the same method described for 48a. The quantities of the reagent and solvents used for 0.45 g (0.43 mmol) starting material 47d were, potassium hydroxide (0.73 g), water (8.7 mL) and n-propanol (4.4 mL), yield 85.4% (0.303 g). M.P. = 159.0 - 161.0°C. 'H-nmr (300 MHz, 8, DMSO-d*): 0.80-0.96(m, 6H, S'-OLCHj), 1.12-1.36(b, 8H, side chain 3, 3'-, 4, 4'-CH2), 1.38-1.56(b, 4H, side chain 2, 2'-CH2), 2.00(s, 3H, NHCOCK,), 150 2.16, 2.20(s, s, 12H, pyrrole 4, 4'-CH3), 2.30(b, 8H, y-CU^U,, side chain 5, 5'-CH2), 2.48(b, 4H, side chain 1, l '-OLJ, 3.90(s, 4H, bridge-CH*), 6.92-7.14(m, 3H, benzene-H), 9.20, 9.48(s, s, 2H, HCO), 11.08(s, 2H, 1-NH), 11.50(s, 2H, l'-NH). Mass spectrum [m/e(relative intensity)]: 819(M+, very weak), 775[(M-COO)+, 2], 731[(M-2COO)+, 3], 702(4), 593(9), 122(70), 109(58), 94(100). 3.6 Cyclization to the capped porphyrins (a) 7.17-Diethyl-2,8,12,18-tetramethyl-3.13-fphenylene-1.4-bis(tetramethvlene)lporphyrin (49a) 49a (i) Decarboxylation of 48a: The dipyrromethane derivative 48a (0.31 g, 0.42 mmol) was dissolved in DMF (100 mL) in a 250 mL Erlenmeyer flask, fitted with a Claisen adapter and a nitrogen inlet. The UV/visible spectrum of a drop of this solution, diluted with methylene chloride, showed two bands, one at 280 nm and the other at 318. nm. The magnetically 151 stirred DMF solution was heated at its reflux temperature (153°C). At regular intervals, an aliquot was removed into methylene chloride and its UV/visible spectrum recorded. The intensity of the 280 nm band decreased considerably relative to that of the 318 nm band. This was taken as an indication of the extent of decarboxylation. In 2 h, the 280 nm band was reduced to a shoulder. The solution was cooled to room temperature under nitrogen and the DMF evaporated in vacuo to -10 mL. Methylene chloride (100 mL) was added. The solution was then extracted with water (3x50 mL), dried with anhydrous sodium sulphate, filtered and evaporated to -10 mL. Distilled THF was added to dilute the solution to 200 mL and the solution was stored in a refrigerator for the subsequent cyclization reaction. (ii) Intramolecular 2+2 coupling: The cyclization was carried out in two 2-Litre Erlenmeyer flasks, covered with aluminum foil, each flask containing toluene-p-sulphonic acid (4.0 g), dissolved in methanol (25 mL) and diluted with distilled methylene chloride (600 mL). The dipyrromethane derivative prepared above was added very slowly to the magnetically stirred acid catalyst solution by means of a syringe pump. The syringe pump, operating at its slowest speed, took approximately 9 h to empty one 50 mL syringe. Once addition was complete (-20 h), the reddish-violet solution was concentrated down to -10 ml and taken into methylene chloride (100 mL) and extracted with saturated aqueous sodium bicarbonate (3x50 mL) to remove the acid catalyst. With removal of the acid the solution turned to dark brown but was fluorescent under 366 nm light. It was then taken to dryness in vacuo and the residue was chromatographed as described below. (iii) Chromatographic purification of the porphyrin 152 The crude porphyrin was first chromatographed on silica gel (120 g, activity I) using methylene chloride as the solvent. Some pink materials which eluted down first were discarded. The solvent was then changed to 1% MeOH/CH2Cl2 and the porphyrin eluted down with some brown impurities. The partially purified porphyrin was chromatographed again on alumina (30 g, 4% water added) with methylene chloride as the eluting solvent. Some brown, fast moving impurities initially eluted out, and then, the porphyrin eluted down cleanly, leaving all other impurities adsorbed at the origin. Crystallization from a solution of MeOH/CH2Cl2 afforded 89.2 mg (34.8%) of the purple porphyrin. M.P. > 300°C. Mol.wt. Calcd. For C^H^N,,: 608.3879; Found, by high resolution mass spectrometry: 608.3883. Anal. Calcd. for CJrLjN^ C, 82.85; H, 7.95; N, 9.20; Found: C, 82.40; H, 7.87; N , 8.95. 'H-nmr (400 MHz, 8, CD2CL.): 9.98(s, 2H, methine protons 10-H and 20-H), 9.77(s, 2H, methine protons 5-H and 15-H), 4.08(q, 4H, J=6.7Hz, -CH^CH,), 3.96-4.05(m, 2H, one proton each at chain termini), 3.63-3.70(m, 2H, one proton each at chain termini), 3.63(s, 6H, 2CH3), 3.44(s, 6H, 2CH3), 2.38(s, 4H, benzene-H), 1.87(t, 6H, J=8Hz, -Cf^CHj), 1.36-1.50(m, 2H, chain protons), 1.18-1.27(m, 2H, chain protons), 0.83-0.91(m, 2H, chain protons), 0.07-0.18(m, 4H, chain protons), -0.08-0.02(m, 2H, chain protons), -3.26(bs, 2H, NH). 153 Visible spectrum (CHjClJ: Xta„ (nm), 404 506 542 572 626 log e, 5.26 4.08 4.10 3.91 3.60 (b) 7.17-Diethvl-2,8,12,18-tetramethvl-3.13-fphenvlene-1.4-bis(pentamethylene)lporphyrin (49b) 49b This porphyrin was prepared and purified in the same way as described for porphyrin 49a. The yield was 19.1%. M.P. > 300°C. Mol.wt. Calcd. for QMH^N^ 636.4192; Found, by high resolution mass spectrometry: 636.4189. Anal. Calcd. for C ^ N , : C, 82.97; H, 8.23; N, 8.80; Found: C, 80.72; H, 8.62; N, 8.57; Calcd. for C ^ N ^ O ^ O H : C, 80.80; H, 8.44; N, 8.38. 154 'H-nmr (400 MHz, 5, CDjCLJ: 10.19(s, 2H, methine protons 10-H and 20-H), 10.09(s, 2H, methine protons 5-H and 15-H), 4.34-4.4 l(m, 2H, one proton each at chain termini), 4.22-4.32(m, 2H, -CJiCHj), 4.06-4.15(m, 2H, -CHjCHs), 3.95-4.05(m, 2H, one proton each at chain terrnini), 3.67(s, 6H, 2CH3), 3.54(s, 6H, 2CH3), 2.68(s, 4H, benzene-H), 2.17-2.27(m, 2H, chain protons), 1.91, 1.87-1.91(t, m, 8H, -CHjCHj, chain protons), 0.86-0.97(m, 6H, chain protons), 0.28-0.35(m, 2H, chain ^ termini), -0.13- -0.24(m, 2H, chain protons), -1.13(m, 2H, chain protons), -3.58(bs, 2H, NH). Visible spectrum (CHjClj): X*„ (nm), 400 498 534 568 622 loge, 5.10 3.90 3.77 3.46 3.13 (c) 7.17-Diethvl-  methylene)! porphyrin (49c) N H C O C H 3 The procedure used in preparation of this porphyrin was the same as that employed in preparation of porphyrin 49a. 155 This porphyrin was found to be adsorbed onto silica gel more strongly than 49a and 49b. For activity I silica gel, 2% MeOH/CILClj was necessary to move the porphyrin. The porphyrin eluate which containing some impurities was purified again using a chromatotron with a 1 mm A\03 plate and 50%-25%hexane/CH2Cl2 as the eluting solvents. The porphyrin was found to be quite clean by an UV/visible spectrum and the product was obtained by evaporating the solvents. The residue was dried under vacuum for 3 days to give 44.8% yield. Crystallization was achieved by evaporating a hexane/CH2Cl2 solution of the porphyrin. M.P. > 300°C Mol.wt. Calcd. for C ^ H J J N J O : 665.4094; Found, by high resolution mass spectrometry: 665.4093. 'H-nmr (400 MHz, 8, CD2C12): 9.94(d, 2H, J=16Hz, methine protons 10-H and 20-H), 9.77(d, 2H, J=16Hz, methine protons 5-H and 15-H), 5.54, 5.36(s, s, 2H, benzene-H), 3.94-4.14(m, 6H, -CH^CHj, one proton each at chain termini), 3.60-3.74(m, d, 9H, one proton each at chain termini, two CH 3 , one benzene-H)), 3.41 (d, 6H, two CH3), 1.86-1.92(m, 6H, -O^CH,), 1.59(s, 3H, NHCOCH,), 1.31-1.59(m, 2H, chain protons), 1.50(s, IH, NHCOCH3), 0.70-0.95(m, 2H, chain protons), 0.01-0.20(b, 4H, chain protons), -0.10- -0.35(b, 4H, chain protons), -3.30(b, 2H, NH). Visible spectrum (CH2Clz): X t o„ (nm), 404 506 542 572 624 log e, 5.23 4.07 4.09 3.89 3.60 156 (d) 7,17-Diethvl-2,8,12.18-tetramethvl-3.13-r2-acetamido-phenylene-1,4-bis(penta  methylene')] porphyrin (49d) This compound was obtained by the same method described for 49c. The yield was 28.6%. M.P. > 300°C Mol.wt. Calcd. C^HsiNjO: 693.4407; Found, by high resolution mass spectrometry: 693.4423. Anal. Calcd. for C ^ N A C, 79.61; H, 7.99; N, 10.09; Found: C, 79.44; H, 8.09; N, 9.86. 'H-nmr (400 MHz, 8, CD.Cy: 10.17(d, 2H, methine protons 10-H and 20-H), 10.07(d, 2H, methine protons 5-H and 15-H), 5.62(m, IH, benzene-H), 4.32-4.42(m, 2H, one proton each at chain termini), 4.21-4.32(m, 2H, -CHjCHj), 4.05-4.16(m, 2H, -QLCF*,), 3.95-4.05(m, 2H, one proton each at chain termini), 3.68(s, 6H, 2CH3), 3.54(s, 8H, NHCOCH3 49d 157 2CH3, two benzene-H), 2.22(b, 2H, chain protons), 1.86, 1.91(m, t, 8H, -CH 2CH, and chain protons), 1.55(s, 3H, NHCOCH3), 1.50(s, IH, NHCOCH3), 0.83-1.03(m, 6H, chain protons), 0.10-0.24(m, 2H, chain protons), -0.23(m, 2H, chain protons), -1.23, -1.38(b, b, 2H, chain protons), -3.82(b, 2H, NH). Visible spectrum (CtLClj): Ajnax (nm), 400 500 536 568 622 loge, 5.26 4.20 4.13 3.96 3.82 3.7 Syntheses of the benzene and amidobenzene-hemin chloride complexes R (50a)Cl, n=4, R=H (50b)Cl, n=5, R=H (50c)Cl, n=4, R=NHCOCH3 (50d)Cl, n=5, R=NHCOCH3 The free base porphyrin (49a-d, 0.035 mmol), dissolved in THF (20 mL), was bubbled with nitrogen for 30 rnin, and then added rapidly to a stirred solution of 158 ferrous chloride (0.35 mmol) in methanol (20 mL) which was previously purged by nitrogen. The mixture was refluxed under nitrogen for 1.5 h. After this time an UV/visible spectrum indicated complete conversion to the hemin chloride. The solution was cooled and stirred in air for 30 min to ensure complete conversion of Fe(II) to Fe(III). The solvent was then evaporated in vacuo, the residue taken up in methylene chloride, washed with water and chromatographed on alurnina (30 g, 4% H 2 0 added) using methylene chloride as the solvent The green p>oxo dimer, eluting from the column, was shaken with aqueous hydrochloric acid (0.2 M, 30 mL) to effect conversion back to the hemin chloride. The solution was evaporated in vacuo and methanol added to crystallize the hemin chloride (yield 80%). (a) Benzene-4/4 hemin chloride (50a)Cl Mol.wt. Calcd. for F e C ^ H ^ C l : 697.2760; Found, by high resolution mass spectrometry: 697.2756. Visible spectrum (toluene): A*, (nm), 374 402(sh) 508 528 630 log e, 4.74 4.67 3.80 3.82 3.56 (b) Benzene-5/5 hemin chloride (50b)Cl: Mol.wt. Calcd. for Fe^rla^Cl: 725.3073; Found, by high resolution mass spectrometry: 159 725.3098. Visible spectrum (toluene): Xta„ (nm), 380 400(sh) 508 532 632 log e, 4.86 4.77 3.73 3.77 3.33 (c) Amidobenzene-4/4 hemin chloride (50c)Cl: Mol.wt. Calcd. for FeC^H^jOCl: 754.2975; Found, by high resolution mass spectrometry: 754.2977. Visible spectrum (toluene): X*. (nm), 380 402(sh) 494 534 630 log e 4.78 4.70 3.98 3.85 3.60 (d) Amidobenzene-5/5 hemin chloride (50d)Cl: Mol.wt Calcd. for FeC^^NjCl: 782.3288; Found, by high resolution mass spectrometry: 782.3267. Visible spectrum (toluene): X^rnm), 380 400 508 532 632 log e 4.88 4.79 3.78 3.82 3.43 160 Chapter 4 Interaction of the capped hemes with CO and 0 2 4.1 Materials and apparatus 4.1.1 General Toluene (1 L) was washed with c. H 2S0 4 (2 x 200 mL) followed by water (2 x 100 mL), and distilled over CaH 2 . Sodium dithionite (sodium hydrosulphite) was purchased from Aldrich. 1,5-Dicyclohexylimidazole (Dclm) was purchased from Aldrich and recrystallized from heptane, m.p. = 113-114°C. Carbon monoxide (99.5%) and dioxygen (99.99%) were passed through KOH columns prior to use. Argon (99.99%) was initially passed through a Redox column and then dried over molecular sieves (3 A) and KOH. The solubilities of CO and 0 2 in toluene used were 1.0 x 10s M/torr and 1.2 x 10'5 M/torr respectively, at 20°C. 1 0 7 4.1.2 Thermostatting equipment (a) For temperatures down to 4°C A thermostatted cell-holder (Fig. 4.1), constructed in the Chemistry Department Mechanical Shop, U.B.C., was used to maintain a constant temperature. It was 161 connected to a Haake (model FK) circulating thermostatting bath filled with 50% EtOH/H20 by tygon tubing (Fig. 4.1). The circulating ethanolic solution was thermally equilibrated with the copper block of the cell-holding compartment. A tonometer (with cell path length 1 cm) could be firmly inserted into the cell-holder. Then, the solution inside the cell was equilibrated to the temperature of the surrounding copper block. The cell-holder could be fixed at the proper position of the UV/visible instrument for the recording of spectra. (b) For low temperature: -45°C A 6 cm path length quartz cell mounted inside a small Dewar (Fig. 4.2) was used for low temperature work. The Dewar was filled with a chlorobenzene-liquid nitrogen slush bath (-45°C). 4.1.3 Electronic absorption spectra A Hewlett Packard 8452A Diode Array Spectrophotometer was used to record all UV/visible spectra in the range of 250-700 nm. 4.2 Solution preparation All solution studies were performed in toluene. The major concern in choosing a suitable solvent is its ability to sustain a six-coordinate heme-02 adduct, during oxygen equilibria studies. Toluene, providing a relatively non-polar environment of dielectric constant 2.38, increases the stability of the oxygen adduct by reducing the rate of its autoxidation (section 1.5.1). For the purpose of consistency, toluene was also employed in the CO binding studies. 162 constant temperature fixed to UV spectrophotometer Fig. 4.1 Thermostatted cell-holder for temperatures above 4°C Because of the tendency for Fe(II) to be oxidized to Fe(m) in the presence of trace dioxygen, preparation of the Fe(TI) five-coordinate complexes and binding experiments must be carried out with complete exclusion of air. The aqueous dithionite reduction method of S. K. David,50 was used to reduce the hemin chloride to the corresponding heme. A solution of the hemin chloride and 1,5-dicyclohexylimidazole, of required concentrations, in toluene (6 mL), was placed in flask A of a set up as shown in Fig. 4.3.A. The entire three flask system and hemin solution were deoxygenated by means of freeze-thaw cycles performed four times; Ar gas was admitted and tap 4 closed. A small volume (-0.5 mL) of freshly prepared 163 joint Q To VACUUM LINE OPTICAL CELL DEWAR FLASK CONSTANT TEMPERATURE BATH L\ SEALED-IN QUARTZ WINDOWS 4.2 6 cm path-length quartz cell5000 164 F l a s k C F l a s k B F l a s k A t e f l o n tap 3 tap 2 Fig. 4.3 (A) Apparatus used for reducing Fem(Por) to Fen(Por) using aqueous dithionite^ (©intO (B) Tonometer for measuring ligand binding constants of five-coordinate hemes, Fe^PorXDcIm)^ to l i M B 165 deoxygenated aqueous NajSA (saturated) was cannulated into the hemin solution via tap 1, by means of a 2' long double-ended needle and a stream of argon. The mixture was stirred for 30 min, during which time reduction to the five-coordinate, Fen(Por)(DcIm), complex was complete. The toluene layer was then decanted into flask B and partially dried over anhydrous NajSO, for 15 min. The solution was finally poured into flask C and dried over CaH2 for 30 min. At this stage, tap 3 was closed and flask A disconnected for convenience. The flask system was then connected to a tonometer (Fig. 4.3.B) by attaching joints 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 solution allowed to filter through the frit into the tonometer. Tap 5 was closed, the flasks removed, and argon admitted through tap 6. During the titration experiment, aliquots of gaseous ligand could be introduced into the tonometer by connecting tap 6 to the vacuum hne. 4.3 Mathematical analyses The equilibrium constant for the reaction of a five-coordinate heme with a ligand as shown in eq. (4.1), K L Fen(Por)(DcIm) + L v v Fen(Por)(DcIm)(L) (4.1) L = CO or O z is defined by: [Fe(Por)(DcIm)(L)] K L = (4.2) [Fe(Por)(DcIm)][L] The equilibrium constant K L is a measure of the affinity of the reactant, the heme 166 complex, towards the ligand L, and is given by the inverse of the concentration of L required to convert half of the reactant heme to the product.108 In the experimental determination of K L , excess imidazole blocks the unhindered side of the heme, while its binding on the capped side is inhibited for steric reasons. Therefore, the binding constant (KL) corresponds exclusively to the coordination of L on the capped face. Eq. (4.2) may be re-written in a form such as: fraction of product y KL[L] = = (4.3) fraction of reactant 1-y If the reaction is followed spectrophotometrically, y can be determined from: A - A 0 y = (4.4) A_ - An where A G = initial absorbance of reactant AM = final absorbance of product and A = absorbance at a specific [L] The logarithmic conversion of eq. (4.3) gives the Hill equation:109 y log( ) = log[L] + logKL (4.5) 1-y y A plot of log( ) versus log[L] should result in a straight hne of slope 1.0 for the 1-y 167 coordination of one ligand at the heme, and the equilibrium constant K L may be determined from the x- or y- axis intercept of the log : log plot. In general, when the value of the slope is not exactly 1.0, the x-axis intercept is used to give a direct 1 estimation of K L =—. For the gaseous ligands CO or 02, the ligand concentration [L] [L] is commonly expressed in terms of partial pressure of the gas over the solution PL. The equihbrium constant for the binding of a gas can then be written as: 1 KCtorr1) = (4.6) r l / 2 Where = the partial pressure of gas (L) required to convert half of the five-coordinate heme into the six-coordinate ligated species. Equilibrium constant determinations for CO binding to the benzene-4/4, and the amidobenzene-4/4 heme systems were carried out at varied temperatures. Van't Hoff plots of ln K versus 1/T(°K), eq. (4.7), afforded the thermodynamic constants A H 0 and AS 0 from the slope and y-intercept, respectively.108 AH° AS° ln K = + (4.7) RT R 168 4.4 Results Binding studies of the durene systems have shown50 that binding of the small 1-methylimidazole (Melm) occurs under the large caps of the durene-7/7 and -5/5 systems. For the 7/7-heme particularly, a typical six-coordinate hemochrome,110 was formed even with a very low [Melm]. In the case of the 5/5-durene heme, binding under the cap was much weaker, but at the high [Melm] required for studying CO and 0 2 binding equilibria, a considerable amount of six-coordination was observed. This excluded Melm as an axial base for the binding studies of these large capped systems. Based on the studies of the durene50 and other systems,111 Dclm was considered unlikely to coordinate under the large caps because of steric reasons. Therefore,, the bulky Dclm was chosen as the base in the CO and 0 2 binding studies for our systems. 4.4.1 Carbon monoxide binding to the five-coordinate hemes: K c o values (a) Solution preparation and the method of CO addition The five-coordinate heme complexes Fe(Por)(DcIm) were prepared and transferred into a tonometer according to the method described in Section 4.2. The 5/5-hemes were found to bind CO too strongly to allow K c o deterrnination using pure CO (P^°2 -0.03 - 0.06 torr). Even for the 4/4-hemes, Pf?2 were several torr and accurate addition of pure CO during titration was difficult. Therefore diluted CO/Ar mixtures prepared in a glass bulb, attached to a manometer, were used for titrations (Fig. 4.4).50 Initially a suitable amount (measured accurately) of CO was admitted into an evacuated bulb, and Ar added to barometric pressure. The total pressure in the bulb was then reduced roughly to half by evacuation (measured accurately on the manometer), and the bulb again filled with Ar. This evacuation procedure was repeated several times until a 169 desired partial pressure of CO remained in the bulb. After being connected to the tonometer containing the five-coordinate heme, the system was completely degassed (taps 1 and 5 closed, taps 3 and 4 open, Fig. 4.4). The line and manometer were allowed to equilibrate to the partial pressure of toluene (tap 4 closed). Appropriate pressures of the prepared CO/Ar mixture were admitted into the tonometer from the bulb, by slowly opening and closing tap 1. The heme solution was stirred briefly and the tonometer disconnected (tap 5 closed). By this Fig. 4.4 Vacuum line set-up used for preparing and admitting CO/Ar mixtures into a tonometer5000 method, CO/Ar mixtures could be prepared in advance, allowing very low partial pressures of CO (-10"3 torr, within ± 10% error) to be admitted into the tonometer. During the titration, a large excess of Dclm was maintained in solution, so that KMm|DcIm1 » Kco[CO]. Thus the equilibrium constant K c o obtained for each heme corresponds exclusively to the binding of CO on the capped side of the heme, without any complication caused by the displacement of the Dclm axial base. 170 (b) Equilibrium constant determination, K 0 0 The spectral properties of the four-, five- and six-coordinate Fe(benzene-4/4) systems are shown in Fig. 4.5. All of the K00 values were obtained via addition of CO/Ar mixtures to the five-coordinate hemes, according to the eq. (4.8): K c o Fe(Por)fDcIm) + CO v N Fe(Por)(DcIm)(CO) (4.8) An example of the isosbestic spectral changes observed for CO binding is shown in Fig. 4.6. The extent of CO binding to the heme is dependent on the partial pressure of the gas, P c o , over the toluene solution. For suitable ranges of P 0 0 (P°° = 1-102 torr for the 4/4-pair and P 0 0 = 0.001-0.1 torr for the 5/5-pair), log(A-Ao)/(A..-A) values were found to be linearly dependent on logP00, with a slope -1.0. After each addition of CO, the solution in the tonometer was shaken vigorously for 5-10 min to ensure equilibration and the tonometer then thermostatted to 20°C for a further 10 min. The volume ratio of solution : tonometer was small (3 : 300 mL), so that changes in P 0 0 with dissolution in the toluene phase were considered negligible. In all cases, the data were analyzed according to the Hill equation109 (Appendix HA). The K 0 0 values together with those of the durene series and open chelated systems are quoted in Table 4.1. A detailed analysis for the Fe(benzene-4/4)(DcIm)(CO) system, corresponding to the spectral changes shown in Fig. 4.6 is given in Appendix IIA(l) and the Hill plot is shown in Fig. 4.7. (c) Temperature variation equilibria: AH£0, AS£o values For the Fe(benzene-4/4)(DcIm) and Fe(amidobenzene-4/4)(DcIm) systems, K c o values were measured at different temperatures within the range 0-30°C (Appendix IIB). 171 K> 400 500 600 700 Fig. 4.5 Spectral trends for Fen(benzene-4/4), Fen(benzene-4/4)(DcIm) and Fen(benzene-4/4)(DcIm)(CXD) systems in toluene +0.910-1 j 300 400 ' ' 500 ' ' ' 600 ' ' ' 700 WAVELENGTH Isosbestic spectral changes for Fen(Por)(DcIm) + CO , ^ Fen(Por)(DcIm)(CO) at 20°C in toluene Por = the benzene-4/4 porphyrin 49a; added P 0 0 = 1.99, 4.30, 7.55, 11.60, 19.03, 34.64 and 51.87 torr in the 300 - 700 nm range; final P 0 0 = 1 atm Van't Hoff plots of ln K°° vs. 1/T(°K), shown in Figs. 4.8 and 4.9, provided the thermodynamic constants, AHgo and AS§o (see Appendix EC and Table 4.3, p. 184). 4.4.2 Dioxygen binding to the five-coordinate hemes, K o z (a) Ambient temperature The five-coordinate heme Fe(II)(Por)(DcIm) solution in toluene (6 mL) was prepared and transferred into a tonometer (Fig. 4.3.B) by the method described in Section 4.2. In order to ensure no nxristure remained, a small volume of toluene 174 Table 4.1 Equilibrium constants for CO binding to the capped hemes compared to those for chelated open hemes in toluene at 20°C Heme (M-1) Heme K°° (M1) chelated protoheme* 4 x 10s benzene-4/4(DcIm) (50a)(DcIm)e 12 x 104 chelated mesoheme* 2 x 108 benzene-5/5(DcIm) (50b)(DcIm)c 1.6 x 106 durene-7/7(DcIm) (13b)(DcIm)b 6.6 x 107 anudobenzene-4/4(DcIm) (50c)(DcIm)c 2.8 x 10* durene-5/5(DcIm) (12b)(DcIm)b 8.6 x 107 amidobenzene-5/5(DcIm) (50d)(DcIm)e 3.3 x 10s durene-4/4(DcIm) (llb)(DcIm)b 4.3 x 106 a Ref. 112; Solvent toluene-CH2Cl2 (90 : 10) b Ref. 50 c Errors <, + 5% 7.0-6.0 5 . 0 -8 + •S 4 . 0 -3.0-AHSo = -15 kealmol 1 ASgo - -44. calmol'K' 1 3.2 3.3 3.4 3.5 1/T(°K) x Iff 3.6 Fig. 4.8 Van't Hoff plot for: FeD(benzene-4/4)(DcIm) + CO ^ Fen(benzene-4/4)(DcIm)(CO) 7.0-, 6.0-5 + fa £ 5.0-4.0-AHgo = -12 kcal mol 1 AS°» = -31 ca lmol 'K 1 3.3 3.4 3.5 1/T(°K) x 103 Fig. 4.9 Van't Hoff plot for. Fen(arrndorjen2«ne-4/4)(DcIm) + CO ^ (DcIm)(CO) 3.6 Fe (amidobenzene-4/4) 176 (~1 mL) was pumped off, while the solution was stirred inside the tonometer. Pure dioxygen was admitted into the tonometer by the procedure outlined in Section 4.2. The UV/visible spectrum was taken immediately. For all of the four hemes, the oxidation of the five-coordinate species was instantaneous and the UV/visible spectrum showed that p>oxo dimer had been formed (Fig. 4.10). Therefore, the studies had to be carried out at lower temperature. (b) Low temperature equilibria: K 0 2 values Several temperatures were exarnined and -45°C was found suitable. A solution of the five-coordinate heme Fe(II)(Por)(DcIm) in toluene (17 mL) was prepared as described in Section 4.2 and transferred into the cell mounted inside a Dewar (Fig. 4.2). During the transfer, the flask system in Fig. 4.3.A was attached to the cell by connecting joints P and Q, and the system evacuated with taps 5 and 6 open (tap 4 closed). With tap 6 then closed and tap 4 opened, the solution was allowed to flow into the cell. Finally, taps 5 and 6 were closed off and the flasks disconnected. The Dewar flask was filled with a slush bath of chlorobenzene-liquid nitrogen, and the solution degased completely by opening tap 6. Dioxygen was admitted into the cell via a flexible stainless steel tube attached to joint Q and the vacuum line. The volume of the solution was maintained well below the narrow neck of the cell, in order to agitate the solution sufficiently by shaking the cell (5 min), to effect equilibration of 0 2 between the solution and gas phases. The Dewar was then disconnected from the vacuum line (taps 5 and 6 closed), taken to the UV/visible instrument and the spectral changes recorded. Prolonged exposure to 0 2 should be avoided in order to minimize oxidation of the hemes by 02; therefore, the spectral changes for only four titrations were measured. A large excess of Dclm had to 177 be maintained, ([Dclm] : [hemeJ-lO4:!), in all cases to effectively thwart irreversible oxidation to the |!-oxo dimer. Spectra of the |i-oxo dimer, five-coordinate and 02-adduct for the Fe(benzene-5/5) system are shown in Fig. 4.10. 0 2 titration for all of the five-coordinate hemes gave absorbance changes with clean isosbestic points, indicating a clean conversion to the oxy-species according to the equation: K 0 2 Fe(Por)(DcIm) + 0 2 v N Fe(Por)(DcIm)(02) (4.9) Deoxygenation, by pumping, after full formation of the 02-complex regenerated the five-coordinate heme with negligible heme oxidation. The data were analyzed according to the Hill equation109 (Appendix UD). An example of the isosbestic spectral changes for 0 2 binding is shown in Fig. 4.11 and a typical plot of log (A-AQ)/(A--A> versus P 0 2 in Fig. 4.12. The affinity constants for 0 2 binding to our systems together with those for the durene systems are quoted in Table 4.2. 4.5 Discussion The five-coordinate high spin Fe(U) system is characterized by a broad absorption in both the visible and Soret region.110 Coordination of CO as the sixth ligand gives rise to a sharp Soret band (Fig. 4.5), whereas the Soret band of the 02-adduct has almost the same shape as that of the five-coordinate species (Fig. 4.10). 4.5.1 Carbon monoxide binding: K c o The data in Table 4.1 show that there has been a remarkable reduction of CO affinities with our systems compared to the "open" chelated meso- and proto-heme 178 VO 400 500 600 700 Fig. 4.10 Spectral trends for Fen(benzene-5/5)(DcIm), Fen(benzene-5/5)(DcIm)(02) and |i-oxo-<Iimer of the Fen(benzene-5/5)(DcIm) systems in toluene oo o o Ji < B = 1,S-Dlcyeloh«i»l-ImidatoU 1 1 1 • 1 1 r - • r 1 1 1 1 1 , , . , , , , 300 400 500 600 700 WAVELENGTH (nm) Fig. 4.11 Isosbestic spectral changes for Fe"(Por)(DcIm) + 0 2 , ^ Fe^orJfDcIm)^ at -45°C in toluene Por = the benzene-4/4 porphyrin 49a; added P 0 2 = 9.76, 30.95, 77.16 and 163.69 torr in the 300 - 700 nm range; final P 0 2 = 1 atm o.e -0.4 --A - Ao log o.o -A . - A -0.2 --0.4 --o.e --0.8-Fig. 4.12 Hill plot for Fen(benzene-4/4)(DcIm) + OlK v Fen(benzene-4/4)(DcIm)(02) at -45°C systems and the corresponding durene series (lib, 12b, 13b). The 5/5- capped complexes exhibit a 100-fold reduction in CO affinities and the 4/4- capped complexes a lfZ-fold reduction compared to those of the "open" chelated hemes. The CO-complexes of the 4/4-series are so unstable that the 5-ccordinate species can be regenerated by pumping. Compared to the durene series, the reduction in CO affinity is still as large as 50- and 350-fold for the 5/5 and 4/4 systems, respectively. The results were unexpected because the caps of the durene systems should be more crowded and access to the central metal should be hindered. However, placement of a more rigid diagonal strap (ie. the durene moiety), relative to the benzene and amidobenzene analogues, presumably induces doming of the porphyrin skeleton and log P 0 2 181 Table 4.2 T?m values for dioxygen binding to the capped hemes in toluene Heme P w (20°C, torr) P w (-45°C, torr) Fe(durene-7/7)(DcIm) (13b)(DcIm)' -64 Fe(durene-5/5)(DcIm) (12b)(DcIm)* -64 Fe(durene-4/4)(DcIm) (HbXDcIm)1 167 0.094b Fe(durene-4/4)(MeIm) (llb^elm)* 82.6 Fe(benzene-5/5)(DcIm) (50b)(DcIm) 22.7 Fe(benzene-4/4)(DcIm) (50a)(DcIm) 55.7 Fe(amidobenzene-5/5) (50d)(DcIm) 2.8 Fe(amidobenzene-4/4) (50c) (Dclm) 9.5 a Ref. 50 b Extrapolated from thermodynamic data resulting in a greater accessibility for an mcorning ligand to the Fe(Tf) atom (Fig. 4.13.A). This would effectively result in an enhanced K c o . On the other hand, the benzene and the amidobenzene moieties are more flexible and may prefer a "squashed" A B Fig. 4.13 (A) "Basket-shaped" conformation of the distorted durene-5/5 and -4/4 systems; (B) "Squashed" conformation of the benzene-5/5 system 182 cap conformation in which the rotating benzene moiety is suspended close to the center of the porphyrin plane (Fig. 4.13.B). Ligand coordination to the sixth position would then be retarded by the benzene moiety. A similar explanation has been proposed for the durene-7/7 complex's low CO association rate relative to those of the smaller 4/4-and 5/5-durene complexes.30 Within our systems, each 5/5- complex has - 100-fold higher CO affinity than that of the corresponding 4/4-complex. This is easily explained by the increased distortion of the porphyrin skeleton and a more crowded distal side (Section 1.2.2) of the 4/4-complexes. The shorter strap over the distal side of the 4/4-hemes should block access to the sixth position and the more distorted porphyrin plane would result in a more "T-state" character, therefore, CO affinity should be lower. Compared to the analogues without amide substitution, the amide groups in the amidobenzene hemes probably fix the distal moiety in a manner similar to the durene system (Fig. 4.13.A), creating a greater accessibility for an incoming ligand to the Fe atom, and therefore an increased CO affinity (-2-fold) results. 4.5.2 Thermodynamic considerations for CO-binding Table 4.3 shows the thermodynamic data for CO binding of the benzene- and the amidobenzene-4/4 hemes together with data for the durene series. It is obvious that the relative decrease in CO affinity in our system results from the contribution of the more unfavorable entropy change. The large entropy loss suggests more rigidity of the CO complexes of the 4/4-benzene and amidobenzene hemes relative to those of the durene systems. After formation of the CO-complex, perhaps the previously flexible benzene moiety might become fixed. The relatively large overall loss of translational entropy (more negative AS°) between reacting species for binding of CO therefore can be 183 rationalized for our systems. Table 4.3 Thermodynamic constants for CO binding1 Heme AH§ 0 (kcahmol1) AS^ (cal-mol-'K1)" chelated protoheme" -17.5 -34 Fe(PocPiv)(l,2-Me2Im)d -13.9 -28 Fe(TPP)(l,2-Me2Im)d -12.8 -26.1 Fe(durene-4/4)(DcIm) (llb)(DcIm)e -9 -12 Fetdurene^XU-M^Im) (Ilb)(l,2-Me2lm)e -14 -32 Fe(benzene-4/4)(DcIm) (50a)(DcIm) -16 -44 Fe(amidobenzene-4/4)(DcIm) (50c)(DcIm) -12 -32 a solvent system is toluene for all model systems with the exception of chelated protoheme. b standard state 1 arm c Ref. 113; aqueous suspension, pH 7 d Ref. 114 e Ref. 50 4.5.3 Dioxygen binding The benzene and amidobenzene capped hemes cannot bind dioxygen reversibly at room temperature. The oxidation of the five-ccoidinate hemes in the presence of 0 2 is instantaneous. This might result from a more flexible cap being able to swing to one side thus allowing for u.-oxo dimer formation. At -45°C, however, the oxidation can be prevented successfully and reversible 0 2 binding achieved. The Fe(II) five-coordinate species could be regenerated by pumping on solutions of the 02-complex. Table 4.2 shows the strikingly high P w values for our system at low temperature (-45°C). But, 184 within each pair of the benzene- and amidobenzene-hemes, a 6-8 fold increase in 0 2 affinity is observed for the amide substituted complexes (a free energy gain of 949 cal/mol for the amidobenzene-4/4 heme and 802 cal/mol for the amidobenzene-5/5 heme, relative to their corresponding nonamido-hemes), suggesting a possible hydrogen bonding stabilization of the 02-complex. In the durene heme series, a hydrophobic "cap" provides a relatively non-polar distal environment, so that (lack of) polarity effects experienced by the bound dioxygen should be identical for all the three durene hemes. Any difference in 0 2 affinities, should therefore, be caused by steric effects alone. Examination of the durene heme systems reveals that at 20°C the K01 values of the Fe(durene-7/7)(DcIm) and Fe(durene-5/5)(DcIm) systems are identical and the difference of K 0 2 values between the Fe(durene-5/5)(DcIm) and Fe(durene-4/4)(DcIm) is only 2-fold,50 indicating very small steric effects on 0 2 binding in spite of the different cap sizes. In the benzene- and amidobenzene-heme systems at -45°C, there is a very small (2- to 3-fold) increase in 0 2 affinity for the 5/5- capped hemes relative to the 4/4-capped hemes. This is consistent with the trend found in the durene series. Perhaps the reduced accessibility to the sixth coordination site due to the smaller cap and the distortion (doming) of the 4/4-hemes could account for this decreased K 0 2 . Porphyrin skeletal doming of this type may be viewed as imparting considerable "T-state" character on the five-coordinate heme complex and therefore leading to the low 0 2 affinity. As the CO binding affinities of the corresponding benzene- and amidobenzene-heme complexes are different (~2 fold increase for the amide-substituted analogues), the possible influence of this steric effect (Fig. 13.A) can be excluded as the only source for the higher 0 2 affinity of the amide substituted analogues. The 0 2 affinity 185 enhancement in the amide analogues is 6-8 fold, too large to be accounted for by the steric effect. Therefore, either the polarity of the distal cage or a direct interaction of the amide proton with bound dioxygen contributes partly to this increased 0 2 affinity for the amidobenzene-hemes. Within each pair of the benzene and amidobenzene hemes, the only difference in structure is the amide functionality. The partial double bond character of the bond between the carbonyl carbon and the nitrogen of an amide due to the resonance structures makes the nitrogen more electropositive. Therefore, the N-H of the amide is capable of forming a strong hydrogen-bonding with bound dioxygen. This electronic effect (or electrostatic effect as often cited in literature) would greatly influence the 02-complex stability. In the free-base amide substituted porphyrin, the N-H of the amide group has been shifted upfield in the nmr spectrum compared to normal aromatic amide protons. It indicates that the H is situated near the center of the porphyrin plane, suggesting that a direct interaction of amide-H with bound 0 2 (H-bonding) is likely in the heme. Compared to the basket handle models 7 of Momenteau et a/.60 (Table 1.4, p. 26) and Chang's porphyrin naphthoic acid series 658 (Table 1.3, p. 25), the 0 2 stability gain (6-8 fold) of the amidobenzene-hemes due to an amide group is not so large. The basket handle model has a 10-fold increased 0 2 affinity and the porphyrin naphthoic acid series has even larger increased 0 2 affinity. The smaller 0 2 affinity increase of our systems can be explained perhaps in terms of H-bonding strength. The strength of a hydrogen bond is largely dependent on steric constraints. For an effective hydrogen bonding to take place, it is essential to minimize the distance and the freedom of motion between the proton source and the acceptor. Only when a close encounter of the H source and the acceptor is realized, and both elements point towards each other, 186 7a R=(CH2)U 7b R=CO(CH2)1oCO can a strong H-bonding result. Various conformational characteristics of each "cap" can influence the direction in which a polar amide function points within the distal cavity. In our systems, space-filling (CPK) models indicate a close encounter of the two groups, but the N-H—O is not in a linear array. The close encounter demonstrates the possibility of H-bonding, but the steric pattern of the N-H—O suggests a relatively weak H-bonding and a relatively small enhancement of 0 2 affinity in the system. Confirmation of this explanation awaits a crystal structure determination. In conclusion, introduction of an amide functionality to the distal side of the heme increases the 0 2 affinity due to the possible hydrogen bonding between the polar amide function and the bound dioxygen. The benzene- and amidobenzene-hemes, therefore, give quantitative support for the hypothesis that hydrogen bonding plays an important role in differential binding of CO and 0 2 by hemoglobin and myoglobin. 187 References 1. (a) J. L. Hoard, in Porphyrins and Metalloporphvrins. K. M. Smith, Ed., American Elsevier, New York, N. Y., 1975, p. 317. (b) R. Bonnett, in The Porphyrins. Vol. I. D. Dolphin, Ed., Academic Press, 1978, p. 1. 2. A. L. Lehninger, Biochemistry. 3rd Ed., Worth Publishers Inc., New York, N.Y., 1975, p. 489. 3. T. P. Wijesekera, J. B. Paine HI, D. Dolphin, F. W. B. Einstein & T. Jones, J. Am. Chem. Soc.. 105. 6747 (1983). 4. W. R. Scheidt & C. A. Reed, Chem. Rev.. 81, 543 (1981). 5. (a) M. F. Perutz, Nature. 228. 726 (1970). (b) M. F. Perutz, Br. Med. Bull.. 32, 195 (1976). (c) M. F. Perutz, Scientific Am.. 239 (6\ 92 (1978). 6. M. F. Perutz, Ann. Rev. Biochem.. 48, 327 (1979). 7. J. M. Baldwin, Br. Med. Bull.. 32, 213 (1976). 8. J. V. Kilmartin, Br. Med. Bull.. 32, 209 (1976). 9. J. V. Kilmartin, FEBS Lett.. 38, 147 (1974). 10. J. C. Kendrew, R. E. Dickerson, B. E. Standberg, R. G. Han, D. R. Davis, D. C. Phillips & V. C. Shore, Nature. 185. 422 (1960). 11. J. Baldwin & C. Chothia, J. Mol. Biol.. 129. 175 (1979). 12. G. Fermi, M. F. Perutz, B. Shaanan & R. Fourme, J. Mol. Biol. 175. 159 (1984). 13. (a) B. R. Gelin & M. Karplus, Proc. Nad. Acad. Sci. USA. 74, 801 (1977). (b) B. R. Gelin, A. W.-M. Lee & M. Karplus, J. Mol. Biol.. 171. 489 (1983). 14. A. Brzozowski, Z. Derewenda, E. Dodson, G. Dodson, M. Grabowski, R. Liddington, T. Skarzynski & D. Vallely, Nature. 307. 74 (1984). 15. R. Liddington, Z. Derewenda, G. Dodson & D. Harris, Nature. 331. 725 (1988). 188 16. B. Shaanan, J. Mol. Biol.. 171. 31 (1983). 17. L. Pauling, Nature. 203. 182 (1964). 18. (a) C. H. Barlow, J. C. Maxwell, W. J. Wallace & W. S. Caughey, Biochem.  Biophys. Res. Commun.. 55, 91 (1973). (b) J. C. Maxwell, J. A. Volpe, C. H. Barlow & W. S. Caughey, Biochem.  Biophys. Res. Commun.. 58, 166 (1974). 19. M. Tsubaki & N.-T. Yu, Proc. Natl. Acad. Sci. USA. 78, 3581 (1981). 20. M. P. Mims, A. G. Porras, J. S. Olson, R. W. Noble & J. A. Peterson, J. Biol. Chem.. 258. 14219 (1983). 21. T. Yonetani, H. Yamamoto & T. Iizuka, J. Biol. Chem.. 249. 2168 (1974). 22. F. A. Walker & J. Bowen, J. Am. Chem. Soc.. 107. 7632 (1985). 23. M. Ikeda-Saito, T. Iizuka, H. Yamamoto, F. J. Kayne & T. Yonetani, J. Biol.  Chem.. 252. 4882 (1977). 24. S. E. V. Phillips & B. P. Schoenborn, Nature. 292, 81 (1981). 25. B. Shaanan, Nature. 296, 683 (1982). 26. J. M. Baldwin, J. Mol. Biol.. 136, 103 (1980). 27. J. S. Olson, A. J. Mathews, R. J. Rohlfs, B. A. Springer, K. D. Egeberg, S. G. Sligar, J. Tame, J.-P. Renaud & K. Nagai, Nature. 336, 265 (1988). 28. Z. Derewenda, G. Dodson, P. Emsley, D. Harris, K. Nagai, M. F. Perutz & J. -P. Renaud, J. Mol. Biol.. 211. 515 (1990). 29. K. Kim, J. Fettinger, J. L. Sessler, M. Cyr, J. Hugdahl, J. P. Collman & J. A. Ibers, J. Am. Chem. Soc.. 111. 403 (1989). 30. B. A. Springer, K. D. Egeberg, S. G. Sligar, R. J. Rohlfs, A. J. Mathews, T. E. Carver & J. S. Olson, J. Biol. Chem.. 265. 3168 (1990). 31. M. F. Perutz, Quarterly Reviews of Biophysics. 22, 139 (1989). 32. L. Pauling & C. D. Coryell, Proc. Natl. Acad. Sci. USA. 22, 210 (1936). 33. J. J. Weiss, Nature, 202. 83 (1964). 34. (a) H. P. Misra & I. Fridovich, J. Biol. Chem.. 247, 6960 (1972). (b) L. S. Demma & J. M. Salhany, J. Biol. Chem.. 252, 1226 (1977). 189 35. A. S. Koster, J. Chem. Phvs. 63, 3284 (1975). 36. (a) W. A. Goddard & B. D. Olafson, Proc. Nad. Acad. Sci. USA. 72, 2335 (1975) . (b) C. A. Reed & S. K. Cheung, Proc. Natl. Acad. Sci. USA. 74, 1780 (1977). 37. I. P. Gerothanassis & M. Momenteau, J. Am. Chem. Soc.. 109. 6944 (1987). 38. I. J. Solomon, J. N. Keith, A. J. Kacmarek & J. K. Raney, J. Am. Chem.  Soc. 90, 5408 (1968). 39. S. E. V. Phillips, J. Mol. Biol.. 142. 531 (1980). 40. F. A. Cotton & G. Wilkinson, Advanced Inorganic Chemistry. 4th Ed., Wiley, New York, N. Y., 1980, p. 82. 41. S.-M. Peng & J. A. Ibers, J. Am. Chem. Soc.. 98, 8032 (1976). 42. J. Kuriyan, S. Wilz, M. Karplus & G. A. Petsko, J. Mol. Biol.. 192. 133 (1986). 43. (a) E. A. Padlan & W. E. Love, J. Biol. Chem.. 249. 4067 (1974). (b) J. C. Norvell, A. C. Nunes & B. P. Schoenborn, Science. 190. 568 (1975). (c) E. J. Heidner, R. C. Ladner & M. F. Perutz, J. Mol. Biol.. 104. 707 (1976) . 44. T. Asakura, P. W. Lau, M. Sono, K. Adachi, J. J. Smith & J. A. McGray, in Hemoglobin and Oxygen Binding, Chien Ho, Ed., Elsevier, New York, N. Y., 1982, p. 177. 45. (a) R. H. Austin, K. W. Beeson, L. Eisenstein, H. Frauenfelder & I. C. Gunsalus, Biochemistry. 14. 5355 (1975). (b) N. Alberding, S. S. Chan, L. Eisenstein, H. Frauenfelder, D. Good, I. C. Gunsalus, T. M. Nordlund, M. F. Perutz, A. H. Reynolds & L. B. Sorensen, Biochemistry. 17, 43 (1978). (c) D. A. Case & M. Karplus, J. Mol. Biol.. 132. 343 (1979). 46. R. D. Jones, D. A. SummerviUe & F. Basolo, Chem. Rev.. 79, 139 (1979). 47. (a) N. Sadasivan, H. I. Eberspaecher, W. H. Fuchsman & W. S. Caughey, Biochem.. 8, 534 (1969). (b) A. L. Balch, Y.-W. Chan, R-J. Cheng, G. N. La Mar, L. Latos-Grazynski & M. W. Renner, J. Am. Chem. Soc.. 106. 7779 (1984). 190 48. (a) W. J. Wallace, J. C. Maxwell & W. S. Caughey, Biochem. Biophvs. Res.  Commun.. 57, 1104 (1974). (b) W. J. Wallace, R. A. Houtchens, J. C. Maxwell & W. S. Caughey, J. Biol.  Chem.. 257. 4966 (1982). 49. I. P. Gerothanassis, M. Momenteau & B. Loock, J. Am. Chem. Soc.. Ill, 7006 (1989). 50. (a) S. K. David, Ph. D Thesis, University of British Columbia, 1985. (b) S. K. David, B. R. James & D. Dolphin, J. Inorg. Biochem.. 28, 125 (1986). (c) S. K. David, D. Dolphin & B. R. James, Front. Bioinorg. Chem., A. V. Xavier, Ed., VCH: Weinheim, Fed. Rep. Ger., 1986. 51. R. S. Drago, J. P. Cannady & K. A. Leslie, J. Am. Chem. Soc.. 102. 6014 (1980). 52. (a) J. P. Collman, R. R. Gagne, T. R. Halbert, J.-C. Marchon & C. A. Reed, J. Am. Chem. Soc.. 95, 7868 (1973). (b) J. P. Collman, R. R. Gagne, C. A. Reed, T. R. Halbert, G. Lang & W. T. Robinson, J. Am. Chem. Soc.. 97, 1427 (1975). 53. G. B. Jameson, G. A. Rodley, W. T. Robinson, R. R. Gagne, C. A. Reed & J. P. Collman, Inorg. Chem., 17, 850 (1978). 54. G. B. Jameson & R. S. Drago, J. Am. Chem. Soc.. 107, 3017 (1985). 55. L. Leiserowitz & G. M. J. Schmidt, J. Chem. Soc. A. 2372 (1969). 56. L. Leiserowitz & A. C. Tuval, Acta. Crvstallogr. Sect. B34. 1230 (1978). 57. C. K. Chang, B. Ward, R. Young & M. Kondylis, J. Macromol. Sci.-Chem., A25, 1307 (1988). 58. C. K. Chang & M. P. Kondylis, J. Chem. Soc.. Chem. Commun.. 316 (1986). 59. E. N. Guryanova, I. P. Goldshtein & T. I. Perepelkova, Russ. Chem. Rev., 45, 792 (1976). 60. (a) M. Momenteau & D. Lavalette, J. Chem. Soc., Chem. Commun., 341 (1982). (b) D. Lavalette, C. Tetreau, J. Mispelter, M. Momenteau & J.-M. Lhoste, Eur.  J. Biochem.. 145. 555 (1984). 61. J. Mispelter, M. Momenteau, D. Lavalette & J.-M. Lhoste, J. Am. Chem. Soc, 191 105, 5165 (1983). 62. (a) J. B. Kim, A. D. Alder & F. R. Longo in The Porphyrins, Vol. I, D. Dolphin, Ed., Academic Press, 1978, p. 85. (b) J. B. Paine HI in The Porphyrins. Vol. I. D. Dolphin, Ed., Academic Press, 1978, p.101. (c) A. W. Johnson in The Porphyrins. Vol. I, D. Dolphin, Ed., Academic Press, 1978, p.235. 63. (a) T. P. Wijesekera, Ph. D. Thesis, University of British Columbia, 1979. (b) T. P. Wijesekera, J. B. Paine TU & D. Dolphin, J. Org. Chem.. 53, 1345 (1988) (c) T. P. Wijesekera, S. K. David, J. B. Paine III, B. R. James & D. Dolphin, Can. J. Chem.. 66, 2063 (1988). 64. B. Morgan, Ph. D. Thesis, University of British Columbia, 1984. 65 T. P. Wijesekera & D. Dolphin, Svnlett. 5, 235 (1990). 66. (a) A. D. Adler, F. R. Longo & W. Shergalis, J. Am. Chem. Soc. 86, 3145 (1964). (b) A. D. Adler, F. R. Longo, J. D. Finarelli, J. Goldmacher, J. Assour & L. Korsakoff, J. Org. Chem.. 32, 476 (1967). 67. J. E. Baldwin, M. J. Crossley & J. Debernardis, Tetrahedron. 38, 685 (1982). 68. G. Jones, Organic Reactions. 15, 204 (1967). 69. Organic Syntheses Collective. Vol. Ill, p.742. 70. W. Carruthers, Some Modern Methods of Organic Synthesis, 2nd Ed.; Cambridge University Press, 1978, p.6. 71. W. M. Weaver in The Chemistry of the Nitro and Nitroso Groups, Part II, Feuer Ed; Interscience Publishers, New York, 1970; pp. 1-48. 72. G. A. Olah & S. J. Kuhn, J. Am. Chem. Soc., 84, 3684 (1962). 73. (a) C. L. Coon, W. G. Blucher & M. E. Hill, J. Org. Chem.. 38, 4243 (1973). (b) Effenberger & Geke, Synthesis. 40 (1975). 74. U. A. Spitzer & R. Stewart, J. Org. Chem.. 39, 3936 (1974). 75. The Sadtler Standard N.M.R. Spectra. Vol. 19. 12083M. 192 76. S. Uemura, A. Toshimitsu & M. Okano, J. C. S. Perkin I„ 1076 (1978). 77. E. S. Gould in Mechanism and Structure in Organic Chemistry. Holt, Rinehart & Winston, New York, 1959; pp. 710-711. 78. J. B. Paine IH & D. Dolphin, Can. J. Chem.. 56, 1710 (1978). 79. H. W. Whidock & R. Hanauer, J. Org. Chem.. 33, 2169 (1968). 80. T. Neilson, H. C. S. Wood & A. G. Wylie, J. Chem. Soc.. 371 (1962) . 81. T. Severin, R. Schmitz & H.-L. Temme, Chem. Ber.. 96, 2499 (1963) . 82. P. Kniel, Helv. Chim. Acta.. 51, 371 (1968). 83. E. J-H. Chu & T. C. Chu, J. Org. Chem.. 19, 266 (1954). 84. A. R. Battersby, E. Hunt, E. McDonald, J. B. Paine Ul & J. Saunders, J. Chem. Soc. Perkin Trans. I. 1008 (1976). 85. A. Hayes, G. W. Kenner & N. R. Williams, J. Chem. Soc. 3779 (1958). 86. J.-J. Delpuech & D. J. Nicole, J. C. S. Perkin I. 570 (1977). 87. P. G. Farrell, F. Terrier & R. Schaal, Tetrahedron Letters. 26, 2435 (1985). 88. G. W. Kenner, J. Rimmer, K. M. Smith & J. F. Unsworth, J. C. S. Perkin I. 332 (1977). 89. R. Chong, P. S. Clezy, A. J. Liepa & A. W. Nichol, Aust. J. Chem.. 22, 229 (1969). 90. H. Fischer and H. Hofelmann, Justus Liebigs Ann. Chem.. 216, 533 (1938). 91. R. B. Woodward, Angew. Chem.. 12, 651 (1960). 92. J. B. Paine III, R. B. Woodward & D. Dolphin, J. Org. Chem.. 41, 2826 (1976). 93. E. Baciocchi & G. muminati, Tet. Lett. 15, 637 (1962). 94. E. Baciocchi, A. Clana, G. Illuminati & C. Pasini, J. Am. Chem. Soc. 87, 3953 (1965). 95. E. Baciocchi & G. Illuminati, Prog. Phys. Org. Chem., 5, 1 (1967). 96. K. H. Lee, Tetrahedron. 25, 4363 (1969). 193 97. P. S. Clezy & A. J. Liepa, Aust. J. Chem.. 23, 2443 (1970). 98. P. S. Clezy, F. D. Looney, A. W. Nichol & G. A. Smythe, Aust. J. Chem.. 19, 1481 (1966). 99. A. Treibs & H. G. Kolm, Liebies Ann. Chem.. 199, 614 (1958). 100 J. A. S. Cavaleiro, A. M. d'A. R. Gonsalves, G. W. Kenner & K. M. Smith, J. Chem. Soc. Perkin Trans I. 1771 (1974). 101. (a) R. J. P. Williams, Chem. Rev.. 56, 299 (1956). (b) K. M. Smith in "Porphyrins and MetaUoporphyrins", K. M. Smith Ed., American Elsevier, New York, N. Y.,1975, p3. 102. (a) R. J. Abraham, P. A. Burbidge, A. H. Jackson & D. B. Macdonald, J. Chem. Soc. B, 620 (1966). (b) T. R. Janson & J. J. Kate, J. Mag. Res.. 6, 209 (1972). 103. (a) W. S. Caughey & W. S. Koski, Biochemistry. 1, 923 (1962). (b) W. S. Caughey & P. K. Iber, J. Org. Chem.. 28, 269 (1963). 104. (a) A. R. Battersby & A. Hamilton, J. Chem. Soc. Chem. Commun.. 117 (1980). (b) T. G. Traylor, C. K. Chang, J. Geibel, A. Berzinis, T. Mincey & J. Cannon, J. Am. Chem. Soc.. 101. 6716 (1979). 105. (a) R. J. Abraham & K. M. Smith, Tet Lett.. 36, 3335 (1971). (b) C. A. Busby & D. Dolphin, J. Magn. Res.. 23, 211 (1976). 106. Beilsteins Handbuch der Organischen Chemie. Springer-Verlag. (a) 9, 914d (b) 9, 888c (c) 9. III. 4324 (d) 6. III. 4724 (e) 9. IV. 3436 107. W. R. Linke & A. Seidell, Solubilities of Inorganic and Metal Organic  Compounds, Van Nostrand: Princeton, N. J., 1958. 108. R. S. Drago, Physical Methods in Chemistry. W. B. Saunders, Philadelphia, 194 1977. 109. (a) A. V. Hill, J. Physiol (London), 40, IV-Vn, (1910). (b) P. E. Ellis, J. E. Linard, T. Szymanski, R. D. Jones, J. R. Budge & F. Basolo, J. Am. Chem. Soc.. 102. 1889 (1980). 110. D. Brault & M. Rougee, Biochemistry. 13, 4598 (1974). 111. (a) T. G. Traylor, S. Tsuchiya, D. Campbell, M. Mitchell, D. Stynes & N. Koga, J. Am. Chem. Soc.. 107. 604 (1985). (b) B. Ward, C. Wang & C. K. Chang, J. Am. Chem. Soc.. 103. 5236 (1981). (c) T. Hashimoto, R. L. Dyer, M. J. Crossley, J. E. Baldwin & F. Basolo, J. Am. Chem. Soc.. 104. 2101 (1982). 112. T. G. Traylor, M. J. Mitchell, S. Tsuchiya, D. H. Campbell, D. V. Stynes & N. Koga, J. Am. Chem. Soc.. 103. 5234 (1981). 113. T. G. Traylor & A. P. Berzinis, Proc. Nad. Acad. Sci. U. S. A.. 77, 3171 (1980) 114 J. P. Collman, J. I. Brauman, B. L. Iverson, J. L. Sessler, R. M. Morris & Q. H. Gibon, J. Am. Chem. Soc.. 105. 3052 (1983). 195 o Appendices Appendix I Spectral data for the iron(U) capped systems Fe(benzene-4/4)(DcIm) ^(nm), 424 556 log e, 4.74 3.94 Fe(benzene-5/5)(DcIm) ^(nm), 420 548 log e, 4.98 4.16 Fe(amidobenzene-4/4)(DcIm) Xta„(nm), 424 548 log e, 4.83 4.20 Fe(amidobenzene-5/5)(DcIm) Unm), 424 548 log E, 4.92 4.30 Fe(benzene-4/4)(DcIm)(CO) Unm), 416 548 log e, 4.93 4.00 Fe(benzene-5/5)(DcIm)(CO) U n m ) , 416 534 log e 5.31 4.14 Fe(amidobenzene-4/4)(DcIm)(CO) 196 Unm). 416 548 log e, 5.00 4.22 Fe(amidobenzene-5/5)(DcIm)(CO) Unm). 416 548 log e, 5.21 4.30 Fe(benzene-4/4)(DcIm)(02) Unm), 406 578 log e, 4.67 3.95 Fe(benzene-5/5)(DcIm)(02) XmM(nm), 408 536 564 log e, 4.96 4.12 4.13 Fe(amidobenzene-4/4)(DcIm)(02) l„(nm), 406 580 log e, 4.79 4.17 Fe(amidobenzene-5/5) Unm), 408 536 564 log e, 4.90 4.05 4.03 197 Appendix II Raw data for the binding of CO and O z to the capped hemes The data were processed by least-squares analysis in all cases. A CO affinity constant determination, K 0 0 in toluene at 20°C [heme] ~ 3 x 10* M, [Dclm] - 0.3 M (1) Fe(benzene-4/4)(DcIm) + CO Preparation of CO/Ar mixture in glass bulb: pco = 84 torr, P ^ = 821 torr = 416 nm, Ao = 0.574, A„ = 0.903 A A 0 - A A - A. logAn- A P •* total pco logP00 A - A. (torr) (torr) 0.634 0.060 0.269 -0.652 19.4 1.985 0.298 0.682 0.108 0.221 -0.311 42.1 4.297 0.633 0.728 0.154 0.175 -0.056 738 7.551 0.878 0.765 0.191 0.138 0.141 113.4 11.602 1.065 0.805 0.231 0.098 0.372 186.0 19.030 1.279 0.847 0.273 0.056 0.688 338.6 34.644 1.540 0.867 0.293 0.036 0.911 507.0 51.873 1.715 slope = 1.1 pco r 112 = 8.2 torr 198 (2) Fe(benzene-5/5)(DcIm) + CO Preparation of CO/Ar mixture in glass bulb: Initial P c o = 25 torr, P ^ = 816 ton-Evacuation procedure: Putsl(after evacuation) (torr) Ptottl(after refilling Ar)(torr) 296.4 827.0 307.6 814.0 281.0 823.6 293.6 821.0 290.0 817.6 Final P™ = 0.1468 torr P ^ = 817.6 torr ^ „ = 416 nm, AG = 0.668, A„ = 1.194 A A 0 - A A - A_ logAo - A p 1 total pco logP00 A - A_ (torr) (torr) 0.698 0.030 0.496 -1.218 20.6 0.00307 -2.432 0.737 0.069 0.457 -0.821 54.0 0.00970 -2.013 0.799 0.131 0.395 -0.479 120.6 0.0217 -1.664 0.876 0.208 0.318 -0.184 227.4 0.0408 -1.389 0.933 0.265 0.261 0.00661 342.0 0.0614 -1.212 0.984 0.316 0.210 0.177 497.0 0.0892 -1.049 slope = 1.0 pco _ r 111 — 0.061 torr (3) Fe(amidobenzene-4/4)(DcIm) + CO Preparation of CO/Ar in glass bulb: P 0 0 = 26 torr, P ^ = 830 torr 199 A^ = 416 nm, A<, = 0.534, A „ = 0.910 A A 0 - A A - A . - A P * total pco logP00 A - A . (torr) (torr) 0.589 0.055 0.321 -0.766 20.4 0.639 -0.194 0.659 0.125 0.251 -0.303 59.0 1.848 0.267 0.747 0.213 0.163 0.116 157.4 4.31 0.63 0.789 0.255 0.121 0.324 245.4 7.687 0.886 0.816 0.282 0.094 0.477 344.8 10.801 1.033 0.848 0.314 0.062 0.705 527.4 16.521 1.218 slope =1.0 Vfl2 = 3.6 torr (4) Fe(amidobenzene-5/5)(DcIm) + CO Preparation of CO/Ar mixture in glass bulb: Initial P c o = 29 torr, P ^ = 873 ton-Evacuation procedure: PIotal(after evacuation)(torr) P^after refilling Ar)(torr) 320 816 287 816 377 828 292 818 283 812 Final P 0 0 = 0.2107 torr, P ^ = 812 torr A^ = 416 nm, AQ = 0.495, A. = 0.937 200 A A 0 - A A - A. loeA„ - A P x total pco logP00 A - A_ (torr) (torr) 0.564 0.069 0.373 -0.733 20.0 0.0052 -2.285 0.641 0.146 0.296 -0.307 53.0 0.0138 -1.862 0.728 0.233 0.209 0.047 122.0 0.0317 -1.500 0.776 0.281 0.161 0.242 208.0 0.0540 -1.268 0.816 0.321 0.121 0.424 325.0 0.0843 -1.074 0.851 0.356 0.086 0.617 536.0 0.1391 -0.857 slope = 0.9 pco rl/2 = 0.03 torr B CO affinity constant determination, K c o , in toluene at varied temperatures [heme] ~ 3 x IO"6 M, [Dclm] - 0.3 M (1) Fe(benzene-4/4)(DcIm) + CO (i) At 30.5°C, P c o in glass bulb = 108.4 torr, P ^ = 834 torr = 418 nm, A Q = 0.519, A. = 0.851 A Ao - A A - A. logA0 - A p total pco logPco A - A. (torr) (torr) 0.545 0.026 0.306 -1.017 21.4 2.78 0.444 0.572 0.053 0.279 -0.721 41.8 5.43 0.735 0.620 0.101 0.231 -0.359 85.4 11.10 1.045 0.654 0.135 0.197 -0.164 128.6 16.71 1.223 0.682 0.163 0.169 -0.016 186.2 24.20 1.384 0.766 0.247 0.085 0.463 532.0 69.15 1.840 slope = 1.1, = 24.9 torr 201 (ii) At 15.5°C, P 0 0 in glass bulb = 58 torr, P ^ = 822.8 torr X = 418 nm, AQ = 0.520, A. =0.894 A A 0 - A A - A_ logA0 - A Ptotal pco logP00 A - A . (torr) (torr) 0.591 0.071 0.303 -0.630 22.8 1.607 0.206 0.627 0.107 0.267 -0.397 37.6 2.650 0.423 0.697 0.177 0.197 -0.046 80.0 5.639 0.751 0.774 0.254 0.120 0.326 184.0 12.970 1.113 0.813 0.293 0.081 0.558 291.0 20.513 1.312 0.846 0.326 0.048 0.832 515.6 36.345 1.560 slope =1.1, p^2 = 6.2 torr (iii) At 9.7°C, P00 in glass bulb = 39.4 torr, P ^ = 841.8 torr X = 418 nm, AQ = 0.334, A. = 0.600 A A 0 - A A - A„ logA0 - A Ptotal pco logP™ A - A_ (torr) (torr) 0.375 0.041 0.225 -0.739 17.0 0.796 -0.099 0.436 0.102 0.164 -0.206 51.8 2.424 0.385 0.489 0.155 0.111 0.145 109.0 5.102 0.708 0.526 0.192 0.074 0.414 198.0 9.267 0.967 0.550 0.216 0.050 0.635 325.0 15.211 1.182 0.564 0.230 0.036 0.805 486.2 22.756 1.357 slope = 1.1, P^2 = 3.8 torr (iv) At 4.7°C, P c o in glass bulb = 16.41 torr, Ptotal = 836.6 torr 202 X = 418 nm, Ao = 0.414, A . = 0.751 A A 0 - A A - A_ togAo - A P x total pco logP00 A - A_ (torr) (torr) 0.458 0.044 0.293 -0.823 16.8 0.329 -0.482 0.536 0.122 0.215 -0.246 62.6 1.228 0.089 0.577 0.163 0.174 -0.028 107.0 2.098 0.322 0.619 0.205 0.132 0.191 173.2 3.397 0.531 0.665 0.251 0.086 0.465 303.0 5.942 0.774 0.701 0.287 0.050 0.759 554.4 10.872 1.036 slope = 1.0, = 2.1 torr (2) Fe(amidobenzene-4/4)(DcIm) + CO (i) At 5.10°C, P c o in glass bulb = 10.7 torr, P ^ = 831.0 torr X = 416 nm, A 0 = 0.376, A„ = 0.734 A A 0 - A A - A„ logAo - A P total pco logPco A - A . (torr) (torr) 0.433 0.057 0.301 -0.723 17.2 0.221 -0.655 0.510 0.134 0.224 -0.223 60.4 0.778 -0.109 0.589 0.213 0.145 0.167 134.0 1.725 0.237 0.628 0.252 0.106 0.376 241.4 3.108 0.493 0.648 0.272 0.086 0.500 333.2 4.290 0.632 0.674 0.298 0.060 0.696 500.4 6.443 0.809 slope = 0.97, P^2 = 1.3 torr 203 (ii) At 10.5°C, P 0 0 = 35.8 torr, P ^ = 835.2 torr X = 416 nm, A 0 = 0.416, A_ = 0.749 A A 0 - A A - A. logAn - A p *• total pco logP00 A - A_ (torr) (torr) 0.475 0.059 0.274 -0.667 14.0 0.600 -0.222 0.545 0.129 0.204 -0.199 33.6 1.440 0.158 0.609 0.193 0.140 0.139 66.8 2.863 0.457 0.659 0.243 0.090 0.431 120.4 5.161 0.713 0.699 0.283 0.050 0.753 244.2 10.467 1.020 0.729 0.313 0.020 1.195 513.6 22.015 1.343 slope = 1-2, PT?2= 2.2 torr (iii) At 15.5°C, P c o in glass bulb =  35.4 torr, P ^ = 820.0 torr X = 416 nm, A D = 0.439, A_ = 0.813 A A 0 - A A - A. logA0 - A p total pco logP00 A - A„ (torr) (torr) 0.511 0.072 0.302 -0.623 16.8 0.725 -0.140 0.564 0.125 0.249 -0.299 35.0 1.511 0.179 0.622 0.183 0.191 -0.019 64.8 2.797 0.447 0.684 0.245 0.129 0.279 126.0 5.440 0.736 0.732 0.293 0.081 0.558 229.0 9.886 0.995 0.777 0.338 0.036 0.973 509.0 21.974 1.342 slope = 2.9 torr (iv) At 29.5°C, P 0 0 in glass bulb = 100 torr, PtoU1 = 846.6 torr 204 X = 416 nm, Ao = 0.590, A. = 1.002 A A 0 - A A - A. logAo - A A - A. P total (ton) pco (torr) logP00 0.6270.037 0.375 -1.006 7.8 0.921 -0.036 0.7250.135 0.277 -0.312 42.0 4.961 0.696 0.7850.195 0.217 -0.046 71.0 8.386 0.924 0.8470.257 0.155 0.220 120.0 14.174 1.152 0.9280.338 0.074 0.660 280.6 33.144 1.520 0.9700.380 0.032 1.075 508.8 60.099 1.779 slope =1.1, P^2 = 8.3 torr C Van't Hoff plots for CO binding to Fe(benzene-4/4)(DcIm) and Fe(amidobenzene-4/4) (Dclm) systems (1) Fe(benzene-4/4)(DcIm) + CO K(torr1) ln K + ln 760 T(°C) 1/T(°K) x 103 0.0402 3.418 30.5 3.293 0.122 4.529 20.0 3.411 0.161 4.809 15.5 3.464 0.263 5.298 9.7 3.535 0.476 5.891 4.7 3.599 slope = -AH°/R = 7.84 x 103 AH 0 = -16 + 2 kcal-mol1 205 Y-intercept = AS°/R = -22.34 AS0 = -44 + 7 cal-mol^ K1 (2) Fe(arnidobenzene-4/4)(DcIrn) + CO KCtorr1) In K + In 760 T(°C) 1/T(°K) x 103 0.7692 6.371 5.10 3.594 0.4545 5.845 10.50 3.525 0.3448 5.569 15.50 3.464 0.2632 5.298 20.00 3.411 0.1205 4.517 29.50 3.304 slope = -AH7R = 6.2 x 103 AH° = -12 + 3 kcal-mol1 Y-intercept = AS°/R = -15.9 AS0 = -32 + 11 cal-mol'K1 D 0 2 affinity constant determination, K 0 2 , in toluene at -45°C [heme] ~ 10"6 M, [Dclm] ~ 0.1 M (1) Fe(benzene-4/4)(DcIm) + 0 2 Preparation of Oj/Ar mixture in glass bulb: P 0 2 = 267.4 torr, P ^ = 838.0 torr X = 420 nm, AQ = 1.039, A_ = 0.652 206 A A 0 - A A - A_ logAo - A p 0 2 logP02 0.992 0.047 0.340 A --0.859 (torr) 30.6 (torr) 9.76 0.990 0.909 0.130 0.257 -0.296 97.0 30.95 1.491 0.814 0.225 0.162 0.143 241.8 77.16 1.887 0.737 0.302 0.085 0.551 513.0 163.69 2.214 slope = 1.1, P°?2 = 55.7 torr (2) Fe(benzene-5/5)(DcIm) + o2 Preparation of O-JAi mixture in glass bulb: p 0 2 _ 265.2 torr, P ^ = 828.8 torr X = 420 nm, A o = 1.182, A. _ = 0.808 A 1.075 A 0 - A 0.107 A - A_ 0.267 logAf, -A --0.397 • A A_ P x total (torr) 28.0 p 0 2 (torr) 9.0 logP02 0.952 0.947 0.235 0.139 0.228 119.8 38.3 1.584 0.893 0.289 0.085 0.531 249.2 79.7 1.902 0.858 0.324 0.050 0.812 469.8 150.3 2.177 slope = 0.98, P?n = 22.7 torr (3) Fe(amidobenzene-4/4)(DcIm) + 0 2 Preparation of CyAr mixture in glass bulb: P 0 2 = 150.0 torr, Ptotal = 822.6 torr X = 420 nm, A 0 = 0.850, A. = 0.543 207 A A 0 - A A - A. iogAp - A P 1 total p02 logP02 A - A, (torr) (torr) 0.742 0.108 0.199 -0.265 28.6 5.22 0.717 0.662 0.188 0.119 0.199 81.0 14.77 1.169 0.598 0.252 0.055 0.661 231.0 42.12 1.625 0.570 0.280 ^ 0.027 1.016 483.6 88.18 1.945 slope = 1.0, p02 _ * 1/2 ~ 9.5 torr (4) Fe(amidobenzene-5/5)(DcIm) + 0 2 Preparation of O^Ar mixture in glass bulb: p 0 2 _ 28.6 torr, P ^ = 829.8 torr X = 420 nm, A 0 = 1.137, A_ = 0.763 A A 0 - A A - A. logA0 - A P ••• total p 0 2 logP02 A - A_ (torr) (torr) 1.038 0.099 0.275 -0.444 27.2 0.937 -0.028 0.966 0.171 0.203 -0.074 66.8 2.302 0.362 0.885 0.252 0.122 0.315 174.8 6.025 0.780 0.837 0.300 0.074 0.608 363.2 12.518 1.098 slope = 0^ 93, P?l2 = 2.8 torr 208 


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