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S-triazolo[3,4-A]phthalazines : implications for C-terminal peptide sequencing 1977

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S-TRIAZ0L0[3,4-A]PHTHALAZINES: IMPLICATIONS FOR C-TERMINAL PEPTIDE SEQUENCING by CARL STANLEY ALLEYNE B . S c . ( H o n s . ) , U n i v e r s i t y of B r i t i s h Columbia, 1970 M . S c , U n i v e r s i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept t h i s thes is as conforming to the requi red standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 © Carl Stanley A l l e y n e , 1977 1 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y of B r i t i s h C o l u m b i a , I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f Chemistry The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 3rd . October 1977 ABSTRACT The syntheses of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e s (15, 3-R-TAP) by reac t ion of hydra laz ine (12, 1 -hydrazinophthalaz ine) with N-protected amino ac ids and d ipept ides under homogeneous (so lu t ion ) and heterogeneous ( "so l id -phase" ) cond i t ions are repor ted . T r a n s i t i o n metal complexes conta in ing the TAP l igand were prepared and t h e i r spect ra l p roper t ies i n v e s t i g a t e d . The use of meta l - ions and a cat ion-exchange r e s i n ( H + form) were considered fo r the mi ld h y d r o l y s i s of s i d e - c h a i n amide bonds in TAP d e r i v a t i v e s . The o b j e c t i v e of these s tud ies was to determine the f e a s i b i - l i t y of reac t ing the carboxyl groups in amino ac ids with hydra laz ine to a f fo rd the TAP d e r i v a t i v e s as a method f o r peptide sequencing from the C-terminal r e s i d u e . Hydralaz ine reacts with c a r b o x y l i c ac ids to form an amide intermediate which undergoes r i n g c l o s u r e with e l i m i n a t i o n of water to form the s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e . To promote the i n i t i a l b inding of hydra laz ine to the a c i d , coupl ing reagents were used to a c t i v a t e the carboxyla te group towards n u c l e o p h i l i c a t tack . N - E t h y l - 5 - p h e n y l i s o x a z o l i u m - 3 ' - s u l f o n a t e (17, NEPIS), 1-ethoxy - c a r b o n y l - 2 - e t h o x y - l , 2 - d i h y d r o q u i n o l i n e (27, EEDQ), var ious phosphorus compounds, ca rbod i im ides , and chloroformates were carboxyl a c t i v a t i n g agents used to synthes ize TAP d e r i v a t i v e s . In s o l u t i o n s t u d i e s , the carbodi imides (EDC, 16 and DCC, Si), NEPIS (17), and a combination of t r ipheny lphosph i te with imidazole are the most success fu l procedures f o r TAP s y n t h e s i s . i i i In s o l i d - p h a s e s t u d i e s , the best procedures f o r a c t i v a t i n g immobilized amino acids, are with isobuty'l ch loroformate , NEPIS ( l ? ) , and DCC (51). T r a n s i t i o n metal complexes were synthesized with the general formula: [M(3-H-TAP) ( H 2 0 ) 6 _ n ] ( C 1 0 4 ) m (n = 4, m = 2, M = Co, N i , Cu; n = 2, m = 2, M = N i ; n = 6, m = 3, M = Co) . The i n f r a r e d and v i s i b l e spectra of these complexes are repor ted . [ C o ( . t r i e n ) C 3 - ( N - A c - g l y ) - T A P ) J ( C 1 0 4 ) 2 was a l s o prepared and under a c i d i c c o n d i t i o n s , no hydro lys is of the s i d e - c h a i n amide bond was observed. There was a l s o no s i g n i f i c a n t hydro lys is of the s i d e - c h a i n with f ree ?+ 2+ 3 - (N-Ac-g ly ) -TAP in the presence of Co and Cu under a c i d i c c o n d i t i o n s , or when i t was e luted through a cat ion-exchange (H + form) column. The decomposit ion of hydra laz ine in non-aqueous media was inves t iga ted and a major product of the decomposit ion was i d e n t i f i e d as d i p h t h a l a z i n y l h y d r a z i n e {82). The i m p l i c a t i o n of our s tud ies i s that the m o d i f i c a t i o n of amino ac ids with hydra laz ine is not ye t a v i a b l e method f o r C-terminal peptide sequencing. Improvements are requi red f o r improving the y i e l d s of the coupled product , and the lack o f a mi ld and s e l e c t i v e method f o r hydrolyz ing the C-terminal peptide bond l i m i t s the method at present to determinat ion of the C-terminal res idue o n l y . i v TABLE OF CONTENTS ABSTRACT i i LIST OF SCHEMES vi LIST OF TABLES ' v i i i LIST OF FIGURES ix LIST OF ABBREVIATIONS x i ACKNOWLEDGEMENTS x iv CHAPTER 1: PROTEIN SEQUENCE DETERMINATION 1 1.1 In t roduct ion 1 1.2 Prote in St ructure and Nomenclature 2 1.3 Prote in Sequence A n a l y s i s 4 1.4 Amino Terminal Peptide Sequencing and End-Group I d e n t i f i c a t i o n 6 1.5 Carboxy Terminal Peptide Sequencing and End-Group I d e n t i f i c a t i o n 9 1.6 Object ives and Out l ine of the Present Work 17 CHAPTER 2: REACTION OF 1-HYDRAZINOPHTHALAZINE WITH N-PROTECTED AMINO ACIDS 21 2.1 Introduct ion ^ 21 2.2 Nomenclature 26 2.3 Pre l iminary Resul ts 27 2.4 Results 32 2.4.1 The Isoxazolium S a l t Method 32 2.4.2 The EEDQ Coupling Reagent 40 2 .4 .3 Acyloxyphosphonium Cat ion 43 2.4.4 Carbodi imides 53 2.5 D iscuss ion 70 V CHAPTER 3: SOLID-PHASE SYNTHESIS OF S-TRIAZ0L0[3,4-A]PHTHALAZINES 84 3.1 Introduct ion 84 3.2 Resul ts 89 3.3 D iscuss ion 97 CHAPTER 4: TRANSITION METAL COMPLEXES WITH THE S-TRIAZ0L0[3,4-A]- PHTHALAZINE LIGAND 113 4.1 In t roduct ion 113 4.2 Resul ts 121 CHAPTER 5: HYDRALAZINE DECOMPOSITION 158 5.1 In t roduct ion 158 5.2 Resul ts and D iscuss ion 160 CHAPTER 6: EXPERIMENTAL 170 6.1 General Methods 170 6.2 Chemicals 172 6.3 Coupling Reactions with the Isoxazolium S a l t Method 181 6.4 Coupl ing Reactions with the EEDQ VCoup!ing Reagent 183 6.5 Coupl ing React ions with the Acyloxyphosphonium S a l t Methods 185 6.5.1 The Kenner-Sheppard Reaction 185 6.5.2 The Azido-Tris(dimethylamino)phosphonium Hexafluorophosphate Method 186 6.5.3 The "Oxidat ion-Reduct ion Condensation" Method 186 6.5.4 The Tr iphenylphosphi te - Imidazo le Method 187 6 .5 .5 The D ipheny lphosphi te -Pyr id ine Method 188 6.6 Coupling Reactions with the Carbodiimide Method 188 6.7 So l id -Phase Studies 193 6.8 T r a n s i t i o n Metal Complexes and Hydro lys is Studies 200 REFERENCES 206 vi LIST OF SCHEMES 1.1 The Edman Degradation 8 1.2 S t a r k ' s Method f o r C-Terminal Sequencing of Pept ides and Prote ins 13 1.3 The T r i t i a t i o n Method fo r C-Terminal Sequencing of Peptides and Prote ins 14 1.4 C-Terminal Peptide Sequencing by Reduction 15 1.5 Peptide C-Terminus Determination v i a Lossen Rearrangement of an O-Subst i tu ted Hydroxamic Ac id 15 1.6 Proposed C-Terminal Peptide Sequencing v i a s - T r i a z o l o [ 3 , 4 - a ] - phthalaz ines 18 2.1 React ion Mechanism f o r the Formation of s - T r i a z o l o [ 3 , 4 - a ] - phthalaz ines 24 2.2 Mechanism of Amide Formation with NEPIS 33 2.3 Formation of 3-(N-Ac-met)-TAP with NEPIS 35 2.4 Mechanism of Amide Formation with EEDQ 41 2.5 Amide Formation v i a an Acyloxyphosphonium Intermediate 43 2.6 Amide Formation v ia an Acyloxydimethylaminophosphonium S a l t 44 2.7 Amide Formation v i a an Acyl Azide 46 2.8 Amide Formation v i a an Acyloxytr iphenylphosphonium S a l t 47 2.9 Amide Formation v ia an Acyl imidazol ium S a l t 49 2.10 Amide Formation v ia an Acyloxyphosphonium S a l t from D ipheny l - phosphite 52 2.11 Amide Formation v i a Acyloxyphosphonium S a l t s from T r i p h e n y l - phosphite 53 2.12 Mechanism of Amide Formation with a Carbodiimide 55 2.13 Reaction of Carboxy l ic Acids with Carbodiimides 59 2.14 Amide Formation by the Phosphazo Method 76 vi i 2.15 Synthesis of Fused s - T r i a z o l e s 78 2.16 Synthesis of a Fused s - T r i a z o l e with an Aminomethyl S ide -Cha in 79 3.1 S t a r k ' s Method f o r Subt rac t ive N-terminal Peptide Degradation 86 3.2 Laursen 's Method fo r N-terminal Peptide Sequencing 87 3.3 So l id -phase Peptide Sequencing by S t a r k ' s Thiocyanate React ion 88 3.4 Preparat ion of Methylchloroformylated Resin 90 3.5 So l id -phase Synthesis of s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e s 93 3.6 Amide Formation with Isobutyl Chloroformate 94 3.7 S i d e - r e a c t i o n s of Mixed-anhydride in Amide Synthesis 95 3.8 M o d i f i c a t i o n of Polystyrene with a Spacer Arm 100 3.9 Amide Formation with DCC/HOBt 105 3.10 S e l e c t i v e Amide Formation at the C-Terminus of Peptides 111 4.1 P o s s i b l e Mechanism f o r the Carboxypeptidase A-Cata lyzed Hydro lys is of G l y c y l - L - t y r o s i n e 115 4.2 Proposed Mechanisms f o r Peptide Hydro lys is 117 4.3 Proposed- Peptide Sequencing with Pept idy l -TAP and Cobal t ( I I I ) Complexes 119 4.4 Synthesis of B - [ C o ( t r i e n ) ( 2 - A M P y ) ] 3 + 144 4.5 M e t a l - a s s i s t e d Hydro lys is of 3 - (N -Ac-g ly ) -TAP 152 5.1 Fragmentation Scheme fo r D iph tha laz iny lhydraz ine 168 vi i i LIST OF TABLES 1.1 The Amino Acid Wheel 3 1.2 Approximate Re la t i ve Rates of Release of Amino Acids by Carboxypeptidase A 11 2.1 Reactions of Hydralaz ine with Various Carboxy l ic Ac id Der iva t ives 25 2.2 Nomenclature f o r the S ide -cha ins of s -T r iazo1o[3 ,4 -a ]ph tha laz ines 28 2.3 Coupling Reagents and t h e i r React ive Intermediates used in Amide Synthesis 71 2.4 Fused s - T r i a z o l e s , t h e i r Proper t ies and Uses 80 4.1 NMR Spectra l Data f o r s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e in DMS0-d g 122 4.2 V i b r a t i o n s of the Perch!orate Group as a Funct ion of Symmetry 127 4.3 Infrared Spectra of Perchlorate Groups 131 4.4 Ligand F i e l d and Nephelauxetic Parameters fo r Octahedral C o b a l t ( l l ) Ions 136 4.5 Crys ta l F i e l d Parameters f o r Octahedral N icke l ( I I ) Der iva t i ves 142 ix LIST OF FIGURES 30 2.1 UV Spectra of Equimolar Amounts of 3-Me-TAP and Hydra laz ine , and a Mixture of Each 2.2 E f f e c t of Excess Tr ie thy lamine on Carbodi imide-mediated Coupl ing Reactions 61 2.3 E f f e c t of Excess Carboxyl Component and Carbodiimide on Carbo- di imide-mediated Coupling Reactions 63 2.4 E f f e c t o f Concentrat ion on Carbodi imide-mediated Coupl ing React ions 64 2.5 Comparison of Pyr id ine and Tr ie thy lamine in Carbodi imide- mediated Coupl ing React ions (1 day) 65 2.6 Comparison of Py r id ine and Tr ie thy lamine in Carbodi imide- mediated Coupl ing Reactions (4 hours) 66 3.1 P a r t i a l S t ructure of a Polyacrylamide .Matrix 101 4.1 Diagrammatic I l l u s t r a t i o n of the Mechanism of Carboxypept idase- Cata lysed Peptide H y d r o l y s i s : (a) Zn-hydroxide Mechanism and (b) Zn-carbonyl Mechanism 116 4.2 Ion iza t ion o f Peptide Amide Hydrogen 118 4.3 100 MHz F o u r i e r - t r a n s f o r m NMR Spectrum of [Co(3 -H-TAP)g] (C10 4 ) 3 in DMS0-d 6 123, 4.4 D i f f u s e Ref lectance Spectrum of [Co (3 -H -TAP)g ] (C10 4 ) 3 125 4.5 Infrared Spectra of TAP Complexes 4.6 Nujol Mull Absorpt ion Spectrum of [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 133 4.7 (a) Tanabe-Sugano Diagram f o r d 7 Ions. (b) T r a n s i t i o n Energy Rat io Diagram f o r Ions with the T-j Ground State 134 Nujol Mull Absorpt ion Spectrum of [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ( C 1 0 4 ) 2 . H 2 0 138 4.8 g 4.9 (a) Tanabe-Sugano Diagram f o r d Ions, (b) T r a n s i t i o n Energy Rat io Diagram fo r Ions with the A 2 Ground State 140 4.10 Nujol Mull Absorpt ion Spectrum of [Cu(3-H-TAP)4(.H20)2]CC104)2 141 3+ 4.11 Absorpt ion Spectra of Purple Complex from [Co(tr ien) (2-AMPy)] Preparat ion 145 3+ 4.12 Components separated from the Preparat ion of [Co(t r ien) (2-AMPy)] by Ion-exchange Chromatography 146 2+ 4.13 Isomers of [ C o ( t r i e n ) ( 3 - A c - g l y ) - T A P ] Isolated by Ion-exchange Chromatography 150 4.14 (a) UV Spectrum of Protonated 3 - (N -Ac-g ly ) -TAP e luted from Cation-exchange R e s i n , (b) UV Spectrum of Neut ra l i zed Eluent 156 5.1 UV Spectrum of the Major Product from Decomposition of Hydralaz ine in DMF 164 5.2 100 MHz Four ie r - t rans fo rm NMR Spectrum of the Major Product from Decomposition of Hydralaz ine in DMF 165 5.3 Mass Spectrum of the Major Product from Decomposition of Hydralaz ine in DMF 167 xi ABBREVIATIONS Ac ace ty l 2-AMPy 2-ami no-methyl p y r i d i ne Ar a ry l asym. asymmetric b broad bipy b i p y r i d i n e bipyz b ipyraz ine BOC butyloxycarbonyl Bu butyl \ C- carboxyl CMC l - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n y l - 4 - e t h y l ) - carbodi imide metho p - to luenesu l fona te CoA coenzyme A CPA carboxypeptidase A CPG c o n t r o l l e d - p o r e g lass DABCO 1 ,4 -d i . azab icyc lo l2 .2 .2 ]oc tane DBu 1 ,5 -d iazab icyc lo [5 .4 .0 ]undec -5 -ene DCC d icyc lohexy lcarbod i im ide def . deformation DMA dimethylacetamide DMF dimethylformami.de DMSO dimethyl s u l f o x i d e DNS dansyl 2-DTP 2 , 2 ' - d i t h i o d i p y r i d i n e XI 1 div inylbenzene 1 -e thy l -3 -d i. methylaminopropyl carbodi imide hydrochlor ide ethylenediamine d iace ta te 1-ethoxycarbonyl -2 -ethoxy- 1 ,2 -d ihydroqu ino l ine 1,8-diami n o - 3 , 6 - d i t h i a o c t a n e ethylenediamine ethyl hexamethylphosphoramide 1-hydroxybenzotr iazol e 1-i s o b u t y l o x y c a r b o n y l - 2 - i sobuty loxy- 1 ,2-d i hydroqui n o l i ne imidazol ium i n f r a r e d medium methyl melt ing po int amino N - e t h y l - 5 - p h e n y l i s o x a z o l i u m - 3 ' - s u l f o n a t e nuclear magnetic resonance polymeric support propyl copo lys ty rene-d iv iny lbenzene support phenylthiocarbamyl phenyl thiohydantoi n p y r i d i n e pyr id ine -N-Ox ide x i i i s strong sh shoulder s t r s t r e t c h i n g sym. symmetric TAP s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e TFA t r i f l u o r o a c e t i c a c i d THF tetrahydrofuran TLC th in layer chromatography tren 2 , 2 ' , 2 " - t r i a m i n o t r i e t h y l e n e t r i e n t r ie thy lenete t ramine UV u l t r a - v i o l e t v very w weak WSC water -so lub le carbodi imide Z benzyloxycarbonyl xiv ACKNOWLEDGEMENTS It is a p leasure to thank the many col leagues and f r i e n d s who have contr ibuted to the r e a l i z a t i o n of t h i s work. F i r s t of a l l , I wish to express my indebtedness to David Do lph in , the guid ing l i g h t of my r e s e a r c h , f o r h is u n f a i l i n g a b i l i t y to s t imulate and to help me keep my perspect ive whenever I was o f f on a tangent ia l course . Th is work has benef i t ted g r e a t l y from the many informal d i s c u s s i o n s and p r a c t i c a l suggestions by var ious members of the Dolphin research group. I thank them a l l , p a r t i c u l a r l y Gene Johnson f o r h is p roof - read ing of t h i s t h e s i s , Bob C a r l s o n , my chromatography c o n s u l t a n t , John B. Paine III f o r h is help on organic chemistry i n g e n e r a l , and Andrew Hamilton who showed me a d i f f e r e n t phi losophy towards c h e m i c a l ' r e s e a r c h . It has been a d i s t i n c t pleasure to work a longside everyone of them. F i n a l l y , but foremost , a deep sense of g ra t i tude and love is •d i rected towards my w i f e , C e c i l i a , who laboured f o r many long hours typing t h i s t h e s i s . Her p a t i e n c e , perseverence, and to le rance was sure ly tested during the course of t h i s work. To her , I dedicate th is t h e s i s . 1 CHAPTER 1 PROTEIN SEQUENCE DETERMINATION 1.1 INTRODUCTION Pro te in sequence a n a l y s i s has , with j u s t i f i c a t i o n , been descr ibed as one of the most important research a c t i v i t i e s o f t o d a y . 1 The informat ion contained in pro te in sequence i s something e s s e n t i a l l y new. The data repre- sent the cova lent chemical s t r u c t u r e o f the prote in molecule and are o f immediate use and importance i n many d i f f e r e n t d i s c i p l i n e s . I t i s c e r t a i n that the importance o f sequence information on prote ins w i l l continue to increase in the same way as the body o f informa- t ion i t s e l f w i l l i n c r e a s e . Sequence data on prote ins are being amassed at an ever i n c r e a s i n g r a t e . This can be seen from the phenomenal growth o f the "At las of Prote in Sequence and S t r u c t u r e " , which approximately doubles every y e a r . 2 The rap id development o f appropr iate a n a l y t i c a l procedures has been one of the fac to rs enabl ing sequence data to be accumulated at the present r a t e . However, a t the present t ime, the determination of the amino a c i d sequence o f a prote in may s t i l l requi re a major e f f o r t and can not be considered a rout ine matter. In 1943, Synge s ta ted : 2 . . . i t seems that the main obstacle to progress in the study of protein structure by methods of organic chemistry is inadequacy of technique rather than any theoretical d i f f i c u l t y . It is l i k e l y that new methods of work in this field will lead us to a very much clearer understanding of the proteins. 3 Today, t h i r t y - f o u r years l a t e r , that statement i s s t i l l v a l i d . 1.2 PROTEIN STRUCTURE AND NOMENCLATURE The prote ins are chains of long unbranched polymers with L-a-amino acids as the monomeric u n i t s , only twenty o f which are commonly found as the b u i l d i n g blocks of p r o t e i n s . The names and s t r u c t u r e s o f these common amino ac ids are given in Table 1.1. The amino ac ids are l i n k e d together in a head to t a i l arrangement through amide bonds c a l l e d peptide bonds, which a r i s e by e l imat ion of the elements o f water from the carboxyl group o f one amino ac id and the a-amino group of the next . The peptide bond i s the repeat ing un i t in p r o t e i n s . The c h a r a c t e r i s t i c bond s t r u c t u r e i s enclosed in the dot ted area: H jo -N-CKR-C-N I H i i 0 CHR'-C- Such polymer chains are c a l l e d polypeptides. In peptide nomenclature, the amino a c i d res idues are wr i t ten as they occur i n the chain s t a r t i n g from the f ree a-amino group, which i s convent iona l ly shown at the l e f t - h a n d part of the s t r u c t u r e . The terminal res idue with the f ree amino group i s r e f e r r e d to as the N-terminal res idue . 3 CONH2 I \ CH2 I \ // > * - V-cy Gin ivdes One letter abbrev iat ions A l a G Gly M Met S~ Ser Cys H His N Asn T Thr Asp 1 l ie P Pro V Val Glu K Lys Q Gin W Trp Phe L Leu R Arg Y Tyr 6>V * Table 1.1 The "amino a c i d wheel". The s t ruc tures of the amino ac id res idues are shown as they normally occur in a polypept ide c h a i n . 4 S i m i l a r l y , the terminal residue with the f ree carboxyl group i s r e f e r r e d to as the C-terminal r e s i d u e . The sequence of the amino a c i d residues in the polypept ide chains which make up the prote ins i s f requent ly c a l l e d the primary structure of the p r o t e i n . . In g e n e r a l , the polypept ide chains of prote ins usua l ly have between 100 to 300 amino a c i d residues (mol. wt. 12,000 to 36,000). A few prote ins have longer c h a i n s , such as serum albumen (^550 res idues) and myosin (^1800 r e s i d u e s ) . U s u a l l y , any prote in having a. molecular weight exceeding 50,000 can be suspected to have two or more c h a i n s . 1.3 PROTEIN SEQUENCE ANALYSIS The approach genera l ly used in determining the primary s t r u c t u r e o f prote ins i s , in p r i n c i p l e , that devised by Sanger in h is epoch-making determinat ion of the amino a c i d sequence of the polypept ide chains of i n s u l i n , f o r which he was awarded a Nobel p r i z e in 1958. S ince that accomplishment, many refinements have been added, and new procedures have been developed. Although each prote in o f f e r s s p e c i a l problems, the fo l low ing sequence of steps i s genera l ly used, though not n e c e s s a r i l y in the order shown. 4 a. I f the prote in chain contains more than one polypept ide c h a i n , the i n d i v i d u a l chains are f i r s t separated and p u r i f i e d . b. A l l the d i s u l f i d e groups are reduced, and the r e s u l t i n g t h i o l groups a l k y l a t e d . c . A sample o f each polypept ide chain is subjected to to ta l h y d r o l y s i s , and i t s amino a c i d composit ion is determined. d. On another sample o f the polypept ide c h a i n , the N-terminal 5 and C-terminal res idues are i d e n t i f i e d . e. The i n t a c t polypept ide chain i s c leaved in to smal le r peptides by enzymatic or chemical h y d r o l y s i s . f . The r e s u l t i n g peptide fragments are separa ted , and t h e i r amino a c i d composit ion and sequence are determined. g. Another sample o f the o r i g i n a l polypept ide chain i s p a r t i a l l y hydrolyzed by a second procedure to fragment the chains at points other than those cleaved by the f i r s t p a r t i a l h y d r o l y s i s . These peptide fragments are separated and t h e i r amino a c i d composit ion and sequence determined. h. By comparing the amino a c i d sequences o f the two sets of peptide fragments, p a r t i c u l a r l y where the fragments from the f i r s t p a r t i a l h y d r o l y s i s over lap the cleavage points in the second, the peptide fragments can be placed in the proper order to y i e l d the complete.ami no a c i d sequence. i . The p o s i t i o n s of the d i s u l f i d e bonds and the amide groups in the o r i g i n a l polypept ide chain are determined. A d i s c u s s i o n o f a l l the steps invo lved in the complete s t r u c t u r e of a pro te in i s beyond the scope of t h i s t h e s i s . For reviews in these a reas , readers are r e f e r r e d to other w o r k s . 5 ' 6 Of d i r e c t re levance to our s tud ies are the methods for e l u c i d a t i n g the primary sequence o f pept ides , i . e . , determining the amino a c i d sequence from the N - , and C - t e r m i n i . \ 6 1.4 AMINO TERMINAL PEPTIDE SEQUENCING AND END-GROUP IDENTIFICATION The general p r i n c i p l e f o r amino end-group determination i s based on the i n t r o d u c t i o n of a marker group ( c o l o r e d , f l u o r e s c e n t , UV absorb ing , e t c . ) onto the amino f u n c t i o n , fo l lowed by the q u a n t i t a t i v e i s o l a t i o n and c h a r a c t e r i z a t i o n o f the d e r i v a t i z e d amino a c i d . In recent y e a r s , methods o f N-terminal ana lys is based on dansyl c h l o r i d e (l3 l -d imethylaminonaphthalene-5-sul fonyl c h l o r i d e , DNS-C1) have found wide a p p l i c a t i o n , l a r g e l y because of the ease with which one can study minute amounts of peptides and l p r o t e i n s . 7 Dansyl c h l o r i d e , which i s i t s e l f n o n - f l u o r e s c e n t , gives a strong ye l low f luorescence upon sulfonamide formation with the amino group of prote ins and peptides as shown in Equation 1. Hydro lys is o f the DNS-peptide with a c i d produces free amino ac ids and the dansyl d e r i v a t i v e o f the N-terminal res idue . A f t e r separat ion from the untagged amino a c i d s , the dansyl-amino a c i d may be i d e n t i f i e d by e l e c t r o - phoresis or th in l a y e r chromatography and v i s u a l i z e d by f l u o r e s c e n c e . The most important and widely used l a b e l l i n g reac t ion for the N-terminal residue o f a pept ide or p ro te in is the Edman d e g r a d a t i o n . 8 Since i t s i n t r o d u c t i o n over 25 years ago, t h i s procedure has undergone 7 almost cont inual m o d i f i c a t i o n , culminat ing in the development o f two auto- mated versions for sequence a n a l y s i s . 9 The f i r s t step of the Edman procedure (Scheme 1.1) involves reac t ing the peptide or prote in N-terminus with phenyl iso th iocyanate (2) at a l k a l i n e pH. A f t e r removal of excess reagents , the r e s u l t i n g pheny l th io - carbamyl peptide [3, PTC-peptide) i s t reated with a c i d , which causes c y c l i z a - t ion and cleavage o f the N-terminal amino a c i d as a 2 - a n i l i n o - 5 - t h i a z o l i n o n e (4). A f t e r separat ion from the pep t ide , the th iazo l inone i s converted to the isomeric 3 -phenyl -2 - th iohydanto in {5, PTH), which can be i d e n t i f i e d by a number o f phys ica l techniques. U s u a l l y , the Edman procedure i s not r e s t r i c t e d to determining the N-terminal r e s i d u e , and the peptide or p ro te in i s subjected to add i t iona l cyc les of the Edman degradat ion , thus e f f e c t i n g a sequential N-terminal a n a l y s i s . In favourable cases , one may expect the manual technique to produce about 30 degradat ion cyc les with c l e a r l y i n t e r p r e t a b l e r e s u l t s . To perform one degradation requi res the best part o f a working day. In 1967, Edman and B e g g 1 0 reported the development o f an automatic Edman Sequenator. S ince that t ime, machines based on t h e i r design have become such an i n t e g r a l par t o f the methodology used f o r sequence a n a l y s i s that less emphasis i s now being placed on manual determinat ion of N-terminal sequences o f p r o t e i n s . The sequence ana lyzer i s i d e a l l y su i ted to degrada- t ion o f prote ins and large peptide fragments, the optimal s i z e being 100-150 res idues . T y p i c a l l y , one may expect the unambiguous e l u c i d a t i o n of between 30 and 70 residues of amino a c i d sequence with the Edman Sequenator, as opposed to about 15-20 res idues with the manual method in not unfavourable c a s e s . 1 1 8 PhNCS + NH 2-CHR-C-NH -PEPTIDE (2) pH 8-9 S 0 PhNH-C-NH-CHR-C-NH PEPTIDE (3) (4) ° H + NH, •PEPTIDE PhNCS, pH 8-9 H + , H 20 \ e t c . - A (5) Scheme 1.1 The Edman Degradation 9 A complementary automatic method i s the so l id -phase sequencer o f L a u r s e n . 1 2 , 1 3 With th is technique, the peptide i s at tached to a s o l i d support and subjected to the Edman degradat ion with rad ioac t i ve phenyl i so th iocyana te . The e f f i c i e n c y o f the so l id -phase method only permits the determination of up to about 30 r e s i d u e s , 1 1 and in t h i s respect i s i n f e r i o r to the Edman Sequenator. However, the so l id -phase instrument may be used with small pept ide fragments (up to 30 r e s i d u e s ) , and i s considerably cheaper in cost and maintenance. The use o f enzymes fo r N-terminal ana lys is of prote ins i s s t i l l i n a f a i r l y i n c i p i e n t s t a t e . Two enzymes are commercially a v a i l a b l e for th is purpose: leuc ine aminopeptidase and aminopeptidase M. These enzymes cata lyze h y d r o l y s i s of the peptide bond o f the N-terminal residue o f prote ins and pep t ides , r e l e a s i n g a f ree amino a c i d . Hydro lys is proceeds s e q u e n t i a l l y from res idue to r e s i d u e , f o r the degradation c o n t i n u a l l y produces a new N-terminus. A p a r t i a l sequence of a pro te in may thus be deduced from a k i n e t i c a n a l y s i s o f the amino ac ids r e l e a s e d . The N-terminal residues o f a l l amino ac ids are cleaved by amino- peptidase M and leuc ine aminopeptidase, with the exception of p r o l i n e fo r the l a t t e r enzyme. Rapid h y d r o l y s i s i s observed with a l i p h a t i c and aromatic amino a c i d r e s i d u e s , slower rates with a l l o t h e r s , with i n d i v i d u a l d i f f e r - ences spanning a range o f several orders of m a g n i t u d e . 1 4 1.5 CARBOXY TERMINAL PEPTIDE SEQUENCING AND END-GROUP IDENTIFICATION While the procedures f o r N-terminal a n a l y s i s o f prote ins and peptides are among the best and most useful methods that have been appl ied in sequence determinat ions, methods f o r the i d e n t i f i c a t i o n o f C-terminal residues have been l e s s s u c c e s s f u l . Only two methods, a chemical and an 10 enzymatic one, have been app l ied to any great extent . The h y d r a z i n o l y s i s method is based on the d iscovery of Akabori e t a l . 1 5 that C-terminal residues of a pro te in are l i b e r a t e d as f ree amino acids by treatment of the prote in with anhydrous hydraz ine . A l l other amino acids in peptide l inkage are converted to amino ac id hydrazides as shown in Equation 2. H 9 N-CHR,-C0•••NH-CHR ,-CO-NH-CHR -C00H + N-hL - 2 - 1 n-1 n d 4 H 2N-CHR 1-C0NHNH 2 + • • • + H2N-CHR ^CONHNHg + H 2N-CHR n-C00H (2) Although the h y d r a z i n o l y s i s method i s simple i n p r i n c i p l e , i t s a p p l i c a t i o n i s f raught with d i f f i c u l t i e s . The y i e l d s o f C-terminal amino acids are only moderate, n e c e s s i t a t i n g the a p p l i c a t i o n of high c o r r e c t i o n f a c t o r s . The quant i ta t iveness o f the method a lso s u f f e r s from the ra ther compl icated methods fo r i s o l a t i n g and est imat ing the f ree amino a c i d s . The technique of Braun and S c h r o e d e r 1 6 , however, appears to be the best improvement of the o r i g i n a l procedure. In th is method the reac t ion o f the pept ide with anhydrous hydrazine i s ca ta lyzed by Amberl i te CG-50 in the hydrogen form. A f t e r removal of c a t a l y s t and excess hydraz ine , the amino acids are separated from hydrazides on a cat ion-exchange res in column, and subsequently analyzed by automatic amino ac id a n a l y s i s . The h y d r a z i n o l y s i s method f a i l s when the C-terminal res idue i s a r g i n i n e , c y s t e i n e , c y s t i n e , asparagine , or glutamine, and may f a i l when the terminal group i s a s p a r t i c a c i d . As with a l l methods fo r the i d e n t i f i c a t i o n of end-groups in macromolecules, po ten t ia l causes o f e r r o r are s u f f i c i e n t l y numerous with hydrazi n o l y s i s t o ' j u s t i f y reservat ions about any r e s u l t s unless i t i s corroborated by other methods. 11 Carboxypeptidases act on prote ins and peptides to re lease L-amino ac ids one residue at a time from the C-termini o f peptides and p r o t e i n s . Several types of enzymes have been charac te r i zed according to t h e i r substra te s p e c i f i c i t i e s . Carboxypeptidase A (CPA) shows a marked preference for C-terminal residues with an aromatic or branched a l i p h a t i c s ide c h a i n , (Table 1.2). Table 1.2 Approximate Re la t ive Rates o f Release o f Amino Acids by Carboxypeptidase A 1 7 Rapid Release: T y r , Phe, T r y , Leu, H e , Met, Thr , G i n , H i s , A l a , V a l , Homoserine Slow Release: A s n , S e r , L y s , MetSC^ Very Slow Release: G l y , Asp , G l u , CySC^H, S-carboxymethylcysteine Not Released: Pro , Arg Note: The presence o f a "very slow" or "not re leased" amino a c i d as penult imate res idue w i l l genera l ly decrease the rate of re lease of the C-terminal amino a c i d . Carboxypeptidase B (CPB) e x h i b i t s a narrower s p e c i f i c i t y , and cleaves the bas ic amino acids l y s i n e , and arg in ine very much f a s t e r than any o f the other common amino a c i d s . In common with a l l carboxypept idases, the rate of re lease o f C-terminal residues i s grea t ly in f luenced by the s t r u c - ture o f the adjacent r e s i d u e . 1 8 Another enzyme ga in ing popu la r i t y f o r C-terminal ana lys is o f peptides i s carboxypeptidase C ( C P C ) . 1 9 This enzyme has a broad s p e c i f i c i t y for a c i d i c , neutral and bas ic amino a c i d s . A great advantage of CPC i s i t s a b i l i t y to hydrolyze both peptide l inkages on e i t h e r s ide o f p r o l i n e . 12 A new commercially a v a i l a b l e exopeptidase i s carboxypeptidase Y ( C P Y ) . 2 0 L ike CPC, i t s s p e c i f i c i t y i s broad fo r a l l common amino a c i d s . In g e n e r a l , when the penult imate and/or C-terminal residues have aromatic or a l i p h a t i c s ide chains c a t a l y s i s is h i g h , bu^when g lyc ine i s p laced in the penultimate p o s i t i o n , the re lease of the terminal amino a c i d i s extremely slow. In cont ras t to CPA and CPB, C-terminal p r o l i n e i s a ra ther good s u b s t r a t e . However, the rate o f s p l i t t i n g of the peptide bond on e i t h e r s ide o f p r o l i n e depends ex tens ive ly upon the s t ruc tu re of the adjacent amino a c i d s . The enzymes of choice fo r C-terminus determinat ion o f prote ins and peptides have t r a d i t i o n a l l y been CPA and CPB. In a t y p i c a l end-group deter - minat ion , these enzymes may be incubated s e p a r a t e l y , o r in combination with the prote in s u b s t r a t e . However, with the increased a v a i l a b i l i t y of CPC and CPY, these l a t t e r exopeptidases w i l l f i n d wider use i n view of t h e i r broader s p e c i f i c i t i e s , and t h e i r a b i l i t y to cleave p r o l i n e . In p r a c t i c e , the rate o f re lease o f amino acids from a peptide by carboxypeptidase i s fo l lowed by ana lyz ing the e n t i r e r e a c t i o n mixture with an amino a c i d ana lyzer . In favorable c i rcumstances, the k i n e t i c measurement can give a f a i r l y r e l i a b l e i n d i c a t i o n o f the C-terminal sequence. However, the chances of m i s i n t e r p r e t a t i o n are l a r g e , and the r e s u l t s should be confirmed whenever p o s s i b l e . The method o f S t a r k 2 1 ' 2 2 f o r C-terminal sequencing o f peptides and prote ins i s based on the r e a c t i o n o f the C-terminal amino ac id with ammonium thiocyanate and a c e t i c anhydride (Scheme 1.2). The C-terminal amino ac id i s a c t i v a t e d to n u c l e o p h i l i c at tack by formation o f a mixed anhydride (6) by r e a c t i o n with a c e t i c anhydr ide. Attack by the thiocyanate a n i o n , a f t e r c y c l i z a t i o n of the product , gives 13 R O R' O I II I II NH,CHCNHCHCOH Peptide A. Addition O II (CH,C)20 O R O II I I CHXNHCHC. Oxazolinone O II -c I ,.CHR' NCS O R O R' O O II I II I II II C H 3 C N H C H C N H C H C O C C H 3 N-Acetyl mixed anhydride (f?) "NCS O R O II I II CH.CNHCHC- Mixed anhydride with isothiocyanic acid R' I O R O HC-II I II I CH-CNHCHC— O II -c I „NH Aminoacylthiohydantoin ( y ) R' I O R O HC-II I II I CH„CNHCHC—N v II s -c I , N H B. Cleavage O II CH.CNHO O R O O II I II II CH3CNHCHCONHCCH3 OH or H O R O II I II C1LCNHCHCOH (8) Scheme 1.2 S t a r k ' s Method for C-Terminal Sequencing of Peptides and Prote ins 14 the pept idy l th iohydantoin (?) which can be cleaved with aqueous a c i d or base, or with acetohydroxamate. A hydantoin (g) i s l i b e r a t e d which i s c h a r a c t e r i s t i c of the C- terminus, and the remaining ace ty la ted peptide or prote in possesses a f ree carboxylate group at the new C-terminus. Degrada- t ion o f peptides may be fo l lowed s u b t r a c t i v e l y by amino ac id a n a l y s i s of the peptide product a f t e r each c y c l e or a l t e r n a t i v e l y , the thiohydantoins may be determined d i r e c t l y by t h i n - l a y e r chromatography. The thiohydantoin method i s somewhat l i m i t e d i n that C-terminal a s p a r t i c a c i d and p r o l i n e are not removed. The rather extreme condi t ions requi red f o r cleavage of the acyl th iohydantoin ( e . g . 12M HC1) l i m i t the method to 2 or 3 cyc les fo r most peptides because o f n o n - s p e c i f i c h y d r o l y s i s o f in te rna l peptide bonds. mi nation o f C-terminal amino acids invo lves s e l e c t i v e exchange o f the hydrogen on the asymmetric carbon o f the C-terminal amino a c i d with t r i t i u m as in Scheme 1.3 The t r i t i a t i o n method o f Matsuo and c o - w o r k e r s 2 2 f o r the deter - Rl Rn-I Rn H,N-CH-C0NH—-CONH-CH-CONH-CH-COjH V 7"-' I AcNH-CH-CONH—C0NH-CH-\ base hydrolysis Scheme 1.3 The T r i t i a t i o n Method f o r C-Terminal Sequencing of Peptides and Prote ins 15 In the presence of a c e t i c anhydride, C-terminal amino acids o f peptides and prote ins s e l e c t i v e l y undergo c y c l i z a t i o n to form oxazalones. The oxazalones contain an ac t ive hydrogen and r e a d i l y incorporate t r i t i u m when t reated with 3 H 2 0 and p y r i d i n e . Hydro lys is with 18% HCl produces a mixture o f amino ac ids which can be separated by paper chromatography. The tagged amino a c i d i s i d e n t i f i e d by i t s r a d i o a c t i v i t y . D i f f i c u l t i e s with the procedure of Matsuo e t a l . 2 3 are that C-terminal a s p a r t i c a c i d and p r o l i n e do not i n c o r p - orate t r i t i u m , and a c i d - c a t a l y z e d condi t ions must be used. Problems may a lso a r i s e with s e r i n e , t h r e o n i n e , 2 h and non-terminal a s p a r t i c a c i d . 2 5 A s tep-wise degradation of peptides from the carboxyl end based on N , 0 -m igra t ion of acyl groups in conjunct ion with a reduct ion procedure was descr ibed by B a i l e y . 2 6 In th is .scheme, peptide esters are reduced to the corresponding a l c o h o l s , which, in the presence of P0C1 3 or S 0 C 1 2 , rearrange to y i e l d the B-amino e s t e r , which can be fu r ther reduced to give the f ree amino a lcohol and res idua l pept ide in a form prepared f o r fu r ther rearrangement (Scheme 1.4). Y - N H - C H R ' - C 0 N H - C H R " - C 0 0 R M H » Y - N H - C H R ' - C 0 N H - C H R " - C H 2 0 H P O C I 3 Y - N H - C H R ' - C H 2 0 H + H 0 - C H 2 - C H R " - N H 2 Y - N H - C H R 1 - C - 0 C H 2 - C H R " - N H 3 + 0 Scheme 1.4 C-Terminal Peptide Sequencing by Reduction The most s t r ingen t requirement in t h i s method i s fo r a mild and s e l e c t i v e reduct ion of the C-terminal es ter bond without any accompanying reduct ion of peptide bonds. Thus, LiBH^ i s p r e f e r a b l e , whi le L i A l i s u n s u i t a b l e . D i f f i c u l t i e s in e s t a b l i s h i n g optimum reac t ion condi t ions f o r the s e l e c t i v e 16 reduct ion of es ter groups have l im i ted i t s a p p l i c a t i o n in peptide sequencing. In a m o d i f i c a t i o n of B a i l e y ' s procedure, Hamada and Yonerni tsu 2 7 recent ly descr ibed the reduct ion of peptide esters with NaBH^ in aqueous s o l u t i o n fol lowed by hydro lys is of the peptide bonds with 6N HCl . Because of the n o n - s p e c i f i c h y d r o l y s i s of the peptide a l c o h o l , the method is l im i ted to determinat ion of the C-terminal res idue o n l y . A new method fo r determinat ion of the C-terminal res idue in peptides descr ibed by Loudon and c o - w o r k e r s 2 8 involves formation of an O-subst i tu ted hydroxamic ac id (9) by reac t ion of the peptide C-terminal carboxyl group with a water -so lub le carbodi imide (WSC) and an O-subst i tu ted hydroxy!amine. The O-subst i tu ted hydroxamic a c i d undergoes, at higher pH, a Lossen rearrangement to the isocyanate (10) leading to degradation of the C-terminal res idue (Scheme 1.5) . 0 0 0 0 0 Pep-C-NH-CHR-COOH + H 9 N-0 -C -Bu- t —*~ Pep-C-NH-CHR-C-NH-O-C-Bu-t (9) OH" 0 0 t - B u - C - 0 " + Pep-C-NH-CHR-N=C=0 (10) H 20 o o H+ j H Q 0 Pep-C-NH, + RCH + NH, — Pep-C-NH-CHR-NH 0 + C0 o (11) Pep = N-terminal por t ion of a peptide Scheme 1.5 Peptide C-Terminus Determination v i a Lossen Rearrangement of an O-subst i tu ted Hydroxamic ac id 17 Hydro lys is of the carboxamide bond to generate a new carboxyl terminus would make a sequent ia l procedure p o s s i b l e . However, the Niaminomethylamide ( l l ) formed on degradat ion of the isocyanate , decomposes under v igorous h y d r o l y t i c condi t ions to the corresponding peptide-amide and aldehyde. I d e n t i f i c a t i o n of the C-terminal residue depends on the d i f f e r e n c e in amino ac id a n a l y s i s of the peptide before and a f t e r degradat ion. Th is r e s t r i c t s the method to r e l a t i v e l y small peptides of a s i z e f o r which t h i s a n a l y t i c a l technique i s a p p l i c a b l e . The method f a i l s when a s p a r t i c or glutamic ac ids are the C-terminal r e s i d u e s . Inter ferences in the method a^e the low degradat ion y i e l d s of asparagine and glutamine and the p a r t i a l l o s s of in te rna l t y ros ine and tryptophan. 1.6 OBJECTIVES AND OUTLINE OF THE PRESENT WORK From the preceding presentat ion of methods c u r r e n t l y in use , or proposed f o r determining the amino ac id sequence of pept ides and p r o t e i n s , i t should be obvious that while some procedures have reached a s ta te of matur i ty ( e . g . Edman degradat ion ) , there are others which are as ye t u n s a t i s f a c t o r y . The statement by Stark seven years ago that no entirely satisfactory chemical method of carboxyl-terminal analysis e x i s t s 2 ^ s t i l l a p p l i e s . Of the several C-terminal methods that have been proposed (vide supra), a l l s u f f e r l i m i t a t i o n s , and few have been useful in actual p r a c t i c e . Vigorous c o n d i t i o n s , so lvent r e s t r i c t i o n s , and f a i l u r e at c e r t a i n amino ac id res idues have a l l cont r ibuted to t h e i r lack of general u t i l i t y . It would be h igh ly advantageous to have a method of sequencing peptides from the carboxyl end s i m i l a r in s e n s i t i v i t y , convenience, and a p p l i c a b i l i t y to the Edman degradation from the amino terminus. 18 The work descr ibed in th is thes is has as i t s o b j e c t i v e s the inves- t i g a t i o n of a reac t ion s p e c i f i c f o r carboxylates which could be used as a method f o r C-terminal residue a n a l y s i s or C-terminal peptide sequencing. The concept of the method which we envisaged f o r determinat ion of the C-terminal res idue in peptides i s shown in Scheme 1.6. 0 Pep-C-NHCHRn_ 1CONH-CHR COOH + N N j j Pep-CONH-CHRn_1CONH-CHR ^ N (13) N H 20 Pep-CONH-CHR -jCOOH N H - N H i Further Degradative Cycles (12) + 2H 20 N N Pep = N-terminal por t ion of a peptide Scheme 1.6 Proposed C-Terminal Peptide Sequencing v i a s -Tr iazo lo l_3 ,4 -aJphtha laz ines The reac t ion involves the coupl ing of 1-hydrazinophthalaz ine (12) with the C-terminal amino ac id of peptides to a f fo rd a p e p t i d y l - s - t r i a z o l o [ 3 , 4 - a ] - phthalaz ine d e r i v a t i v e (13). Hydro lys is of the terminal peptide bond removes the C-terminal res idue as a 3 -aminomethy l -s - t r i azo lo [3 ,4 -a ]ph tha laz ine (14) d e r i v a t i v e which i s c h a r a c t e r i s t i c of the terminal amino a c i d . The 19 remaining peptide contains a f ree carboxyla te which can undergo fu r ther degradative c y c l e s with 1 -hydraz inophtha laz ine . The choice of 1-hydrazino- phthalazine as the reagent f o r m o d i f i c a t i o n o f the C-terminal amino ac id a r i s e s from several c o n s i d e r a t i o n s : a . 1-Hydrazinophthalazine reacts with carboxylates to form an amide intermediate which spontaneously c y c l i z e s with l o s s o f water to form an s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (TAP). The formation of t h i s c y c l i c product i s s p e c i f i c f o r carboxyl groups among those f u n c t i o n a l i t i e s which are commonly found in amino a c i d s , and there can be no ambiguity in r e s u l t s a r i s i n g from p o s s i b l e r e a c t i o n o f 1-hydrazinophthalazine with other c l a s s e s o f e l e c t r o p h i l e s . b. An advantage o f the TAP system i s that i t e x h i b i t s a strong blue f luorescence when i r r a d i a t e d with short wavelength u l t r a - v i o l e t l i g h t . This property of TAP d e r i v a t i v e s should permit i d e n t i f i c a t i o n and de tec t ion o f the TAP-modif ied amino acid with high s e n s i t i v i t y , analogous to the Dansyl method fo r N-terminal amino a c i d s . c . The e s s e n t i a l components f o r formation of a fused 1 , 2 , 4 - t r i a z o l e r i n g are a carboxyl component and a hydrazino compound with the s t r u c t u r e : NH-NH, We chose the hydrazino d e r i v a t i v e o f phthalazine s ince i t s chemistry i s well known, and in almost a l l o f i t s reac t ions with c a r b o x y l i c a c i d s , the f u l l y c y c l i z e d products are formed, un l ike those o f other r i n g systems which may stop at the amide product . 20 The major thrust of t h i s work was in d e l i n e a t i n g the cond i t ions necessary f o r coupl ing 1-hydrazinophthalaz ine with N-protected amino ac ids and d i p e p t i d e s . 1-Hydrazinophthalazine does not reac t with c a r b o x y l i c ac ids under mi ld c o n d i t i o n s , hence we explored several methods o f promoting t h i s r e a c t i o n . In view of the p o s s i b l e a p p l i c a t i o n of our r e a c t i o n f o r pept ide sequencing, we a l s o explored the s o l i d - p h a s e synthes is of TAP d e r i v a t i v e s . We considered the use of metal complexes to promote the h y d r o l y s i s of pept ide bonds. In t h i s contex t , severa l t r a n s i t i o n metal coord ina t ion complexes of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e s were syn thes i zed . 21 i CHAPTER 2 REACTION OF 1-HYDRAZINOPHTHALAZINE WITH N-PROTECTED AMINO ACIDS 2.1 INTRODUCTION The reac t ions of hydra laz ine (12, 1 -hydraz inophtha laz ine , Apreso l ine ) NH-NH 2 (12) have been studied only s ince 1 9 5 0 . 3 0 Studies with th is chemical were prompted by the d iscovery that in hypertensive man, hydra laz ine lowers blood pressure by reducing vascu la r r e s i s t a n c e through d i r e c t r e l a x a t i o n of a r t e r i o l a r smooth muscle. These c h a r a c t e r i s t i c s made i t an idea l agent f o r the treatment of severe a r t e r i a l h y p e r t e n s i o n . 3 1 The e f f e c t on blood pressure was so s t r i k i n g that i n t e r e s t in the drug surged , and wi th in 8 y e a r s , there were some 920 references to hydra laz ine in the l i t e r a t u r e . Today, despi te the advent of many other d rugs , hydra laz ine i s s t i l l widely used fo r the treatment of high blood p r e s s u r e , and i s f requent ly administered in combination with s y m p a t h e t i c - i n h i b i t i n g and/or d i u r e t i c an t ihyper tens ive a g e n t s . 3 2 The c e l l u l a r mechanism respons ib le f o r the r e l a x a t i o n of vascu la r 22 smooth muscle remains unknown, but the a b i l i t y of hydra laz ine to chelate c e r t a i n t race metals perhaps requi red fo r smooth-muscle c o n t r a c t i o n may be i m p o r t a n t . 3 3 Hydralaz ine was f i r s t synthesized and tested in the Ciba L a b o r a - t o r i e s at Basle by Gross , Druey, and Meir as one of a s e r i e s of experimental a n t i h i s t a m i n e agents conta in ing a hydrazino g r o u p . 3 0 As i t turned out , th is compound was not an a n t i h i s t a m i n e , but i t s i n j e c t i o n in to animals caused long ac t ing vasodepressor responses un l ike those of any h i ther to known drug. In response to the p o t e n t i a l i t i e s of such an agent,[ s tud ies were immediately i n i t i a t e d in to the chemical and biochemical reac t ions of hydra laz ine with the i n t e n t i o n of e l u c i d a t i n g i t s mode of ac t ion Druey and R ing ie r made the f i r s t systemat ic study of the chemistry of h y d r a l a z i n e . 3 4 They found that a c y l a t i n g agents , such as c a r b o x y l i c a c i d s , ac id c h l o r i d e s , anhydr ides , and e s t e r s , d id not give the expected N-acyl d e r i v a t i v e s with hydra laz ine . Instead of the amide product being i s o l a t e d , r i n g c losure occurred with e l i m i n a t i o n of water and a new type of compound, the s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (TAP) d e r i v a t i v e was always obta ined . NH-NH 2 (12) Hydralaz ine X = -OH, - O R ' , -0C0R", ha l ide 23 Subsequent ly , two reports appeared which i d e n t i f i e d N - a c e t y l - hydra laz ine as a major metabol ic product o f hydra laz ine in jec ted in to a n i m a l s . 3 5 ' 3 6 These i d e n t i f i c a t i o n s were l a t e r shown to be erroneous, and the metabol i te i s o l a t e d was in f a c t , 3 - m e t h y l - s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e [15, R = C H 3 ) . 3 7 > 3 8 Other metabol i tes of hydra laz ine i d e n t i f i e d s ince that time inc lude the compounds shown in Chart 2 . 1 . 3 9 - 1 4 2 N N R 1 = OH Chart 2.1 Metabol i tes of 1-Hydrazinophthalazine The non-enzymatic convers ion of hydra laz ine to 3-CH 3 -TAP was shown to occur in the presence of ace ty l CoA in human plasma u l t r a f i l t r a t e . 1 * 3 Independently of the pharmacological and c l i n i c a l s tudies on hydra laz ine and i t s me tabo l i t es , Love le t te and Potts inves t iga ted the synthesis and chemical proper t ies of var ious s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e s as part of a general program on the study of bridgehead n i t rogen heteroaromatic r ing s y s t e m s . 1 * 4 * 1 * 5 24 While the work descr ibed in th is thes is was in p rogress , Zimmer and co-workers reported t h e i r i n v e s t i g a t i o n s on the react ion between hydra laz ine and a v a r i e t y of a c y l a t i n g a g e n t s . 4 6 The i r ob jec t i ve was to determine whether the r i n g c losure to g ive 3 -subst i tu ted-TAP d e r i v a t i v e s was a c h a r a c t e r i s t i c of a c y l a t i o n reac t ions on ly . In every reac t ion they s t u d i e d , the c y c l i z e d product was obta ined . The range of react ions from which TAP d e r i v a t i v e s may be formed are shown in Table 2 .1 . The r e a c t i o n mechanism f o r the coupl ing and r i n g c losure suggested by Druey and R ing ie r i s shown in Scheme 2 . 1 . 3 4 • H (15) Scheme 2.1 Reaction Mechanism f o r Formation of s -T r iazo1o[3 ,4 -a ]ph tha laz ines It has been shown with the 1-hydrazinophthalazine system, that once the ac id component was coup led , the amide intermediate c y c l i z e d spontaneously , even in those cases where attempts were made to synthesize the a c y l h y d r a l a z i n e . 3 8 > 4 4 > 4 5 The ease by which t h i s c y c l i z a t i o n occurs seems to be unique f o r the phthalaz ine system. In other c a s e s , e . g . the a c y l a t i o n of 2 -hydraz inopyr id ine , the amide d e r i v a t i v e s could be obtained as s tab le and i s o l a b l e compounds 4 7 which c y c l i z e under dehydrat ive c o n d i - 25 Table 2.1 Reactions of Hydralazine with. Various Carboxy l ic Ac id Der iva t ives N N A c i d Der iva t i ve R Reference HCOOH H 45 CH3COOH CH 3 45 CC1 3 C00H c c i 3 46 HSCHpCOOH CH2SH 46 PhCH(0H)C00H CH(0H)Ph 46 COOH H 45 COOH ( C F 3 C 0 ) 2 0 C F 3 46 0 ( C H 2 ) 2 C 0 0 H 46 (CH 3 ) 3 C-C0C1 C ( C H 3 ) 3 46 CC13CN c c i 3 46 H C ( 0 E t ) 3 H 45 C H 3 C ( 0 E t ) 3 C H 3 45 c s 2 SH 44 CNBr NH 3Br" 44 26 t i o n s , such a s , r e f l u x i n g with phenol or phosphorous o x y c h l o r i d e . 4 7 - 1 + 9 The f i r s t goal of t h i s work was to de l inea te the cond i t ions required f o r coupl ing hydra laz ine with c a r b o x y l i c acids under as mild cond i t ions as p o s s i b l e . L i t e r a t u r e methods c a l l f o r t h i s coupl ing to be e f fec ted at r e f l u x temperatures with neat l i q u i d c a r b o x y l i c a c i d s , or under melt cond i t ions f o r s o l i d a c i d s . 4 5 * 4 6 If the coupl ing reac t ion is to be appl ied to amino acids or to pept ides , such severe cond i t ions are not appropr ia te . The f i r s t step in the coupl ing reac t ion i s the formation of an amide bond from carboxyl and amine components. Amide formation i s an endoergic reac t ion so that energy must be s u p p l i e d , e . g . in the form of heat. An a l t e r n a t i v e is that one of the r e a c t i o n components i s introduced in an ac t iva ted form. Most of the publ ished a p p l i c a t i o n s of t h i s r e a c t i o n are der ived from t h i s common technique: they occur through an a c t i v a t e d form of the carboxyl group, in general through acyl d e r i v a t i v e s such as h a l i d e s , anhydrides and e s t e r s . 5 0 2.2 NOMENCLATURE The fo l lowing naming and numbering systems, both IUPAC and t r i v i a l , are used in t h i s work. 1-Hydrazinophthalazine (Hydralazine) 27 s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (TAP) 2 N N The naming of the s i d e - c h a i n in the 3 - p o s i t i o n fo l lows normal procedures when the TAP d e r i v a t i v e i s der ived from hydra laz ine and a simple c a r b o x y l i c a c i d , e . g . , When the TAP d e r i v a t i v e i s der ived from an amino a c i d , we depart from IUPAC ru les and give the s i d e - c h a i n subs t i tuen t the same name as the parent carboxyl component. The naming of TAP d e r i v a t i v e s i s i l l u s t r a t e d in Table 2 .2 . 2.3 PRELIMINARY RESULTS The coupl ing r e a c t i o n of hydra laz ine hydrochlor ide with neat g l a c i a l a c e t i c ac id to give 3 - m e t h y l - s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e hydro- c h l o r i d e requi res up to 4 hours under r e f l u x cond i t ions to be completed. Carboxy l ic ac ids are not always l i q u i d s however, and the quest ion a r i s e s whether the coupl ing can s t i l l occur when the reactants are d i l u t e d in a so lvent medium. In aqueous acetate buf fe r s o l u t i o n s (pH 2 . 9 - 4 . 8 ) , no coupl ing reac t ion occurred a f t e r 9 hours at 100°C which suggested that the Hydralaz ine + CH^COOH Hydralaz ine + HCOOH *• s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (3-H-TAP) *- 3 - m e t h y l - s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (3-CHLTAP) Table 2.2 Nomenclature f o r the S ide-cha ins of s - T r i a z o l o [ 3 , 4 - a ] p h t i i a l a z i n e s Parent Carboxy l ic Acid Accepted Name of 3-Subst i tuent on TAP T r i v i a l Name of 3 -Subst i tuent on TAP TAP Abbrev ia t ion Formic Ac id HCOOH 3-H-TAP A c e t i c Acid CH3C00H N - A c e t y l - g l y c i n e CH3C0NHCH2C00H Methyl Acetarnidomethy1 3-Me-TAP N - A c e t y l - g l y c y l 3 - (N-Ac-g ly ) -TAP N- t -Buty loxycarbony lg lyc ine N-t-Butyloxycarbonylaminomethyl N - t -Buty loxycarbony l - 3 - (N-B0C-gly) -TAP (CH3)3COCONHCH2COOH g i y c y i N- t -Buty loxycarbonyla l anine (CH3)3C0C0NHCHC00H l - (N- t -Buty loxycarbonyl )amino- N - t -Buty loxycarbony l - 3- (N-B0C-ala) -TAP ethyl a lanyl CH. u -Acety l -methionine CH3C0NHCHC00K ( C H 2 ) 2 SCH, l -Acetamido-3-methyl thiopropyl N-Acetyl -methionyl 3-(N-Ac-met)-TAP 29 coupl ing of a c a r b o x y l i c ac id with hydralaz ine was unfavourable under d i l u t e c o n d i t i o n s . The a l t e r n a t i v e mode of a c t i o n , that of a c t i v a t i n g the carboxyl group was then cons idered . The approach taken was to generate the a c t i v a t e d carboxyl interme- d ia te in situ by means of a 'coupl ing reagent , ' and to react the a c t i v a t e d species d i r e c t l y with the amine component without p r i o r i s o l a t i o n of the intermediate . The coupl ing reagent used f o r exp lora tory s tud ies was l -e thy l -3 -d imethy laminopropyl carbodi imide h y d r o c h l o r i d e 5 1 (16„ EDC). + Et -N=ON- (CH 2 ) 3 NMe 2 C l " H (16) The carbodi imide reacts with c a r b o x y l i c ac ids to form an 0 - a c y l i s o u r e a which is attacked very r e a d i l y by amine nucleophi les to form the amide product (see Sect ion 2 . 4 . 4 ) . The f i r s t t r i a l s were performed with hydra laz ine HC1 and aqueous a c e t i c ac id in the presence of EDC at ambient temperature. The r e a c t i o n was monitored by UV spectroscopy (F igure 2 .1 ) . The molar a b s o r p t i v i t y (e) of the most intense absorpt ion of 3-CH 3 -TAP i s almost four times greater than that of hydra laz ine . Thus, as the coupl ing reac t ion proceeds, i t i s p o s s i b l e to monitor the growth of the 231 nm band of 3-CH 3 ~TAP, concomitant with the decrease in i n t e n s i t y of the hydralaz ine bands. In aqueous s o l u t i o n , the reac t ion d id proceed with the formation of 3 -CH 3 -TAP. However, the reac t ion is slow, and a f t e r two weeks there was s t i l l a s i g n i f i c a n t amount of unreacted hydra laz ine HC1. Coupl ing occurred much f a s t e r using dry methanol as s o l v e n t , the react ion being e s s e n t i a l l y complete wi th in two days. 30 o c (0 Xi o < 2 0 0 2 5 0 3 0 0 Wavelength (nm) 3 5 0 Figure 2.1 U l t r a v i o l e t Spectra of Equimolar Amounts of (a) Hydra laz ine , (b) 3-M.e-TAP, and (c) a Mixture in \\n0 S o l u t i o n — — — — — — — — — — — — — 2 — — — — — 31 Although the reac t ion time was not yet s a t i s f a c t o r y , these experiments showed that with carboxyl a c t i v a t i n g agents mi lder reac t ion condi t ions than previous l i t e r a t u r e methods could be used. Th is se t the stage fo r the coupl ing of hydra laz ine with N-protected amino a c i d s . In p r i n c i p l e , the problems faced in t h i s work were s i m i l a r to those found in peptide s y n t h e s i s . The synthes is of peptides requi res a c t i v a t i o n of the carboxyl group of an N-protected amino a c i d such that i t w i l l react with the amino func t ion of another C-protected amino a c i d molecule to give a new peptide l i n k . In our c a s e , the nuc leoph i le is the amino group of a monosubstituted hydraz ine , raNther than the amino group of an amino a c i d . Previous work has shown that once the carboxamide l inkage is formed with hydra laz ine and c a r b o x y l i c a c i d s , r ing c losure i s immediate to form the s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e . 4 5 ' 4 6 One expects t h e r e f o r e , that coupl ing methods used in peptide synthes is should be a p p l i c a b l e to the formation of TAP compounds from hydra laz ine and amino a c i d s . Because of the mult i tude of coupl ing methods used in peptide s y n t h e s i s 5 2 " 5 4 i t i s necessary to be h igh ly s e l e c t i v e i n the choice of methods f o r coupl ing the amino and carboxyl components. In pept ide s y n t h e s i s , the choice of coupl ing reagents i s f requent ly d i c t a t e d by the ease of racemizat ion produced by the method. Racemization is unimportant in our system. In view of the promise of coupl ing reagents f o r e f f e c t i n g TAP format ion under mi ld c o n d i t i o n s , we d i r e c t e d our a t ten t ion to f i n d i n g t In peptide s y n t h e s i s , coupl ing reagents are genera l ly considered to be reagents added to the amino and carboxyl components to e f f e c t amide bond formation in a one-pot r e a c t i o n , without i s o l a t i o n of in termedia tes . 32 appropr ia te coupl ing reagents . The use of coupl ing reagents s i m p l i f i e s the synthe t ic procedures c o n s i d e r a b l y , and makes the method more amenable to p o s s i b l e automation of the procedure. 2.4 RESULTS 2.4.1 The Isoxazolium S a l t Method N - E t h y l - 5 - p h e n y l i s o x a z o l i u m - 3 ' - s u l f o n a t e (17, NEPIS, Woodward's Reagent K) was f i r s t used as an amide-forming reagent in 1 9 6 1 . 5 5 ' 5 6 (17) This reagent reacts with carboxyl groups to form a c t i v e enol es ters which condense with amine nuc leophi les with amide bond formation as shown in Scheme 2 .2 . React ion i s i n i t i a t e d by the base induced a b s t r a c t i o n of a proton in 17 with a concerted r ing opening rearrangement to form the h igh ly r e a c t i v e ketoketenimine ( i s ) . The reac t ion of t h i s species with f ree c a r b o x y l i c ac id produces an iminoanhydride (19b) which i s converted to the enol es te r (20). This intermediate can react with amine nuc leophi les to form the amide product , or i t can undergo 0 N acyl migrat ion to y i e l d the unreact ive keto- amide, 21. 5 7 The enol es te r i s a good a c y l a t i n g agent towards nuc leophi les s ince the leav ing group i s s t a b i l i z e d as the an'ion of a B-ketoamide. Some add i t iona l s t a b i l i z a t i o n of the leav ing group might be der ived from i n t r a - 33 Ar ,N-Et Base Ar (17) Ar 0 H ,0-CO-R NH-Et (19a) o o (21) R-CO-NH-R Ar = m - S 0 3 C 6 H 4 H ^ C . (18) ^N-Et RCOOH Ar. .OH O-CO-R NH-Et (22a) A r ^ O H - V NH-Et (22b) Scheme 2.2 Mechanism of Amide Formation with NEPIS 34 molecular hydrogen bonding to the forming 0-anion during attack by the nuc leophi le (:N). R i I 0 The high r e a c t i v i t y of the a c t i v e enol ester intermediate together with i t s f a c i l e generat ion in situ, recommended the isoxazol ium s a l t method f o r f a c i l i t a t i n g the coupl ing of hydra laz ine with amino a c i d s . Another f a c t o r in favour of t h i s method was the reputed ease of separat ion of by-products from the des i red coupled product . NEPIS was designed by incorpora t ing a su l fonate group onto the phenyl subs t i tuen t to provide an i o n i c ketoamide by-product (22), S0 3 HNEt3 (22) The procedure recommended fo r product i s o l a t i o n in peptide synthes is involves simple t r i t u r a t i o n with water to remove the water -so lub le i m p u r i t i e s . 5 6 Any unconsumed enol es te r or s i d e - r e a c t i o n products connected to the sul fonated framework would be removed from the coupled product along with 22. 35 L i t e r a t u r e procedures f o r synthesis of peptides c a l l f o r i n i t i a l l y suspending the isoxazol ium s a l t with the ac id component in the presence of EtgN, in e i t h e r a c e t o n i t r i l e or nitromethane so lvent u n t i l the isoxazol ium s a l t d i s s o l v e d complete ly . At t h i s s tage , the c a r b o x y l i c ac id i s assumed to be completely converted to the enol e s t e r . The amine component i s then added and the a c y l a t i o n reac t ion i s usua l l y allowed to proceed overnight (15-18 hours) before work-up of the r e a c t i o n mixture. For our t r i a l s with the isoxazol ium s a l t method, NEPIS was the coupl ing reagent used. The amine component was hydra laz ine as the HC1 s a l t because of the i n s t a b i l i t y of the f ree base (c f . Chapter 5 ) . I n i t i a l e x p e r i - ments were conducted with N-acety l -DL-methionine because of i t s s o l u b i l i t y in a c e t o n i t r i l e and a l s o because, at t h i s ear ly stage of the work, there were few other N-protected amino ac ids on hand. The r e a c t i o n of the above components i s shown in Scheme 2 .3 . (22) Scheme 2.3 Formation of 3-(N-Ac-met)-TAP with NEPIS 36 The r e a c t i o n cond i t ions used i n i t i a l l y were s i m i l a r to those recommended fo r peptide s y n t h e s i s , with a d d i t i o n of hydra laz ine HCl and Et-^N in a c e t o n i t r i l e suspension a f t e r formation of the enol e s t e r . However, i t became apparent that s t r i c t adherence to l i t e r a t u r e procedures was i m p r a c t i c a l . In the f i r s t i n s t a n c e , high concentrat ions of a l l reagents could not be used to promote the coupl ing r e a c t i o n because of the low s o l u - b i l i t y o f the hydra laz ine s a l t in a c e t o n i t r i l e . A yel low p r e c i p i t a t e was formed during the coupl ing r e a c t i o n which appeared to be a decomposit ion product of hydra laz ine . I s o l a t i o n of the TAP d e r i v a t i v e was not s p e c t a c u l a r l y s u c c e s s f u l . T r i t u r a t i o n of the r e a c t i o n mixture with water a f t e r the p r e c i p i t a t e was f i l t e r e d o f f , and the so lvent removed, not only d i s s o l v e d the su l fonated by -products , but a lso some TAP product as w e l l . Ex t rac t ion of a C H ^ C ^ s o l u t i o n of the r e a c t i o n mixture with water a l s o r e s u l t e d in loss of product in to the aqueous phase. On an encouraging note however, the CH2CT2 s o l u t i o n a f t e r e x t r a c t i o n showed c lean UV spectra c h a r a c t e r i s t i c of the TAP chromophore. The i d e n t i t y of the product i s o l a t e d from the C H g C ^ s o l u t i o n was confirmed by mass spectrometry to be the des i red 3 - ( N - a c e t y l - m e t h i o n y l ) - TAP (23). In an e f f o r t to improve the r e a c t i o n c o n d i t i o n s , the next s e r i e s of experiments were conducted with N-buty loxycarbonylg lyc ine (N-BOC-gly ) . The expected reac t ion product is 3 - ( N - B 0 C - g l y ) - T A P (24). 37 N-Buty loxycarbonylg lyc ine i s very so lub le in a c e t o n i t r i l e , and i s thus s u i t a b l e f o r our needs. Of more importance, the TAP d e r i v a t i v e should be r e l a t i v e l y i n s o l u b l e in water, thus f a c i l i t a t i n g i s o l a t i o n of product . Coupling reac t ions were performed under the same cond i t ions as p r e v i o u s l y , with the product i s o l a t e d by ex t rac t ion of C h ^ C ^ s o l u t i o n with water. As expected, 3 - (N-B0C-gly) -TAP showed low s o l u b i l i t y in water, and very l i t t l e TAP was l o s t in the aqueous phase during e x t r a c t i o n s . The i s o l a t i o n procedure was a lso modif ied by e x t r a c t i n g the C f - ^ C ^ s o l u t i o n f i r s t with d i l u t e HCl to expedite removal of unreacted h y d r a l a z i n e , and then with aqueous NaHC0 3 s o l u t i o n and water. The ex t rac t ions were u s u a l l y monitored by record ing the UV spectra of the organic and aqueous phases, and ex t rac t ions were repeated u n t i l the UV spectra of the aqueous phases showed no UV absorbing m a t e r i a l . However, during the ex t rac t ions with 5% H C l , some compound remained in the aqueous phase in approximately constant concent ra - t i o n even a f t e r most of the su l fonated products were removed. Th is compound showed a d i f f e r e n t UV band s t ruc tu re from the TAP chromophore. When an HCl ex t rac t was t i t r a t e d with base, the c h a r a c t e r i s t i c TAP band s t ruc tu re was generated. Thus , protonat ion o f one of the r i n g n i t rogen atoms o c c u r r e d , and the protonated product was more s o l u b l e than the bas is TAP molecule in aqueous media. When IN or ~\% HCl were used f o r e x t r a c t i o n s , no protonat ion of TAP was observed. To obta in more homogeneous cond i t ions f o r amide-bond format ion , the so lvent system was modi f ied . As be fore , the enol es ter of the N-protected amino ac id was prepared in a c e t o n i t r i l e s o l u t i o n . Hydralaz ine HCl and Et^N. v/ere added in DMF suspension . When the r e a c t i o n mixture was s t i r r e d overnight almost a l l the hydra laz ine HCl had d i s s o l v e d . An attempt was made to i s o l a t e the TAP d e r i v a t i v e by s o n i c a t i n g the reac t ion mixture with 0.5N HCl a f t e r 38, organic so lvent had been removed. The s o n i c a t i o n procedure acce le ra ted the ex t rac t ion of wa te r -so lub le mater ia ls in to aqueous s o l u t i o n , l eav ing TAP as a s o l i d . When t h i s mixture was l e f t s t a n d i n g , a gas was l i b e r a t e d and a l l s o l i d g radua l ly d i s s o l v e d . Very l i t t l e of the d i s s o l v e d product could be extracted in to C H 2 C 1 2 or EtOAc. Ev ident ly the work-up procedure deblocked the butyloxycarbonyl group from 3- (N-B0C-gly) -TAP with l i b e r a t i o n of C 0 2 (Equation 3) . The deblocking o f . t h e BOC- group under such mi ld cond i t ions C M e 3 _djl_HCl_ (24) CI C 0 2 + Me3C0H (3) i s in cont ras t with the observed s t a b i l i t y in IN HC1, and the usual deblock- ing procedure with HBr in g l a c i a l a c e t i c a c i d . 5 8 When the coupl ing r e a c t i o n was done e n t i r e l y in DMF s o l u t i o n , a l l hydra laz ine HC1 d i s s o l v e d on overnight r e a c t i o n , but i t s disappearance was masked by the simultaneous p r e c i p i t a t i o n of E t^N.HCl . Moni tor ing the r e a c t i o n by TLC showed s i g n i f i c a n t unreacted reagents a f t e r 5 hours, but the reac t ion appeared to be e s s e n t i a l l y complete wi th in 22 hours. The product was obtained by e x t r a c t i o n with IN or 2N HC1 and H 20 of a methylene c h l o r i d e s o l u t i o n of the reac t ion a f te r DMF was removed. Th is procedure removed almost a l l i m p u r i t i e s . R e c r y s t a l l i z a t i o n of the product from methanol gave 3- (N-B0C-gly) -TAP pure by TLC c r i t e r i a . 39 In cont ras t to t h e i r recommended use in peptide s y n t h e s i s , aceto- n i t r i l e and nitromethane were unsui tab le fo r the synthes is o f TAP d e r i v a t i v e s due to the low s o l u b i l i t y o f hydra laz ine H C l . Incomplete d i s s o l u t i o n o f hydra laz ine HCl has an obv ious ly detr imental e f f e c t on TAP y i e l d s , and delayed or prolonged add i t ion o f the amine component to the reac t ion s o l u t i o n encourages compet i t ion o f rearrangement and s i d e - r e a c t i o n s . 5 6 N,N-Dimethylformamide was used as a so lvent medium with some reserva t ions in view of i t s reported u n s u i t a b i l i t y f o r carboxamide formation with NEPIS 5 6 and the i n s t a b i l i t y o f hydra laz ine in DMF s o l u t i o n . Water and ethanol are not recommended as so lvents because they react with the keten- imine intermediate {18). Low coupl ing y i e l d s in dioxane, tetrahydrofuran (THF) and ethyl acetate are a t t r i b u t e d to incomplete enol es ter format ion. Lowered y i e l d s are found with DMF and d imethy lsu l fox ide (DMSO) probably due to an increase in the rate o f ketoamide formation caused by the increased p o l a r i t y o f the s o l v e n t . Our r e s u l t s show that whereas the occurrence o f the s i d e - r e a c t i o n (20*21) d iscourages the use o f po lar s o l v e n t s , po lar so lvents are required to s o l u b i l i z e hydra laz ine H C l . Indeed, i t i s poss ib le that the y i e l d - l i m i t i n g f a c t o r for TAP formation in DMF s o l u t i o n i s the rearrangement 20-+21. No extensive e f f o r t s were made to optimize the reac t ion c o n d i t i o n s , and our maximum observed TAP y i e l d o f 73% does not represent an optimum va lue . C e r t a i n l y , the y i e l d should be improved with a mixed CH3CN/DMF system by decreasing the extent o f competing 0->N acyl m igra t ion . S u b s t i t u t i o n o f DMF with dimethylacetamide (DMA) should decrease the extent o f hydralaz ine decomposit ion in s o l u t i o n ( c f . Chapter 5 ) . Another means o f improving the coupl ing reac t ion i s to use an isoxazol ium s a l t with l e s s tendency to undergo rearrangement o f the enol 40 e s t e r . Woodward and Woodman showed that s u b s t i t u t i n g the N-a lky l s u b s t i - tuent with a b u l k i e r group slowed down the rea r rangement . 5 9 They succeeded in b locking the enol es te r rearrangement completely in N - t - b u t y l - 5 - m e t h y l - isoxazol ium perch lora te (25) which bears the bulky N- t -buty l group. (25) Unfor tunate ly , the i s o l a b l e t - b u t y l ketenimine (26) der ived from t h i s ca t ion was so unreact ive that i t d id not e a s i l y y i e l d enol es ters with peptide a c i d s ; the a c t i v a t i o n step was so slow that s ide react ions became s i g n i f i c a n t . 6 0 * 6 1 (26) 2.4.2 The EEDQ Coupling Reagent \ l - E t h o x y c a r b o n y l - 2 - e t h o x y - l , 2 - d i h y d r o q u i n o l i n e (27, EEDQ) was o r i g i n a l l y developed by Bel leau and his a s s o c i a t e s 6 2 as a depressor of the cent ra l nervous system. EEDQ was a l s o shown to be an e f f i c i e n t and s e l e c t i v e coupl ing reagent . Be l leau and Malek proposed the mechanism for amide bond formation shown in Scheme 2 . 4 . 6 3 41 I R 7 N H 2 O EtOH + C 0 2 + R - C - N H - R ' Scheme 2.4 Mechanism of Amide Formation with EEDQ 42 The c a r b o x y l i c a c i d replaces the ethoxy group of EEDQ to form an intermediate (28) which i s converted to a mixed carbonic anhydride in situ. This mixed anhydride (30) reacts with amine nuc leophi les to form the amide compound. The by-products of the reac t ion ( q u i n o l i n e , e t h a n o l , and carbon d ioxide) are reported to be r e a d i l y removed by f l a s h evaporat ion of the r e a c t i o n mixture . The r e a c t i o n may be c a r r i e d out in so lvents such as benzene, absolute e t h a n o l , or te t rahydro furan . An a t t r a c t i v e fea ture of t h i s method i s the promise of easy i s o l a t i o n of the des i red product . T r i a l s with EEDQ were conducted with N - a c e t y l - g l y c i n e and hydra laz ine in methanol s o l u t i o n . UV spect ra l a n a l y s i s of the r e a c t i o n mixture showed very l i t t l e of the TAP product when the r e a c t i o n was allowed to proceed at room temperature f o r up to three days. The presence of qu ino l ine i n ' t h e r e a c t i o n mixture ind ica ted that the mixed carbonic anhydride had been formed, but apparent ly much of the a c t i v e intermediate was l o s t through h y d r o l y s i s with water present in the s o l v e n t . Other t r i a l s in a c e t o n i t r i l e s o l u t i o n were only m i l d l y s u c c e s s f u l due to the spar ing s o l u b i l i t y of N - a c e t y l - g l y c i n e in a c e t o n i t r i l e and the tendency of hydra laz ine^to p r e c i p i t a t e from s o l u t i o n when EEDQ was added. In s p i t e of these problems, the y i e l d of 3 - ( N - a c e t y l - g l y c y l ) - T A P was higher in a c e t o n i t r i l e than in methanol. Using N - a c e t y l - L - a l a n i n e , which was more s o l u b l e in a c e t o n i t r i l e than the g l y c i n e d e r i v a t i v e , formation of 3 - ( N - a c e t y l - L - a l a n y l ) - T A P was much improved fo r overnight reac t ions at room temperature. Although some hydra laz ine s t i l l p r e c i p i t a t e d from s o l u t i o n , the tendency to do so was much less than with N - a c e t y l g l y c i n e , notwithstanding the use of more concentrated s o l u t i o n s with the a lan ine d e r i v a t i v e . Coupl ing reac t ions with EEDQ in THF or DMSO s o l u t i o n r e s u l t e d in l i t t l e or no TAP formed. Good amide formation was e f fec ted with N-BOC-L- 43 a lan ine and hydra laz ine in methylene c h l o r i d e s o l u t i o n . The product could be i s o l a t e d by e x t r a c t i n g the methylene c h l o r i d e so lu t ion with d i l u t e aqueous ac id and base. 2 .4 .3 Acyloxyphosphonium Cations Acyloxyphosphonium d e r i v a t i v e s [zo) are very suscept ib le to n u c l e o p h i l i c at tack at the carbonyl carbon atom [Scheme 2 .5 ) , and such species feature as intermediates in several r e c e n t l y developed procedures f o r amide bond format ion. 0 . 0 . R'NH 2 0 II + n + Z II + R-C-0" + R 3 P-X ^ . R - C - 0 - P R 3 - R - C - N H R ' + "0 -PR 3 (30) Scheme 2.5 Amide Formation v i a an Acyloxyphosphonium Intermediate Much e f f o r t has been expended in developing acyloxyphosphonium s a l t s as v i a b l e a c y l a t i n g agents in peptide synthes is with encouraging s u c c e s s , and several methods show promise f o r p r a c t i c a l use in peptide s y n t h e s i s . 5 k Phosphorus-containing reagents were considered f o r the fo l low ing reasons: 1. Reaction condit ions, are genera l l y mi ld and s u i t a b l e f o r ' e i n t o p f ver fahren ' o f TAP d e r i v a t i v e s . 2. Reagents are inexpensive and r e a d i l y a c c e s s i b l e . . 3. The reagents, were deyeloped f o r peptide synthes is and l i t t l e i s known about t h e i r more general a p p l i c a b i l i t y in carboxamide s y n t h e s i s . 44 In 1969, Kenner and a s s o c i a t e s reported on the resonance- s t a b i l i z e d acyloxyphosphonium s a l t s der ived from hexamethylphosphoramide (31, HMPA) (the Kenner-Sheppard Reaction) . 6 l * » 6 5 They showed amide formation according to the sequence in Scheme 2 .6 . + (Me 2 N) 3 P0 + TsCl * [ ( M e ^ P - O T s j C l " (31) (32) + + [•(Me 2 N) 3 P-0-P(NMe 2 ) 3 3TsO"CV (33) 1RC00H 0 •i + [RC-0-P(NMe 2 ) 3 ]Cl" (OTs") (34) R'NH 2 RC-NHR' + (Me 2N) 3P0 T s - = M e ^ ~ y S 0 , Scheme 2.6 Amide Formation v i a an Acyloxydimethylaminophosphonium S a l t The a d d i t i o n of tosy l c h l o r i d e to excess HMPA y i e l d s the d i c a t i o n (33), presumably by way of the i n i t i a l adduc t .(32) . The d i c a t i o n (33) i s r e l a t i v e l y s t a b l e , and the c r y s t a l l i n e d i - t e t r a f l u o r o b o r a t e s a l t has been used as a coupl ing reagent in peptide s y n t h e s i s . 6 6 Two routes can be v i s u a l i z e d f o r the a c y l a t i o n s tep . Th is step can proceed e i t h e r by d i r e c t a t tack of the amino component at the a c t i v a t e d carboxyl group with anchimeric ass is tance by a hydrogen-bonded c y c l i c t r a n s i t i o n s ta te (35) or v ia the add i t ion of the amino component to the 45 eg H_N:3 yc ...2 R 1/ H-N (35) •NMe, 0" I R ~ , C - 0 W N M e 2 7 W ^ Me, phosphorus atom of 34 to form 55, which then decomposes to the amide and 0 II R-C-0x R'-I/ I H P(NMe 9 ) 2'3 (36) HMPA in a manner not d i s s i m i l a r to the Wi t t ig r e a c t i o n . 5 5 A c t i v a t i o n of HMPA can a l s o be achieved with t o s i c anhydride or th iony l c h l o r i d e in place of t o s y l c h l o r i d e . In a t r i a l with HMPA and tosy l c h l o r i d e , we used N - a c e t y l - g l y c i n e as the carboxyl component and hydra laz ine HCl with Et^N as the amine component with reac t ion at room temperature. TAP was detected in the r e a c - t i o n mixture w i th in two hours , but a f t e r overnight r e a c t i o n , there s t i l l remained a f a i r amount of undissolved hydra laz ine HCl . E f f o r t s at i s o l a t i n g the TAP product were u n s u c c e s s f u l . Ex t rac t ion of the HMPA s o l u t i o n with benzene, petroleum^ether, d ie thy l e t h e r , and methylene c h l o r i d e was u n s a t i s f a c t o r y because of the high s o l u b i l i t y of 3<-.(N-acetyl-glycyl)-TAP in HMPA. The high b o i l i n g point of HMPA (^290°C) precluded removal of so lvent by d i s t i l l a t i o n . 46 No f u r t h e r work was done with th is system because of the problems of i s o l a t i n g the product from HMPA s o l u t i o n . A lso d iscouraging any recon- s i d e r a t i o n of t h i s method f o r TAP s y n t h e s i s , was a repor t that HMPA is a potent ia l c a r c i n o g e n . 6 7 Since the Kenner-Sheppard method proved to be u n s a t i s f a c t o r y f o r TAP s y n t h e s i s , we considered an a l t e r n a t i v e means of generat ing acy loxy - phosphonium in termedia tes , i . e . the coupl ing reagent , a z i d o - t r i s ( d i m e t h y l - amino)phosphonium hexafluorophosphate ( 3 7 ) . 6 8 + [ ( M e 2 N ) 3 P - N 3 ] P F 6 " (37) In so lu t ion ,37 reacts r a p i d l y at -10°C with triethylammonium s a l t s of c a r b o x y l i c ac ids to give the acyl azide (39) v ia an acy loxy - phosphonium azide (38) which i s not i s o l a b l e . The a c t i v e intermediate reacts with amines to form the carboxamide (Scheme 2 .7 ) . 0 [ ( M e 2 N ) 3 P - N 3 ] P F 6 + RCOOH + E t 3 N " 1 Q ° C > [R -C -0 -P (NMe 2 ) 3 ]N 3 + [Et 3 NH]PFg (37) (38) \ 0 R-N=C=0 + N 2 [ R - C - N 3 ] + (Me 2 N) 3 P0 (40) (39) R'NH 2 - 1 0 ° 0 R'NH 2 R-NH-CO-NH-R' RC-NHR1 (41) Scheme 2.7 Amide Formation v i a an Acyl Azide 47 At higher temperatures, the acyl az ide may undergo the Curt ius rearrangent to the isocyanate (40) which w i l l condense with the amine component to g ive the urea (41). Our e f f o r t s with the coupl ing reagent (37) involved reac t ion o f N-BOC-glycine with hydra laz ine at -15°C in DMF s o l u t i o n . A f t e r one hour, TLC a n a l y s i s o f the r e a c t i o n mixture ind ica ted only a l i t t l e TAP present , and UV spectra showed mainly unreacted hydralaz ine in s o l u t i o n . Overnight r e a c t i o n at room temperature d id not s i g n i f i c a n t l y increase the y i e l d s o f TAP. Coupl ing reac t ions in Methyl C e l l o s o l v e s o l u t i o n under the same cond i t ions a lso gave low y i e l d s of 3 - (N -B0C-g lycy l ) -TAP. The coupl ing reac t ion with [(Me2N) 3P-N2]PFg was not pursued fu r ther because o f the low TAP y i e l d s and d i f f i c u l t i e s in mainta in ing low temperatures to minimize the Curt ius rearrangement. Another coupl ing method used was the s o - c a l l e d "ox ida t ion - reduct ion condensat ion" r e a c t i o n o f Mukaiyama and c o - w o r k e r s , 6 9 ' 7 0 i n which the dehydrat ion reac t ion involved in amide synthes is i s coupled with ox ida t ion o f an ary l phosphine and reduct ion of a d i s u l f i d e (Scheme 2 .8 ) . (45a) (45b) Scheme 2.8 Amide Formation v ia an Acyloxytriphenylphosphonium Sa l t 48 The e f f e c t i v e n e s s of t h i s combination of oxidant and reductant can be envisaged as a r i s i n g from formation of an acyloxyphosphonium t h i o l a t e (44) as the a c t i v e a c y l a t i n g in termediate . Th is s a l t , by v i r t u e of the e l e c t r o n withdrawing proper t ies of the phosphonium e n t i t y , reac ts r a p i d l y with incoming amino components to a f f o r d amides, t r iphenylphosphine oxide and the thione as depicted below. o o / ° - c - R #T "CNH-R1 A f 3 P \ R'-NH, A'3P /H R-CO-NH-R1 + Ar 3P=0 + ( f ^ N S H A dec id ing f a c t o r in using 2 , 2 ' - d i t h i o d i p y r i d i n e (42, 2-DTP) as the oxidant i s the isomer iza t ion in s o l u t i o n of the hydrogenated product (45a) to the thione form (45b). In the absence of t h i s i s o m e r i z a t i o n , the t h i o l formed would reac t with the acyloxyphosphonium s a l t intermediate to g ive undesi rab le s ide products thus n e c e s s i t a t i n g the add i t ion of a t h i o l scavenger to the r e a c t i o n mixture . A coupl ing r e a c t i o n with the Mukaiyama procedure was attempted using N - a c e t y l - g l y c i n e and hydra laz ine HC1 (+ Et 3 N) in dioxane s o l u t i o n . A f t e r overnight r e a c t i o n at 4 0 ° C , UV spec t ra l and TLC a n a l y s i s of the r e a c t i o n s o l u t i o n showed only a l i t t l e of the TAP d e r i v a t i v e in s o l u t i o n . A s o l i d p r e c i p i t a t e during the r e a c t i o n showed Et^N.HCl and a decomposit ion product of h y d r a l a z i n e . 49 The o x i d a t i o n - r e d u c t i o n condensation r e a c t i o n showed l i t t l e promise f o r our purposes although the y i e l d of TAP would probably be improved by conducting the r e a c t i o n in a so lvent where hydra laz ine decom- p o s i t i o n is l ess pronounced. No fu r ther ac t ion was taken with t h i s system because more encouraging r e s u l t s were being obtained elsewhere. considered the method developed by Mi t i n 7 1 fo r the one-step synthes is of peptides from s u i t a b l y protected amino ac ids (Scheme 2 .9 ) . Continuing our e f f o r t s with phosphorus-containing reagents , we P(PhO) 3 + H N ^ N ( P h O ) 2 P - N ^ N + PhOH (46) RCOOH 0 0 ^ ' R - C - N 0 N H (PhO) 2 P-0" (47b) II PhOH R-C-OPh (48) 0 n ^ R C - N H - R ' Scheme 2.9 Amide Formation v i a an Acyl imidazol ium S a l t On the bas is of i n d i r e c t data o n l y , M i t i n et a l . 7 2 assumed the r e a c t i o n mechanism shown. The r e a c t i o n sequence i s considered to c o n s i s t of i n i t i a l formation of imidazo ly l d iphenylphosphi te (46), which on r e a c t i o n 50 with the carboxyl component gives the a c t i v a t e d compound of the l a t t e r (47a=^±: 47b). The acy l imidazo l ium intermediate (47b) reacts with the amino component to a f fo rd the carboxamide. In the absence of the amine component, the a c t i v e intermediate (47b) reacts with the phenol formed at the f i r s t stage to give the phenyl e s t e r o f the c a r b o x y l i c a c i d . Our f i r s t experiment with the Mi t in procedure involved the coupl ing of N-BOC-glycine with hydra laz ine HCl suspended in dioxane with Et^N added to n e u t r a l i z e the s a l t . When the react ion was c a r r i e d out in the presence of t r ipheny l phosphite and imidazole f o r one day at 4 0 ° C , UV spect ra l a n a l y s i s of the reac t ion s o l u t i o n showed a high concentra t ion o f the des i red TAP product . Th is r e s u l t was most g r a t i f y i n g ; un fo r tuna te ly , a s i g n i f i c a n t amount o f hydra laz ine HCl remained und isso lved . The s i n g l e t r i a l with t h i s procedure showed more promise than any of the previous reac t ions i n v o l v i n g other phosphorus-containing reagents. We there fore set about to opt imize the cond i t ions fo r TAP format ion. The reac t ion of c a r b o x y l i c ac ids with t r i a r y l p h o s p h i t e s leads to the formation of ary l e s t e r s , but high temperatures ( 1 5 0 ° - 2 0 0 ° C ) are requ i red . In the presence of t e r t i a r y amines, however, the react ion proceeds r a p i d l y at room t e m p e r a t u r e . 7 3 A s e r i e s o f coup l ing r e a c t i o n s were performed in which the base was v a r i e d . It became apparent that the i n i t i a l choice of bases in the exp lora tory experiment was the optimum one. The e f f i c i e n c y of bases in promoting TAP formation fo l lows the sequence: Imidazole + EtgN >> DBU, Imidazole , EtgN » Morphol ine , 2 , 4 , 6 - C o l l i d i n e , DABCO, t - B u 3 N , Proton Sponge where DBU = 1 , 5 - d i a z a b i c y c l o [ 5 . 4 . 0 ] u n d e c - 5 - e n e DABCO = 1 , 4 - d i a z a b i c y c l o [ 2 . 2 . 2 ] o c t a n e Proton Sponge = 1 ,8-bis(dimethylamino)naphthalene J 51 One conc lus ion from the above sequence i s that i t i s not based on base st rength a l o n e , s ince the s t rong ly basic Proton Sponge (pK 12.37) a i s i n e f f e c t i v e in the coupl ing r e a c t i o n , w h i l s t imidazole (pK 6.95) in a combination with t r i e t h y l amine (pK 11.0 ) i s s a t i s f a c t o r y . a Coupling reac t ions were performed at ambient temperature ( ^ 2 3 ° C ) , 40°C and 70°C to determine the e f f e c t of temperature on the y i e l d of TAP. Not s u r p r i s i n g l y , higher y i e l d s of TAP were obtained at the higher tempera- t u r e s . However, some deblocking of the BOC-group from 3-(N-B0C-gly)TAP was observed at 7 0 ° C . The e f f i c i e n c y of TAP formation was h igh ly so lvent dependent. A s e r i e s of experiments was conducted with var ious so lvents using imidazole and t r ipheny lphosphi te as the carboxyl a c t i v a t i n g agents, and EtgN to n e u t r a l i z e the hydra laz ine HC1. The most s u i t a b l e of the so lvents t r i e d was DMF. A c e t o n i t r i l e , THF, and dioxane gave good to moderate y i e l d s of TAP with d i s s o l v e d h y d r a l a z i n e , but were l im i ted because of incomplete s o l u t i o n of hydra laz ine HCI. even a f t e r one day 's r e a c t i o n . Dimethyl - s u l f o x i d e , water and Methyl C e l l o s o l v e were unsui tab le even though hydra- laz ine HCI d i s s o l v e d completely in the s o l v e n t s . An expected consequence of slow or incomplete s o l u t i o n of hydralaz ine HCI is formation of the phenyl es ter {48) of the N-acylamino a c i d . In f a c t , when the amino component i s absent , t h i s procedure may be u t i l i z e d f o r the synthes is of phenyl e s t e r s . 7 2 Thus, in those so lvents where re lease of hydra laz ine in to s o l u t i o n i s slow, a c y l a t i o n of hydra laz ine v ia the a c t i v e phenyl es te r should become more prominent. In the coupl ing reac t ion of ca rboxy l i c ac ids with amines, promoted by t r ipheny lphosphi te in the presence of im idazo le , M i t in et al.71*72 reported the by-products of the r e a c t i o n to be diphenylphosphi te and phenol . 52 They d id not consider the p o s s i b i l i t y of fu r ther coupl ing reac t ions with the d iphenylphosphi te . Yamazaki and H i g a s h i 7 4 re-examined the reac t ion with t r i p h e n y l - phosphite and determined the e f f e c t of var ious t e r t i a r y bases on the synthesis of an amide. In agreement with our r e s u l t s , they found that imidazole was s i g n i f i c a n t l y more e f f e c t i v e than other bases. An unexpected r e s u l t was the r e a c t i o n with d ipheny lphosphi te , in which p y r i d i n e was the super ior base. As a consequence, Yamazaki and Higashi explored the use of d iphenylphosphi te and p y r i d i n e f o r peptide s y n t h e s i s . 7 k They showed that the coupl ing reac t ion proceeds as shown in Scheme 2.10 H0-P(0Ph) 9 n 2 0 ^|\| n l N u 0 0 u > R-C-0-P-H "OPh R-C-IMHR" + H-'P-OPh + PhOH py / \ i HO OPh OH H-P(0Ph) 9 II <- 0 (49) \\ (H0) 2 P-0Ph Scheme 2.10 Amide Formation v i a an Acyloxyphosphonium S a l t from Diphenylphosphite The acyloxyphosphonium s a l t of py r id ine (49) i s formed f i r s t , with re lease of a- phenolate anion from the phosphi te . In the presence of an amino component, t h i s a c t i v e ingred ient reacts r e a d i l y to form the amide d e r i v a t i v e . When no amino component i s present the phenyl es ter of the • c a r b o x y l i c ac id i s formed by in teramolecular reac t ion with the phenolate anion re leased from the phosphorus atom. If t r ipheny l phosphite i s used in the r e a c t i o n , then only h a l f an equiva lent amount i s r e q u i r e d , s ince the pyr id in ium s a l t of d i p h e n y l - phosphite (50) i s formed which can undergo fu r ther r e a c t i o n (Scheme 2.11) . 53 "OPh + ( + + py II i \t P(OPh) 3 + RCOOH ^ R - C - O - P - H P H ° M ° P H RS-NHR'^+ PhOH P h 0 ' ° P h "OPh O-P-H hO Ol (50) RCOOH ^ R'NH 2 0 0 II it (50) ^ R - C - O - P - H — R C - N H R 1 + PhOH + H-P-OPh HO OPh OH Scheme 2.11 Amide Formation v ia Acyloxyphosphonium S a l t s from Tr iphenylphosphi te The r e a c t i o n mechanism proposed by Yamazaki and Higashi f o r the coupl ing r e a c t i o n with phosphites i s at var iance with that of M i t i n , which assumes that the a c t i v e intermediate i s an acy l imidazo l ium s a l t . It would be i n t e r e s t i n g to t e s t these assumptions to determine which i s the actual a c t i v e a c y l a t i n g agent: acyloxyphosphonium s a l t or acy l imidazol ium s a l t . To complete our i n v e s t i g a t i o n s with phosphorus-conta in ing reagents , we attempted a coupl ing r e a c t i o n between N-BOC-alanine and hydra laz ine HCI with diphenyl phosphite in p y r i d i n e suspens ion , f o l l o w i n g the procedure of Yamazaki and H i g a s h i . 7 4 A f t e r two days , the reac t ion s o l u t i o n showed no t race of the des i red 3- (N-B0C-alany1) -TAP. The f a i l u r e of t h i s r e a c t i o n was a t t r i b u t e d p a r t l y to the i n e f f e c t i v e n e s s of py r id ine in n e u t r a l i z i n g hydra laz ine HCI. No f u r t h e r t r i a l s were attempted under more e f f e c t i v e c o n d i t i o n s . 2.4.4 Carbodiimides In 1955, Sheehan and H e s s , 7 5 and K h o r a n a 7 6 working independently showed that s u i t a b l y blocked amino ac ids could be jo ined through an amide l inkage under the in f luence of d icyc lohexy lcarbod i im ide (51, R = C f i H 1 1 , DCC). 54 Since that t ime, DCC has become perhaps the most useful and popular coupl ing reagent in peptide s y n t h e s i s , and i t has a lso gained great importance as a v e r s a t i l e reagent in organic s y n t h e s i s . 7 7 The accepted mechanism f o r amide bond formation i s shown in Scheme 2 . 1 2 . 7 8 The f i r s t step of the reac t ion sequence invo lves the add i t ion of the carboxyl component to a carbodi imide to form an 0 - a c y l - isourea (52) which i s h igh ly r e a c t i v e . If the external nuc leoph i le i s d i l a t o r y then in te rna l n u c l e o p h i l i c at tack may occur to produce an N-acylurea (53). For amide fo rmat ion , two pathways are p o s s i b l e : a . D i r e c t at tack of the amino component on the r e a c t i v e intermediate with formation of the amide bond. b. Attack by the carboxyl component to g ive a symmetrical anhydride (54) which in turn acy la tes the amine. Delayed a d d i t i o n of the amine component decreases the importance of path (a) and increases the extent of anhydride p a r t i c i p a t i o n . 7 9 - 8 1 In cont ras t to many other methods f o r carboxyl a c t i v a t i o n , the carbodi imide reac t ion i s reported to be r e l a t i v e l y i n s e n s i t i v e to moisture . The coupl ing r e a c t i o n may even be c a r r i e d out in aqueous s o l u t i o n , a l b e i t with lowered y i e l d . The by-product of the r e a c t i o n , d icyc lohexy lurea ( 5 5 , R=CgH-j-|, DCU), has low s o l u b i l i t y in most organic and aqueous s o l v e n t s , and i s u s u a l l y removed by f i l t r a t i o n . However, the d i s s o l v e d DCU i s sometimes troublesome to remove, and to overcome the problems of separat ing the by -product , the water -so lub le ca rbod i im ides , l - e t h y l - 3 - ( 3 - d i m e t h y l - aminopropyl) carbodi imide h y d r o c h l o r i d e , (16, EDC) and 1 - c y c l o h e x y l - 3 - (2 -morpho l iny l -4 -e thy l )ca rbod i im ide metho p - to luenesu l fona te (56, CMC) may be u s e d . 5 1 > 8 2 The corresponding urea and acylurea d e r i v a t i v e s are 55 R'COOH + R-N=C=N-R (5J) RHN-C-NHR RHN-C-NHR C55) C55) Scheme 2.12 Mechanisms of Amide Formation with a Carbodiimide 56 •Et-r =C=N-(CH2)3NHMe2 (16) "CI O - N=C=N-(CH 2) 2N 0 "OTs (56) Me usua l ly s o l u b l e in water and may be removed by e x t r a c t i o n with d i l u t e ac id or water. The extent of the carbodi imide-mediated coupl ing of hydra laz ine with N-protected amino ac ids was determined from UV spec t ra and TLC of the reac t ion mix tures , u s u a l l y a f t e r overnight r e a c t i o n . F igure 2.1 shows the UV spect ra of equimolar amounts of 3-Me-TAP and h y d r a l a z i n e , and a mixture of the two i n g r e d i e n t s . The spectrum (c) shows a spectrum of a r e a c t i o n mixture in which hydra laz ine has been h a l f converted to the TAP d e r i v a t i v e . I f no other components in the r e a c t i o n mixture show s i g n i f i c a n t UV absorp- t ions in the useful wavelength r e g i o n , then the i n t e n s i t y of the most in tense TAP band (^230-240 nm) r e l a t i v e to those of hydra laz ine (^270 nm) can be used as a q u a l i t a t i v e guide to the r e l a t i v e proport ions of TAP and hydra laz ine in the r e a c t i o n mixture. In g e n e r a l , the components of the carbodi imide coupl ing r e a c t i o n show l i t t l e in te r fe rences in the UV region of i n t e r e s t . The only s i g n i f i c a n t in te r fe rences in the 250-290 nm region a r i s e from degradation products o f hydra laz ine . These decomposit ion products have higher molar a b s o r p t i v i t i e s than hydra laz ine i t s e l f and can make the coupl ing r e a c t i o n appear l e s s complete than i t a c t u a l l y i s , the extent depending on the degree of hydra laz ine decomposit ion. The f i r s t s e r i e s of experiments with the carbodi imide method involved a comparison of coupl ing react ions run under i d e n t i c a l cond i t ions 57 2 0 0 2 5 0 3 0 0 3 5 0 Wavelength (nm) Figure 2.1 U l t r a v i o l e t Spectra of Equimolar Amounts of (a) H y d r a l a z i n e , (b) 3-Me-TAP, and (c) a Mixture i n " H O S o l u t i o n 58 (hydra laz ine : N-BOC-glycine : DCC = 1 : 1 : 1; 0.05M) except f o r the solvent used. S ix solvents were compared under these c o n d i t i o n s . Good y i e l d s of TAP were obtained when C H 2 C 1 2 , CHC1 3 , CH 3 CN, and THF were used. Methyl C e l l o s o l v e , d ioxane, and DMF gave poor y i e l d s of TAP. Other e x p e r i - ments with N - a c e t y l - g l y c i n e as the carboxyl component showed methanol to be capable of mediating good y i e l d s of TAP. Water on the other hand, gave poor r e s u l t s , e s p e c i a l l y under bas ic c o n d i t i o n s , because of the ox ida t ion of hydra laz ine to phthalaz ine which was detected in the r e a c t i o n mixture . Reaction in DMA s o l u t i o n gave a moderate y i e l d of TAP. These r e s u l t s are c o n s i s t e n t with the observat ions of Sheehan et a Z . 7 5 > 8 3 who found that rearrangement of the O-acy l isourea intermediate (52) to the i n a c t i v e N-acylurea {53) i s suppressed in solvents l i k e methyl - ene c h l o r i d e and a c e t o n i t r i l e . K h o r a n a 7 6 showed that when d ioxane, t e t r a - hydrofuran, and chloroform were solvents in peptide s y n t h e s i s , N-acylurea was always obta ined. I s d e b s k i , 8 4 in a study of the formation of N-acylurea in the r e a c t i o n of N -benzy l - l euc ine with g lyc ine ethyl e s t e r , found that reac t ion in DMF gave 22% of the s ide -product whi le r e a c t i o n in THF and C H g C l 2 gave 4 and 2% N-acy lurea , r e s p e c t i v e l y . In the synthesis o f TAP d e r i v a t i v e s , we a l s o i s o l a t e d s i g n i f i c a n t amounts of the N-acylurea from C H g C ^ s o l u t i o n . Two sets of comparisons were made to determine the e f f e c t of e levated temperature on the formation of TAP. The coupl ing of hydra laz ine HCI with N-BOC-glycine using DCC in methanol s o l u t i o n in the r a t i o 1 : 1 : 1 and 1 : 5 : 5 r e s p e c t i v e l y were studied at ambient and a t r e f l u x temperatures. The comparisons ind ica ted tha^t there were l i t t l e advantages to be gained from pursuing the carbodi imide reac t ion at high temperature. Indeed, the accepted p r a c t i c e i s to i n i t i a l l y lower the temperature to 0°C in order to minimize N-acylurea f o r m a t i o n . 8 5 When hydra laz ine HCI in the presence of 59 Et-jN i s the amine component in so lvents where the s a l t has only l im i ted s o l u b i l i t y , there i s a s i g n i f i c a n t increase in the rate of TAP formation at higher temperature, presumably because o f increased s o l u b i l i t y of hydra laz ine HCl . To compensate f o r the greater p r o b a b i l i t y of N-acylurea formation at e levated temperature an excess of c a r b o x y l i c a c i d and carbodi imide must be used. Another danger assoc ia ted with e levated temperatures i s the increased decomposit ion of f ree base hydra laz ine . In view of the low s o l u b i l i t y of hydra laz ine HCl in most organic s o l v e n t s , we wished to determine the e f f e c t of varying the proport ion of Et^M in s o l u t i o n . The f i r s t s e r i e s of comparisons involved a 1 : 1 : 1 reac t ion mixture of hydra laz ine H C l , N -BOC-g lyc ine , and EDC in methanol, with e i t h e r 0, 1, 1.3, 1.6, 3 .4 , 5 .3 , 16, or 50 equiva lents of E t 3 N added to the r e a c t i o n mixture . Coupling reac t ions conta in ing more than 5 equiva lents of Et^N showed l i t t l e evidence of TAP formati 'on. I n h i b i t i o n of the reac t ion by excess Et^N a r i s e s in several ways. In the mechanism f o r reac t ion of c a r b o x y l i c ac ids with carbodi imides (Scheme 2 .13 ) , the f i r s t step involves formation of the protonated carbodi imide (51a) which i s added to a H + + R'COO" R_N=C=N-R ' R-NH=C=N-R R-NH-ON-R R'COO (51) (51a) (52) + j. 0 0 , % H + R'COO" (52) „ R-NH-C=NHR - R-C-0-C-R + R-NH-C-NH-R R'COO 0 (52a) (54) (55) Scheme 2.13 Reaction of Carboxy l ic Acids with Carbodi imides 60 carboxylate anion to form the 0 - a c y l i s o u r e a (52). In the presence of excess a c i d , a s i m i l a r protonat ion e q u i l i b r i u m fs set up, and the protonated species (52a) reacts f u r t h e r with carboxylate to give an anhydride {54).78 When hydra laz ine HCI i s the amino component in the coupl ing r e a c t i o n , i t reacts opt ima l ly only when i t s amino group i s unprotonated. This requirement c o n f l i c t s with the i n i t i a l need to protonate both carbo- di imide and the 0 - a c y l i s o u r e a . As a r e s u l t , the y i e l d of the coupled product should be very pH-dependent, and add i t ion of a t e r t i a r y base w i l l reduce the concentra t ion of the protonated carbodi imide {51a) and the adduct (52a), and thereby reduce the rate of r e a c t i o n . Another consequence of excess t e r t i a r y base i s the i n c r e a s e d - y i e l d of N -acy lurea . For example, reac t ion of N-protected amino ac ids with DCC in the presence of py r id ine or t r i e t h y l amine may a f f o r d the N-acylurea as the main p r o d u c t . 8 5 * 8 7 We detected subs tan t i a l amounts of N-acylurea by-products in r e a c t i o n mixtures conta in ing excess t r i e t h y l amine. A d i s t r e s s i n g feature of reac t ions conducted in the presence of excess t r i e thy lamine i s the ready decomposit ion of hydra laz ine ca ta lyzed by the t e r t i a r y base. Th is decomposit ion i s charac te r i zed in UV spect ra by an absorpt ion band centered at 278 nm ( c f . Chapter 5 ) . Carbodiimide coupl ing reac t ions performed with 1.3 , 1.6, or 3.4 equiva lents of t r i e thy lamine showed good formation of TAP. In a coupl ing reac t ion of N - a c e t y l - g l y c i n e with hydra laz ine HCI and one equiva lent of t r i e thy lamine in methanol s o l u t i o n , unreacted hydra laz ine HCI was i s o l a t e d from the r e a c t i o n mixture a f t e r 2 days. Thus, in p r a c t i c e , an excess of t e r t i a r y base is needed when EDC i s the carbodi imide used. The e f f e c t of excess t r ie thy lamine on the y i e l d of TAP in carbodi imide-mediated reac t ions i s evident in F igure 2 .2 . urc 2.2 E f f e c t of Excess Tr ie thy lamine on Carbodiimide-mediated Coupling React ions . Hydralaz ine : ac id : Carbodiimide : Et (a) 1 :5:5:5 , (b) 1:5:5:1.3 Spectra normalized with respect to bands at 270 nm. 62 The reac t ion between hydra laz ine HCl and N - a c e t y l - g l y c i n e in methanol s o l u t i o n i s slower without added t r ie thy lamine but is unexpectedly f a c i l e cons ider ing that hydra laz ine is present in the protonated form. The rearrangement o f O-acy l isourea to the i n a c t i v e N-acylurea is always a danger in carbodi imide coupl ing r e a c t i o n s . A consequence of the rearrangement i s loss of the carboxyl component to the i n a c t i v e by-product and hence a lowering of the y i e l d of the des i red product . F igure 2.3 shows coupl ing reac t ions with s t o i c h i o m e t r i c and with excess amounts of the carboxyl component and carbodi imide . The s u p e r i o r i t y of the r e a c t i o n with excess reagents is ev ident . S i m i l a r r e s u l t s are a l s o obtained in comparison reac t ions with added t r i e t h y l a m i n e . Since the reac t ion of hydra laz ine with the N-acylamino a c i d - carbodi imide adduct (52) i s i n t e r m o l e c u l a r , whereas the rearrangement of the adduct to the N-acylurea (53) i s i n t r a m o l e c u l a r , keeping the volume of the s o l u t i o n to a minimum favors formation of the des i red TAP product . The concentra t ion e f f e c t i s evident in a comparison of coupl ing react ions at d i f f e r e n t concentrat ions (Figure 2 .4 ) . S ince hydra laz ine was of ten used as the hydrochlor ide s a l t in the carbodi imide coupl ing r e a c t i o n s , i t was i n t e r e s t i n g to compare the e f f e c t of d i f f e r e n t t e r t i a r y bases in the reac t ion mixture. Two coupl ing reac t ions were conducted under i d e n t i c a l condi t ions of reagent concentra t ion and s to ich iometry with Et^N or py r id ine as the added t e r t i a r y base. F igure 2.5 of UV spectra of the reac t ions a f t e r one day shows d e f i n i t e l y super io r TAP formation with the t r i e t h y l a m i n e - c o n t a i n i n g r e a c t i o n . Py r id ine i s a ra ther i n e f f e c t i v e base f o r n e u t r a l i z i n g the HCl s a l t s ince the s p e c t r a l fea tures at 300-320 nm in F igure 2.5(b) a r i s e s from protonated hydra laz ine . 63 Wavelength, (nm) Figure 2.3 E f f e c t of Excess Carboxyl Component and Carbodiimide on Carbodiimide Mediated Coupling React ions . Hydralaz ine : A c i d Carbodi imide , (a) 1:1:1 , (b) 1:5:5. Spectra normalized with respect to band at 240 nm. 64 2 5 0 3 0 0 Wavelength (nm) Figure 2.4 E f f e c t of Concentrat ion on Carbodi imide-mediated Coupling Reac t ions . Hydralazine.HCI : A c i d ; Carbodi imide : E U N = 1 :1 :1 :1 .5 . Concentrat ion (a) 0.016 M,. (b) 0.029 M. Spectra normalized with respect to band at 240 nm. 65 Figure 2.5 Comparison of Py r id ine and Tr ie thy lamine in Carbodi imide- Mediated Coupling React ions . Hydralazine.HCI : Ac id : Carbodi imide : Base = 1:1:1:1 . (a) Et^N, (b) P y r i d i n e . Reaction f o r 1 day. Spectra normalized with respect to band at 240 nm. 66 I 1— 2 5 0 3 0 0 Wavelength (nm) Figure 2.6 Comparison of Pyr id ine and Tr ie thy lamine in Carbodi imide- Coupling React ions . Hydralaz ine.HCl : Ac id : Carbodiimide : Base = 1 :1:1:1 . (a) Et^N, (b) P y r i d i n e . Spectra normalized with respect to band at 240 nm. Reaction f o r 4 hours. 67 The y i e l d of TAP a f t e r one day can be r e a d i l y expla ined by the r e l a t i v e base strengths of E t 3 N and p y r i d i n e , but the y i e l d a f t e r 2 hours of reac t ion can not . F igure 2.6 of the react ion a f t e r four hours shows a d i f f e r e n t order of r e a c t i v i t y . Reaction with added pyr id ine i s i n i t i a l l y f a s t e r than with E t 3 N . There i s l i t t l e change in the r e a c t i o n with pyr id ine between 4 and 24 hours w h i l s t the react ion with added E t 3 N shows a s lower , more even growth o f TAP. The d i f f e r e n c e in reac t ion rates suggests somewhat d i f f e r e n t reac t ion mechanisms in both cases . A poss ib le explanat ion of these observa- t ions i s formation of an acylpyr id in iurn d e r i v a t i v e from the 0 - a c y l i s o u r e a and pyr id ine which condenses with hydra laz ine to form the TAP product . There i s good evidence f o r the acety lpyr id in iurn ion as an intermediate in the p y r i d i n e - c a t a l y z e d h y d r o l y s i s o f a c e t i c a n h y d r i d e . 8 8 When the carboxyl component i s in excess r e l a t i v e to the amine component as in the case with the pyr id ine r e a c t i o n , in which hydra laz ine i s r e l a t i v e l y i n a c c e s s i b l e as the protonated base, then carbodi imide coupl ing may proceed v i a the mixed anhydride pathway (Scheme 2.12) . If the above specula t ions are c o r r e c t , then t h i s may be the f i r s t case , to the au thor 's knowledge, o f amide formation proceeding v i a an a c y l - pyr id ine in termedia te . A c y l a t i n g agents der ived from other bases such as imidazo le , p y r a z o l e , 1 , 2 , 4 - t r i a z o l e , and t e t r a z o l e are well known. 8 9 Since the subst i tuents on the carbodi imide molecule are expected to in f luence the r e a c t i v i t y of the 0 - a c y l i s o u r e a in termediate , we wanted to compare the s u i t a b i l i t y of some carbodi imides (DCC, EDC, and CMC) f o r TAP s y n t h e s i s . Three pa i rs o f comparison coupl ing react ions were made, each p a i r under i d e n t i c a l cond i t ions of reagent s to ichiometry and concen t ra t ion . In 68 the coupl ing o f hydra laz ine HCl with N - a c e t y l - g l y c i n e in methanol s o l u t i o n , EDC i s somewhat more e f f e c t i v e than CMC in TAP format ion . In the presence of t r i e t h y l a m i n e , the reac t ion with CMC i s much more s u s c e p t i b l e to s i d e - react ions than with EDC. This may a r i s e from increased N-acylurea formation with CMC. Faced with a d e f i c i e n c y of the a c t i v e O-acy l i sourea with which to c o u p l e , hydra laz ine showed greater tendency to undergo base-ca ta lyzed decomposit ion. Comparison react ions with DCC and EDC show that the l a t t e r carbodi imide gives super ior y i e l d s of TAP. The e f f i c i e n c y of the carbo- di imides in the formation of TAP d e r i v a t i v e s thus fo l low the order: EDC > DCC > CMC. A f a c t o r to be considered in the choice of carbodi imide is that DCC i s a l l e r g e n i c and should be handled with c a r e . Cases o f dermat i t i s have been ascr ibed to i t s use. There i s l i t t l e d i f f e r e n c e in the rate of formation of TAP whether hydra laz ine i s added to the r e a c t i o n mixture as the f ree base, or as the hydrochlor ide s a l t with one equiva lent of t r i e t h y l a m i n e . Thus, from that c o n s i d e r a t i o n , i t matters l i t t l e which form of hydra laz ine i s used in the r e a c t i o n - f r e e base or HCl s a l t with added Et^N. One caut ion with the l a t t e r system i s that a large excess of t e r t i a r y base cannot be t o l e r a t e d due to hydra laz ine decomposi t ion , but a s l i g h t excess of Et^i i s needed to ensure complete n e u t r a l i z a t i o n of the hydra laz ine HCl s a l t . An unusual fea ture of the carbodi imide coupl ing r e a c t i o n was observed when N - a c e t y l - g l y c i n e reacted with hydra laz ine in methanol or aqueous s o l u t i o n . In a d d i t i o n to the expected product , 3 - ( N - a c e t y l - g l y c y 1 ) - TAP, another product was i n v a r i a b l y formed which possessed t y p i c a l charac- t e r i s t i c s of a TAP d e r i v a t i v e : blue f luorescence under short wavelength 1 UV l i g h t , and s i m i l a r UV spectrum. The s ide -p roduc t was i d e n t i f i e d by i t s TLC behaviour , NMR and mass spectra to be 3-methyl-TAP. Its formation 69 appears to be favored by EDC r e l a t i v e to DCC and CMC. I t forms under a c i d i c and basic cond i t ions apparent ly simultaneous with 3 - ( N - a c e t y l - g l y c y l ) - T A P . Pure 3 - ( N - a c e t y l - g l y c y l ) - T A P i s i t s e l f i n d e f i n i t e l y s tab le i n methanol s o l u t i o n . The expected product from hydro lys is o f the s i d e - c h a i n amide bond i s 3-aminomethyl-TAP and no evidence o f th is compound was found in c a r b o d i - imide coupl ing r e a c t i o n s . Authent ic 3-aminomethyl-TAP appears to be s tab le in methanol s o l u t i o n . The fo l low ing summarizes the r e s u l t s obtained fo r the r e a c t i o n o f hydralaz ine with N-protected amino acids using carbodi imide coupl ing reagents . Appropr iate so lvents f o r the coup l ing r e a c t i o n are CH^Cl^ . CHCl^ , CH 3 CN, THF, CH30H, and DMA. The l e a s t po lar of these solvents are p re fe r red as r e a c t i o n media in order to minimize formation o f the N-acylurea s i d e - product . Low r e a c t i o n temperatures, high reagent concentrat ions and a s l i g h t excess o f carbodi imide and carboxyl component are advantageous f o r the same reason. I f the HCl s a l t o f hydralaz ine i s used as the amino component, then a s l i g h t excess o f a t e r t i a r y base such as Et^N i s needed. However, to avoid compl icat ions a r i s i n g from the presence o f excess t e r t i a r y base, f r e s h l y - prepared, f ree base hydralaz ine i s p r e f e r r e d . As f a r as coupl ing e f f i c i e n c i e s and ease o f i s o l a t i o n o f the TAP product are concerned, EDC i s more s u i t a b l e than DCC as the coupl ing reagent. 70 2.5 DISCUSSION We have shown that hydra laz ine w i l l couple with N-protected amino ac ids under a v a r i e t y of reac t ion cond i t ions to a f f o r d s - t r i a z o l o [ 3 , 4 - a ] - phthalaz ine d e r i v a t i v e s . The rate l i m i t i n g step of th is coupl ing i s forma- t ion of an amide intermediate which undergoes spontaneous dehydrat ive c y c l i z a t i o n to y i e l d the t r i a z o l o compound. We addressed ourse lves p r i m a r i l y to the problem of s u i t a b l y a c t i v a t i n g the r e a c t i o n components to give the des i red product . To lend the proper t ies of an a c y l a t i n g agent to an amino ac id or pept ide , the e l e c t r o p h i l i c character of the carbonyl carbon of the r e a c t i n g carboxyl group must be enhanced - usua l l y by s u b s t i t u t i o n of an e l e c t r o n - withdrawing group on the carboxyl f u n c t i o n : 0 Z-NH-CHR-C X 6+ <5- This s t r u c t u r a l m o d i f i c a t i o n of a carboxyl group must r a i s e i t s r e a c t i v i t y to the ' r i g h t ' l eve l so that coupl ings w i l l occur r a p i d l y and complete ly , without the in tervent ion of i n t e r - or i n t r a - m o l e c u l a r s i d e - r e a c t i o n s . O v e r a c t i v a t i o n may lead to r e a c t i o n with amino a c i d s i d e - c h a i n s , whi le low a c t i v a t i o n leads to low coupl ing r a t e s . The coupl ing reagents and t h e i r r e a c t i v e a c y l a t i n g intermediates which were used in t h i s work are shown in Table 2 .3 . The r e a c t i v e intermediates inc lude such types as mixed and symmetric anhydr ides, a c t i v a t e d e s t e r s , acyloxyphosphonium s a l t s , acyl a z i d e s , and h e t e r o c y c l i c amides. In mixed anhydr ides , the strong e lectron-wi thdrawing e f f e c t of the carboxyl group a c t i v a t e s the carbonyl carbon of the amino a c i d d e r i v a t i v e . 71 Table 2.3 Coupling Reagents and t h e i r Reactive Intermediates used in Amide Synthesis Coupl ing Reagent Isoxazolium S a l t N - e t h y l - 5 - p h e n y l i s o x a z o l i u m - 3 ' - su l fonate (NEPIS) 1-Ethoxycarbonyl -2-ethoxy- 1 ,2 -d ihydroquino l ine (EEDQ) I c=u I OEt Acyloxyphosphonium Sa l ts ( i ) [ ( C H 3 ) 2 N ] 3 P 0 + TsCl Hexamethylphosphoramide (HMPA) + tosy l c h l o r i d e + ( i i ) [ ( C H 3 ) 2 N ] 3 P - N 3 P F 6 " Az ido t r i s (d imethy l ami no)- phosphonium hexaf1uorophosphate ( i i i ) Ph^p + (fil fj Triphenylphosphine + 2 , 2 ' - d i t h i o d i p y r i d i n e Reactive Intermediate 0 0 0 R-C-0-C-OEt 2 • R - C - 0 - P [ N ( C H 3 ) 2 ] 3 TsO" ( C T ) 0 n R -C -N . v ia 0 % R - C - 0 - P [ N ( C H 3 ) 2 ] 3 N 3 ~ 72 Coupling Reagent Reactive Intermediate 0 N or ( iv ) (PhO) 3P + HN^N R - C - N ^ f Diphenylphosphite + py r id ine 4. Carbodiimide 0 Imid-H + Tr iphenylphosphi te + imidazole R-C-O-P-H OPh PhO OPh (v) (Ph0) 2 P-0H + ( [ ̂ ) R_C_0 -P-H "OPh HO* OPh 0 R'-N=C=N-R" R-C-0 or R'NH-C=NR" ( i ) R ^ R - ' - C g H , , 0 1 1 0 0 n n Dicyc lohexy lcarbodi imide (DCC) R-C-O-C-R + ( i i ) R ' = - C 2 H 5 , R"=- (CH 2 ) 3 NH(CH 3 ) 2 CI" l - E t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - carbodi imide hydrochlor ide (EDC) ( i i i ) R ' = - C 6 H i r R"=- (CH 2 ) 2 N / \ "OTs M e - 7 l - C y c l o h e x y l - 3 - ( 2 - m o r p h o l i n y l - 4 - e t h y l ) carbodi imide metho-p- to luenesul fonate (CMC) 73 However, there are two e l e c t r o p h i l i c s i t e s , and n u c l e o p h i l i c s u b s t i t u t i o n at 0 0 Z-NH-CHR-C-O-C-R' + t a b s i t e b w i l l r e s u l t in the undesired amide product . To minimize t h i s compet i t ion r e a c t i o n , an e lec t ron-donat ing s t ruc tu re i s requi red in the 'par tner ' a c i d . The ethoxy moiety in the mixed anhydride der ived from EEDQ f u l f i l s t h i s requirement. The isobuty loxy mixed anhydr ide , der ived from l - i s o b u t y l o x y c a r b o n y l - 2 - i s o b u t y T o x y - l , 2 - d i h y d r o q u i n o l i n e {57, IIDQ) has been suggested to be pre ferab le for reducing reac t ion at the a c t i v a t i n g group by 0-CH 2 CH(CH 3 )2 C=0 6-CH 2 CH(CH 3 ) 2 {57) v i r t u e of the s t e r i c hinderance of the isobuty l g r o u p . 9 0 In symmetrical anhydr ides , the s i t e s for n u c l e o p h i l i c s u b s t i t u t i o n are i d e n t i c a l , and only one amide product is p o s s i b l e . The es te r intermediates der ived from carbodi imides and NEPIS rece ive t h e i r a c t i v i t y from the e lectron-withdrawing nature of the m u l t i p l e - bonded C=N and C=C attached to the ester oxygen. The migrat ion of e l e c t r o n dens i ty from the carbonyl oxygen in v acyloxyphosphoniurn s a l t s i s enhanced by l o c a l i z a t i o n of a p o s i t i v e charge on the phosphorus atom. 74 Although the azide group is not a powerful e lectron-wi thdrawing group, the r e a c t i v i t y of acyl az ides towards amines can be explained by formation of a hydrogen-bonded c y c l i c t r a n s i t i o n state which enhances the a c y l a t i o n r e a c t i o n . 9 1 * 0 i % H-N-H. . .N i R' The high r e a c t i v i t y of N-acy l imidazole i s connected with the aromatic nature of the h e t e r o c y c l e . As a r e s u l t of p a r t i c i p a t i o n of the e l e c t r o n - p a i r on the amide n i t rogen in the i r-system of the r i n g , t h i s n i t rogen becomes more p o s i t i v e , exer t ing an a t t r a c t i o n on the e lec t rons of the e x o c y c l i c bond towards the r i n g , thus enhancing the rate of n u c l e o p h i l i c reac t ion at the carbonyl carbon of the acyl group. Of the ten reagents inves t iga ted f o r a c t i v a t i n g the carboxyl group of amino a c i d s , four of these were s u f f i c i e n t l y success fu l in TAP synthes is to meri t f u r t h e r c o n s i d e r a t i o n . These reagents are the carbodi imides - EDC and DCC, the isoxazol ium s a l t - NEPIS, and the combination of t r i p h e n y l - phosphite with imidazo le . As we a n t i c i p a t e d when t h i s work was i n i t i a t e d , the use of coupl ing reagents d id permit the synthes is of TAP d e r i v a t i v e s under r e l a t i v e l y mi ld c o n d i t i o n s . The r e a c t i o n of hydra laz ine with amino ac ids to a f f o r d TAP compounds i s s p e c i f i c f o r carboxyl groups, but some care must be taken when r e a c t i v e s i d e - c h a i n s in the amino ac ids are present . These s i d e - c h a i n s show 75 d i f f e r i n g s e n s i t i v i t i e s to coupl ing reagents. Thus, the carboxyl groups of s e r i n e , th reon ine , and ty ros ine can be ac t iva ted by NEPIS without previous protec t ion of the s i d e - c h a i n hydroxyl groups. Asparagine and glutamine can be ac t iva ted without ser ious p r o b l e m s ; 5 6 t h i s i s e s p e c i a l l y s i g n i f i c a n t s ince the co-amide group in these amino acids i s the only r e a c t i v e amino acid s ide -group ing which cannot o r d i n a r i l y be b locked. No problems are observed with h i s t i d i n e and arg in ine using the t r ipheny lphosphi te and imidazole combinat ion, but s ide react ions may sometimes occur with asparagine and glutamine. The hydroxyl groups of ser ine and threonine must be p r o t e c t e d . 7 1 When the carboxyl groups of N-protected asparagine or glutamine are ac t i va ted by DCC, dehydrat ion of the e- or y-carboxamide moiety to the corresponding cyano group occurs , asparagine being more s u s c e p t i b l e to the r e a c t i o n . Dehydration does not occur when these amino acids occur w i th in a peptide c h a i n . In a d d i t i o n , the formation of adducts between the imidazole r ing of h i s t i d i n e and DCC has been o b s e r v e d . 5 3 The successfu l use s e r i n e , th reon ine , and t y r o s i n e in peptide synthesis i s n o t e w o r t h y , 9 2 however, in aqueous s o l u t i o n s carbodi imides have been reported to react with t y r o s i n e and ser ine s i d e - c h a i n s in p r o t e i n s . 9 3 An unfortunate feature of the methods developed f o r coupl ing hydralaz ine to amino acids i s the long reac t ion times r e q u i r e d ; in per iods of l ess than 15 hours, coupl ing reac t ions were often incomplete. While times of t h i s durat ion are not unreasonable from a syn the t ic v iewpoint , they do impose l i m i t a t i o n s on the p r a c t i c a l i t y of the react ion of hydra laz ine with carboxyl groups as a method of peptide sequencing. We have i s o l a t e d pure TAP d e r i v a t i v e s from the coupl ing reac t ions in y i e l d s up to about 80% which are l ess than ideal f o r a peptide sequencing method though s a t i s f a c t o r y f o r a new syn the t i c procedure. Thus, assuming 76 80% coupl ing y i e l d in each cyc le of a sequencing procedure, the y i e l d of TAP a f t e r 5 cyc les i s 33%. Several modes of ac t ion are a v a i l a b l e f o r improving the hydra- l a z i n e coupl ing r e a c t i o n with amino a c i d s . The coupl ing methods used in t h i s work are based on an increase in the r e a c t i v i t y of the carboxyl group of the carboxyl component due to enhanced e l e c t r o p h i l i e p roper t ies of i t s carbonyl carbon. A counterpart of th is approach would be enhancement of the nuc leo- p h i l i c p roper t i es of the amino group o f hydra laz ine . This approach has been considered in peptide s y n t h e s i s , but only a few a p p l i c a t i o n s are known. Although the a v a i l a b l e methods do invo lve a r e a c t i v e d e r i v a t i v e of the amine, in the f i n a l a n a l y s i s , they proceed through an ac t i va ted carboxyl component. They a re , in f a c t , s p e c i a l cases of the mixed anhydride method. An example i s the phosphazo method which invo lves reac t ion of the amino component with PCI3 to g ive the phosphazo intermediate which i s present i n a d imer ic s t a t e , 5 2 (Scheme 2 .14 ) . The r e a c t i o n of the phosphazo compound with the 2 P C 1 3 + 4R ' -NH 2 Scheme 2.14 Amide Formation by the Phosphazo Method carboxyl component leads to formation of the amide product v i a a mixed anhydride. The method presents the same problems with regard to p o s s i b l e a p p l i c a t i o n s as d i r e c t mixed anhydride methods. R'-N R'-N / \ 4R"r00H y - N H - R ' 4R"C0-NHR' + [ P H O ^ P=N-R' 77 A less d i r e c t method of i n c r e a s i n g the r e a c t i v i t y of the primary amino group in hydra laz ine is s u b s t i t u t i o n of a Tr -e lectron donating group onto the phthalaz ine r i n g system. Considered by i t s e l f , the s u b s t i t u t i o n may have only a marginal e f f e c t on the r e a c t i v i t y of the hydrazino moiety , but i f s u i t a b l y p l a c e d , i t may a l s o enhance the coupl ing r e a c t i o n by r e s t r i c t i n g the r o t a t i o n a l freedom of the hydrazino group. Thus, placement of a group on the 8 - p o s i t i o n of the aromatic r i n g system (55) e . g . a lkoxy , d i r e c t s the hydrazino subst i tuent in to a favorab le o r i e n t a t i o n f o r coupl ing with carboxylates which i s fo l lowed by dehydrat ive c y c l i z a t i o n . The hydrazino group can be " f rozen" in to a s i n g l e conformation by hydrogen- bonding to the alkoxy oxygen atom. H I (58) Cohen has shown that as a r e s u l t of a lky l s u b s t i t u t i o n in both the aromatic r i n g and the s i d e - c h a i n (c f . 59), the rate constant f o r a c i d - ca ta lyzed l a c t o n i z a t i o n of hydrocoumaric ac id (60) i s increased by f a c t o r s as high as 5 x 1 0 ^ . 9 4 The r a t e - a c c e l e r a t i o n e f f e c t s are a t t r i b u t e d p r i m a r i l y to a cons iderab le increase in the populat ion of a conformer h igh ly favorab le to the l a c t o n i z a t i o n r e a c t i o n . (60) (59) 78 The procedures we have developed f o r the synthes is of s - t r i a z o l o - [3 ,4 -a ]phtha laz ine d e r i v a t i v e s from hydra laz ine and N-protected amino ac ids have potent ia l a p p l i c a t i o n s in the general synthesis of fused r i n g s - t r i a z o l e systems. The o v e r a l l procedures which we used in t h i s work fo r phthalaz ine are a p p l i c a b l e to other r i n g systems as well (Scheme 2 .15) . 0 0 NH-NH 2 + RC-X NH-NH-C-R ^ N N ^ ( « ) " < ^ N X = OH, OR, OCOR', Scheme 2.15 Synthesis of Fused s - T r i a z o l e s ha l ide If the carboxyl component i s a c a r b o x y l i c a c i d , r e a c t i o n cond i t ions involve heat ing a mixture of the a c i d and hydrazino components to high temperatures. If the ac id i s a s o l i d , melt cond i t ions may be r e q u i r e d . Reactions with other more r e a c t i v e carboxyl d e r i v a t i v e s may a lso involve e levated temperatures, but these are l ess common than with a c i d s , presumably because the d e r i v a t i v e s are genera l ly l e s s a c c e s s i b l e . As a r e s u l t of the severe reac t ion condi t ions and the low a v a i l a b i l i t y of a large v a r i e t y of carboxyl d e r i v a t i v e s , the s i d e - c h a i n subst i tuent in fused s - t r i a z o l e systems i s often l i m i t e d to groups such as a l k y l s , h a l o a l k y l s and a r y l s . Although we have not pursued the a p p l i c a b i l i t y of coupl ing reagents to hydrazino d e r i v a t i v e s of heterocycles other than ph tha laz ine , or to carboxyl components other than amino a c i d s , there i s no reason why the mild r e a c t i o n condi t ions made p o s s i b l e by our procedures should not al low the synthes is of a much wider v a r i e t y of fused s - t r i a z o l e s with s ide chain subst i tuents than i s now p o s s i b l e . Thus, a wide v a r i e t y of c a r b o x y l i c 79 ac ids w i t h , or without other s e n s i t i v e f u n c t i o n a l i t i e s can now be used, and t h i s opens up the p o s s i b i l i t y of s i d e - c h a i n s in the t r i a z o l e with r e a c t i v e groups ( e . g . Scheme 2 .16) . (61) + H0-C-CH 2 -NH-B0C BOC N N Scheme 2.16 Synthesis of a Fused s - T r i a z o l e with an Aminomethyl S i d e - c h a i n Reaction of a hydra laz ino compound with N-BOC-glycine can give a s - t r i a z o l e d e r i v a t i v e with an aminomethyl s i d e - c h a i n a f t e r removal of the BOC-group. We have done t h i s with 1 -hydraz inophtha laz ine . One might well ask what i s the s i g n i f i c a n c e of extending the scope of s - t r i a z o l e s y n t h e s i s , beyond that of a purely academic achievement. The uses and poten t ia l uses of t r i a z o l e d e r i v a t i v e s are many and v a r i e d , and even l i m i t i n g our cons idera t ion to fused s - t r i a z o l e s o n l y , the number of a p p l i c a t i o n s remains c o n s i d e r a b l e . Table 2.4 shows a sample of condensed s - t r i a z o l e systems and t h e i r p roper t ies and uses , taken from the recent l i t e r a t u r e (mostly 1976-1977). In many of the examples shown in Table 2.4 the t r i a z o l e r ing was formed by coupl ing a hydrazino d e r i v a t i v e with a carboxyl compound and subsequent c y c l i z a t i o n of the r e s u l t i n g carboxamide. Other methods involve a preformed s - t r i a z o l e which bind to r e a c t i v e groups in the molecule to form the m u l t i c y c l i c s t r u c t u r e . The t r i a z o l e d e r i v a t i v e s in Table 2.4 show proper t ies and uses which inc lude a p p l i c a t i o n s as f u n g i c i d e s , in cancer chemotherapy, and a wide v a r i e t y of pharmacological uses . A s i g n i f i c a n t aspect of these uses i s that most of them are descr ibed in the patent l i t e r a t u r e . E v i d e n t l y , the 80 t Table 2.4 Fused s - T r i a z o l e s - Proper t ies and Uses Fused s - T r i a z o l e Proper t ies Reference 62 63 N N (CH 2'n N N 1 A Central nervous system and 95 r e s p i r a t o r y system st imulants A n t i - c o n v u l s a n t and t r a n q u i l - 96 i z e r . Ant i - in f lammatory agent 64 CF Ant i - in f lammatory , a n a l g e s i c , 97 3 and a n t i - p y r e t i c a c t i v i t i e s 65 N N. l L h 3 N Ant i - in f lammatory agent 98 66 Control of plant-pathogens 99a 67 N — N Control of plant-pathogens 99b 68 N N X X R" R Ant i - in f lammatory , ana lges ic and anti . -yi .ral a c t i v i t i e s 100 81 Fused s - T r i a z o l e Proper t ies Reference 69 70 HO N N X J L | \ r ^ N ^ S H Ant i - tumor and a n t i - c a n c e r 101 a c t i v i t y A n t i - d e p r e s s i v e and a n x i o l y t i c 102 a c t i v i t i e s . Hypothermia an tagon is t , t r a n q u i l i z e r , s e d a t i v e , muscle re laxant 71 N N Jl A Control of p lant -pathogens. 103 72 Control of fungal f o l i a r pathogens 104 75 N N Sedat ive and ana lges ic a c t i v i t i e s . An t i -hypotens ive ac t ion 105 82 synthes is of these condensed s - t r i a z o l e s are s u f f i c i e n t l y important to warrant p ro tec t ion of the procedures and a p p l i c a t i o n s under the patent laws. An in tegra l part of any screening program fo r b i o l o g i c a l a c t i v i t y of a compound i s the synthesis of a s e r i e s of compounds wi th in the same general c l a s s . V a r i a t i o n of the s i d e - c h a i n subst i tuent on the t r i a z o l e r i n g i s a common denominator in most of the examples shown i n Table 2 .4 . Our work would permit the synthes is of t r i a z o l e s with a wider range of s i d e - c h a i n s than p rev ious ly prepared, and hence, p o s s i b l y fused t r i a z o l e s with a broader spectrum of a c t i v i t i e s . Only a few of the b i o l o g i c a l p roper t ies of the condensed s - t r i a z o l e s have been descr ibed in any d e t a i l . 3 - T r i f l u o r o m e t h y l - s - t r i a z o l o [ 3 , 4 - a ] i s o q u i n o l i n e (64) shows a pharmacological p r o f i l e which suggests that i t would be useful in the treatment of edema, pain and fever assoc ia ted with inflammatory d i s e a s e s , such as rheumatoid a r t h r i t i s . The minimal g a s t r i c a c t i v i t y in the ra t i n d i c a t e s that i t causes l e s s g a s t r i c d i s t r e s s than the ant i inf lammatory agents now in use. Its ana lges ic a c t i v i t y in mice i s greater than that of a c e t y l s a l i c y l i c a c i d . 9 7 3 - T r i f l u o r o m e t h y l [ 3 , 4 - a ] p h t h a l a z i n e (65) was r e c e n t l y reported to have a higher ant i inf lammatory e f f e c t than the corresponding i s o q u i n o l i n e d e r i v a t i v e (64).98 s - T r i a z o l o [ 4 , 3 - a ] q u i n o l i n e s (66) are used fo r the cont ro l of p lant pathogenic organisms. Thus, the methyl d e r i v a t i v e (F^CH^) c o n t r o l l e d anthracnose of cucumbers and r i c e b l a s t of r i c e . 9 9 a A lso a c t i v e f o r the contro l of r i c e b l a s t are s - t r i a z o l o [ 4 , 3 - a ] q u i n o x a l i n e (67), the parent t r i a z o l e (R ,RpH) being most e f f e c t i v e against Piriaularia oryzaef9^ and s - t r i a z o l o [ 3 , 4 - b ] b e n z o t h i a z o l e s (71).103 83 l -Mercapto -5 -hydroxy -6 ,7 - te t ramethy lene -s - t r i azo1o[3 ,4 -b ]pyr imid ine (69) prevents metastasis of human epidermoid carcinoma and e x h i b i t s antitumor a c t i v i t y aga inst primary human epidermoid carcinoma and other tumors, such as adenocarcinoma and s a r c o m a . 1 0 1 84 CHAPTER 3 SOLID-PHASE SYNTHESIS OF S-TRIAZQLO[3,4-AjPHTHALAZINES 3.1 INTRODUCTION The process of assembling a peptide chain in a s tep-wise manner while i t i s attached at one end to an i n s o l u b l e support i s known as the " s o l i d - p h a s e " method f o r polypept ide s y n t h e s i s . The technique was f i r s t used by two groups operat ing independent ly . The f i r s t a p p l i c a t i o n was by M e r r i f i e l d in 1963 to the synthesis o f a t e t r a p e p t i d e . 1 0 6 Soon a f t e r , Le ts inger reported the synthes is of a d ipept ide on a "popcorn" polymer s u p p o r t . 1 0 7 The major d i f f e r e n c e in the two methods was that M e r r i f i e l d attached the polymer to an amino ac id as the carboxylate es te r whi le Lets inger bound the amino ac id v i a the amino group as an amide. Since i t s i n t r o d u c t i o n , the s o l i d - p h a s e method has been s u c c e s s - f u l l y app l ied to pept ides of increas ing s i z e . These developments have been crowned by the s o l i d - p h a s e preparat ion of r ibonuc lease A (124 r e s i d u e s ) 1 0 8 and human growth hormone (188 r e s i d u e s ) , 1 0 9 both with a s i g n i f i c a n t degree of enzymic a c t i v i t y . A f t e r the o r i g i n a l successes of the s o l i d - p h a s e method in polypept ide s y n t h e s i s , p a r a l l e l a c t i v i t y developed in the a p p l i c a t i o n of 85 polymer attachment to non-peptide organic syntheses. Polymer supports have now been app l ied to the synthes is of carbohydrates-and t h e i r d e r i v a t i v e s , and to p o l y n u c l e o t i d e s . 1 1 0 . 1 1 1 Only very r e c e n t l y have s o l i d supports been used in general organic synthes is unrelated to r e p e t i t i v e ' s e q u e n t i a l - t y p e ' syntheses of po lypep t ides , p o l y n u c l e o t i d e s , and p o l y s a c c h a r i d e s . It has now been found that i n s o l u b l e polymers can be used fo r many purposes to so lve s p e c i f i c synthe t ic problems. We considered the coupl ing of hydra laz ine with N-protected amino ac ids on an i n s o l u b l e support f o r two primary reasons. The f i r s t stems from syn the t i c advantages of the s o l i d - p h a s e method. The most appeal ing advantage w i th in the context of t h i s work i s that the s o l i d - p h a s e method al lows excesses of reagents to be separated from the reac t ion product by simple f i l t r a t i o n , thus avoiding tedious chromatographic or so lvent ex t rac t ion procedures. For example, we have shown in Chapter 2 that s - t r i a z o l o [ 3 , 4 - a ] - phthalaz ine (TAP) d e r i v a t i v e s can be obtained by a v a r i e t y of coupl ing methods. D i f f i c u l t i e s i n some of these methods arose from i s o l a t i n g the des i red product from s o l u t i o n . Thus, e f f o r t s to i s o l a t e the TAP product were f r u s t r a t e d when HMPA + tosy l c h l o r i d e were used as a coupl ing method ( c f . Sect ion 2 . 4 . 3 ) . The other reason f o r cons ider ing the s o l i d - p h a s e method i s connected with the p o s s i b l e a p p l i c a t i o n of the hydra laz ine r e a c t i o n with carboxylate groups of peptides as a C-terminal peptide sequencing method. Several bene f i t s are obtained by anchoring the peptide onto a polymeric support : a . The peptide m a t e r i a l , once attached to the s o l i d suppor t , i s not l o s t dur ing the degradat ive procedures. Th is permits sequencing to be performed with small amounts of peptide samples or fu r ther in to the peptide c h a i n . 86 b. A f t e r each reac t ion s t e p , excess reagents may be r e a d i l y removed by f i l t e r i n g and washing the support . c . The s o l i d - p h a s e method i s amenable to automation because of the r e p e t i t i v e nature of peptide sequencing. The f i r s t a p p l i c a t i o n to the step-wise degradation of peptides by the s o l i d - p h a s e method was that of S t a r k , 1 1 2 whose procedure invo lved a t tach ing the peptide at i t s N-terminus to an i n s o l u b l e Edman reagent , p o l y s t y r y l i s o t h i o c y a n a t e , c y c l i z i n g the adduct to form the th iohydanto in , i s o l a t i n g and ana lyz ing the res idua l pept ide , and rea t tach ing the peptide to the support (Scheme 3 .1 ) . 0 S 0 repeat degradat ion c o p o l y s t y r e n e - d i v i n y l benzene support Scheme 3.1 S t a r k ' s Method for Subt rac t ive N-terminal Peptide Degradation 87, Using a somewhat d i f f e r e n t approach, Laursen attached the peptide by i t s C-terminal amino ac id to a polystyrene support and then p e r f o r m ' ^ the degradation in the usual manner with phenyl i s o t h i o c y a n a t e . The l i b e r a t e d t h i a z o l i n o n e i s removed in each c y c l e by f i l t r a t i o n . A f t e r the t h i a z o l i n o n e i s converted to a phenyl th iohydantoin i t i s i d e n t i f i e d by a number of procedures (Scheme 3 . 2 ) . 1 3 In keeping with the success of s o l i d - 0 X-NH-CHR-C-NH-peptide-COOH 0 P S - N H 2 X-NH-CHR-C-NH-peptide-CONH-P c deblock NH 2 -CHR-C-NH-peptide-CONH-P c 0 PhNCS PhNH-C-NH-CHR-C-NH-peptide-CONH-P H H Ph-I \K^ + NrL-peptide-CONH-P n 4 R 2 H repeat degradat ion P s = c o p o l y s t y r e n e - d i v i n y l benzene support Scheme 3.2 Laursen 's Method fo r N-Terminal Peptide Sequencing 88 phase peptide s y n t h e s i s , peptide sequencing by the Laursen method has been automated and commercial vers ions of the so l id -phase sequencer are a v a i l - a b l e . 1 1 3 a peptide attached to an i n e r t support were descr ibed by Laursen over 10 years a g o , 1 1 4 the method i s s t i l l not as commonly used as the l i q u i d - p h a s e techniques of Edman and B e g g . 1 0 The reasons f o r t h i s a r i s e mainly from the development of only a few s u i t a b l e supports , and the problems assoc ia ted with the procedures f o r coupl ing peptides to supports . Some progress is being made i n overcoming these problems, and the s o l i d - p h a s e sequencing method i s r a p i d l y increas ing in p o p u l a r i t y . i n fancy , sequencing from the C-terminus i s barely past the embryonic s tage . During the time that th is work was in progress , two C-terminal s o l i d - p h a s e methods were reported based on S t a r k ' s thiocyanate reac t ion and using copoly (s tyrene-d iv iny lbenzene) or porous-g lass supports (Scheme 3 . 3 ) . 1 1 ' 1 ' 1 1 5 Although the p r i n c i p l e s of determining the amino ac id sequence of While so l id -phase N-terminal peptide sequencing i s s t i l l in i t s 0 0 II II P-C-NH-peptide-C-NH-CHR-COOH a c e t i c anhydride P = polymeric support repeat degradation H Scheme 3.3 So l id -phase Peptide Sequencing by S t a r k ' s Thiocyanate Reaction 89 3.2 RESULTS The choice of an appropr ia te support mater ia l and the means of a t tach ing the substra te onto the support in the s o l i d - p h a s e method of organic synthes is are major d e c i s i o n s a f f e c t i n g the success of the s y n t h e s i s . In s p i t e of the enormous polymer technology which has developed in recent y e a r s , a very l i m i t e d amount of polymer types have been examined f o r use in s o l i d - p h a s e s y n t h e s i s . The most widely used polymer, and the one used in t h i s work, i s the copolymer of styrene and d iv iny lbenzene ( D V B ) . 1 0 6 The r e s i n ^ used was a polymer in the form of small (200-400 mesh) beads with 1% c r o s s - l i n k i n g by d iv iny lbenzene . The low degree of c r o s s - l i n k i n g allows the polymer to swell in non-polar so lvents and permits penetrat ion of reagents in s o l u t i o n to react with substra te molecules bound to in te rna l sur faces of the polymer. For the synthes is of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e s on i n s o l u b l e suppor ts , e i t h e r hydra laz ine or the amino ac id may be anchored to the suppor t . If hydra laz ine i s attached to the suppor t , the hydrazino group must remain f ree f o r r e a c t i o n with amino a c i d s . The attachment i s most r e a d i l y achieved v ia a f u n c t i o n a l i t y introduced onto the a ry l r i n g of the phthalaz ine moiety. However, synthes is of r i n g - s u b s t i t u t e d hydra laz ine s u i t a b l e f o r s o l i d - p h a s e synthes is i s not a t r i v i a l undertak ing, and the more d i r e c t a l t e r n a t i v e of immobi l iz ing the amino ac id v ia i t s a-amino group i s p r e f e r r e d . Several opt ions are a v a i l a b l e f o r anchoring amino groups onto a polymeric matr ix . The method used by L e t s i n g e r , 1 0 7 and by M e r r i f i e l d 1 1 6 t The s o l i d - p h a s e terminology which has become es tab l i shed f o r d e r i v a t i v e s of copolystyrene-DVB and f o r var ious l inkages with amino ac ids or pep t ides . 90 was adopted in t h i s work. Commercial chloromethylated copolystyrene-1% DVB was t reated with potassium ace ta te . The product was converted to the hydroxymethylated r e s i n by s a p o n i f i c a t i o n with aqueous sol ium hydroxide. Treatment of t h i s r e s i n with phosgene in benzene a f forded the methyl - ch loroformylated r e s i n (Scheme 3 .4 ) . By th is procedure, complete conversion * KOAc NaOH C0C1 9 ?, P -CrLC l - P -ChLOAc » P -CrLOH C P -CrLOC-Cl S <: S c s c s c . P s = copo lys ty rene-d iv iny lbenzene support Scheme 3.4 Preparat ion of Methylchloroformylated Resin of c h l o r i d e to a c i d c h l o r i d e groups was obtained to g ive a r e s i n capac i ty of 1.36 meq c h l o r i d e per gram dry r e s i n . The f u n c t i o n a l i z e d po lystyrene r e s i n thus obtained can couple r e a d i l y to the amino groups of C-protected amino ac ids v i a an amide bond which i s normally s tab le to syn the t i c operat ions with the r e s i n . Solvents such as benzene, p y r i d i n e , DMF, and chloroform penetrate the polymer e f f e c t i v e l y and are therefore good r e a c t i o n media, whereas with water, methanol, and e the r , regions of the polymer appear to be i n a c c e s s i b l e s ince there i s l i t t l e swel l ing in these s o l v e n t s . Dahlmans suggested the use of a polystyrene r e s i n conta in ing s u l f o n y l c h l o r i d e groups which would bind amino groups by means of a Schotten-Bauman r e a c t i o n . 1 1 7 A b e n e f i t of t h i s method is the high s t a b i l i t y of the P S-S02"NH bond. The r e s i n may be s p l i t o f f from the bound amino a c i d by treatment with phosphonium iod ide in t r i f l u o r o a c e t i c a c i d . Th is method however, has found very l i m i t e d use in s o l i d - p h a s e s y n t h e s i s . 91 In our f i r s t e f f o r t s at coupl ing amino ac ids to the carboxyl ac t iva ted r e s i n we used the procedure of Le ts inger and K o r n e t . 1 0 7 The methylchloroformylated r e s i n was s t i r r e d with excess g l y c i n e ethyl es ter hydrochlor ide and t r ie thy lamine in dry DMF. The product was sapon i f i ed with base in methanol-acetone s o l u t i o n . The f i n a l r e s i n product , expected to be immobil ized g l y c i n e ethyl e s t e r , showed strong i n f r a - r e d bands at 3580 cm" 1 and 3440 cm" 1 which were a t t r i b u t e d to f ree and hydrogen-bonded 0-H groups, r e s p e c t i v e l y . A f t e r es te r h y d r o l y s i s , the lower wavenumber v(O-H) band s h i f t e d to 3390 c m " 1 . The carbonyl bands were broad f o r both products , and exh ib i ted a s h i f t from 1724 cm" 1 to 1718 cm" 1 upon r e s i n h y d r o l y s i s . By comparison, f ree g l y c i n e ethyl es te r hydroch lor ide e x h i b i t s a sharp carbonyl s t r e t c h i n g band at 1746 c m " 1 , and g l y c i n e shows a very broad band about 1582 c m - 1 . Le ts inger and Kornet observed a s h i f t of s i m i l a r magnitude from 1730 cm" 1 to 1724 c m - 1 on ester h y d r o l y s i s of P s ~ C H 2 0 - l e u c i n e ethyl e s t e r . Hydro lys is of a sample of the ac id c h l o r i d e r e s i n with sodium hydroxide in methanol-acetone showed v(O-H) bands s i m i l a r to those obtained in the attachment r e a c t i o n . However, there were no carbonyl bands i n d i c a t i n g that any a c i d which might have been formed by h y d r o l y s i s of the ac id c h l o r i d e had decomposed f u r t h e r to the hydroxymethyl r e s i n . If h y d r o l y s i s of the a c i d c h l o r i d e r e s i n was c a r r i e d out i n the presence of excess p y r i d i n e , i n f r a - r e d spect ra of the product showed bands at 3575 and 3360 c m " 1 , and 1738 c m " 1 , suggesting that both P s -CH 2 0C00H and P s -CH 2 0H were formed. On the basis of i n f r a - r e d s p e c t r a , the coupl ing react ion of the ac id c h l o r i d e r e s i n with g l y c i n e ethyl es te r was in terpre ted as g i v i n g a mixture of P s -CH 2 0C00H, P s - C H 2 0 H , and the des i red P s - C H 2 0 C 0 - g lyc ine ethyl e s t e r . The low i n t e n s i t y of the v(0-H) bands in the spect ra of the g l y c i n e ethyl es te r r e s i n from the i n i t i a l attachment reac t ions suggested low s u b s t i t u t i o n y i e l d s . Further t r i a l s using chloroform in place 92 of DMF as the r e a c t i o n medium appeared to g ive somewhat bet ter y i e l d s , and t h i s so lvent was used in subsequent immobi l izat ion r e a c t i o n s . The cond i t ions u l t ima te ly used f o r a t taching amino ac ids to the support were s t i r r i n g the methylchloroformyl r e s i n (1.36 mmol C l / g ) with two-fo ld excesses of the amino ac id ethyl es te r hydrochlor ide and t r i e t h y l - amine in dry chloroform at room temperature f o r one day. A f t e r f i l t e r i n g and washing, the r e s i n was resuspended in chloroform and reacted with excess diethylamine to block any unreacted ac id c h l o r i d e groups. The r e s i n thus obtained was s a p o n i f i e d with potassium hydroxide in methanol-acetone s o l u t i o n . S u b s t i t u t i o n s of up to 0.63 mmoles of amino ac id per gram of r e s i n were obtained with a lan ine and g lyc ine ethyl es ter h y d r o c h l o r i d e s . A s u b s t i t u t i o n of 0.19 mmol/g r e s i n was obtained with g l y c y l g l y c i n e ethyl es ter hydroch lo r ide . These r e s u l t s compare with a s u b s t i t u t i o n of up to 0.50 mmol L - l e u c i n e BOC-hydrazide/g r e s i n from P S-CH 20C0C1 (0.72 mmol C l / g ) by F e l i x and M e r r i f i e l d . 1 1 6 S u b s t i t u t i o n c a p a c i t i e s up to about 0.6 mmol d i p e p t i d e s / g were obtained by Darbre and R a n g a r a j a n . 1 1 5 We focussed our a t ten t ion on u t i l i z i n g the immobil ized substrate once cond i t ions fo r a t tach ing amino ac ids to the support were e s t a b l i s h e d . A search of the l i t e r a t u r e showed that the number of carboxyl a c t i v a t i o n methods used in s o l i d - p h a s e synthesis was qui te l i m i t e d . The f i r s t e f f o r t s at coupl ing hydra laz ine with immobil ized g l y c i n e were with the carbodi imide method (c f . Sect ion 2 . 4 . 4 ) . The g l y c i n e r e s i n was s t i r r e d with 2 . 5 - f o l d excesses of hydra laz ine HCI, t r i e t h y l a m i n e , and d icyc lohexy lcarbod i im ide in dimethylacetamide suspension under an argon atmosphere. A f t e r r e a c t i o n overn igh t , the r e a c t i o n products were cleaved from the polystyrene support with HBr/CFgCOOH. The des i red product should be obtained as the HBr of 3 -aminomethy l -s - t r i azo lo [3 ,4 -a ]ph tha laz ine (Scheme 3 .5 ) . Scheme 3.5 So l id -phase Synthesis of s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e s Monitor ing the progress of the reac t ion i s a d i f f i c u l t problem, as i t i s in s o l i d - p h a s e peptide s y n t h e s i s . In a developmental s i t u a t i o n i t i s important to know, at l e a s t q u a l i t a t i v e l y , the rate of r e a c t i o n and when the reac t ion is completed. Unfor tunate ly , we d id not f i n d any e n t i r e l y s a t i s f a c t o r y monitor ing method. In p r i n c i p l e , i n f r a - r e d spectroscopy should be u s e f u l . Reaction of hydra laz ine with the immobil ized amino ac id should show a disappearance of the carbonyl band a r i s i n g from the c a r b o x y l i c a c i d . In p r a c t i c e , the over lap o f strong bands a r i s i n g from amide carbonyls of the l inkage bonds makes any changes in shape and/or frequency of the broad carbonyl bands of dubious d iagnos t ic va lue . In f ra - red bands a r i s i n g from the t r i - c y c l i c TAP product are superimposed on the ra ther crowded spectrum exh ib i ted by the copolystyrene-DVB matr ix . Furthermore, in our hands, polystyrene beads almost i n v a r i a b l y d id not give t rans lucent KBr d i s c s . A s t ra ight forward monitor ing procedure i s to measure the decrease of hydra laz ine in s o l u t i o n by u l t r a - v i o l e t spectroscopy. This procedure however, i s inaccurate and u n r e l i a b l e by v i r t u e of the decomposit ion of h y d r a l a z i n e , e s p e c i a l l y in the presence of t r i e t h y l a m i n e . 94 In view of the d i f f i c u l t i e s , l i t t l e e f f o r t was made to fo l low the rate of the coupl ing r e a c t i o n , although IR spectra of the r e s i n , and UV spectra of the s o l u t i o n were recorded r o u t i n e l y . Coupling reac t ions were usua l ly terminated a f t e r one day and the products examined f o r the presence of TAP product . unsuccessful in de tec t ing any TAP products . Th is i s not s u r p r i s i n g as ample oppor tun i t ies were a v a i l a b l e for f a i l u r e at every stage o f the s o l i d - phase procedure. A p o t e n t i a l l y ser ious d i f f i c u l t y with the carbodi imide methods i s the p r o b a b i l i t y that some of the a c t i v e 0 - a c y l i s o u r e a intermediate might be shunted o f f to the i n e r t N-acylurea (Scheme 2.12) . In s o l i d - p h a s e peptide synthes is the s i d e - r e a c t i o n i s rap id in DMF s o l u t i o n 1 1 8 and t h i s may be a c o n t r i b u t i n g f a c t o r to f a i l u r e of the hydra laz ine coupl ing reac t ion in DMA. Chloroform was used in subsequent reac t ions s ince the s i d e - r e a c t i o n i s not favored in t h i s s o l v e n t . However, the coupl ing reac t ion remained r e l a t i v e l y u n s u c c e s s f u l , presumably because of the low s o l u b i l i t y of hydra- laz ine HCl in ch loroform. Confronted with the lack o f success with the ca rbod i im ides , s a l v a t i o n was sought with the mixed-anhydride method used by Le ts inger and K o r n e t . 1 0 7 The mixed anhydride intermediate may be prepared in situ by the r e a c t i o n of i sobuty l chloroformate with the carboxyl component, and then coupled d i r e c t l y with the amino reagent (Scheme 3 . 6 ) . 1 1 9 I n i t i a l e f f o r t s with the carbodi imide method were t o t a l l y 0 0 R-C n C„H n 0 -C-C l + RCOOH C 4 H g 0 -C . » RCONHR' + C 4 H 9 0H + C 0 2 Scheme 3.6 Amide Formation with Isobutyl Chloroformate 95 Isobutyl and other a lky l chloroformates react through s i m i l a r intermediates as the EEDQ and IIDQ coupl ing reagents (Sect ion 2 . 4 . 2 ) . In a s o l i d - p h a s e peptide synthesis by extension from the C- terminus, T i l a k and H o l l i n d e r 1 2 0 reported that mixed anhydride coupl ing with i sobuty l c h l o r o - formate proceeded f a s t e r than with DCC and required shor ter coupl ing peri ods. Mixed-anhydride coupl ing was e f fec ted by reac t ing an excess of isobuty l chloroformate with the amino ac id res in in chloroform suspension f o r an hour at - 1 0 ° C . A f t e r the amino ac id anhydride was freed of excess ch loro formate , the r e s i n was immediately resuspended in a chloroform s o l u t i o n of hydra laz ine HCI and t r i e t h y l a m i n e . The product was cleaved from the support a f t e r the reac t ion mixture was allowed to react f o r about 18 hours at room temperature. Two undesired s i d e - r e a c t i o n s are assoc ia ted with amide formation by the mixed-anhydride method. F i r s t , anhydride (74) can d ispropor t iona te to y i e l d a symmetrical anhydride (75) and-a d ia lky lpyrocarbonate (76) (Scheme 3 .7 ) . The l a t t e r can i r r e v e r s i b l y block the amino component 0 0 R - C - 0 - C - 0 C 4 H 9 Scheme 3.7 S i d e - r e a c t i o n s of Mixed-anhydride in Amide Synthesis (route l). In s o l i d - p h a s e s y n t h e s i s , aminolys is of the symmetrical anhydride r e s u l t s in lowered y i e l d of the coupled product s ince ha l f of the carboxyl (1) R'NH, t (2) 0 0 II II R-C-O-C-R + (75) 0 C 4 H g 0-C-NHR' 0 n 0 II C 4 H 9 0 - C - 0 - C - 0 C 4 H g (76) + R-COOH 96 component is l o s t as the f ree a c i d . This d i s p r o p o r t i o n a t e reac t ion i s minimized by a c t i v a t i n g the amino ac id at low temperature. Second, wrong s ide aminolys is of the anhydride (74) can r e s u l t in a mixture of products (route 2). Th is reac t ion i s made less favorable by using a bulky alkoxy group, e . g . i s o b u t y l o x y . We were f i n a l l y success fu l with the mixed-anhydride method in preparing the TAP d e r i v a t i v e s by coupl ing hydra laz ine with immobil ized g l y c i n e . Only one f l u o r e s c e n t product was i s o l a t e d i n d i c a t i n g that nuc leo- p h i l i c at tack by hydra laz ine at the isobuty loxy carbonyl carbon atom (route 2) was i n s i g n i f i c a n t . The product of the coupl ing reac t ion was p u r i f i e d by preparat ive TLC on s i l i c a g e l , and i t s i d e n t i t y was confirmed by mass spectrometry. Coupling of hydra laz ine with a lan ine r e s i n under s i m i l a r r e a c t i o n condi t ions a lso gave the des i red TAP product in moderate y i e l d . Buoyed by the success with isobuty l ch loroformate , we next t r i e d the coupl ing reagent , l - e t h o x y c a r b o n y l - 2 - e t h o x y - l , 2 - d i h y d r o q u i n o l i n e (EEDQ, Sect ion 2 . 4 . 2 ) . EEDQ generates an ethoxy mixed carbonic anhydride on reac - t ion with c a r b o x y l i c a c i d s . G lyc ine r e s i n in TilF suspension was allowed to react with excess EEDQ at room temperature f o r 45 minutes under a n i t rogen atmosphere, and then f i l t e r e d f ree of excess reagents . A f t e r the r e s i n had reacted with hydra laz ine in Th'F s o l u t i o n f o r about 18 hours, the product was cleaved from the r e s i n with HBr/HOAc. UV spec t ra l a n a l y s i s of the product showed that TAP was s u c c e s s f u l l y formed, but in r e l a t i v e l y low y i e l d . In s o l i d - p h a s e peptide synthes is and in th is work, the usual l inkage of amino ac ids to the polystyrene r e s i n is a benzyl es te r bond. For a long t ime, the standard procedure f o r c l e a v i n g t h i s bond was treatment 97 of the r e s i n in t r i f l u o r o a c e t i c a c i d suspension with HBr. However, t r i - f l u o r o a c e t i c ac id swel ls the polystyrene r e s i n very p o o r l y , and i n f r a - r e d spectra of the r e s i n (a f te r 4 hours treatment) showed incomplete cleavage of product . This has a lso been one of the more aggravating disadvantages of the HBr/TFA cleavage in peptide s y n t h e s i s . 1 2 1 When t r i f l u o r o a c e t i c a c i d was rep laced by a c e t i c a c i d , swel l ing of the polystyrene support was improved, and the cleavage reac t ion appeared to be more complete by IR c r i t e r i a (absence of v(C=0) bands). The experiments with d icyc lohexy lcarbod i imide as a coupl ing reagent were performed at an e a r l y stage of t h i s work when experience with the s o l i d - p h a s e procedure, and with i n t e r p r e t a t i o n of i n f r a - r e d spectra o f the r e s i n s were l i m i t e d . A coupl ing reac t ion in chloroform s o l u t i o n with DCC was repeated under the same condi t ions as descr ibed p r e v i o u s l y except f o r the change in cleavage procedure to treatment with HBr/HOAc. UV spec- t r a l a n a l y s i s of the product showed the des i red 3 -aminomethy l -s - t r i azo lo - [3 ,4 -a ]ph tha laz ine ! Thus, the apparent f a i l u r e of the carbodi imide method in e a r l i e r e f f o r t s probably r e s u l t e d not only from the coupl ing s t e p , but from incompleteness of the cleavage reac t ion as w e l l . 3.3 DISCUSSION One f a c e t of our s tudies on the s o l i d - p h a s e synthes is of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e d e r i v a t i v e s that i s c l e a r l y evident i s the interdependence o f the var ious f a c t o r s : support m a t e r i a l , at tachment, c o u p l i n g , and cleavage methods, in determining the success of the s o l i d - phase method f o r chemical s y n t h e s i s . The i n s o l u b l e support i s the backbone of the s o l i d - p h a s e method i t s e l f . A good support should possess c e r t a i n d e s i r a b l e phys ica l and 98 chemical , c h a r a c t e r i s t i c s . The support should be in the form of homogeneous, r e l a t i v e l y r i g i d , porous beads. An appropr ia te p o r o s i t y permits rap id d i f f u s i o n of reagents in to the r e a c t i v e s i t e s and easy removal of reagents by f i l t r a t i o n and washing. It a l s o provides fo r a large e f f e c t i v e sur face area which is d e s i r a b l e f o r obta in ing a high degree of m o d i f i c a t i o n . I d e a l l y , the support should have good mechanical s t rength and chemical s t a b i l i t y toward extremes of temperature and pH, and toward organic s o l v e n t s . The support should a l s o be h i g h l y i n s o l u b l e in the so lvent media of r e a c t i o n . The support must possess chemical c h a r a c t e r i s t i c s which al low i n t r o d u c t i o n of r e a c t i v e f u n c t i o n a l i t i e s under mild cond i t ions and permit a c t i v a t i o n of the support without dest roy ing i t s s t r u c t u r a l i n t e g r i t y . L a s t , but not l e a s t , a good support should be reasonably p r i c e d , otherwise l a r g e - s c a l e syntheses would be economical ly p r o h i b i t i v e . A v a i l - a b i l i t y from e i t h e r supply houses or from simple chemical synthes is i s advantageous. The cost f a c t o r w i l l be minimized i f the a c t i v e support can be regenerated a f t e r use. The support mater ia l which we used in t h i s work was a copolymer of styrene and d iv iny lbenzene which i s an amorphous gel with a random net- work of l o o s e l y c r o s s l i n k e d polystyrene c h a i n s . We pre fer red the 1% c r o s s - l inked polymer over the 2% c r o s s l i n k e d r e s i n used in the major i ty of s o l i d - phase r e a c t i o n s . Copolystyrene-1% DVB swel ls apprec iab ly more in non-polar solvents than polymers with higher c r o s s l i n k i n g , and can lead to more complete reac t ions in peptide s y n t h e s i s . 1 2 2 In h igh ly c r o s s l i n k e d copo ly - styrene-DVB polymers, the beads are too r i g i d to permit easy penetrat ion of reagents , and slower and less complete react ions may r e s u l t . The c r o s s - l inked polystyrene beads are not a "sur face support" . Due to t h e i r 99 swe l l ing in organic so lvents they are f r e e l y permeable to reagents . M e r r i - f i e l d and L i t t a u 1 2 3 showed by autoradiography of beads conta in ing t r i t i a t e d peptides that the d i s t r i b u t i o n i s qui te uniform throughout the bead. Recent s tudies with polymer-bound t r a n s i t i o n metal c a t a l y s t s and complexes s t rong ly suggest that the degree of swel l ing of the polymer l a t t i c e i s an important f a c t o r in determining the chemical r e a c t i v i t y of immobil ized molecu les , and there in l i e s one of the main d e f i c i e n c i e s of c r o s s l i n k e d p o l y s t y r e n e . 1 2 4 * 1 2 5 Supports are needed which are more compatible with po lar s o l v e n t s , or are l ess a f f e c t e d by the s w e l l a b i l i t y of the r e s i n . In N-terminal peptide sequencing with an i n s o l u b l e Edman reagent , Dowling and S t a r k 1 1 2 synthesized a copolystyrene-O.25% DVB r e s i n with cova len t l y l inked glucosaminol to increase i t s h y d r o p h i l i c charac te r . Rangarajan and D a r b r e 1 1 5 introduced methylthiocarbamoyl groups in to the benzene r ings of copolystyrene-2% DVB to achieve the same end. Th is modif ied polymer approximately doubled i t s swel l ing in aqueous media and showed improved attachment y i e l d s of t e t r a - and hexapeptides to the support . The improvement was ascr ibed to the increased p o l a r i t y of the polymer a l lowing e a s i e r a c c e s s i b i l i t y of r e a c t i v e s i t e s . A f f i n i t y chromatography has in common with sol i -d-phase synthes is the use of i n s o l u b l e supports f o r immobi l iz ing a l i g a n d . In the former a p p l i c a t i o n , i t i s f requent ly advantageous to at tach the l igand to the support at a d is tance from the s u r f a c e . Spacer arms provide more e f f i c i e n t i n t e r a c t i o n between the immobil ized l igand and the so lu te molecules due to the increased s t e r i c a v a i l a b i l i t y of the l igand to the s o l u t e . The length of the spacer i s chosen e m p i r i c a l l y and must be optimized f o r each use. The same p r i n c i p l e s a lso apply to the s o l i d - p h a s e r e a c t i o n of amino ac ids with hydra laz ine . The b i f u n c t i o n a l molecules B-alanine methyl 100 ester HCl and 3 -a lan ine isopropyl es ter HCl were therefore synthesized to be used as spacer arms as shown in Scheme 3.8. 0 0 0 0 H n P S -CH 2 0C-C1 + H 2 N - ( C H 2 ) 2 - C - 0 R P s - C H 2 0 C - N H - ( C H 2 ) 2 - C - 0 R R=CH OH" n ' II P s - C H 2 0 C - N H - ( C H 2 ) 2 - C - 0 H Scheme 3.8 M o d i f i c a t i o n of Polystyrene with a Spacer Arm The advantages of a spacer arm were a l s o apparent to Rangarajan and D a r b r e 1 1 5 who attached a y -amino-n -bu ty r ic ac id methyl e s t e r spacer arm to a carboxylate polystyrene support . An improvement in the y i e l d of peptide attachment was a t t r i b u t e d to a r i s e p a r t l y from t h i s polymer m o d i f i - c a t i o n . In a s i m i l a r v e i n , S p a r r o w 1 2 6 modif ied a M e r r i f i e l d r e s i n by in t roducing a long spacer arm f o r use in peptide s y n t h e s i s . A t h r e e - f o l d improvement in the o v e r a l l y i e l d of a 19-residue peptide was r e a l i z e d with th is support . EPR studies of a s p i n - l a b e l attached to the N-terminus of a peptide on t h i s r e s i n suggested an increased m o b i l i t y of the N-terminal res idue over the unmodified commercial r e s i n , and hence decreased pept ide - r e s i n i n t e r a c t i o n s . In s p i t e of mod i f i ca t ions to the usual polystyrene mat r ix , the inherent hydrophobic!'ty and dependence on swel l ing by so lvents of styrene Br 101 > based polymers l i m i t t h e i r usefu lness in peptide sequencing. An ideal support should be capable of coupl ing e f f e c t i v e l y a l l s i z e s of p e p t i d e s , inc lud ing p r o t e i n s , and the func t iona l groups should be a c c e s s i b l e f o r chemical reac t ion in a v a r i e t y of aqueous and organic media. Polyacrylamide appears to be an e x c e l l e n t matr ix f o r s o l i d - p h a s e s y n t h e s i s . The beads are mechanical ly and chemica l ly s t a b l e , and are r e l a t i v e l y h y d r o p h i l i c . The neutral polyacrylamides are e n t i r e l y s y n t h e t i c ge ls formed by copolymer izat ion of acrylamide with the A f u n c t i o n a l c r o s s - l i n k i n g agent , N ,N 1 -methy lene -b is -acry lamide . The r a t i o of the concentra - t ion of acrylamide in the reac t ion mixture to that of the c r o s s l i n k i n g agent can be var ied to g ive an i n f i n i t e s e r i e s of i n s o l u b l e gel products which d i f f e r in t h e i r average pore s i z e . 1 2 7 F igure 3.1 shows part of a polyacrylamide matr ix . The p r i n c i p a l advantage of polyacrylamide i s that -CH-CH 0 -CH-CH 0 -CH-CH 0 -CH- CONH CONHo CONH, 2 HN-CO C H 2 HN-CO -CH-CH 2 -CH-CH 2 -CH- C0NH9 OC-NH CH 2 OC-NH - C H 2 - C H - C H 2 - C H - C H 2 - C H - CONH, CONH, F igure 3.1 P a r t i a l S t ructure of a Polyacrylamide Matr ix 102 i t possesses a very abundant supply of modi f iab le carboxamide groups which, together with a v e r s a t i l i t y in d e r i v a t i z a t i o n techniques , al lows the covalent attachment of a v a r i e t y of f u n c t i o n a l i t i e s . 1 2 7 L im i ta t ions of polyacrylamide as an i n s o l u b l e matrix are the low degree of poros i ty of the beads c u r r e n t l y a v a i l a b l e , and the shrinkage observed during the chemical mod i f i ca t ions requi red for attachment of func t iona l groups. Polyacrylamide ge ls have found t h e i r most f requent a p p l i c a t i o n s in the immobi l izat ion of prote ins and other b ioorganic mater ia ls in a f f i n i t y chromatography, immunosorbent s y n t h e s i s , e t c . 1 2 7 " 1 3 0 A p p l i c a t i o n s in s y n t h e t i c organic chemistry are l i m i t e d to the recent use of a po lyd imethy l - acrylamide support in peptide s y n t h e s i s . 1 3 1 A notable feature of t h i s support i s the large swel l ing of the r e s i n in po lar media such as DMF, HOAc, and l-^O. Very much less swel l ing occur in methylene c h l o r i d e and less polar organic s o l v e n t s . These proper t ies are the reverse of those of po ly - styrene-based r e s i n s . In peptide s y n t h e s i s , the polydimethyl 'acrylamide support gave r e s u l t s c l e a r l y super ior to those obtained with the customary polystyrene support . In N-terminal p ro te in sequencing, polyacrylamide-based supports are j u s t beginning to a t t r a c t a t t e n t i o n . Pre l iminary r e s u l t s show s i g n i f i c a n t advantages over polystyrene r e s i n s . 1 3 2 > 1 3 3 C l e a r l y , p o l y a c r y l - amide supports show s u f f i c i e n t promise to warrant f u r t h e r i n v e s t i g a t i o n s in a wider range of a p p l i c a t i o n s . B io-Gel CM-2 i s a f u l l y carboxylated polyacrylamide matrix a v a i l - able from Bio-Rad Labora tor ies with a capac i ty of 5 meq/gram. We used t h i s mater ia l to prepare the a c i d - c h l o r i d e r e s i n by reac t ion with th iony l c h l o r i d e , with the in ten t ions of modifying the support with a 3 -a lanine ester spacer arm, and using t h i s support to evaluate i t s s u i t a b i l i t y f o r the s o l i d - p h a s e synthes is of TAP d e r i v a t i v e s . These experiments however, remain incomplete , and they deserve cons idera t ion by other workers. 103 Other types of supports are in use fo r s o l i d - p h a s e prote in sequencing and prominent among them are those based on c o n t r o l l e d - p o r e g lass (CPG). The term c o n t r o l l e d - p o r e r e f e r s to a rather narrow pore d i s t r i b u t i o n . Th is type of support was f i r s t introduced f o r Edman degradat ion of peptides and prote ins by Machleidt and co-workers in 1 9 7 3 . 1 3 4 Porous g lass i s an a t t r a c t i v e support mater ia l f o r a number of reasons. It i s r i g i d , w i th - stands organic s o l v e n t s , i s regenerable , and i s r e s i s t a n t to microb ia l a t tack . Porous g lass may be ac t i va ted by s i l a n i z a t i o n with y -aminopropyl - t r i e t h o x y s i l a n e . 1 3 5 The amino groups of the r e s u l t i n g alkylamine g lass may o be s u c c i n y l a t e d to give an extension arm approximately 10 A long . The carboxyl g lass may be converted to the acyl c h l o r i d e with th iony l c h l o r i d e . 0 0 - S i - ( C H 2 ) 3 - N H - C - ( C H 2 ) 2 - C - 0 H This product can be used d i r e c t l y f o r coupl ing with the amino groups p r o t e i n s . The s u c c i n y l a t e d CPG i s commercial ly a v a i l a b l e from P ie rce Chemical Company, as are the N-hydroxysuccinimide and p -n i t ropheny l e s t e r s . For an e x c e l l e n t review on CPG, the reader i s re fe r red to the P ierce General C a t a l o g . 1 3 6 Recent C-terminal peptide and prote in sequencing s tud ies have used N-hydroxysucciniinide CPG b e a d s . 1 1 4 * 1 1 5 Good attachment y i e l d s of lysozyme and r ibonuclease to the porous g lass support were obta ined. Wi l l iams and K a s s e l l 1 1 4 achieved average attachment y i e l d s varying from 49 to 85% with e ight d i f f e r e n t peptides possessing between two and f i v e amino a c i d s . Porous g lass possesses many of the r e q u i s i t e s for a good support m a t e r i a l , and i t i s g radua l ly rep lac ing the conventional po lystyrene-based support in prote in sequencing. There are severa l inherent l i m i t a t i o n s in 104 the suppor t , however. The S i - 0 bonds which hold the l igand to the g lass are not completely s tab le to an ac id ( p a r t i c u l a r l y aqueous ac id ) and small amounts of peptide tend to be l o s t in each degradat ive c y c l e . Erosion of the g l a s s , e s p e c i a l l y at high pH i s a lso a problem, and severe l igand leakage can be encountered. A coat of z i rconium oxide s t a b i l i z e s the g lass but t h i s r a i s e s fu r ther the cos t of an a l ready expensive m a t e r i a l . The Corning CPG/N-hydroxysuccinimide ester d i s t r i b u t e d by P ierce costs $60/25 m l . 1 3 6 A f u r t h e r l i a b i l i t y of CPG i s i t s rather low binding capac i ty (CPG/N-hydroxysuccinimide, ^0.036 m e q / m l ) 1 3 6 which makes i t somewhat imprac t ica l f o r preparat ive s c a l e syntheses. in the s o l i d - p h a s e coupl ing r e a c t i o n of hydra laz ine with amino a c i d s . Using d icyc lohexy lca rbod i im ide as a coupl ing reagent , the des i red TAP product i s formed, but y i e l d s are u n s a t i s f a c t o r y . Perhaps one of the most ser ious problems with the carbodi imide method i s the p o s s i b i l i t y of forma- t ion of the i n a c t i v e N-acy lurea . A t a c t i c used to reduce the amount of N-acylurea i s the a d d i t i o n of N-hydroxy compounds such as 1-hydroxybenzo- t r i a z o l e ( H O B t ) 1 3 7 (77) and 3 - h y d r o x y - 4 - o x o - 3 , 4 - d i h y d r o - l , 2 , 3 - b e n z o t r i a z i n e (78).138 These reagents are used p r i m a r i l y f o r the suppression of racemiza- At the present s ta te of development, there are many d e f i c i e n c i e s OH (77) (78) t ion in peptide synthesis with DCC. They combine r a p i d l y with the carboxyl component to form h igh ly r e a c t i v e es ters which in turn couple with the amino component. Formation of a complex between the amino component and 105 the HOBt a c t i v e es te r was proposed to expla in the proper t ies of these a d d i t i v e s 1 3 9 (Scheme 3 . 9 ) . 0 R-C-OH + OH 0 DCC n " J\lx R-C-O-N *N R'NH, 5 0 R - C RCONHR1 + HOBt Scheme 3. 9 Amide Formation with DCC/HOBt N-hydroxy a d d i t i v e s are good nuc leophi les and they compete with the amino component f o r the acyl group of the 0 - a c y l i s o u r e a intermediate to form a c t i v e e s t e r s . Furthermore, the presence of the amine and of the a d d i t i v e r e s u l t s in a higher concentra t ion of n u c l e o p h i l e s , thus reducing the l i f e t i m e of the 0 - a c y l i s o u r e a , and hence decreasing the p r o b a b i l i t y of in t ramolecu lar rearrangement. S ing le coupl ing reac t ions were attempted with the HOBt add i t i ve f o r both s o l i d - p h a s e and s o l u t i o n r e a c t i o n s . In both c a s e s , r e s u l t s were i n c o n c l u s i v e and no d e f i n i t e improvement in coupl ing y i e l d s could be demonstrated. The best coupl ing y i e l d s were obtained when isobuty l chloroformate was used to a c t i v a t e the immobil ized amino a c i d . This suggested that EEDQ and IIDQ ( c f . Sect ion 2.4.2) which a l s o react v i a mixed-anhydride i n t e r - mediates should a lso be s u c c e s s f u l . Thus, Yajima and c o - w o r k e r s 9 0 obtained near ly q u a n t i t a t i v e coupl ing of a hexapeptide with H - G l y - A l a - P s after~43 hours r e a c t i o n with IIDQ in DMF. Good coupl ing y i e l d s with EEDQ were a lso obtained by Sipos and Gaston in s o l i d - p h a s e peptide s y n t h e s i s . 1 4 0 106 The N - e t h y l - 5 ' - p h e n y l i s o x a z o l i u m - 3 1 - s u l f o n a t e (NEPIS), (c f . Sect ion 2.4.1) coupl ing reagent was s u c c e s s f u l l y app l ied to the synthesis of TAP d e r i v a t i v e s under convent ional l i q u i d - p h a s e c o n d i t i o n s . However, t h i s reagent has a t t rac ted very l i t t l e a t ten t ion f o r s o l i d - p h a s e r e a c t i o n s , and i t would be worthwhile to i n v e s t i g a t e i t s e f f i c i e n c y in s o l i d - p h a s e TAP s y n t h e s i s . Of the other reagents used in t h i s work f o r coupl ing hydra laz ine with N-protected amino ac ids in homogeneous s o l u t i o n , only the " o x i d a t i o n - reduct ion" reagents of llukaiyama et al. ( c f . Sect ion 2.4 .3) appear to have been used in s o l i d - p h a s e s y n t h e s i s . This method i s p a r t i c u l a r l y re levant to our s tudies s ince there are very few examples in the l i t e r a t u r e f o r carboxyl a c t i v a t i o n of an amino ac id or peptide attached to a support at i t s amino group. These s tudies in peptide synthesis by chain e longat ion from the N-terminal amino a c i d use isobutoxy mixed anhydride (from isobuty l c h l o r o f o r m a t e ) 1 0 7 and a z i d e 1 1 6 in termediates . Matsueda and co-workers succeeded in syn thes iz ing porcine l u t e i n i z i n g hormone-releasing hormone (LH-RH, 10 res idues) by coupl ing three fragments with excess 2 , 2 ' - d i t h i o - d i p y r i d i n e and t r i p h e n y l p h o s p h i n e . 1 4 1 Th is synthes is was accomplished without the necess i ty of pro tec t ing the s ide chains of t ryptophan, g lutamine, h i s t i d i n e , t y r o s i n e and s e r i n e . In a fu r ther demonstration of the u t i l i t y of the o x i d a t i o n - r e d u c t i o n method, a 24 residue segment of a d r e n o c o r t i c o - t r o p i c (ACTH) was synthesized in good y i e l d by e longat ion from the N-terminus in f i v e fragment coupl ing s t e p s . 1 4 2 The s u p e r i o r i t y of the o x i d a t i o n - r e d u c t i o n process c e r t a i n l y l i e s in the advantage i t possesses of minimizing s i d e - r e a c t i o n s . By comparison the carbodi imide method, which i s by f a r the most widely used in s o l i d - p h a s e peptide s y n t h e s i s , i s s u s c e p t i b l e to (a) N-acylurea format ion , and 107 (b) n i t r i l e formation from s i d e - c h a i n carboxamide groups during a c t i v a t i o n of glutamine or asparagine d e r i v a t i v e s . While we were unsuccessful with the o x i d a t i o n - r e d u c t i o n method f o r synthes is of TAP d e r i v a t i v e s in s o l u t i o n , perhaps t h i s was due to the experimental condi t ions used, and not to the method i t s e l f . The o x i d a t i o n - reduct ion method c e r t a i n l y deserves some cons idera t ion f o r the s o l i d - p h a s e coupl ing of hydra laz ine with immobilized amino a c i d s . We have shown new avenues f o r fu r ther development of our s o l i d - phase s t u d i e s , but with the present system, a modicum of changes in the reac t ion cond i t ions can br ing about s i g n i f i c a n t improvements in the r e a c t i o n e f f i c i e n c i e s observed. The f i r s t change which suggests i t s e l f i s the use of f ree base hydra laz ine ra ther than hydra laz ine HCI with t r i e t h y l a m i n e . The hydrochlor ide s a l t requi res polar so lvents f o r d i s s o l u t i o n whereas p o l y - styrene-based supports are most e f f i c i e n t l y swelled by non-polar s o l v e n t s . With f resh ly -p repared hydra laz ine non-polar so lvents such as methylene c h l o r i d e can be used. In a d d i t i o n , hydra laz ine decomposes r a p i d l y in the presence of excess Et^N. This i s e s p e c i a l l y a problem when the d i s s o l u t i o n of hydra laz ine HCI i s slow, and there i s a low concentrat ion of hydra laz ine in s o l u t i o n r e l a t i v e to Et^N. If hydra laz ine decomposit ion i s ex tens ive , any of the decomposition products conta in ing primary or secondary amine groups may couple with the amino ac id to give undesired s i d e - p r o d u c t s . Our observat ion o f a ye l low co lour in the r e s i n during coupl ing reac t ions may be evidence of t h i s s i d e - r e a c t i o n . Several approaches are a v a i l a b l e f o r improving the coupl ing e f f i c i e n c y of any given coupl ing method. Reaction times of 15-18 hours were genera l l y used in these s t u d i e s . Even these extended r e a c t i o n times may be i n s u f f i c i e n t . In s o l i d - p h a s e peptide s y n t h e s i s , the standard 108 reac t ion time i s two hours. However, the a p p l i c a t i o n of i n c r e a s i n g l y longer reac t ion times has been a not iceab le t rend . In some c a s e s , reac t ion times have been extended to 24 hours or g r e a t e r . 1 2 1 One of the i n t r i n s i c problems with the s o l i d - p h a s e procedure i s that the phase separat ion may o f ten r e s u l t in slower r e a c t i o n . A p p l i c a t i o n of l a rger excesses of hydra laz ine and 'coupl ing reagent would seem to be an obvious remedy f o r incomplete c o u p l i n g . We have genera l ly used a two- to t h r e e - f o l d excess of these reagents in the coupl ing r e a c t i o n . In a s o l i d - p h a s e synthesis of cytochrome C, Sano and Kuihara used amounts of 30- to 7 0 - f o l d excess of reagents in a rout ine manner through the f i n a l eighteen steps of t h e i r s y n t h e s i s . 1 4 3 In these c a s e s , excesses of reagents would be f i l t e r e d o f f and reused. The r e a c t i v i t y of any peptide carboxylate group i s somewhat dependent on the nature of the amino ac id s i d e - c h a i n and the adjacent amino a c i d sequence. Th is v a r i a b i l i t y in r e a c t i v i t y advises aga ins t a standard reac t ion time fo r hydra laz ine coupl ing with amino ac ids and pept ides . There must there fore be some means of monitor ing the progress of the r e a c t i o n . The d i f f i c u l t i e s assoc ia ted with a p p l i c a t i o n of i n f r a - r e d spectroscopy to polymeric mater ia ls have been p rev ious ly d iscussed (vide supra). F e a s i b l e a l t e r n a t i v e s inc lude cleavage of the peptide from the support and subject ing i t to amino a c i d a n a l y s i s to determine the extent of TAP m o d i f i c a t i o n . The amount of TAP present in a hydrolyzate of the peptide under study may a lso be determined by q u a n t i t a t i v e f luorescence a n a l y s i s of the TLC i s o l a t e d product . The extent o f peptide m o d i f i c a t i o n by hydra laz ine can a l s o be monitored by microana lys is of the peptide r e s i n . However, low s u b s t i t u t i o n of pept ide on the suppor t , and the presence of the support i t s e l f , can make th is a somewhat i n s e n s i t i v e method. 109 If the so l id -phase method is to be used as a purely syn the t ic procedure, then a cleavage of the benzyl es te r bond i s required by which the TAP d e r i v a t i v e i s attached to the support . We have found that s c i s s i o n of th is bond i s s a t i s f a c t o r y under a c i d i c condi t ions with HBr/HOAc. However, i f se r ine and threonine are present in the peptide c h a i n , the hydroxyl groups may be ace ty la ted under these c o n d i t i o n s . 1 1 8 Th is s i d e - reac t ion is avoided i f a c e t i c ac id i s subs t i tu ted by anhydrous t r i f l u o r o - a c e t i c a c i d , but extended reac t ion times may be requi red s ince we observed incomplete cleavage with HBr/TFA. A l t e r n a t i v e l y , anhydrous, l i q u i d hydrogen f l u o r i d e may be u s e d . 1 1 8 Peptide sequencing from the carboxyl terminus by the s o l i d - p h a s e method requ i res attachment of the N-terminal amino a c i d to the support at the a-amino group. A f requent ly used method f o r producing peptides from prote ins i s treatment of the prote in with t r y p s i n . This enzyme cleaves peptide bonds at the C-end of l y s i n e and a r g i n i n e . A s i g n i f i c a n t number of peptides used in peptide sequencing may therefore contain l y s i n e . If such a peptide i s attached to a support , binding w i l l occur at the N-terminus (a-amino group) and at the C-terminus (e-amino group) . C-terminal sequenc- ing of the peptides thus anchored w i l l show gaps in the amino ac id sequence at the N-terminus and at l y s i n e s s ince these res idues w i l l remain bound to the r e s i n . The i r i d e n t i t i e s may be deduced from the d i f f e r e n c e between the amino ac id composit ion of the peptide and the TAP d e r i v a t i v e s de tec ted , or by h y d r o l y s i s and a n a l y s i s of the support a f t e r degradat ion. I f bas ic amino ac ids are present in the p e p t i d e s , attachment of the peptide at the amino s i d e - c h a i n s may be prevented by car ry ing out one step of Edman N-terminal degradation on the f ree p e p t i d e . 1 1 4 This procedure protects e-amino groups of l y s i n e as t h e i r phenylthiocarbamyl (PTC) d e r i v a - no t i v e , and at the same time permits i d e n t i f i c a t i o n of the N-terminal res idue . A f t e r t h i s s t e p , the a-amino group of the second residue i s the only amino group f ree f o r attachment to the support . A p o s s i b l e compl icat ion in C-terminal residue a n a l y s i s i s the reac t ion of hydra laz ine with s i d e - c h a i n carboxyl groups of a s p a r t i c and glutamic a c i d s . Th is s i d e - r e a c t i o n may create d i f f i c u l t i e s where the hydra laz ine m o d i f i c a t i o n reac t ion is used f o r C-terminal determinat ion o n l y . Tota l h y d r o l y s i s of the modi f ied peptide w i l l y i e l d TAP d e r i v a t i v e s from both the C-terminal amino ac id and in te rna l amino a c i d s . The s i t u a t i o n may be more complex in the case of a s p a r t i c a c i d ; the 3-carboxyl group a f t e r a c t i v a t i o n by coupl ing reagent can react with a neighbouring amide ni t rogen to form a B-lactam which may stop peptide degradation complete ly . Several s o l u t i o n s to the s i d e - c h a i n carboxyl group problem are p o s s i b l e . Prev iero et al.ll*k have shown that carbodi imides can e f f e c t both p ro tec t ion of s i d e - c h a i n carboxyl groups and a c t i v a t i o n of the C-terminus under s u i t a b l e experimental cond i t ions (Scheme 3.10) . Peptides conta in ing a carboxyl group only at the C-terminal reach a degree of a c t i v a t i o n which remains constant as a func t ion of t ime, while peptides conta in ing a s i d e - chain carboxyl show an i n i t i a l maximum of a c t i v a t i o n which subsequently decreases . Thus, a c i d i c peptides which were incubated with l - e t h y l - 3 - dimethylaminopropyl carbodi imide hydrochlor ide (EDC) f o r 90 minutes a t 40°C in the absence of nuc leoph i les before reac t ing with the amino component showed coupl ing with only the C-terminal amino a c i d . During the incubat ion p e r i o d , the 0 - a c y l i s o u r e a intermediate isomerizes to the i n e r t N-acylurea d e r i v a t i v e at the s i d e - c h a i n c a r b o x y l , whi le the C-terminus remains ac t i va ted as an oxazol inone which subsequently reacts with the amino component. I l l 0 O R ' P -NH-CH-C-peptide-C-NH-CH-COOH COOH RN=C=NR 7NR 0 n 0-(/ " // I NHR P - N H - C H - C - p e p t i d e - C 7 C=0 I N n K S i I I RN=C-0-C=0 NHR HN—CHR 1 S / O v xO ,NHR P -NH-CH-C-pept ide-C C / + 0=C 0 ( C H 2 L HN-C-N-C=0 N—CHR' NHR R R R"NH, 0 - 0 0 P s -NH-CH-C-pept ide-C-NH-CHR'-C-NHR" HN-C-N-C=0 i i R R Scheme 3.10 S e l e c t i v e Amide Formation at the C-Terminus of Peptides 112 A d i f f e r e n t approach to t h i s problem i s to e s t e r i f y a l l the carboxyl groups in the pept ide , and then to l i b e r a t e the C-terminal a -carboxyl group with t r y p s i n , which can act as a f a i r l y s p e c i f i c es te rase . Mross and D o o l i t t l e have suggested convert ing peptide carboxyl groups to amides and then c leav ing the C-terminal amide s e l e c t i v e l y with t r y p s i n . 1 4 5 3.4 SUMMARY We have inves t iga ted the reac t ion of hydra laz ine with amino ac ids attached to i n s o l u b l e supports under a v a r i e t y of c o n d i t i o n s . Using a copolystyrene-1% div iny lbenzene matrix subs t i tu ted with methylchloroformyl groups, amino ac id and peptide esters can be attached to the support v i a t h e i r a-amino groups. The immobil ized amino ac ids react with hydra laz ine to form the s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (TAP) d e r i v a t i v e s when isobuty l ch loroformate , l - e t h o x y c a r b o n y l - 2 - e t h o x y - l , 2 - d i h y d r o q u i n o l i n e (EEDQ), and d icyc lohexy lcarbod i imide (DCC) are used f o r carboxyl a c t i v a t i o n . The TAP d e r i v a t i v e s may be c leaved from the r e s i n under a c i d i c cond i t ions with HBr-HOAc. The work on s o l i d - p h a s e react ions i s s t i l l in a pre l iminary s ta te of development, but we have demonstrated the f e a s i b i l i t y of using the s o l i d - phase method f o r the synthesis of TAP d e r i v a t i v e s . Under the present reac t ion c o n d i t i o n s , the products of the coupl ing r e a c t i o n are contaminated with unreacted amino a c i d s , and they requi re chromatographic techniques f o r p u r i f i c a t i o n . The coupl ing reac t ion of hydra laz ine with amino ac ids i s not ye t p r a c t i c a l f o r so l id -phase C-terminal sequencing of pept ides because of the unacceptably low coupl ing y i e l d s . 113 CHAPTER 4 TRANSITION METAL COMPLEXES WITH THE S-TRIAZ0L0[3,4-A]PHTHALAZINE LIGAND 4.1 INTRODUCTION Perhaps the most ser ious l i m i t a t i o n which prevents the extension of most peptide C-terminal amino ac id determinat ion methods to sequent ia l a n a l y s i s i s a s a t i s f a c t o r y method of s e l e c t i v e l y and q u a n t i t a t i v e l y hydro lyz ing the terminal peptide bond under mild c o n d i t i o n s . In the thiocyanate method of S t a r k , 2 1 * 2 2 (Scheme 1.2) the th iohydantoin group which i s formed from the C-terminal amino a c i d i s cleaved by n u c l e o p h i l i c c a t a l y s i s with acetohydroxamate, or ac id c a t a l y s i s with 12M HCI. The use of h igh ly a c i d i c condi t ions f o r cleavage of the th iohydan- to in group i s a r i s k y p r o p o s i t i o n , and cleavage of in terna l peptide bonds i s a l s o l i k e l y . The p r o b a b i l i t y of t h i s occurr ing prompted Y a m a s h i t a 1 4 6 to look f o r a m i l d e r , more s e l e c t i v e cleavage method. He found that shaking the pept idy l th iohydanto in with the a c i d i c form of a cat ion-exchange r e s i n was success fu l in hydrolyz ing the bond between the peptide and t h i o h y d a n t o i n , but not the other peptide bonds. With t h i s m o d i f i c a t i o n , Yamashita was able to determine the amino acid sequence up to around 10 res idues from the C-termini of p o l y p e p t i d e s . 1 4 7 . 114 The very nature of the h y d r a z i n o l y s i s method 1 5 precludes i t s use as a C-terminal sequent ia l procedure s ince on treatment of peptides with hydraz ine , the peptide chain i s s p l i t and a l l the in terna l amino ac ids are converted to hydraz ides , except the C-terminal amino ac id which remains as the f ree amino a c i d . The t r i t i a t i o n method o f Matsuo et al.12 (Scheme 1.3) and the reduct ion method of B a i l e y 2 6 (Scheme 1.4) r e l y on concentrated h y d r o c h l o r i c ac id to hydrolyze the terminal peptide bond. These methods s u f f e r from the same l i m i t a t i o n s as the Stark method. In the method descr ibed by Loudon and c o - w o r k e r s 2 8 (Scheme 1.5) the N-aminomethylamide formed from the C-terminal residue degrades under h y d r o l y t i c cond i t ions (6N HC'i) to the peptide-amide which prevents f u r t h e r C-terminal amino ac id ana lys is of the pept ide . In another C-terminal amino ac id a n a l y t i c a l method, Maekawa and Kumano 1 4 8 converted peptides to the corresponding p e p t i d e - t r i a z i n e s by react ing the carboxyl groups with d imethylb iguanid ine . However, in the absence of a m i l d , s e l e c t i v e method f o r c leav ing the terminal bond they resorted to t o t a l hydro lys is of the peptide with Streptomyoes griseus protease, and i d e n t i f i c a t i o n of the modif ied C-terminal d e r i v a t i v e in the hydro lyzate . NMe2 HN< peptide-NH-CHR-COOH + NH p e p t i d e - N H - C H R - r ^ N V ' 2 HN=< J. U. NH 2 N ^ N N H 2 From the above e x p o s i t i o n , i t i s c l e a r that the lack of a s u i t a b l e method of hydro lyz ing the C-terminal peptide bond is an impediment 115 which i s common to most of the C-terminal methods. The approach which we considered was the meta l - ion a s s i s t e d h y d r o l y s i s of the peptide bond. The r o l e that metal ions can play in c a t a l y z i n g the h y d r o l y s i s of peptide bonds was long u t i l i z e d by Nature in incorpora t ing a z inc ( I I ) ion in to the a c t i v e s i t e of carboxypeptidase A (CPA). This enzyme, as i t s name i m p l i e s , ca ta lyzes the h y d r o l y s i s of the peptide bonds at the carboxyl end of a polypept ide s u b s t r a t e . The z inc l igands have been i d e n t i f i e d as two h i s t i d i n e s , glutamic a c i d , and water in d i s t o r t e d te t rahedra l c o o r d i n a t i o n ! 4 9 In the presence of a peptide s u b s t r a t e , the carbonyl group of the C-terminal peptide bond d i s p l a c e s the water l igand from the z i n c ion in the complex. A nearby a rg in ine s i d e - c h a i n binds the terminal carboxyl group present in the s u b s t r a t e . A p o s s i b l e mechanism f o r the ac t ion of CPA given by Lipscomb and c o - w o r k e r s , 1 5 0 i s shown in Scheme 4 . 1 . H i s ^ 6 9 H i s - 1 9 6 — Z n — G l u ^ 7 2 N H G l u - 2 7 0 H i s ^ 9 H i s - 1 S 6 — Z n — G l i / 7 2 H O^-hQ) - T y r - 2 4 8 C H 2 V l H . G l u - 2 7 0 Scheme 4.1 Poss ib le Mechanism f o r the Carboxypeptidase A- Catalyzed Hydro lys is of G l y c y l - L - t y r o s i n e 116 Lipscomb has suggested that coord ina t ion of the metal ion by the carbonyl oxygen atom of the substrate r e s u l t s in p o l a r i z a t i o n of the C=0 bond of the carbonyl group, render ing the carbon atom more s u s c e p t i b l e to n u c l e o p h i l i c a t tack . In a d d i t i o n to the ' z i n c - c a r b o n y l 1 mechanism, the ' z i n c - h y d r o x i d e ' mechanism can a lso be cons idered , in which a z inc-bound hydroxide ion acts as a n u c l e o p h i l e . The two mechanisms are i l l u s t r a t e d in F igure 4 . 1 . S t ruc tu ra l evidence from X-ray studies however, favors the z i n c - c a r b o n y l mechanism. H—0 > R—d-^-NH—R' II o 0 H _ 270 OH lyr 248 V to) R—C—NH—R Co H p~H H 0—| glu 270 lyr 248 (b) Figure 4.1 Diagrammatic I l l u s t r a t i o n of the Mechanism of Carboxypeptidase- Catalysed Peptide H y d r o l y s i s : (a) Zn-hydroxide Mechanism and (b) Zn-carbonyl Mechanism The e f f e c t of metal ions on the h y d r o l y s i s of amide bonds has been known f o r over twenty y e a r s . Lawrence and M o o r e 1 5 1 found that C o C ^ almost doubled the rate of ac id h y d r o l y s i s of g l y c y l g y c i n e . Meriwether and W e s t h e i m e r 1 5 2 examined the e f f e c t s of copper ( I I ) , c o b a l t ( I I ) , and n i c k e l ( I I ) ions on the hydro lys is of glycinamide and pheny la lany lg lyc inamide . Copper ( l l ) ions were the most e f f e c t i v e c a t a l y s t s , and between pH 7.9 and 9.25 at 7 5 ° C , they increased the rate of h y d r o l y s i s of glycinamide by a f a c t o r of t h i r t y over the uncatalyzed h y d r o l y s i s . The mechanism of these h y d r o l y s i s reac t ions i s not ye t e s t a b l i s h e d , but i t i s l i k e l y that the c a t a l y t i c a l l y a c t i v e spec ies i s the carbonyl-bonded complex. 117 The copper ( I I ) - ca ta lyzed hydro lys is of g l y c y l g l y c i n e a t t a i n s a maximum rate at pH 4 . 2 . 1 5 3 The decrease in rate at higher pH values is assoc ia ted with the formation of a c a t a l y t i c a l l y i n a c t i v e complex produced by i o n i z a t i o n of the peptide hydrogen atom (Figure 4 . 2 ) . 1 5 4 R U • M-^ C NH, I + H1 CH, I NH2 act ive inactive Figure 4.2 Ion izat ion of Peptide Amide Hydrogen Extensive work, mainly by the Buckingham and Sargeson groups in Canberra , with k i n e t i c a l l y i n e r t c o b a l t ( I I l ) complexes has g r e a t l y c l a r i f i e d the mechanist ic pathways in m e t a l - a s s i s t e d amide h y d r o l y s i s . It has been found that a number of complexes of the type [CoL 4(()H) (H 2 0)] = 2en, t r i e n , t r e n , edda, eee) s t o i c h i o m e t r i c a l l y and s p e c i f i c a l l y c leave the N-terminal amino a c i d from p e p t i d e s . 1 5 5 , 1 5 9 Buckingham et al.155 were the f i r s t to demonstrate the s e l e c t i v e N-terminal h y d r o l y s i s of simple peptides by s t o i c h i o m e t r i c reac t ion with the g - [Co( t r ien)0H(H 2 0) j i o n . The proposed mechanisms f o r the h y d r o l y s i s are shown in Scheme 4 .2 . The ra te determining step invo lves the replacement of a coordinated water molecule by the terminal amino group of the pept ide . Then e i t h e r the carbonyl group i s attacked by the adjacent coordinated hydroxide group (path A) or the carbonyl group becomes ac t iva ted to attack by external t en = ethylenediamine, t r i e n = t r i e t h y l e n e t e t r a m i n e , t ren = 2 , 2 ' , 2 " - t r i - . aminotr ie thylamine, edda = e thy lened iamine -d iace ta to , eee = 1,8-diamino- 3 ,6 -d i th iaoc tane 118 i fast N H C H j C O - P e p [Coltnerrt AAJ [Cof t r ienXHptOH] 2 * + N H 2 C H / ; O N H C H p D - P e p NH j fast 0 = 0̂  \ N H j C O - P e p Scheme 4.2 Proposed Mechanisms f o r Peptide Hydro lys is hydroxide through p r i o r coord ina t ion of the carbonyl oxygen (path B) . These mechanisms are analogous to the " z i n c - c a r b o n y l " and "z inc -hydrox ide" mechanisms proposed f o r carboxypeptidase A. Buckingham, Sargeson, and t h e i r c o l l a b o r a t o r s have shown that both of these mechanisms cont r ibu te s i g n i f i - can t ly to the h y d r o l y s i s reac t ions although the exact extent of t h e i r c o n t r i b u t i o n may depend on the reac t ion condi t ions e m p l o y e d . 1 5 0 } 1 6 1 d i r e c t p o l a r i z a t i o n of the coordinated carbonyl func t ion by metal ion (carbonyl mechanism) i s l O 4 - 10 5 over that found in the absence of the meta l . Intramolecular at tack of bound hydroxide (hydroxide mechanism), in the absence of buf fers i s somewhat l e s s e f f e c t i v e at pH 7 ( l C ^ - l O 4 ) , but buf fers ( e . g . HPO^ ) r e s u l t in a tremendous rate enhancement, 1 0 1 0 - 1 0 n a t pH7. Under s l i g h t l y more a c i d i c condi t ions (pH 4-5) where the bound aquo group i s i n v o l v e d , there is a s i m i l a r ra te increase ( 1 0 1 1 ) . Such rates match, or exceed, the turn -over number found in carboxypeptidase and t r y p s i n The a c c e l e r a t i o n of amide and peptide h y d r o l y s i s provided by U9 proteases under i d e n t i c a l condi t ions of pH and t e m p e r a t u r e . 1 6 2 Ci s-B-coba11(111) complexes are -appea l ing as c a t a l y s t s fo r N-terminal peptide sequencing because of the amino ac id s p e c i f i c i t y and the large rate enhancement e f f e c t . Indeed, the h y d r o l y s i s of peptides and 2+ prote ins by c i s - B - [ C o ( t r i e n ) 0 H ( H 2 0 ) ] has been developed into an N-terminal amino a c i d determinat ion and peptide sequencing method by several w o r k e r s . 1 6 3 - 1 6 7 Comparisons of the e f f i c i e n c y of var ious c i s - [ C o L 4 ( 0 H ) ( H 2 0 ) ] species ind ica ted the order of La f o r peptide h y d r o l y s i s : 1 6 4 t r i e n > t ren > 2en In a comparison of t r i e n and edda, Oh and S t o r m 1 5 8 found that the rates of h y d r o l y s i s of d ipept ides were somewhat slower f o r edda complexes, but that there was a smal ler ra te v a r i a t i o n between d i f f e r e n t pept ides . The success of coba l t ( I I I ) complexes in s e l e c t i v e l y promoting the h y d r o l y s i s of peptide bonds prompted us to consider the f e a s i b i l i t y of using metal complexes to f a c i l i t a t e the h y d r o l y s i s of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e modif ied pept ides . The o v e r a l l scheme envisaged f o r pept ide sequencing with hydra laz ine and coba l t ( I I I ) complexes i s shown in Scheme 4 .3 . In t h i s scheme, we a n t i c i p a t e displacement of the aquo and hydroxo 2+ l igands in c i s - B - [ C o ( t r i e n ) 0 H ( H 2 0 ) ] by the l ^ - n i t r o g e n of the TAP l igand and the deprotonated amide ni t rogen of the C-terrninal peptide bond. Ac id h y d r o l y s i s of the e x o c y c l i c imide bond should l i b e r a t e the peptide minus the C-terminal r e s i d u e . 120 Scheme 4 .3 . Proposed Peptide Sequencing with Pept idy l -TAP and Cobal t ( I I I ) Complexes There are two l i k e l y s i t e s in the TAP group at which the TAP- peptide can coordinate to the matal i o n : N-2 of the t r i a z o l e r i n g ( I ) , or N-5 of the phthalaz ine r ing ( I I ) . CPK s p a c e - f i l l i n g models of both c o o r d i n a - t ion p o s s i b i l i t i e s show that the t -o complexes are f e a s i b l e and there is no undue s t e r i c s t r a i n i n v o l v e d . A l k y l a t i o n of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e s give only the N-2 a l k y l p r o d u c t 4 5 i n d i c a t i n g that N-2 i s the most bas ic ni t rogen atom and hence the most l i k e l y to form a strong coordinate bond. 121 Since our envisaged scheme f o r peptide sequencing involves metal complexes with TAP l i g a n d s , we saw our f i r s t task as studying the c o o r d i n a - t ion proper t ies of the TAP l igand s ince these are t o t a l l y unknown. The next p r i o r i t y was then to prepare metal complexes with a s u i t a b l e TAP d e r i v a - t i v e to determine whether the presence of the metal ion a c t u a l l y a ided i n hydrolyz ing amide bonds in the TAP s i d e - c h a i n . 4.2 RESULTS The fo l lowing t r a n s i t i o n metal complexes conta in ing the s - t r i a z o l o - [3 ,4 -a ]phtha laz ine (3-H-TAP) l igand were synthes ized: [M (3 -H -TAP) n (H 2 0 ) 6 _ n ] ( C 1 0 4 ) 2 (n = 4, M = Co, N i , Cu ; n = 2, M = Ni) and [Co(3-H-TAP) 6 ] ( C 1 0 4 ) 3 . [Co(3-H-TAP)g] ( C 1 0 4 ) 3 was prepared from a 6:1 molar mixture of s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e and sodium t r i s ( c a r b o n a t o ) c o b a l t a t e ( I I I ) i n an ethanol suspension conta in ing d i l u t e p e r c h l o r i c a c i d . N a 3 [ C o ( C 0 3 ) 3 ] i s a convenient intermediate f o r the synthesis of coba l t ( I I I ) complexes s ince i t avoids the d i f f i c u l t i e s assoc ia ted with other methods of synthes is which involve in situ ox idat ion of coba l t ( I I ) to c o b a l t ( I I I ) . 1 6 8 The hexakis- ( s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e ) c o b a l t ( I I I ) perchlorate complex i s formed as a l i g h t brown amorphous s o l i d . 6 3+ The octahedral s p i n - p a i r e d d Co ion is s u i t a b l e f o r nmr studies of the coordinated l igand without in ter fe rences from paramagnetism of the metal i o n . [Co(3-H-TAP)g] ( C 1 0 4 ) 3 was synthesized p r i m a r i l y f o r nmr s t u d i e s . On coord ina t ion of 3-H-TAP, per turbat ion by the metal ion of the e lec t ron dens i ty about the protons^is expected to be most evident in the chemical s h i f t s of protons adjacent to the locus of coord ina t ion on the l i g a n d . Thus, in 3-H-TAP, coord ina t ion of metal to N-2 should desh ie ld the proton at the C-3 p o s i t i o n which would show up in a s h i f t of the nmr resonance to lower f i e l d . There may be a smal ler d e s h i e l d i n g e f f e c t at other p o s i t i o n s a r i s i n g from an induct ive e f f e c t . ^ - n m r s tud ies of [Co(3 -H-TAP)g] (C10 4 ) 3 are hindered by i t s low s o l u b i l i t y in s u i t a b l e organic s o l v e n t s . A Four ie r - t rans fo rm nmr spectrum of a d imethy lsu l fox ide -dg s o l u t i o n of the complex is shown in F igure 4 . 3 . It i s i d e n t i c a l in appearance with that of f ree 3-H-TAP. The sharp s i n g l e t at lowest f i e l d i s assigned to H-3 and the adjacent sharp resonance at h igher f i e l d a r i s e s from H-6. The chemical s h i f t s of s e l e c t e d resonances of f r e e , complexed, and protonated 3-H-TAP are l i s t e d in Table 4 . 1 . Table 4.1 NMR Spectra l Data f o r s -T r iazo lo [3 , . 4 -a ]ph tha laz ine in DMS0-d c Chemical S h i f t s , ppm b a 1 0 7 . 8 , 9 3-H-TAP 9.58 9.07 8.53 8.44 8.06 3-H-TAP.DC! 9.80 9.21 8.68 8.60 8.14 3-H-TAP.HCl 9.71 9.17 8.63 8.56 8.15 [ C o ( 3 - H - T A P ) f i ] 3 + 9.77 9.16 8.54 8.46 8.12 a Chemical s h i f t s f o r 6 and 6 are given f o r resonances ind ica ted 1 0 7 > 8 > 9 in F igure 4 .3 . b Doublet with superimposed f i n e s t r u c t u r e . o M u l t i p l e t 123 Figure 4.3 100 MHz. Four ie r - t rans form NMR Spectrum of . [ C o ( 3 - H - T A P ) 6 ] ( C 1 0 4 ) 2 in DMS0-d g 124 It i s apparent from Table 4.1 that the greatest change in chemical s h i f t s on.complexation and protonat ion of 3-H-TAP occurs f o r H-3. Th is suggests that complexation and protonat ion do occur at the N-2 s i t e . We cannot s tate unequivocal ly that coord inat ion d id not occur at N-5, but the small s h i f t of 6 6 to lower f i e l d ind ica tes that t h i s is not very s i g n i f i c a n t . Small changes in the chemical s h i f t s may a r i s e from an induct ive or concen- t r a t i o n e f f e c t . [ C o ( 3 - H - T A P ) 6 ] ( C 1 0 4 ) 3 i s not completely s tab le in DMSO or methanol so lu t ion s ince f ree l igand c r y s t a l l i z e s from a saturated s o l u t i o n of the complex . The d i s s o c i a t i o n of l igands i s not evident in nmr spect ra of the complex which were obtained wi th in one hour of s o l u t i o n prepara t ion . For a s p i n - p a i r e d d^ ion in an octahedral f i e l d , two e l e c t r o n i c t r a n s i t i o n s are expected: -<- and ^ T ^ ^ i g - The d i f f u s e re f l ec tance spectrum of [Co(3 -H-TAP)g] (C10 4 ) 3 i s shown in F igure 4 .4 . Only one d-d t r a n s i t i o n i s resolved in the v i s i b l e spectrum, and t h i s i s super- imposed on an intense background. A nujol mull absorpt ion spectrum of the complex has the same appearance as the d i f f u s e re f l ec tance spectrum. The band at 17.1 kK (584 nm) i s assigned to the H-j ^ A ^ t r a n s i t i o n . An i n d i c a t i o n of the higher energy t r a n s i t i o n is apparent in F igure 4 . 4 , but an estimate of the energy of band i s not poss ib le due to the intense background 3+ absorpt ion . For comparison, the absorpt ion spectrum of the [Co(en) 3 ] ion in aqueous s o l u t i o n shows bands at 21.4 kK (467 nm) and 29.4 kK (340 n m ) . 1 6 9 The tetraaquobis(3-H-TAP) and d iaquotetrak is(3-H-TAP) complexes were a l l prepared by the same procedure. The hexaquo metal( I I ) perch lora tes (metal = c o b a l t , n i c k e l , copper) were dehydrated with 2 ,2 -d imethoxypropane 1 7 0 according to the equat ion: ( C H 3 0 ) 2 C ( C H 3 ) 2 + H 20 2CH30H + CH 3 C0CH 3 350 400 450 500 550 600 650 700 Wavelength (nm) Figure 4.4 D i f fuse .Re f lec tance Spectrum of [Co(3-H-TAP) , . ] (CIO, ) q 126 The s o l u t i o n s of the so lvated metal perch lora tes thus obtained were used d i r e c t l y f o r react ion with the s t o i c h i o m e t r i c amount of 3-H-TAP. Infrared spectra of the 3-H-TAP complexes are shown in Figure 4 .5 . In g e n e r a l , the i n f r a r e d spect ra of the complexes are not p a r t i c u l a r l y d i f f e r e n t from those of the f ree s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (a l lowing f o r the peaks due to v/ater and the an ion) . However, some of the TAP bands change i n t e n s i t i e s on coord ina t ion or are s p l i t . A number of the v i b r a t i o n a l modes of TAP show s i g n i f i c a n t s h i f t s to higher f r e q u e n c i e s , and these may be c h a r a c t e r i s t i c of coord ina t ion of the TAP l i g a n d . These bands are the C-H s t r e t c h i n g modes oa. 3120-3030 cm"'' , and a l s o the bands at 1530, 1476, the doublet 1356 and 1352, 1184, 776, and 625 c m " 1 . The two absorpt ion bands between 3600 and 3400 may be a t t r i b u t e d • to the antisymmetric and symmetric 0-H s t re tch ing modes of coordinated and l a t t i c e - b o u n d water. A n a l y t i c a l evidence supports the presence of l a t t i c e water in a l l of the complexes except [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . The broad band about 1640-1600 c m - 1 may be assigned to the H0H bending mode. Other v i b r a t i o n s a r i s i n g from coordinated water are not v i s i b l e because of the superimposed TAP v i b r a t i o n s . The band s t ruc ture about 630 cm" 1 in [ N i ( 3 - H - T A P ) 2 ( H 2 0 ) 4 ] ( C 1 0 4 ) 2 may a r i s e from overlap of a N i - 0 H 2 wagging v i b r a t i o n on a TAP r ing deformation mode and the CI0^ bending modes of p e r c h l o r a t e . The f ree perch lora te ion possesses te t rahedra l symmetry and belongs to the point group having nine v i b r a t i o n a l degrees of freedom d i s t r i b u t e d between four normal modes of v i b r a t i o n . The assignments of these modes are l i s t e d in Table 4 . 2 . 1 7 1 In the s o l i d s tate i n f r a r e d spectra of p e r c h l o r a t e s , the non- degenerate f requency, v-,, which should be i n f r a r e d i n a c t i v e usua l l y occurs 127 Table 4.2 V i b r a t i o n s of the Perchlorate Group as a Funct ion o f Symmetry State of Anion CIO," Symmetry -0-C10, 3v ' l v 2 E(R) A (R ) . sym.s t r . sym.bend v 2 A-j (r,R). C10*s t r . v 3 F 2 ( I , R ) asym.s t r . v 6 v l £ ( I , R ) A(I ,R) rock sym.s t r . CIO. v 4 E(I ,R) asym.st r . C10 o v 4 F 2 ( I , R ) asym.bend v 3 v 5 A( I ,R) E( I ,R) sym.bend asym.bend CIO, CIO, A , non-degenerate E, doubly degenerate F, t r i p l y degenerate I, i n f ra red ac t ive R, Raman a c t i v e as a weak absorpt ion owing to d i s t o r t i o n o f the ion in a c r y s t a l f i e l d of lower symmetry than i t s e l f . Where the in f ra red spect ra of perchlorates d i f f e r from those of the f ree i o n , one of three f a c t o r s i s u s u a l l y i n v o l v e d . These f a c t o r s are: lowering of the s i t e symmetry o f the a n i o n , per turbat ion of the anion' by water mo lecu les , and coord ina t ion o f the anion to the meta l . The in f ra red data of the perchlorate groups in the complexes are given in Table 4 .3 . Only in the copper(I I ) and the coba l t ( I I I ) complexes do the perch lora te groups show symmetry. In the othef complexes there i s a s l i g h t lowering of symmetry as evidenced by a small s p l i t t i n g of the degenerate v 3 band, and in one case , the band. Agreement of the band Figure 4.5 Nujol Mull Infrared Spectra of 3-H-TAP and Metal Complexes (a) 3-H-TAP, (b ) . [Co(3H-TAP) 6 ] ( .C10 4 ) 3 .H 2 0 Figure 4.5 IR Spectra , (c) [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 , . ( d ) [ N i ( . 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 Figure 4.5 IR Spect ra , . ( e ) [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 , ( f ) [ N i ( 3 - H - T A P ) 2 ( H 2 0 ) 4 ] ( C l 0 4 ) . o 131 Table 4.3 Infrared Spectra of Perchlorate Groups ( c m - 1 ) * v 3 v l v 4 v 2 [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C I 0 4 ) 2 l lOOvsb 93 lw 621s - [ C o ( 3 - H - T A P ) 6 ] ( C 1 0 4 ) 3 1093vsb 950w 622s 448vwb [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 l l l O v s b 1070vsb 929w 623s 446vw [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 l l l O v s b 1070vsb 928w 619s 446vw [ N i ( 3 - H - T A P ) 2 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 l l l O v s b 1080vsb 935 626s 620s , - [ C o ( p y ) 4 ( C 1 0 4 ) 2 ] 1 7 2 1137s 1034s 931m 631m 617m 425w c i o 4 " 1 7 1 1110 932 626 460 * Band assignments in terms of symmetry v s , very strong, s , strong . . m, medium, vw, very weak, w, weak. b, broad. f requencies with those of i o n i c C10 4 ~ i n d i c a t e s that perch lora te i s uncoord i - nated in the complexes s t u d i e d . The lowering of the perch lora te group symmetry from y ^ t o C S y probably a r i s e s from d i s t o r t i o n of the anion by the c r y s t a l l a t t i c e or from i n t e r a c t i o n with water molecules with which the C10 4 ~ ion may form hydrogen-bonds. These e f f e c t s are common in the spectra of metal p e r c h l o r a t e s . 1 7 3 By comparison, in the t e t r a g o n a l l y d i s t o r t e d [ C o ( p y ) 4 ( C l 0 4 ) 2 ] where the perch lora te groups are coord ina ted , there i s c l e a r s p l i t t i n g o f the (103 c m - 1 ) and v 4 (14 c m - 1 ) bands un l ike the complexes studied here which show incomplete r e s o l u t i o n of the s p l i t band, and removal of degeneracy of the v 4 band is observed in only one case . The e l e c t r o n i c spect ra of the b is and te t rak is (3 -H-TAP) complexes in the s o l i d s ta te were obtained by d i f f u s e r e f l e c t a n c e and from nujol mul ls at room temperature. So lu t ion spectra were not obtained because of the l i m i t e d s o l u b i l i t y of the complexes in appropr iate s o l v e n t s . 132 [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 i s obtained as an amorphous tangerine s o l i d . Nujol mull v i s i b l e spectra of the complex are charac te r i zed by a weak, very broad band centered at 10.5 kK (950 nm) and a st ronger asymmetric band at 21.1 kK (475 nm) with a shoulder about 19.1 kK (523 nm), (Figure 4 .6 ) . Three sp in -a l lowed t r a n s i t i o n s are expected f o r h i g h - s p i n d 7 coba l t ( I I ) complexes in an octahedral f i e l d , and the observed absorpt ions in the v i s i b l e spectrum are given the assignments: 4 T 2 g ( F ) <- 4 T l g ( F ) 10.5 kK V ] 4VP) - 4VF) 21J kK v 3 The shoulder about 19.1 kK probably a r i s e s from s p i n - f o r b i d d e n t r a n s i t i o n s to doublet s ta tes or from low symmetry s p l i t t i n g of the ^T-jg(P) term. The 4 A 2 ^ -<- ^ T ^ t r a n s i t i o n i s not normally o b s e r v e d . 1 7 4 By a p p l i c a t i o n of the Tanabe-Sugano diagram and T r a n s i t i o n Energy Rat io diagram f o r ions with the T-j ground s ta te (Figure 4.7) to the e l e c - t r o n i c spectrum, i t i s p o s s i b l e to c a l c u l a t e the l igand f i e l d s p l i t t i n g parameter, Dq, and the Racah e lec t ron repu ls ion parameter, B. 1 7 5 The r a t i o of the upper and lower bands, v^/v^ = 2 .0 , when f i t t e d to a p l o t of t r a n s i - t ion energy r a t i o v s . Dq/B corresponds to Dq/B = 1.50 and to 4 w f ) - V F ) . , 3 . 5 B = 10,500 B t kK = k i lokayser = 10 3 cm" 1 I I I 1 I I 1 1 1 — 400 600 800 1000 1200 Wavelength (nm) F i g u r e . 4 . 6 Nujol Mull V i s i b l e Spectrum of iCo(3-ti-TI\P) Jti?0)^(ClO,)9.Ho0 134- Figure 4.7(a) Energy Level -Diagram (Tanabe-Sugano) f o r d Ions in a Octahedral F i e l d " F igure 4.7(b) T r a n s i t i o n Energy Ratio Diagram fo r T-. Ground State 135 Whence, f o r [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 : B = 780 cm" 1 10 Dq = 11,800 cm" 1 Using the c a l c u l a t e d values of B and 10 Dq the f i t of the upper and lower band to the Tanabe-Sugano diagram pred ic ts that the A 2 (F) «- 4 T-|g(F) t r a n s i t i o n should be at 22.1 kK (452 nm). This t r a n s i t i o n i s not observed in the v i s i b l e spectrum because of the over lapping 4 T ^ ( P ) 4T-jg(F) t r a n s i t i o n in t h i s r e g i o n . The Racah e l e c t r o n repu ls ion parameter, B, i s a func t ion of l i g a n d , centra l i o n , and s to ich iomet ry . The l a r g e r the metal i o n , the smal ler i s the mutual i n t e r e l e c t r o n i c r e p u l s i o n . Since the s i z e of the ion is re la ted to the e f f e c t i v e nuclear charge experienced by the d - e l e c t r o n s , B i s not only a measure of s i z e , but a lso of e f f e c t i v e nuclear charge. A comparison of the B value f o r the Co(11) complex with that of the f ree ion (B = 971 cm" 1 ) shows a reduct ion in B upon complexat ion, A mechanism respons ib le f o r t h i s e f f e c t i s covalency in the meta l - l i gand bond. Thus, the greater the reduct ion in B, as represented by the r a t i o B in complex B in f ree ion the greater the covalency in the meta l - l i gand bond. The s e r i e s obtained f o r 3 with d i f f e r e n t l igands i s c a l l e d the nephelauxet ic s e r i e s . The l igand f i e l d and nephelauxet ic parameters f o r octahedral coba l t ( I I ) complexes are shown in Table 4 .4 . 136 Table 4.4 Ligand F i e l d and Nephelauxetic Parameters f o r Octahedral C o b a l t ( l l ) Ions * Complex 10 ^ ( c n f 1 ) B ( c n f 1 ) 3 [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] 2 + 11,800 780 0.80 [ C o ( H 2 0 ) 6 ] 2 + 9,200 825 0.85 [ C o ( N H 3 ) 6 ] 2 + 10,200 885 0.91 [ C o ( D M S 0 ) 6 ] 2 + 8,480 824 0.85 [ C o ( P y O ) 6 ] 2 + 10,195 766 0.79 C o C l 2 7,640 775 0.80 CoBr 2 6,490 786 0.81 * Parameters f o r a l l complexes, except those of the TAP complex are taken from reference 176. 2+ The covalency of the meta l - l igand bonds in [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] thus appear to be s i m i l a r to complexes conta in ing C l " , B r " , and p y r i d i n e - N - ox ide . The l igand f i e l d parameter, 10 Dq(b) i s a measure of the s p l i t t i n g of the t 0 and the e d - o r b i t a l s of a metal ion in an octahedral f i e l d . For 2g g a given meta l , and s tereochemist ry , a spectrochemical s e r i e s of the l igands can be e s t a b l i s h e d . There may be a number of f a c t o r s such as p o l a r i z a b i l i t y , d i p o l e moment, l igand charge or e l e c t r o n e g a t i v i t y which make up the ' s t r e n g t h ' of a l i g a n d , and t h i s makes i t d i f f i c u l t to r a t i o n a l i z e the order of l igands in the s e r i e s . However, the l igand f i e l d parameter f o r the TAP complex probably r e f l e c t s the extent of n-bonding ,of the l igand with the meta l . In a mixed l igand complex such as [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 i t i s appropr ia te 137 to apply the law of average environment, which s ta tes that the 10 Dq(k) value is given by the weighted average of the 10 R v a l u e s f o r each of the i n d i v i d u a l l i q u i d s . Thus, f o r the complex [ M A ^ ] we have A = 1 / 6 [ 4 A A + 2 A B ] Using the appropr iate A values from Table 4.4 we obtain a A f o r 2+ -1 [Co(3-H-TAP)g] of 13,100 cm . When comparing the parameters der ived from the v i s i b l e spectrum of the TAP complexes, i t must be remembered that the symmetry i s not 0^, but a c t u a l l y c l o s e r to D ^ , and thus the l igand f i e l d and nephelauxet ic parameters are only approximat ions. The pale blue [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 J ( C I 0 4 ) 2 shows two c l e a r l y resolved bands in the nujol mull e l e c t r o n i c spectrum at 11.2 kK (890 nm) and 17.7 kK (564 nm) (F igure 4 .8 ) . The n i c k e l ( I I ) ion has a 3d valence e lec t ron 3 con f igura t ion which gives r i s e to a F ground term. In an octahedral c r y s t a l 3 f i e l d , the degeneracy of the F term i s removed, and the v i s i b l e spectrum 3 involves three spin allowed t r a n s i t i o n s from the A 2 g(F ) s ta te to the 3 3 3 T 2 g ( F ) , T- |g(F) , and T]g(P) l e v e l s . The bands observed are assigned as f o l l o w s : 3 T 2 g ( F ) - % g ( F ) H . 2 kK v} 3 T l g ( F ) - 3 A 2 g ( F ) 17.7 kK v 2 3 3 The highest energy t r a n s i t i o n (v^) T-^^(P) .-«- ^ ( F ) appears as a shoulder about 27.8 kK (360 nm). A weak absorpt ion at 13.7,k 1< (730 nm) probably a r i s e s frcm a s p i n - f o r b i d d e n t r a n s i t i o n to the V l e v e l . CO CO 400 600 800 1000 1200 Wavelength (nm) Figure 4.8 Nujol Mull V i s i b l e Spectrum of [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 139 The accuracy of the nephelauxet ic parameter, B, c a l c u l a t e d from incomplete spec t ra l data may be suspect . Thus, using the Tanabe-Sugano diagram f o r ground state ions and the appropr iate t r a n s i t i o n energy r a t i o diagram (Figure 4 . 9 ) 1 7 5 and the v i and v 2 bands, we obtain B = 826 c m - 1 and pred ic t that v 3 [ 3 T l g ( P ) 3 A 2 g ( F ) ] should appear at 28.4 kK (352 nm). Th is shows reasonable agreement with the shoulder about 27.8 kK observed in the v i s i b l e spectrum. A problem with using the vj and v 2 bands to c a l c u l a t e the e lec t ron repu ls ion parameter i s that a comparat ively small e r r o r in measuring the band maxima can cause qui te a large change in the c a l c u l a t e d value of B. In a mixed l igand complex such as [Ni ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C l O ^ the lowered symmetry from 0^ may be r e f l e c t e d in a s p l i t t i n g of the v i band, and p o s s i b l y of the v 2 and v 2 bands as w e l l . These s p l i t t i n g s are another source of e r r o r . However, the v i s i b l e spectrum of the TAP complex shows l i t t l e s p l i t t i n g of these absorpt ion bands, i n d i c a t i n g that d i s t o r t i o n from 0^ symmetry i s s m a l l . By comparison, in the D^ molecules [ N i ( p y ) 4 ( C 1 0 4 ) 2 ] or [ N i f p y J ^ S O g F j g L d e f i n i t e s p l i t t i n g o f a l l absorpt ion bands are observed. The c r y s t a l f i e l d parameter, 10 Dq, i s equal to the energy separa - 3 3 t ion between the A 2 g ( F ) and the T 2 g ( F ) l e v e l s . The 10 Dq values f o r var ious n i c k e l ( I I ) complexes are compared in Table 4 . 5 . Apply ing the law of average environment using the data from Table 4.5 the c a l c u l a t e d 10 Dq f o r [ N i ( 3 - H - T A P ) g ] 2 + is 12,550 c m - 1 . The nujol mull e l e c t r o n i c spectrum of [Cu(3-H-TAP) / j (H 2 0) 2 ] ( C 1 0 4 ) 2 c o n s i s t s of a s i n g l e asymmetric band centered at 18.3 kK (546 nm) which spans the e n t i r e v i s i b l e region (Figure 4 .10) . 140 Figure 4.9(a) Energy Level •Diagram (Tanabe-Sugano) f o r d Ions in an Octahedral F i e l d ure 4.9(b) T r a n s i t i o n Energy Rat io Diagram f o r Ions with the A 2 Ground State  142 Table 4.5 Crysta l F i e l d Parameters f o r Octahedral N icke l ( I I ) Der iva t ives * Complex 10 Dq(cm~^ ) [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] 2 + 11,200 [ N i ( H 2 0 ) 5 ] 2 + 8,500 [ N i ( C H 3 0 K ) 6 ] 2 + 8,430 [ N i ( N H , ) J 2 + 10,800 '3'6- >6-[ N i ( D M S 0 ) - ] 2 + 7,730 CNi(Py0)6J2+ 8 j 4 0 0 L N i ( C H 3 N H 2 ) 6 J 2 + 1 0 > 0 0 0 [ N i ( C H 3 C N ) 6 ] 2 + 1 0 > 7 0 0 C N i ( B i P y ) 3 J 2 + 1 2 j 6 5 Q C N i ( B i p y z ) 3 J 2 + 1 2 } 9 0 0 * Values taken from r e f . 178 except f o r 3-H-TAP complex. In an octahedral cub ic l igand f i e l d , the f i v e - f o l d degenerate 3 d - o r b i t a l s s p l i t in to the lower t 2 g o r b i t a l s and the upper e g o r b i t a l s . 9 The s i n g l e unpaired e l e c t r o n in d copper( I I ) could be in e i t h e r of the components of the eg s ta te thus g iv ing r i s e to a s i n g l e t r a n s i t i o n 2 2 Eg T 2 g in the absorpt ion spectrum. The J a h n - T e l l e r e f f e c t requi res any n o n - l i n e a r system with a degenerate ground s ta te to undergo such a d i s t o r t i o n as w i l l remove the degeneracy. For copper ( I I ) , the degeneracy is of ten removed by elongated tetragonal d i s t o r t i o n s of the octahedron. A s i n g l e asymmetrical absorpt ion band is f requent ly observed f o r c o p p e r ( l l ) compounds because the r e l a t i v e energies of the d-d t r a n s i t i o n s involved genera l l y occur wi th in 5.0 kK of each o t h e r . 1 7 9 143 2 2 Hathaway 1 8 0 has observed that the E g B-j t r a n s i t i o n i s the most intense t r a n s i t i o n , and l i e s at the higher energies in tetragonal compounds. The band maximum at 18.3 kK (545 nm) f o r [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] 2 2 therefore probably represents a c lose approximation to the E g t r a n s i t i o n with the remaining t r a n s i t i o n s hidden on the low energy s ide of the band to give an asymmetric band shape. The f a c i l e synthesis and spectra l p roper t ies of t r a n s i t i o n metal complexes conta in ing the 3-H-TAP l igand ind ica ted that the l igand possesses a f a i r l y strong and s p e c i f i c coord inat ion to metal i o n s . We therefore turned our a t tent ion to the bidentate coord inat ion of the h e t e r o c y c l i c N-2 n i t rogen of TAP and the amide n i t rogen of a s i d e - c h a i n at the C-3 p o s i t i o n . In Scheme 4.3 we envisaged complexation. of a metal ion with a TAP-modif ied peptide to give a f t e r h y d r o l y s i s , a complex with the general s t r u c t u r e : As a model fo r the bidentate TAP l i g a n d , we considered that 2-amino- methylpyr id ine (2-AMPy) would possess s i m i l a r coord ina t ion p r o p e r t i e s . Both l igands possess h e t e r o c y c l i c and primary amine ni t rogens s i m i l a r l y or iented f o r metal c o o r d i n a t i o n . By using the 2-AMPy l igand we were able to conserve the precious supply of TAP l igand during pre l iminary studies on e s t a b l i s h i n g the protocol f o r the complexation reac t ion and i s o l a t i n g the product from the react ion mixture. 144 The react ions studied i n v o l v i n g formation of a cobalt(111) - 2-AMPy complex i s shown in Scheme 4 .4 . The procedures of Bentley and C r e a s e r 1 6 5 H 3+ OH 2+ 6 - [ C o ( t r i e n ) C 0 3 ] + B - [ C o ( t r i e n ) ( H 2 0 ) 2 ] B- [Co( t r ien)0H(H 2 0) ] 2-AMPy B-[Co(tr ien)(2-AMPy)] 3+ Scheme 4.4 Synthesis of B-[Co(tr ien)(2-AMPy)] 3+ f o r the formation of [Co ( t r ien) (amino a c i d ) ] complexes were modif ied to our purposes. A s o l u t i o n of [CoUr ienJCOgjC lO^ was a c i d i f i e d to form the red diaquo complex which was then converted to the hydroxoaquo complex. A f t e r add i t ion of 2-aminomethylpyridine to the s o l u t i o n and heating to 60°C or being l e f t at room temperature f o r a couple of hours , the reac t ion mixture turns orange. This i s ev ident in absorpt ion spect ra by s h i f t s of the two bands v i s i b l e f o r the hydroxoaquo-cobalt ( I I I ) complex to lower wavelength. On heating the s o l u t i o n , an intense purple complex p r e c i p i t a t e s which d i s s o l v e s in ch loro form, acetone, and methanol to give a blue s o l u t i o n , and in water to give a b l u e - v i o l e t - s o l u t i o n . V i s i b l e spectra of t h i s complex in var ious so lvents are shown in Figure 4 .11. No e f f o r t was made to i d e n t i f y the purple complex. of the reac t ion mixture were separated by ion-exchange chromatography on Carboxymethyl Sephadex CM-25, e luted with sodium perchlorate s o l u t i o n (0 .1-1.5M). Four components were i s o l a t e d by the cat ion-exchange column. V i s i b l e spect ra of these components are shown in Figure 4.12. A f t e r removing the purple p r e c i p i t a t e by f i l t r a t i o n , the components  300 400 500 600 Wavelength (nm) Figure 4.12 Components Separated from the Preparat ion of [Co(tr ien)(2-AMPy)] by Ion-exchange Chromatography (a) Red, (b) Brown Figure 4.12 (cont inued) , (c) 1st Orange, (d) 2nd Orange 148 The f i r s t complex e luted from the column as a red band with absorpt ions at 28.0 kK (357 nm) and 19.8 kK (505 nm) in the v i s i b l e spectrum. 2+ This was probably B- [Co( t r ien)0H(H 2 0) ] . The next component of the reac t ion mixture was a brown complex with a s i n g l e absorpt ion band at 28.2 kK (355 nm) in the v i s i b l e spectrum. Its i d e n t i t y was not determined, but by i t s e l u t i o n behaviour appears to be a 3+ charged s p e c i e s . C l o s e l y fo l lowing the brown complex was a major orange band in the ion-exchange column which s p l i t in to two well resolved components. The two orange components showed s i m i l a r v i s i b l e s p e c t r a , the f i r s t orange complex e l u t i n g exh ib i ted absorpt ion band at 29.2 kK (342 nm) and 21.2 kK (471 nm); the other orange complex showed bands at 29.5 kK (339 nm) and 21.4 kK (467 nm). These complexes were taken 3+ to be the two isomers of the 3- [Co(tr ien)(2-AMPy)] i o n : I n f r a r e d , UV, and mass spect ra of the complexes ind ica ted the presence of both t r i e n and 2-AMPy l igands in the orange complexes. Chela t ion of 3 - (N-Ac-g ly ) -TAP to e - [ C o ( t r i e n ) 0 H ( H 2 0 ) ] 2 + was e f fec ted in a manner s i m i l a r to that of 2-AMPy, the main d i f f e r e n c e in procedure being the add i t ion of a lcohol to the aqueous suspension to d i s s o l v e 3 - (N-Ac-g ly ) -TAP complete ly . The reac t ion components were separated a f t e r overnight reac t ion by ion-exchange chromatography. On developing an ion-exchange column with 0.1M NaClO^, a red band was r a p i d l y e luted which appeared to conta in two metal components and 149 uncomplexed TAP. Well separated from the red bands were two orange components which were e luted with 0.2-0.3M NaClO^ and showed absorpt ion bands at 21.2 kK (472 nm) and 21.1 kK (475 nm) r e s p e c t i v e l y . Other bands %28.2 kK (355 nm) were not resolved f o r both complexes because of high absorpt ion in the UV reg ion . UV spectra of the orange components showed band s i m i l a r in shape to protonated TAP, with maxima at 41.8 kK (239 nm) and 41.5 kK (241 nm) respec - t i v e l y . A brown band was e luted with 0.5M NaClO^ which showed an absorpt ion at 28.5 kK (351 nm). This component d id not appear to conta in any TAP by i t s UV spectrum. In another attempt at c h e l a t i o n of 3 - ( N - A c - g l y ) - T A P , only two major coloured bands were obtained - a red band which could be e lu ted from the ion-exchange column with pure water with v i s i b l e absorpt ions at 28.2 kK (355 nm) and 19.8 kK (505 nm). An orange band was e lu ted with 0.2M NaC10 4 with only one resolved band in the v i s i b l e region at 21.1 kK (474 nm), and a T A P - l i k e band in the UV region at 41.3 kK (239 nm). The two orange compo- nents were not resolved in t h i s case because a smal ler column with less r e s o l v i n g power was used f o r the l a t t e r separa t ion . Some brown mater ia l always remained f i r m l y stuck at the top of the ion-exchange column which could not be e lu ted even with 1M NaClO^ s o l u t i o n ( F i g . 4 .13 ) . 3+ By comparison with the i s o l a t i o n of the 3- [Co( t r ien) (2-AMPy)] isomers, the two orange bands separated from the reac t ion mixture were 2+ i d e n t i f i e d as two isomers of the 3 - [ C o ( t r i e n ) ( S - ( N - A c - g l y ) - T A P ] i o n : Figure 4.13 Isomers of [Co ( t r i en ) (3 - (N -Ac -g ly ) -TAP) ;p Iso la ted by Ion-exchange Chromatography, (a) 1st Orange, (b) 2nd Orange 151 A d i f f e r e n c e between the coba l t ( I I I ) complexes of the two hetero- c y c l i c l igands i s that the complexes with 2-AMPy are obtained as 3+ ions whereas those with 3 - (N-Ac-g ly ) -TAP are obtained as 2+ i o n s . Th is i s ev ident in t h e i r e l u t i o n behaviour on an ion-exchange column. Sodium perch lora te s o l u t i o n s of concentrat ion c a . 1.0M were needed to e lu te the [ C o ( t r i e n ) ( 2 - A M P y ) ] 3 + isomers, whereas [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) ] 2 + c o u l d be e luted with c a . 0.25M NaC10 4 < In [Co(tr ien)(3-(N-Ac-gly)-TAP)] 2t che la t ion of 3 - (N-Ac-g ly ) -TAP to the metal i s expected to occur at N-2 of the t r i a z o l e r i n g , and at the ion ized amide ni t rogen of the s i d e - c h a i n s u b s t i t u e n t . Coordinat ion of an amide or peptide ni t rogen atom to a metal ion a f t e r deprotonat ion is well e s t a b l i s h e d . 1 8 1 It seems l i k e l y that the propensi ty of a coordinated amide to lose a proton from the bound amido group i s a func t ion of the combined e lectron-wi thdrawing capac i ty of the metal ion and the C = 0 group. With the condi t ions e s t a b l i s h e d f o r the c h e l a t i o n of 3 - ( N - A c - g l y ) - TAP to a coba l t ( I I I ) complex, and the presence of the des i red product demon- s t ra ted by i t s i s o l a t i o n from the reac t ion mixture , we next considered what would happen to the complex under a c i d i c or bas ic c o n d i t i o n s . Hydro lys is of an amide under a c i d i c cond i t ions involves at tack by water on the protonated amide: 0 OH „ n OH 0 R-C-NHR1 — - — - R-C+ • R-C-OHo R-C-OH + R'NH 0 1 . • • . i 1 NHR' NHR' 0 R-C-0" R ' N H 0 + 152 A s i m i l a r mechanism may be a p p l i c a b l e f o r metal complexes with a che la t ing TAP l igand conta in ing an amide s i d e - c h a i n , e . g . the hydro lys is , of [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) ] 2 + (Scheme 4 .5 ) . The most bas ic s i t e in an Scheme 4.5 M e t a l - a s s i s t e d Hydrolys is of 3 - (N-Ac-g ly ) -TAP amide l inkage i s the carbonyl oxygen so that protonat ion occurs at that atom. This has been demonstrated in an X-ray c r y s t a l study o f . t h e protonated product of the [ C o I I I ( g l y - g l y ) 2 J " anion (79) which showed that protonat ion occurred at the amide oxygen atom to form the iminol tautomer of g l y c y l g l y - c i n e , [ C o I I I ( g l y - g l y H ) 2 ] + (80).182 P o l a r i z a t i o n of the iminol group in 2+ protonated [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) ] by the metal ion should promote n u c l e o p h i l i c at tack at the iminol carbon by water. In th is r e s p e c t , the metal ion i s l i k e a "super -proton" to enhance the hydro lys is r e a c t i o n . H (79) (80) 153 We a n t i c i p a t e that chelated 3 - (N-Ac-g ly ) -TAP complex with coord inat ing imide ni t rogen would be i n a c t i v e toward base h y d r o l y s i s due to resonance s t a b i l i z a t i o n of the coordinated imide group. A s i m i l a r s i t u a t i o n e x i s t s with C o ( e n ) 2 ( g l y N H R ) . 1 8 3 At high pH (^11) deprotonat ion of the amide ni t rogen o c c u r s , and the deprotonated species does not undergo base h y d r o l y s i s (Figure 4 .2 ) . The peptide bond in B - [ C o ( t r i e n ) ( Z - g l y p h e - H ) ] + (81) has been shown •NH. ^HN. HN • "Co. I N :NH 2 CHR ^ L ^ C H 2 - N H - Z - (81) to be s tab le under cond i t ions (pH 7 .5 , 65°C) which r a p i d l y hydrolyze the 2+ amide bond in 8 - [Co( t r ien ) (g lyphe) ] with coordinated amino terminus and the amide carbonyl group ( c f . Scheme 4 . 2 ) . 1 5 5 To i n v e s t i g a t e the m e t a l - a s s i s t e d h y d r o l y s i s of amide bonds under a c i d i c c o n d i t i o n s , [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) ] ( C 1 0 4 ) 2 was d i s s o l v e d in 0.05M HCI and incubated at 50°C. Under these cond i t ions of a c i d i t y and temperature we expect h y d r o l y s i s of the amide bond in the s i d e - c h a i n 154 acetamidomethyl group. The reac t ion was checked by TLC on a sample of the s o l u t i o n t reated with IH NaCN which d isp laced the TAP l igand from the Co(111) coord inat ion sphere. A l t e r n a t i v e l y , the Co(II I ) complex was reduced with s o l i d NaBH^ to the more l a b i l e Co(11) complex. Thin layer chromatograms of the l igands re leased by the metal were obtained a f t e r n e u t r a l i z a t i o n of the s o l u t i o n . There was no evidence of 3-aminomethyl-TAP from TLC a f t e r [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) ] 2 + was heated f o r 4 hours. In view of the apparent f a i l u r e of Co(II I ) to f a c i l i t a t e the hydro lys is of s i d e - c h a i n amide groups in TAP d e r i v a t i v e s we a l s o considered h y d r o l y s i s by in situ formation of metal-TAP complexes. An equimolar mixture of 3 - (N-Ac-g ly ) -TAP and C o C l 2 . 6 H 2 0 s o l u t i o n s (0.0125M) in 1.0M HCl was incubated at 4 4 ° C . Another s o l u t i o n of 3 - (N-Ac-g ly ) -TAP (0.0125M) in 1.0M HCl was a l s o heated to 44° as a cont ro l experiment. A f t e r 3 hours at e levated temperature, the h y d r o l y s i s and contro l reac t ions were neu t ra l i zed and analyzed by TLC on s i l i c a g e l . The th in layer chromatograms showed that most of the 3 - (N-Ac-g ly ) -TAP remained unhydrolyzed with only a l i t t l e 3-NH 2CH 2~TAP present . The i d e n t i t i e s of the TAP d e r i v a t i v e s were confirmed by comparison with s tandards, and by r e a c t i o n with n i n h y d r i n . From v i s u a l inspec t ion of the T L C ' s under UV l i g h t , we concluded that there was no s i g n i f i c a n t d i f f e r e n c e between the hydro lys is mixture conta in ing metal ions and the contro l s o l u t i o n . It thus appears that under the h y d r o l y s i s reac t ions with C o C l 2 3- (N-Ac-g ly ) -TAP i s coordinated to the metal only at the N-2 s i t e of the TAP moiety. At low pH, the hydrogen on the amide n i t rogen of the s i d e - c h a i n i s un- ion ized and c h e l a t i o n does not occur , hence there i s no m e t a l - a s s i s t e d hydro lys is of the amide bond. Thus, coba l t ( I I ) ion promoted i o n i z a t i o n of the amide hydrogens in peptides does not appear to occur below pH 10-11 (c f . pH 4-6 f o r copper( I I ) and pH 7-8 f o r n i c k e l ( l l ) i o n s ) . 1 8 4 155 To ensure that che la t ion of 3 - (N-Ac-g ly ) -TAP a c t u a l l y took p l a c e , an hydro lys is reac t ion was conducted with equimolar amounts of 3 - ( N - A c - g l y ) - TAP and CuSO^. The mixture was adjusted to pH 10 to ion i ze the s i d e - c h a i n amide hydrogen and then a c i d i f i e d to pH 0.8 f o r hydro lys is at 50°C. Compari- son of the h y d r o l y s i s mixture with a cont ro l hydro lys is s o l u t i o n without 2+ Cu a f t e r 5 hours showed that the s o l u t i o n contained predominantly unhydro- lyzed S-CH-jCONHCh^-TAP. However, the concentrat ion of the hydro lys is product 2+ 3-NH2CH0 -TAP, was s l i g h t l y higher in the presence of Cu than in the contro l s o l u t i o n . Attempts to study the hydro lys is of 3 - (N-Ac-g ly ) -TAP under bas ic 2+ 2+ condi t ions with Co and Cu were f r u s t r a t e d by p r e c i p i t a t i o n of the metal hydroxides on a d d i t i o n of base to neutral so lu t ions of the metal s a l t and 1igand. In add i t ion to our attempts with metal ions f o r a s s i s t i n g in the hydro lys is of amide bonds we a lso considered the method of Y a m a s h i t a 1 4 6 in which ion-exchangers in the hydrogen form were used to e f f e c t the hydro lys is of peptides under mild c o n d i t i o n s . An aqueous s o l u t i o n of 3 - (N-Ac-g ly ) -TAP was e lu ted through a chromatography column conta in ing an excess of Dowex 50W-X8 (B io -Rad , 200-400 mesh, 5.1 meq/dry g ) . The TAP compound bound s t rong ly to the cat ion-exchange mater ia l and was removed from the column with saturated NaCl s o l u t i o n . UV spectra of the s o l u t i o n e luted from the column ind ica ted the presence of protonated TAP [Figure 4 .14(a) ] . A f t e r n e u t r a l i z a t i o n of the s o l u t i o n , UV spectra showed the band s t ruc ture c h a r a c t e r i s t i c of the TAP chromophore [F igure 4.13(b)] and t h i n - l a y e r chromatography of the product showed only one product from ion-exchange chromatography which was i d e n t i c a l with 3 - (N-Ac-g ly ) -TAP standard. The product a l s o showed no reac t ion with n inhydr in conf i rming the absence of the h y d r o l y s i s product , 3 -NH ? CH ? -TAP. 156 I 1 1 —I 2 0 0 2 5 0 3 0 0 3 5 0 Wavelength (nm) Figure 4.14 U l t r a v i o l e t Spectra of 3 - (N-Ac-g ly ) -TAP Eluted from Cation-exchange R e s i n , (a) Protonated, (b) Neut ra l i zed 157 From our i n v e s t i g a t i o n s with metal ions we have shown that TAP 3+ 2-t* 2"f" 2~f~ d e r i v a t i v e s coordinate s t rong ly with metal ions (Co , Co , . N i , Cu ) to 3+ 2+ form well def ined complexes. Furthermore, Co and Co ions do not promote the a c i d - h y d r o l y s i s of amide bonds in s i d e - c h a i n s of 3 -subs t i tu ted TAP compounds. Copper(II) ions show a s l i g h t l y b e n e f i c i a l e f f e c t but not to any great extent under the condi t ions s t u d i e d . We therefore conclude that meta l - a s s i s t e d a c i d - h y d r o l y s i s of peptide-TAP d e r i v a t i v e s does not o f f e r any s i g n i f i c a n t advantages f o r s e l e c t i v e l y and m i l d l y c leav ing the C-terminal peptide bond. Relevant to our s tudies is the observat ion by Buckingham that the peptide bond in [ C o t e n ^ Z - g l y g l y ) ] * i s s tab le to hydro lys is in IM [H + ] over a per iod of w e e k s . 1 8 5 Broader imp l i ca t ions of our l i m i t e d success with m e t a l - a s s i s t e d hydro lys is are that our method f o r modifying carboxylates with hydra laz ine i s present ly r e s t r i c t e d to C-terminal amino ac id ana lys is of peptides on ly . Extension to a sequencing method awaits development of an appropr ia te hydro lys is procedure. 158 CHAPTER 5, HYDRALAZINE DECOMPOSITION 5.1 INTRODUCTION Hydralazine (12, 1 -hydrazinophthalazine) is commercial ly a v a i l a b l e as a s tab le hydrochlor ide s a l t , the form in which i t is u s u a l l y used f o r most of i t s a p p l i c a t i o n s , s ince the f ree base i s unstable both in the s o l i d s tate and in s o l u t i o n . Hydralazine is very unstable in basic s o l u t i o n with many f a c t o r s cont r ibu t ing to th is behavior: a . Oxygen i s necessary f o r the breakdown to occur . b. pH i s a c r i t i c a l f a c t o r in the r e a c t i o n . c . Type and concentra t ion of ions in aqueous s o l u t i o n are i m p o r t a n t . 1 9 3 Very l i t t l e hydralaz ine decomposition occurs at neutral or a c i d i c pH, whereas decomposition is rapid at basic pH, e . g . , 30-50% decomposit ion in 1/2 hr . in pH 7.4 S0rensen buf fer at 3 7 ° C . t f 0 Phosphate bu f fe r causes more rapid disappearance of hydra laz ine than borate or g l y c i n e b u f f e r s , 1 9 3 but I'^EDTA i n h i b i t s the decomposition in aqueous s o l u t i o n . 4 0 The rate of breakdown i s independent of the concentrat ion of hydra laz ine . 159 The major product obtained from degradation of hydra laz ine in aqueous s o l u t i o n i s ph tha laz ine , which is hydra laz ine minus i t s hydrazine s i d e - c h a i n . 4 0 ' 1 9 3 Oxidat ion of hydra laz ine with oxygen in ethanol a l k a l i or with copper s u l f a t e at pH8 i s a l s o reported to f u r n i s h p h t h a l a z i n e . 1 9 4 In apparent c o n f l i c t with other work {vide supra), Mclsaac and Kanda observed a major product which does not appear to be p h t h a l a z i n e . 3 6 Using 1 - h y d r a z i n o p h t h a l a z i n e - l - C 1 4 , they observed a new r a d i o a c t i v e compound which had a much lower than phthalaz ine on paper chromatography with n-Bu0H-H0Ac-H20(4 : 1 : 5 ) , and which d id not give co lour reac t ions with d i a z o t i z e d s u l f a n i l i c ac id or p-dimethylaminobenzaldehyde, i n d i c a t i n g the absence of f ree amine or hydrazine groups. To exp la in the negative r e s u l t s with the co lour r e a c t i o n s , they speculated that the product might be d iph tha laz iny lhydraz ine (82). However, in the vast l i t e r a t u r e on hydra laz ine (82) there i s no f u r t h e r evidence to support t h i s s p e c u l a t i o n . The i n s t a b i l i t y of f ree base hydra laz ine has plagued the work descr ibed in t h i s t h e s i s from the beginning. While hydra laz ine degradation in aqueous media has been amply descr ibed in the l i t e r a t u r e , nothing is reported on degradat ion i n non-aqueous s o l u t i o n , or in the s o l i d s t a t e . We there fore considered i t to be a worthwhile d i g r e s s i o n to i n v e s t i g a t e the decomposit ion process s ince i t would be b e n e f i c i a l for determining a p p r o p r i - ate cond i t ions fo r reac t ions i n v o l v i n g h y d r a l a z i n e , and f o r i n t e r p r e t i n g UV and NMR s p e c t r a . 160 5.2 RESULTS AND DISCUSSION The decomposit ion of hydra laz ine in the s o l i d s ta te i s charac te r - ized v i s u a l l y by a change in co lour from l i g h t lemon-yellow to dark orange. This change occurs gradua l ly at room temperature wi th in minutes of the s o l i d being exposed to the atmosphere, and very much slower (days) in vacuo. Storage o f the f r e e base below 0°C re tards the decomposit ion but does not stop i t complete ly . The decomposit ion rate of hydra laz ine in s o l u t i o n var ies with the s o l v e n t . To compare these rates q u a l i t a t i v e l y , the UV spectra of f r e s h l y - prepared hydra laz ine in var ious so lvents were monitored at var ious time i n t e r v a l s . The changes in the spect ra observed were band s h i f t s , band shapes and i n t e n s i t i e s , and the appearance of new bands. The so lvents used in t h i s i n v e s t i g a t i o n were: water (pH 7 ) , methanol, DMSO, DMF, DMA, THF, d ioxane, a c e t o n i t r i l e , methylene c h l o r i d e , and ch loroform. Hydralaz ine decomposition was monitored up to 42 hours a f t e r the s o l u t i o n s were prepared, and care was taken to ensure that condi t ions were standardized f o r a l l the s o l u t i o n s . The same batch of f r e s h l y - p r e p a r e d hydra laz ine was used f o r a l l the degradat ion s t u d i e s . Some general conc lus ions can be drawn concerning the s u i t a b i l i t y of c e r t a i n so lvents as media f o r reac t ions with h y d r a l a z i n e . Hydralaz ine showed the grea tes t s t a b i l i t y in CHClg and C H ^ C ^ , and UV spectra of hydra- laz ine in these so lvents were e s s e n t i a l l y unchanged wi th in the time per iod s t u d i e d . In aqueous s o l u t i o n at pH 7, hydra laz ine showed s l i g h t decomposi- t i o n , presumably due to the formation of ph tha laz ine . The decomposit ion was ind ica ted in UV spectra by a reduct ion i n r e l a t i v e i n t e n s i t y of the 263 nm band of hydra laz ine . A methanol s o l u t i o n showed s l i g h t l y increased absorpt ion in the 240-250 nm r e g i o n . Th is change was a t t r i b u t e d to the 161 formation of 1,2(H)-phthalazone with support ing evidence from TLC. However, the product was not i s o l a t e d f o r d e f i n i t i v e conf i rmat ion of i t s i d e n t i t y . Hydralaz ine s o l u t i o n s in CH 3 CN, DMSO, DMA, and DMF showed the growth of new bands at 286 nm and <395 nm. These bands were most evident in DMF s o l u t i o n where the 286 nm band became the s t rongest band in the UV spectrum. In CH^CN, DMSO, and DMA s o l u t i o n s , the 286 nm absorpt ion was only s l i g h t l y resolved above the background. Dioxane and THF s o l u t i o n s showed d i f f e r e n t UV band shapes from the other s o l u t i o n s . In the UV spectra of the s o l u t i o n s , an absorpt ion at 286 nm appeared wi th in one minute of preparat ion of the s o l u t i o n as a shoulder on a more intense 272 nm band and gradua l ly decreased in i n t e n s i t y , and was replaced by a strong unresolved band ^278 nm. Dioxane s o l u t i o n s showed the presence of t h i s 278 nm band from the onset and, in common with THF s o l u t i o n s , the 263 nm band of hydra laz ine decreased to a weak shoulder on the 273 nm band. Another s e r i e s of s tud ies on hydra laz ine decomposit ion in var ious so lvents using hydra laz ine HCI in the presence of excess Et^N under an argon atmosphere gave s l i g h t l y d i f f e r e n t r e s u l t s from the one where f ree base hydra laz ine was used with t e r t i a r y base absent . The UV spectra of the hydra- l a z i n e s o l u t i o n s a l l showed the 286 nm band to varying degrees with the exception of a dioxane s o l u t i o n which, as b e f o r e , showed a strong 278 nm band, and a THF s o l u t i o n which in cont ras t with previous r e s u l t s , showed no 286 nm band at a l l . The v a r i a b i l i t y of these r e s u l t s serves to demonstrate that hydra laz ine degradation is dependent on f a c t o r s other than the s o l v e n t . Thus, decomposit ion occurs even under an argon atmosphere, and oxygen has been reported to be e s s e n t i a l f o r the degradat ion to o c c u r . 1 9 3 It appears that d i s s o l v e d oxygen in the solvent i s s u f f i c i e n t . Presumably, impur i t i es in the so lvent may a l s o play a r o l e in the decomposi t ion, e . g . d iethylamine in DMF. 162 Some conclus ions may be drawn regard ing the s t a b i l i t y of hydra- laz ine in s o l u t i o n although d i f f e r e n t decomposit ion products may be formed in d i f f e r e n t s o l v e n t s . The s t a b i l i t y o f hydra laz ine in var ious so lvents appears to fo l low the order : C H ? C 1 ? H ?0 DMSO THF > > ChLCN > > Dioxane > CHC1 3 CH30H J DMA DMF Methyl C e l l o s o l v e (2-methoxyethanol) was not inc luded in any of the s e r i e s of hydra laz ine decomposition s t u d i e s , but hydra laz ine showed l i t t l e tendency to decompose in t h i s s o l v e n t , which may there fore be p laced c lose to the c h l o r i n a t e d hydrocarbons in the s t a b i l i t y order . Of the so lvents ind ica ted above, d ioxane, THF, and DMF are not recommended as media f o r reac t ions i n v o l v i n g h y d r a l a z i n e . The remaining solvents are s a t i s f a c t o r y provided that the hydra laz ine r e a c t i o n i s completed wi th in 1 day, or that an excess of hydra laz ine i s used in the r e a c t i o n . Other sol vents w h i c h are unsui table as reac t ion media are morpholine and N-methy l -pyrro l idone. The decomposit ion of hydra laz ine in aqueous and organic so lvents i s acce lera ted in the presence of a t e r t i a r y base. In a d d i t i o n , the degrada- t ion product respons ib le f o r the 286 nm band in the UV spectrum i t s e l f breaks down f u r t h e r to g ive a species absorbing at 278 nm in the UV spectrum. Thus, in 1 ,5 -d iazab icyc lo [5 .4 .0 ]undec-5 -ene (DBU) s o l u t i o n , the i n t e n s i t y of the 286 nm band grows to a maximum and then decreases which i s concomitant with growth of the 278 nm band. The UV spectra showed i s o s b e s t i c points at 277 nm and 291 nm. In the presence of a large excess of strong base, e . g . , Et^N, the species absorbing at 286 nm has a very shor t l i f e t i m e s ince i t was 163 not observed, and UV spectra showed only the decomposit ion product absorbing at 278 nm. Clean i s o s b e s t i c points were observed in the UV s p e c t r a . From the m u l t i p l i c i t y of changes seen in the UV spectra of hydra- laz ine during so lvent - induced decomposi t ion, i t is c l e a r that the decomposi- t ion phenomenon is a complex one. Thin layer chromatography of the decom- p o s i t i o n products from DMF s o l u t i o n on s i l i c a gel with CHCl^/MeOH (10:1) showed at l e a s t f i v e components, the major ones being a ye l low compound at Rp0.76, and f l u o r e s c e n t components at R^0.47 and 0.18. A l a r g e - s c a l e degradation of hydra laz ine was performed in DMF s o l u t i o n in order to i d e n t i f y the major decomposit ion products . The products were separated by preparat ive s c a l e TLC on s i l i c a g e l . The major component of the decomposit ion was i s o l a t e d as an orange amorphous s o l i d whose UV spectrum showed strong bands at 286 nm and 393 nm. However, i t was unstable in chloroform and methanol s o l u t i o n , and i t s UV spectrum changed even during the time i t was being scanned. N o t w i t h - standing these changes, the 286 nm band remained, and changes were mainly of i n t e n s i t y , a red s h i f t of the higher wavelength band, and the appearance of two new bands at 255 nm and 265 nm. The UV spectrum of the major decomposition product i s o l a t e d by preparat ive TLC, and p u r i f i e d by column chromatography on s i l i c a gel i s shown in F igure 5 .1 . A 100MHz Four ie r - t rans fo rm NMR spectrum of the major decomposit ion product f r e s h l y obtained by column chromatography i s shown in F igure 5.2. The resonances between 0.8 and 2.0 ppm probably a r i s e from degradat ion of the "286 nm" s p e c i e s , s ince aged s o l u t i o n s show new bands in t h i s region and a growth of the bands evident in F igure 5.2. Whi lst i t was d i f f i c u l t to obtain c lean UV and NMR s p e c t r a , good mass spect ra of the major decomposit ion product were c o n s i s t e n t l y obta ined . 164 250 300 350 Wavelength, (nm) 400 450 Figure 5.1 UV Spectrum of a C H 2 C 1 2 So lu t ion of the Major Product from Decomposition of Hydralazine in DMF Figure 5.2 100 MHz Four ier - t ransform NMR Spectrum of the Major Product from Decomposition of Hydralazine in DMF 166 H i g h - r e s o l u t i o n mass spectra showed a parent m/e at 288.1118 i n d i c a t i n g a composit ion of C-jgH-^Ng (ca lcu la ted mass. 288.1123). The low- reso lu t ion mass spectrum obtained with a probe temperature of 200°C is shown in F igure 5.3. The mass spectrum is charac te r i zed by intense fragment ions at m/e 273, 272, and 171 in the higher m/e r e g i o n . At probe temperatures below 2 0 0 ° C , the m/e 171 ion i s the base peak in the mass spectrum on the bas is of high and low r e s o l u t i o n mass s p e c t r a , the p r i n c i p a l decomposition product of hydra- l a z i n e was i d e n t i f i e d as d iph tha laz iny lhydraz ine (82). A fragmentat ion scheme c o n s i s t e n t with t h i s assignment i s shown in Scheme 5 .1 . The fragmen- t a t i o n steps notated with an a s t e r i s k (*) were v e r i f i e d by the appearance of metastable peaks at the appropr ia te (m^/nip) va lues . D iphtha laz iny lhydraz ine i s unstable in the s o l i d s t a t e , and apparent ly undergoes a i r o x i d a t i o n . Preparat ive sca le TLC of the ox id ized product on s i l i c a gel with CHCl^/MeOH (10:1) g ives two components about R^'s 0.12 and 0.22 which e x h i b i t blue f luorescence under short wavelength UV l i g h t . These components in CHCl^ s o l u t i o n show strong bands in t h e i r UV spectra at 280 and 267 nm, r e s p e c t i v e l y . V a r i a b i l i t y of the band i n t e n s i t i e s in the UV spectra ind ica ted that the components were not pure. The R^ 0.22 component showed fragment ions in the mass spectrum at m/e 149 (100), 167 (54) , 185 (22) , 200 (6 ) , and 279 (48). The numbers in brackets c o r r e s - pond to the r e l a t i v e i n t e n s i t i e s - of the i o n s . H i g h - r e s o l u t i o n mass s p e c t r o - metry d id not g ive unambiguous atomic composit ions f o r these i o n s , but the parent ion at m/e 279 appears to have the composit ion C ^ ^ i N ^ O - j (observed 279.1569, c a l c u l a t e d 279.1583). The mass spectrum of the Rf 0.12 component showed the same fragment i o n s , but with d i f f e r e n t r e l a t i v e i n t e n s i t i e s : m/e 149 (53) , 167 (35) , 185 (100), 200 (26) , and 279 (37). No subs tan t i a l e f f o r t was expended in attempting to obtain these secondary decomposit ion products in a pure s t a t e , or to determine t h e i r i d e n t i t i e s . 100 <_> ro -a c - Q <C CD > 300 m/e Figure 5.3 Mass Spectrum of the Major Product from Decomposition of Hydralazine in DMF Scheme 5.1 . Fragmentation Scheme f o r P inh tha laz iny lhydraz ine 169 We have there fore shown that d iph tha laz iny lhydraz ine i s a major decomposition product of 1 -hydrazinophthalaz ine in non-aqueous s o l v e n t s . It there fore appears that the specu la t ion by Mclsaac and K a n d a 3 6 i s co r rec t a f te r a l l . While we d id not observe d iph tha laz iny lhydraz ine under the condi t ions they r e p o r t e d , we have been able to i s o l a t e and c h a r a c t e r i z e i t fo r the f i r s t t ime. The formation of an N , N ' - d i s u b s t i t u t e d hydrazine from a N-mono- subst i tu ted hydrazine i s not without precedence. I s o n i c o t i n y l h y d r a z i n e (83, I son iaz id ) is ox id i zed at a l k a l i n e pH to give d i i s o n i c o t i n y l h y d r a z i n e (84) as a major product . CONHNH 2 CONHNHCO (83) (84) , From our s tudies on the decomposit ion of hydra laz ine in s o l u t i o n we can recommend c e r t a i n condi t ions to minimize the degradat ion. If f r e e - base hydra laz ine i s obtained by n e u t r a l i z a t i o n of the HCl s a l t , i t should be f r e s h l y prepared immediately before use. C h ^ C ^ and CHC1^ should be used as so lvent media whenever p o s s i b l e s ince hydra laz ine shows l i t t l e tendency to decompose in these s o l v e n t s . Methanol , water (pH 7 ) , a c e t o n i t r i l e , dimethylacetamide and d imethy lsu l fox ide may a l s o be used. Reactions should pre ferab ly be conducted under an i n e r t atmosphere, s ince hydra laz ine i s s e n s i t i v e to oxygen, and the s o l u t i o n s should be de-oxygenated. An excess of strong base ought to be avoided i f p o s s i b l e s ince t h i s promotes decomposi- t ion of h y d r a l a z i n e . Hydra laz ine i n excess of that required f o r r e a c t i o n should be used to compensate f o r decomposed reagent i f strong base cannot be avoided. 170 CHAPTER 6 EXPERIMENTAL 6.1 GENERAL METHODS E l e c t r o n i c Spectroscopy Cary recording spectrophotometers Model 17, 14 or 15 were used to obtain u l t r a - v i o l e t , v i s i b l e and n e a r - i n f r a r e d s p e c t r a . So lu t ion spect ra were obtained with matched s i l i c a g lass c e l l s of 1 mm path l eng th . S o l i d s ta te mull spectra were run using nujol mul ls pressed between s i l i c a windows. L ight scat te red by the mull was compensated f o r with a nujo l -soaked f i l t e r paper in the reference beam of the spectrophotometer. The l i g h t i n t e n s i t y in the sample and reference beams were balanced by p lac ing appropr ia te at tenuators c o n s i s t i n g of metal screens of varying meshes in the reference beam. D i f fuse r e f l e c t a n c e spectra were recorded on a Bausch and Lomb Spect ron ic 600 spectrophotometer equipped with a v i s i b l e r e f l e c t a n c e a t t a c h - ment and a Sargent r e c o r d e r , Model SR. Spectra were obtained over the wave- length range 740-350 nm. Magnesium carbonate was used as the r e f l e c t a n c e standard. 171 Infrared Spectroscopy Infrared spect ra were recorded on a Perkin-Elrner Model 457 grat ing spectrophotometer cover ing the frequency range 4000-250 cm~\ The c e l l windows used to conta in the sample were KBr and NaCl . The c u t - o f f points with these c e l l s were approximately 450 c m - 1 and 550 c m - 1 r e s p e c t i v e l y . Nujol and hexachlorobutadiene were used as mul l ing agents. IR spectra were c a l i b r a t e d with polystyrene f i l m at 1601.4 c m - 1 and 906.7 c m " 1 . Nuclear Magnetic Resonance Nuclear magnetic resonance spectra were obtained at 60 MHz with a Varian T-60 or Varian EM-360A spectrometer , and at 100 MHz with a Var ian HA-100 spectrometer f o r continuous-wave spectra and a Varian XL-100 or N i c o l e t Model NIC-80 spectrometer f o r F o u r i e r - t r a n s f o r m s p e c t r a . The chemical s h i f t s are recorded in the <s(ppm) s c a l e with te t ramethy ls i l ane (TMS) as an in te rna l s tandard . Mass Spectrometry Mass spectra were recorded on an A t l a s CH-4 spectrometer or an A . E . I . MS-902 spectrometer , high r e s o l u t i o n measurements being obtained on the l a t t e r instrument. Melt ing Point Determination Mel t ing points of s o l i d s were measured with a Thomas-Hoover c a p i l l a r y melt ing point apparatus and are uncorrec ted . Elemental A n a l y s i s Elemental analyses f o r carbon, hydrogen and n i t rogen were performed by Mr. P. Borda of the M i c r o a n a l y t i c a l Laboratory , U. b. C. 172 Chromatography Column chromatography was performed using s i l i c a gel obtained from 1CN Pharmaceuticals ( s i l i c a gel woelm f o r A d s o r p t i o n , A c t . I) or Baker (column chromatography grade) . Thin l ayer chromatography (TLC) was performed using S i l i c a Gel GF precoated p la tes (Ana l tech -Un ip la te , 250y). Precoated p lates 2000M th ick were used f o r preparat ive sca le TLC. Drummond Microcap c a p i l l a r y tubes were used f o r spot t ing samples to a n a l y t i c a l TLC p l a t e s . Compounds were detected by UV l i g h t (254 nm) or iod ine a b s o r p t i o n , or by spraying the p la tes with n inhydr in s o l u t i o n in ethanol fol lowed by warming at 110°C. Unless s p e c i f i e d otherwise , the so lvent system used f o r developing TLC p la tes was CHCl^-MeOH (10:1) . Ion-exchange chromatography was performed using Carboxymethyl- Sephadex CM-25 (Pharmacia) with a capac i ty of 4.5 meq/g. 6.2 CHEMICALS A l l chemicals were reagent grade unless otherwise i n d i c a t e d . Spect ra l grade solvents were used in a l l so lu t ions f o r u l t r a - v i o l e t and v i s i b l e s p e c t r a . Tr ie thy lamine Tr ie thy lamine was p u r i f i e d by d i s t i l l a t i o n from sodium hydroxide fo l lowed by double d i s t i l l a t i o n with 2% 1-naphthyl i s o c y a n a t e , and a s i n g l e d i s t i l l a t i o n from sodium t u r n i n g s . 173 N, N-Dimethylformamide N,N-Dimethylformamide was p u r i f i e d by d i s t i l l a t i o n from anhydrous copper( I I ) s u l f a t e at reduced pressure under a n i t rogen atmosphere. The middle f r a c t i o n b o i l i n g a t 53°C at 20 nm pressure was c o l l e c t e d and s tored over molecular s ieves 4A in serum-capped b o t t l e s . Tetrahydrofuran Tetrahydrofuran was d r ied by r e f l u x i n g over l i t h i u m aluminum hydride f o r 15 hours and d i s t i l l i n g . The f r a c t i o n d i s t i l l i n g at 6 4 . 9 ° C was c o l l e c t e d . To remove perox ides , THF was e luted through a column of Alumina Brockman A c t i v i t y I which had been d r ied at 110°C. Hexamethylphosphoramide Hexamethylphosphoramide (HMPA) was d i s t i l l e d , from ca lc ium hydride at water -asp i ra tor pressure ( b . p . ^ l 3 5 ° C ) under an argon atmosphere. The d i s t i l l e d mater ia l was stored under argon. Chloroform Chloroform was re f luxed over ca lc ium hydr ide , f r a c t i o n a l l y d i s t i l l e d , and stored over molecular s ieves 3A. Ethanol 1 8 6 Absolute ethanol (1 a) was s t i r r e d with c lean sodium turnings (14 g) u n t i l the sodium had reacted complete ly . Ethyl formate (40 g) was added and the mixture re f luxed f o r 3 hours before being d i s t i l l e d . The f i r s t 25 ml of d i s t i l l a t e was d i s c a r d e d . The dry ethanol was s tored in a t i g h t l y stoppered b o t t l e with Para f i lm wrapped around the b o t t l e cap. 174 N-Ethy1-5-pheny1isoxazo1ium-3' -su1fonate (17, NEPIS, Woodward's Reagent K ) 5 6 NEPIS (Aldr ic l i ) was d i s s o l v e d in excess IN HCI, p r e c i p i t a t e d with acetone, f i l t e r e d , v/ashed with acetone, and d r ied in vacuo to give a f l u f f y white product . 0 + II Glyc ine Isopropyl Ester H y d r o c h l o r i d e , 1 8 7 H 3 NCH 2 C0C 3 H 7 CI" Fresh ly d i s t i l l e d th iony l c h l o r i d e (15 ml) was added slowly to a s t i r r e d suspension of g lyc ine (15.1 g , 0.250 mol) in isopropyl a lcohol (150 ml) which was maintained at 0 ° C . The suspension was heated to 80°C f o r 12 hours protected from moisture by D r i e r i t e . The c l e a r s o l u t i o n thus obtained was concentrated to about 50 ml in vacuo and ether added u n t i l the s o l u t i o n became t u r b i d . On c o o l i n g , the product c r y s t a l l i z e d from s o l u t i o n and was c o l l e c t e d by suct ion f i l t r a t i o n , and r e c r y s t a l l i z e d twice from absolute ethanol /anhydrous e ther . The t w i c e - r e c r y s t a l l i z e d product weighed 26.5 g (86%). NMR (CDC1 3 ) : 1.27 (double t , J = 6 Hz, 6, 2 C H 3 ) , 3.92 ( s i n g l e t , 2 , C H 2 ) , 5.05 (septe t , 1, CH), 8.40 (broad s i n g l e t , 3, NH 3 ) . 0 + n DL-Alanine Ethyl Es te r Hydroch lo r ide , H 3 NCHC0C 2 H 5 CI" C H 3 DL-Alanine (18.8 g , 0.211 mol) was suspended in absolute ethanol (350 ml) and dry hydrogen c h l o r i d e was bubbled through the s t i r r e d suspen- sion u n t i l no more gas appeared to be absorbed. The c l e a r s o l u t i o n thus obtained was cooled to 0°C and re -sa tu ra ted with hydrogen c h l o r i d e . The s o l u t i o n was s t i r r e d at room temperature f o r 15 hours protected from the atmosphere with a D r i e r i t e dry ing tube. The solvent was removed under reduced p ressure , and the r e s u l t i n g syrup was twice d i s t i l l e d with 200 ml 175 absolute ethanol to remove water as the a lcoho l -water azeotrope. On t r i t u r a t i n g with dry e ther , the syrup c r y s t a l l i z e d to a white s o l i d . Y i e l d of crude product - 31.7 g (98%). The product was r e c r y s t a l l i z e d from ethanol-petroleum ether and d r ied under reduced pressure over D r i e r i t e . 0 G l y c y l g l y c i n e Ethyl Ester Hydroch lo r ide , H 3NCH 2C0NHCH 2C0C 2H 5 CI" G l y c y l g l y c i n e (9.98 g , 75.5 mmol) was suspended in dry ethanol (200 ml) and hydrogen c h l o r i d e was bubbled through the s t i r r e d suspension. Within 10 minutes, almost a l l the g l y c y l g l y c i n e had d i s s o l v e d and a white p r e c i p i t a t e appeared. Hydrogen c h l o r i d e treatment was continued f o r 5 minutes before the reac t ion mixture was cooled to 0°C and resatura ted with HCl f o r an add i t iona l 5 minutes. The mixture was s t i r r e d f o r 21 hours and the s o l i d was c o l l e c t e d by suc t ion f i l t r a t i o n . Another crop of product was obtained from the concentrated f i l t r a t e . The product was r e c r y s t a l l i z e d from absolute ethanol in 82 % y i e l d . 0 N - B u t y l o x y c a r b o n y l - g l y c i n e , 1 8 8 Me3C0CNHCH2C00H A s o l u t i o n of butyloxycarbonyl az ide (17 ml) in 150 ml dioxane was slowly added to a s t i r r e d s o l u t i o n of g l y c i n e (7.51 g , 100 mmol) in 150 ml water and 42 ml t r i e t h y l a m i n e . A f t e r 2 hours , suspended mater ia l was removed by f i l t r a t i o n , and dioxane was d i s t i l l e d o f f from the s o l u t i o n under reduced pressure . The aqueous s o l u t i o n was a c i d i f i e d with IN HCl and then extracted with ethyl ace ta te . The organic ex t rac t was d r i e d with anhydrous magnesium s u l f a t e and taken to dryness to g ive an orange syrup . On c o o l i n g , the syrup c r y s t a l l i z e d . The crude product r e c r y s t a l l i z e d from ethyl acetate /petro leum ether as la rge t rans lucen t needles in 75% y i e l d . Caut ion: B0C-N 3 i s shock s e n s i t i v e and has a thermal i n s t a b i l i t y range of 1 0 0 ° - 1 3 5 ° C and an a u t o i g n i t i o n temperature of 1 4 3 ° C . 1 8 9 176 NMR (CDC1 3 ) : 1.45 ( s i n g l e t , 9, C ( C H 3 ) 3 ) , 3.87 (doublet , J = 6 Hz, 2, C H 2 ) , 5.32, 6.43 (broad s i n g l e t s , 1, NH), 11.38 ( s i n g l e t , 1, COOH). Mass spectrum, m/e (Re l . i n t e n s i t y ) : Parent at 175 not observed, Base at 120; 160 (3 ) , 130 (9) , 120 (100), 116 (3) , 102 (5 ) , 86 (1 ) , 76 (17) , 75 (6) , 74 (8). N - T r i f l u o r o a c e t y l - g l y c y l g l y c i n e , 1 9 0 CF3C0NHCH2C0NHCH2C00H S-Ethy l t h i o l t r i f 1 u o r o a o e t a t e (1.60 ml , 12.5 mmol) was s t i r r e d with g l y c y l g l y c i n e (1.06 g , 8.00 mmol) d i s s o l v e d in IN NaOH (8 ml) f o r 24 hours. The reac t ion was quenched with cone. HCI (5 ml) whereupon a white p r e c i p i t a t e appeared. The product was c o l l e c t e d by suct ion f i l t r a t i o n a f t e r being placed in a r e f r i g e r a t o r f o r 2 hours. Add i t iona l product was obtained by e x t r a c t i n g the f i l t r a t e with ethyl ace ta te . Weight of the crude product was 1.53 g (84%). R e c r y s t a l l i z a t i o n from 95% ethanol gave small c o l o u r l e s s c r y s t a l s . Azidotris(dimethylaminophosphoniurn) H e x a f l u o r o p h o s p h a t e , 6 8 (37) [ ( M e 2 N ) 3 P - N 3 ] P F 6 Tris(dimethylaminophosphine) (10 g , 0.061 mol) was d i s s o l v e d in i c e - c o l d anhydrous ether (250 m l ) , and the temperature maintained at 0°C in an ice -water bath. Bromine (10 g , 0.063 mol) was added slowly and with e f f i c i e n t mixing (Caut ion: t h i s i s an extremely vigorous r e a c t i o n , and appropr ia te safe ty precaut ions must be taken) . A f t e r a few minutes, sodium hexafluorophosphate (10.4 g , 0.061 mol) in 250 ml water was added. The p r e c i p i t a t e of [ (Me 2 N) 3 P-Br ]PFg was f i l t e r e d , and washed with water and ether u n t i l a l l the excess bromine was removed. A f t e r the l i g h t ye l low s o l i d was d r ied overn ight in a vacuum desiccator- over i t was d i s s o l v e d in 200 ml acetone ( d i s t i l l e d from KMn04) and an excess of sodium az ide (5 g) was added. 177 The s o l u t i o n was s t i r r e d overnight and NaBr and excess NaN 3 f i l t e r e d o f f . A f t e r so lvent was removed with a ro ta ry evaporator , the product was obtained as a white s o l i d which was r e c r y s t a l l i z e d from acetone/e ther i n 83% y i e l d . Mel t ing point > 250°C . IR (nujol mu l l ) : v (N 3 ) = 2173 cm" 1 ( l i t . 2176 c m " 1 ) . 6 8 NMR (DMS0-d 6): 2.82 (double t , J = 11 Hz, 6, C-(-CH 3 ) 2 ) ( l i t . 2.79 ppm, J = 11 H z ) . 6 8 l - E t h o x y c a r b o n y l - 2 - e t h o x y - l , 3 - d i h y d r o q u i n o l i n e , 1 9 1 (27, EEDQ) 6=0 OEt A s o l u t i o n of absolute ethanol (92 ml , 2.0 mol) and t r i e thy lamine (155 m l , 1.07 mol) was added dropwise to a s t i r r e d and we l l - coo led ( - 5 ° C ) mixture of ethyl chloroformate (97 m l , 1 mol) and qu ino l ine ( tech . grade) (130 g , 1.0 mol) in benzene (300 ml ) . A f t e r s t i r r i n g f o r 1 hour more, the mixture was washed with water and the aqueous layer ext racted with chloroform (300 ml ) . The combined organic s o l u t i o n was evaporated to dryness under reduced pressure . On the add i t ion of ether (^50 ml) to the r e s i d u e , an o f f -whi te s o l i d separated and a f t e r standing in the c o l d , i t was c o l l e c t e d and washed with co ld e ther . R e c r y s t a l 1 i z a t i o n from ether a f forded large c o l o u r l e s s c r y s t a l s in 65% y i e l d . s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e 4 4 178 N N A mixture of hydra laz ine .HCl (5.668 g, 28.8 mmol), t r i e t h y l orthoformate (60 m l ) , and t r i e thy lamine (4 ml) v/as re f luxed f o r 3 hours , and s t i r r e d at room temperature f o r 1 day. The s o l i d was f i l t e r e d o f f , washed with t r i e t h y l or thoformate, and r e c r y s t a l l i z e d from water and from methanol. The product was obtained as long c o l o r l e s s needles . A n a l . C a l c d . f o r 3-H-TAP: C, 63 .5 ; H, 3 .6; N, 33.0. Found C, 63.5; H, 3 .6; N, 33.0. Mel t ing po in t : 186.5 - 1 8 7 . 5 ° C . UV ( C H 9 C 1 9 ) , X m 3 v (nm): 234 s h , 239, 246, 264, 274, 284 s h . L. l max NMR (CDC1 3 ) : 7.92 ( m u l t i p l e t , 3, H-7, 8, 9 ) , 8.65 ( s i n g l e t - m u l t i p l e t , 2, H-6, 10) , 9.04 ( s i n g l e t , 1, H-3). Mass spectrum, m/e (Re l . I n t e n s i t y ) : Parent and Base at 170; 170 (100),129 (2) , 116 (4) , 115 (33) , 114 (9) , 102 (2) , 88 (15) , 76 (4) . 0 M 3-Aminomethyl -s -Tr iazolo[3 ,4 -a ]phtha1az ine . HOCCF, 3- (N-B0C-gly) -TAP (100 mg) was d i s s o l v e d in 50% (V/V) t r i f l u o r o a c e t i c ac id (2 ml) in C H ^ C ^ . The deprotect ion of the BOC-group was monitored by TLC on s i l i c a gel GF with CHCl 3-MeOH (10:1) developer . When deprotect ion was completed (approx. 10 mins ) , the s o l u t i o n was concent ra ted , and the product • was p r e c i p i t a t e d by adding ^ C ^ . The y i e l d was 95%. UV (H 2 0) , X m a x (nrn): 233 s h , -238, 244, 262, 271 , 281. Mass spectrum, m/e (Re l . I n t e n s i t y ) : Parent and Base at 199; 199 (100), 198 (53) , 133 (10), 171 (47), 149 (9) , 145 (10), 129 (22), 117 (11), 115 (16) , 102 (11). 179 3 - M e t h y l - s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e Hydrochlor ide N N.HCI _ II J l 1-Hydrazinophthalazine hydrochlor ide (0.485 g , 2.47 mmol) in g l a c i a l a c e t i c ac id (50 ml) was re f luxed f o r 4 hours. Excess a c e t i c ac id was removed from the l i g h t blue s o l u t i o n under reduced p r e s s u r e , and the product d r i ed in vacuo f o r 18 hours. Y i e l d of 3-Me-TAP.HCl = 0.505 g (93%). Mel t ing po in t : 233 - 245°C dec. NMR (DMS0-d 6 ): ^8.15 ( m u l t i p l e t , 3, H-7, 8 , 9 ) , 8.59 (doublet , J i 7 Hz, 1, H-10), 9.17 ( s i n g l e t , 1, H-6), 9.71 ( s i n g l e t , 1, H-3). 3-Methyl - s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e t t ' + N N 1-Hydrazinophthalazine (0.993 g , 5.05 mmol) in t r i e t h y l o r t h o a c e t a t e (15 ml) was heated to r e f l u x f o r 1 hour. The product separated from the g o l d - co lored reac t ion mixture as ye l low ish needles and was r e c r y s t a l l i z e d from water conta in ing a t race of NaOH, as c o l o r l e s s needles in 96% y i e l d . Mel t ing po in t : 167 - 169°C. UV (MeOH), A (nm): 236 s h , 240, 248, 265, 275, 285 s h . max NMR (CDC1 3 ) : 2.78 ( s i n g l e t , 3, C H 3 ) , 7.84 ( m u l t i p l e t , '3, a romat ic ) , 8.58 (poss ib le double t , 2, a romat ic ) . Mass spectrum, m/e (Re l . I n t e n s i t y ) : Parent and Base at 184; 184 (100), 156 (7) , 155 (8) , 129 (11), 116 (11), 115 (82), 114 (20), 102 (8) , 88 (26), 76 (9) . 180 0 + II M l anine Methyl Es te r Hydroch lor ide , H 3 N(CH 2 ) 2 C0CH 3 CI" Hydrogen c h l o r i d e was bubbled through a suspension of B-a lanine (17.8 g , 0.200 mol) in dry methanol (300 ml) at 0°C u n t i l the s o l i d had d isso lved completely and the s o l u t i o n was saturated with HCI. The s o l u t i o n was s t i r r e d at room temperature f o r 18 hours and then ro tary evaporated to dryness. The product was r e c r y s t a l l i z e d from e thano l -e ther in 80% y i e l d . 0 3-Alanine Iso-Propyl Ester Hydroch lor ide , H 3 N ( C H 2 ) 2 C 0 C H ( C H 3 ) 2 CI" Hydrogen ch lo r ide was bubbled through a suspension of e -alanine (17.8 g , 0.200 mol) in iso-propanol (200 ml) at 0°C f o r 2 hours. The mixture was then s t i r r e d f o r 1 day at room temperature when a l l s o l i d d i s s o l v e d . The solvent was removed by ro tary evaporat ion and the product r e c r y s t a l l i z e d from e thanol -e ther in 85% y i e l d . 1-Hydrazinophthalazine (Hydralazine) 1-Hydrazinophthalazine HCI (Hydralazine HCI, Apreso l ine HCI ; Sigma, CIBA Pharm Co.) (washed with CHC1 3 and r e c r y s t a l l i z e d from CH-jOH) was d i s s o l v e d in a minimum volume of hot water and n e u t r a l i z e d with a s t o i c h i o m e t r i c amount of IN NaOH s o l u t i o n . The s o l u t i o n was immediately extracted with chloroform under an i n e r t atmosphere (Note: the e x t r a c t i o n procedure i s not very e f f i c i e n t ) . The organic phase was d r ied with anhydrous Na 2S0^ and concentrated to dryness by ro ta ry evaporat ion with as l i t t l e heating as p o s s i b l e . Hydralazine decomposit ion products may be sometimes seen as a dark band of mater ia l in the f l a s k . The impur i t ies p r e c i p i t a t e from s o l u t i o n before the bulk of hydra laz ine when the s o l u t i o n i s concentrated and may thus be separated e a s i l y from the hydra laz ine . The f ree -base hydra laz ine was d r ied under reduced pressure and was used immediately , or stored at 5°C in vacuo or under an i n e r t atmosphere. 181 6.3 COUPLING REACTIONS WITH THE ISOXAZOLIUM SALT METHOD Synthesis of 3-(N-Ac-met)-TAP in A c e t o n i t r i l e So lu t ion N-acety l -DL-methionine (0.319 g , 1.67 mmol) was d i s s o l v e d in warm a c e t o n i t r i l e (10 ml) conta in ing t r ie thy lamine (0.175 g , 1.72 mmol), cooled to 0°C and s t i r r e d with a suspension of p u r i f i e d NEPIS (17) (0.424 g , 1.68 mmol) in i c e - c o l d a c e t o n i t r i l e (10 ml ) . A f t e r a l l s o l i d had d i s s o l v e d (1 hour ) , hydralaz ine HCI (0.326 g , 1.66 mmol) was added together with t r ie thy lamine (0.166 g , 1.64 mmol), and the reac t ion mixture s t i r r e d at room temperature. A f t e r 2 days a l i g h t yel low s o l i d was removed by f i l t r a t i o n . UV spect ra of the s o l i d in methanol showed bands at 263, 278, and 318 nm. The s o l u t i o n was concentrated to an orange syrup which was d i s s o l v e d in methylene c h l o r i d e and extracted with water. Most of the co lour i s t rans fe r red to the aqueous phase. On r e - e x t r a c t i n g the organic l ayer with water, some product i s l o s t in to the aqueous phase. The CH^Cl^ s o l u t i o n was extracted with saturated NaCl s o l u t i o n , dr ied with anhydrous sodium s u l f a t e , and concentrated to dryness. The crude product was obtained in 50-60% y i e l d . Mel t ing po in t : 166 - 167°C. UV ( C H 2 C 1 2 ) , A m a x (nm): 237 s h , 243, 252, 264, 278. Mass spectrum, m/e (Re l . I n t e n s i t y ) : Parent at 315, Base Peaks at 240 and 197; 315 (11) , 253 (11), 245 (5 ) , 241 (16) , 240 (100), 229 (6 ) , 223 (5) , 211 (17), 198 (17) , 197 (100), 183 (9) , 170 (25) , 144 (5) , 129 (11) , 117 (5 ) , 115 (9) , 102 (7 ) , 89 (3) . High r e s o l u t i o n mass spectrum: C a l c d . for C ^ H ^ N g O S , 315.1154. Found 315.1152. 182 Synthesis of 3 - (N -B0C-g ly ) -TAP in DMF So lu t ion A s o l u t i o n of N-BOC-glycine (0.300 g , 1.71 mmol) in d r y , p u r i f i e d DMF (2 ml) conta in ing t r ie thy lamine (0.24 m l , 1.7 mmol) was s t i r r e d with a suspension of p u r i f i e d NEPIS [17) (0.434 g , 1.71 mmol) in dry DMF (3 ml) at 0 ° C . A f t e r 1 hour, a l l the s o l i d d i s s o l v e d and hydralazine.HCI (0.337 g , 1.71 mmol) and t r ie thy lamine (0.24 m l , 1.7 mmol) was added to the reac t ion mixture. The s o l u t i o n was s t i r r e d f o r 2 days at room temperature, and then f i l t e r e d . The white s o l i d thus obtained was shown by nmr and mass s p e c t r o - metry to be Et^N.HCI. The lack of any UV absorpt ions i n d i c a t e d the absence of undissolved hydra laz ine .HCI . A f t e r DMF was removed from the f i l t r a t e under reduced p ressure , the res idue was d i s s o l v e d in CH2d2> and extracted with IN HCI and water. The C H 2 C I 2 s o l u t i o n was dr ied over anhydrous MgSO^, reduced to dryness , and the l i g h t ye l low res idue washed with ether to remove most of the coloured i m p u r i t i e s . Y i e l d of crude 3 - (N -B0C-g ly ) -TAP = 0.450 g (88%). R e c r y s t a l l i z a t i o n from methanol-hexane gave f l u f f y white f l akes of 3 - (N -B0C-g ly ) -TAP which showed only one spot by TLC on s i l i c a gel GF with CHCl 3/MeOH (10:1) . Mel t ing po in t : 166 - 167°C. UV ( C H 2 C 1 2 ) , X m a x (nm): 238 s h , 243, 251.5, 267, 277, 288 s h . NMR ( C D C I 3 ) : 1.47 ( s i n g l e t , 9, C ( C H 3 ) 3 ) , 4.93 (double t , J = 6 Hz, 2, C H 2 ) , 5.51 (broad s i n g l e t , 1, NH), 7.9 ( m u l t i p l e t , 3 , a romat ic ) , 8.66 ( s i n g l e t - m u l t i p l e t , 2, a romat ic ) . Mass spectrum, m/e ( R e l . I n t e n s i t y ) : Parent at 299, Base at 243; 299 (11) , 244 (22), 243 (100), 226 (20) , 199 (23), 198 (39), 183 (22) , 172 (10), 171 (16), 145 (9) , 129 (10), 115 (9 ) , 102 (5) , 88 (5) . High r e s o l u t i o n mass spectrum: C a l c d . f o r C-j^H-j^N^O^, 299.1442. Found 299.1412. 183 6.4 COUPLING REACTIONS WITH THE EEDQ COUPLING REAGENT Synthesis of 3 - (N -Ac-g ly ) -TAP in A c e t o n i t r i l e So lu t ion EEDQ (27) (51 mg, 0.21 mmol) was added to a mixture of hydra laz ine (32 mg, 0.20 mmol) and N - a c e t y l - g l y c i n e (27 mg, 0.23 mmol) in a c e t o n i t r i l e (10 m l ) , and s t i r r e d at room temperature f o r 4 1/2 hours. The reac t ion mixture was f i l t e r e d to remove a yel low s o l i d which showed UV absorpt ion bands at 265 nm and 278 nrn, i n d i c a t i v e of decomposed h y d r a l a z i n e , and bands at 239 nm and 248 nm i n d i c a t i v e of the TAP chromophore. Due to the apparent ly low concentra t ion of TAP in s o l u t i o n and in the s o l i d , no e f f o r t was made to work up the reac t ion mixture . Synthesis of 3 - (N-Ac-DL-a la ) -TAP in A c e t o n i t r i l e So lu t ion EEDQ (70 mg, 0.28 mmol) was s t i r r e d with a s o l u t i o n of hydra laz ine (45 mg, 0.28 mmol) and N -ace ty l -DL -a lan ine (48 mg, .37 mmol) in a c e t o n i t r i l e (10 ml ) . A f t e r 18 hours at room temperature, the s o l u t i o n was f i l t e r e d to remove p r e c i p i t a t e d s o l i d . UV spect ra of the s o l u t i o n showed a high concen- t r a t i o n of TAP in add i t ion to the qu ino l ine by-product . The s o l u t i o n was reduced to d ryness , the r e s u l t a n t res idue d i s s o l v e d in water, and ext racted with ^ C ^ . UV spectra and TLC showed both q u i n o l i n e and TAP in the organic phase.- Solvent was removed from the CHgClg s o l u t i o n under reduced pressure , and the s o l i d obtained was washed with cyclohexane to remove a l l t races of q u i n o l i n e . A small amount of yel low impuri ty was removed from the crude 3 - (N-Ac-DL-a la ) -TAP by preparat ive sca le TLC on s i l i c a gel G with e thano l . Mel t ing Point : ^215° dec. UV ( C H 9 C 1 9 ) , \ v (nm): 237 s h , 243, 251.5, 267, 287, 288 s h . c c max Mass spectrum, m/e ( R e l . I n t e n s i t y ) : Parent at 255, Base at 212; 255 (47), 213 (18) , 212 (100), 198 (33), 197 (12) , 184 (7) , 172 (12), 171 (79) , 145 (6) , 144 (7) , 129 (26), 117 (10 ) , 115 (14), 102 (11), 89 (11) , 76 (6) , 69 (7) . 184 Synthesis of 3-(N-B0C-l_-ala)-TAP in Methylene Ch lor ide So lu t ion EEDQ (0.136 g , 0.552 mmol) w a s . s t i r r e d with a s o l u t i o n of hydra la - z ine (0.088 g , 0.55 mmol) and N-BOC-L-alanine (0.103 g , 0.546 mmol) in CH2CI2 (20 ml ) . Within 4 hours the reac t ion mixture showed good formation of TAP by UV spect roscopy, never the less , the reac t ion was continued f o r 23 hours at room temperature. The C F U C ^ s o l u t i o n was extracted with d i l u t e HCT u n t i l a l l qu ino l ine was removed, then with 5% NaHCOg s o l u t i o n , and f i n a l l y d r ied over anhydrous sodium s u l f a t e . A f t e r so lvent was removed, crude 3 - ( N - B 0 C - L - a l a ) - T A P was obtained as a yel low sol id iim -150% y i e l d . UV ( C H 9 C 1 9 ) , X m a v (nm): 237 s h , 243, 252, 268, 278, 288 s h . L. c. max 6.5 COUPLING REACTIONS WITH THE 185 ACYLOXYPHOSPHONIUM SALT METHODS 6.5.1 The Kenner-Sheppard Reaction A s o l u t i o n of N - a c e t y l - g l y c i n e (0.340 g , 2.91 mmol) in d r y , d i s t i l l e d HMPA (2.9 ml) was added to a s o l u t i o n of r e c r y s t a l l i z e d tosy l c h l o r i d e (0.549 g , 2.88 mmol) in dry HMPA (4.64 g , 25.9 mmol) at 0 ° C . A f t e r 10 minutes, hydra laz ine .HCl (0.568 g , 2.89 mmol) and t r ie thy lamine (0.51 g , 5.0 mmol) were added, and the mixture was allowed to warm to room temperature. A f t e r 2 hours the reac t ion was checked by UV spectroscopy and TLC on s i l i c a gel G with ri-BuOH developer . Thin l a y e r chromatograms showed a large f luorescent spot i n d i c a t i v e of the TAP product . The TAP bands in UV spectra are p a r t i a l l y obscured by strong absorpt ions of the tosy l group. The r e a c t i o n was allowed to proceed overnight before undissolved s o l i d was f i l t e r e d . The s o l i d was predominantly hydra laz ine .HCl by i t s UV spectrum. Attempts to ex t rac t the 3 - (N-Ac-g ly ) -TAP product by ex t rac t ion of the HMPA s o l u t i o n with benzene, petroleum e ther , and d ie thy l ether were l a r g e l y u n s u c c e s s f u l . On ex t rac t ion of the HMPA s o l u t i o n with ^ C ^ some TAP was t r a n s f e r r e d into the C H 2 C I 2 l ayer together with a l l the co lored components of the reac t ion mixture. However, the ex t rac t ion of TAP in to CHgCl 2 s o l u t i o n was i n e f f i c i e n t , and no f u r t h e r e f f o r t was made to i s o l a t e the product . 186 6.5.2 The A z i d o - Tris(dimethylamino)phosphoniurn Hexaf1uorophosphate Method [ ( M e 2 N ) 3 P - N 3 ] P F 6 (37) (0.072 g , 0.21 mmol) was added over a per iod of 1 - 1 1/2 hours to a s t i r r e d s o l u t i o n of N -BOC-glycine (0.036 g , 0.21 mmol) and t r ie thy lamine (33 y l ) in DMF (10 ml) at - 1 5 ° C . A f t e r 1 hour, hydra laz ine HCl (0.040 g , 0.20 mmol) and t r ie thy lamine (60 pi) were added, and the coupl ing reac t ion continued under an argon atmosphere f o r an add i t iona l 4 hours at -15°C and 15 hours at room temperature. Thin layer chromatograms of the reac t ion mixture showed a low concentrat ion of the f l u o r e s c e n t TAP product . There was no evidence of 3 - ( N -B0C-g ly ) -TAP in UV spect ra because of the high UV c u t - o f f of DMF. However, hydra laz ine decomposit ion products were evident by absorpt ion bands at 278 nm and 286 nm. A coupl ing reac t ion in Methyl C e l l o s o l v e was conducted under i d e n t i c a l cond i t ions as the reac t ion in DMF and s i m i l a r r e s u l t s were obta ined. However, UV spectra of the Methyl C e l l o s o l v e reac t ion showed very l i t t l e decomposit ion of hydra laz ine a f t e r 20 hours of r e a c t i o n . 6 .5 .3 The "Oxidat ion-Reduct ion Condensation" Method A suspension of N - a c e t y l - g l y c i n e (0.039 g , 0.34 mmol) and 2 , 2 ' - d i t h i o d i p y r i d i n e (42, 2-DTP) (0.077 g , 0.32 mmol) in dioxane (2 ml) was s t i r r e d with a mixture of hydra laz ine .HCl (0.064 g , 0.32 mmol), t r i e t h y l - amine (0.039 g , 0.38 mmol), and t r iphenylphosphine (0.110 g , 0.419 mmol) in dioxane (5 ml) at 40°C f o r 20 hours. P r e c i p i t a t e d s o l i d was removed by s o l u t i o n f i l t r a t i o n and was shown by i t s UV spectrum to conta in predominantly decomposed hydra laz ine U m a x (nm): 267, 278, 317). NMR and mass spect ra ind ica ted that the s o l i d a l s o contained 3 - ( N - A c - g l y ) - T A P and E t o N . H C l . TLC on S i l i c a Gel GF developed with n-BuOH showed a low concentra t ion of TAP product in the s o l u t i o n . 187 6.5.4 The Tr iphenylphosphi te - Imidazole Method In view of the large number (24) of reac t ions conducted with the M i t i n 7 1 procedure under s i m i l a r c o n d i t i o n s , only one coupl ing r e a c t i o n t y p i c a l of those used in t h i s study w i l l be d e s c r i b e d . Synthesis of 3 - (N-Z -g ly ) -TAP A mixture of N -Z -g lyc ine (0.307 g , 1.47 mmol), hydralazine.HCI (0.290 g , 1.48 mmol), t r ipheny lphosphi te (0.478 g , 1.54 mmol), and t r i e t h y l - amine (0.21 ml) was d i s s o l v e d in dry DMF (5 ml ) . A s o l u t i o n of imidazole ( r e c r y s t . toluene) (0.202 g , 2.96 mmol) in DMF (7 ml) was added to the other reagents , and the mixture s t i r r e d at 4 0 ° C . TLC of the reac t ion mixture showed the presence of TAP even before the s o l u t i o n was heated . / A f t e r heating f o r one day, the s o l u t i o n was f i l t e r e d and reduced to dryness under reduced pressure . UV spect ra of the s o l i d f i l t e r e d from the s o l u t i o n showed the presence of undissolved hydra!azine.HCI and a phosphorus d e r i v a t i v e . The syrup obtained from concentra t ing the s o l u t i o n was d i s s o l v e d in CHgClg and extracted with 1M Ua^CO^ and 0.7N HCI r e s p e c t i v e l y . The o i l y res idue which was obtained a f t e r CHgCl 2 was removed by rotary evaporat ion c r y s t a l - l i z e d on s tand ing . The crude product was r e c r y s t a l l i z e d from ethanol s o l u t i o n . i n 60% y i e l d . 188 6 .5 .5 The Pi pheny lphosphi te -Pyr id ine Method A mixture of N-BOC-L-alanine (0.289 g , 1.53 mmol), hydralazine.HCI (0.301 g , 1.53 mmol), and diphenyl phosphite ( p r a c t i c a l grade) (0.362 g , 1.55 mmol) was s t i r r e d with pyr id ine (10 ml) f o r 2 days. Undissolved s o l i d (hydralazine.HCI) was f i l t e r e d o f f and the gold s o l u t i o n concentrated under reduced pressure . A s o l i d which c r y s t a l l i z e d from s o l u t i o n was shown by UV spectroscopy to be predominantly hydralazine.HCI a f t e r washing with acetone. The acetone wash contained mostly phosphorus d e r i v a t i v e s . There was no evidence fo r TAP product in the reac t ion mixture . 6.6 COUPLING REACTIONS WITH THE CARBODIIMIDE METHOP It would be impract ica l to descr ibe a l l the coupl ing reac t ions conducted with carbodi imides s ince they t o t a l l e d some f i f t y in number. Instead, several experiments w i l l be descr ibed to i l l u s t r a t e c e r t a i n features and c h a r a c t e r i s t i c s of the procedures i n v o l v e d . Synthesis of 3 - ( N - Z - g l y c y l ) - T A P with PCC in DMA So lu t ion Tr ie thy lamine (1.10 m l , 7.89 mmol) was added to a mixture of N -Z -g lyc ine (1.076 g , 5.141 mmol), hydralazine.HCI (1.034 g , 5.257 mmol), and d icyc lohexy lcarbod i imide (si) (2.137 g , 10.36 mmol) in dry dimethylacetamide (50 ml) at 0 ° C . A f t e r 1 hour, a d d i t i o n a l hydralazine.HCI (1.015 g , 5.162 mmol) and t r ie thy lamine (1.10 m l , 7.89 mmol) were added. The reac t ion mixture was s t i r r e d at 0°C f o r 4 hours before being al lowed to warm to room temperature. The reac t ion was continued f o r 1 day at room temperature, and 5 hours to 60°C . On coo l ing the s o l u t i o n , d icyc lohexy lurea (DCU) c r y s t a l l i z e d o u t , and was removed by f i l t r a t i o n . Rotary evaporat ion of the DMA s o l u t i o n gave a syrup which s o l i d i f i e d when the l a s t t races of DMA were removed by 189 ro tary evaporat ion of the syrup d i s s o l v e d in methanol or C F L C ^ . The res idua l s o l i d was d i s s o l v e d in CI-^Cl^j the i n s o l u b l e DCU f i l t e r e d o f f , and the s o l u t i o n extracted with 2% MCI, 5% Nal-ICOg, and saturated NaCl s o l u t i o n s , r e s p e c t i v e l y . When the C H 2 C 1 2 s o l u t i o n was concent ra ted , more DCU c r y s t a l - l i z e d from s o l u t i o n which was removed by f i l t r a t i o n . The product was f u r t h e r p u r i f i e d by column chromatography on s i l i c a qel with CHCl^-MeOH (50:1) as the developing s o l u t i o n . The crude 3 - (N-Z -g ly ) -TAP thus obtained was leached with benzene to remove a l l remaining co lored i m p u r i t i e s . A n a l . C a l c d . f o r 3 - ( N - Z - g l y ) - T A P : C, 64 .9; H, 4 . 5 ; N, 21.0 . Found C, 65 .0; H, 4 .5 ; N, 21.0. Mel t ing p o i n t : 147 - 148°C dec . U V i ( C H 2 C l 2 ) , A m a x (nm): 236 s h , 242.5, 251, 266, 276, 287 s h . NMR (CDC1 3 ) : 5.00 (doublet , J = 6 Hz, 2, C H 2 ) , 5.15 ( s i n g l e t , 2, C H 2 ( b e n z y l ) ) , 6.03 (broad s i n g l e t , 1, NH), 7.32 ( s i n g l e t , 5, C g H 5 ) , 7.95 ( m u l t i p l e t , 3, a romat ic ) , 8.59 ( s i n g l e t - m u l t i p l e t , 2, a romat ic ) . Mass spectrum, m/e ( R e l . I n t e n s i t y ) : Parent at 333, Base at 198; 333 (63) , 242 (15), 225 (17), 199 (22) , 198 (100), 197 (47), 184 (95) , 183 (32) , 171 (28) , 155 (6) , 129 (17) , 115 (14) , 103 (11), 102 (10) , 91 (71) , 79 (18) , 77 (19), 66 (44), 51 (12). High r e s o l u t i o n mass spectrum: C a l c d . f o r C^gH^Ng0 2 , 333.1226. Found 333.1208. 190 Synthesis of 3 - (N-Ac-q ly ) -TAP with EDC in Methanol So lu t ion Tr ie thy lamine (0.071 m l , 0.51 mmol) was added to a mixture of N -Ac -g lyc ine (0.0399 g , 0.513 mmol), hydra laz ine .HCl (0.100 g , 0.510 mmol), and 1 -e thy l -3 - (3 -d imethy laminopropyl )carbodi imide hydrochlor ide {16, EDC) in dry methanol (15 ml) at 0 ° C . A l l the s o l i d s d i s s o l v e d to give a l i g h t ye l low s o l u t i o n . Reaction was continued f o r 4 hours at 0 ° C , and 18 hours at room temperature. The solvent was removed from the s o l u t i o n by ro tary evaporat ion to give a dry. s o l i d . The residue was shaken and sonicated with C H 2 C 1 2 u n t i l a l l TAP product was leached into s o l u t i o n . The C H 2 C 1 2 s o l u t i o n was streaked onto a s i l i c a gel TLC p la te (2000 y) and developed with CHCl 3-MeOH (10:1) . Two we l l -separa ted f l u o r e s c e n t bands were obtained which were i d e n t i f i e d as 3-CH 3~TAP (upper band) and 3 - (N-Ac-g ly ) -TAP (lower band) by t h e i r nmr and mass s p e c t r a . Synthesis of 3 - (N-B0C-gly) -TAP with DCC in Methylene Ch lo r ide S o l u t i o n D icyc lohexy lcarbodi imide (51, DCC) (0.373 g , 1.81 mmol) was s t i r r e d with a mixture of f r e s h l y - p r e p a r e d hydra laz ine (0.255 g , 1.59 mmol) and N-BOC-glycine (0.280 g , 1.60 mmol) in dry C H 2 C 1 2 (25 ml) at 0 ° C . The r e a c t i o n was allowed to proceed f o r 1 hour at 0°C and 18 hours at room temperature before i t was f i l t e r e d to remove d i c y c l o h e x y l u r e a . The s o l u t i o n was extracted with IN H C l , 5% N a 2 C 0 3 , and saturated NaCl s o l u t i o n , respec - t i v e l y . A f t e r so lvent was removed by ro tary evapora t ion , the crude l i g h t - ye l low 3- (N-B0C-gly) -TAP thus obtained was r e c r y s t a l l i z e d from methanol in 76% y i e l d . 191 Synthesis of 3 - (N -Ac-g ly ) -TAP with DCC in Methanol So lu t ion Tr ie thy lamine (0.85 m l , 6.1 mmol) was added to a mixture of N - a c e t y l - g l y c i n e (1.444 g , 1.233 mmol), hydralazine.HCI (1.176 g , 5.983 mmol) and d icyc lohexy lcarbod i imide (51, DCC) (2.602 g , 12.61 mmol) in dry methanol (45 ml) at 0 ° C . UV spectra of the s o l u t i o n a f t e r 15 minutes showed the presence of protonated hydra laz ine . Excess t r ie thy lamine (0.85 ml , 6.1 mmol) was therefore added. The reac t ion was conducted at 0°C f o r 2 hours , and at ambient temperature f o r 18 hours. During t h i s time the c o l o r of the s o l u t i o n changed from ye l low to reddish-orange . When the r e a c t i o n mixture was re f luxed f o r 1 hour, UV spectra showed ^ 3x increase in the concentra t ion of TAP. However, TLC ind ica ted that r e f l u x i n g a lso e f fec ted the formation of 3-CH.j-TAP s i d e - p r o d u c t . The s o l u t i o n was concentrated to dryness and the res idue suspended in CHCl^. Undissolved d icyc lohexy lu rea (DCU) was f i l t e r e d o f f . The TAP product i s only moderately so lub le in CHClg. The chloroform s o l u t i o n was p u r i f i e d by column chromatography on s i l i c a gel developed i n i t i a l l y with pure CHC1 ^» then with a CHClg-MeOH mixture whose composit ion var ied from a r a t i o of 50:1 to 20:1. Crude 3 - (N-Ac-g ly ) -TAP was obtained as a l i g h t ye l low s o l i d . C r y s t a l l i z a t i o n from HgO-EtOH a f f o r d white needles of pure 3 - ( N - A c - g l y ) - T A P . A n a l . C a l c d . f o r 3 - ( N - A c - g l y ) - T A P . 1/2 H 2 0: C, 57.6; H, 4 .8 , N, 28.0 . Found C, 57.4; H, 4 .8 ; N, 28.0. Mel t ing po in t : 209 - 210°C dec. UV ( C H 2 C 1 2 ) , A m a x (nm): 236 s h , 242.5, 251, 267, 277, 287 s h . ' NMR (CDC1 3 ) : 2.11 ( s i n g l e t , 1, C H 3 ) , 5.07 (double t , J = 5 Hz, 2, C H 2 ) , 6.78 (broad s i n g l e t , 1, NH), 7.9 ( m u l t i p l e t , 3, a romat ic ) , 8.67 ( s i n g l e t - m u l t i p l e t , 2, a romat ic ) . Mass spectrum, m/e ( R e l . I n t e n s i t y ) : Parent at 241, Base at 198; 241 (77), 199 (16) , 198 (100), 183 (7 ) , 171 (32) , 129 (11) , 117 (4) , 115 (4) , 102 (4) . High r e s o l u t i o n mass spectrum: C a l c d . f o r C - ] 2 H 1 1 N 5 ° , 241.0964. Found 241.0965. 192 Synthesis of 3 - (N -B0C-g1y ) -TAP in A c e t o n i t r i l e So lu t ion A s o l u t i o n of N-BOC-glycine (0.258 g , 1.47 mmol) in a c e t o n i t r i l e (10 ml) conta in ing t r ie thy lamine (0.21 m l , 1.5 mmol) was- s t i r r e d with a suspension of p u r i f i e d NEPIS (17) (0.373 g , 1.47 mmol) in a c e t o n i t r i l e (10 ml) at 0 ° C . A f t e r 1 1/4 hours, hydra laz ine hydrochlor ide (0.307 g , 1.56 mmol) and t r ie thy lamine (0.22 m l , 1.6 mmol) were added and the mixture allowed to warm to room temperature. Undissolved h y d r a l a z i n e H C l was f i l t e r e d a f t e r 18 hours, and the so lvent removed from the s o l u t i o n with a rotary evaporator . The residue -as d i s s o l v e d in C H 2 C 1 2 and extracted with 5% MaHCO^, 5% HCI, and H^O, r e s p e c t i v e l y . Ex t rac t ions were repeated u n t i l the aqueous phases showed no UV absorbing m a t e r i a l . However, during ex t rac t ions with 5% HCI, the TAP product was protonated and l o s t in to aqueous s o l u t i o n . Y i e l d of crude product = 0.132 g (30%). The work-up procedure in which 5% HCI was replaced with 1% or IN HCI gave higher y i e l d s of 3 - ( N - A c - g l y ) - T A P . Attempted Synthesis of 3 - (N-B0C-gly) -TAP in CH^CN-DMF So lu t ion A s o l u t i o n of N-BOC-glycine (0.265 g , 1.5. mmol in a c e t o n i t r i l e (5 ml) conta in ing t r ie thy lamine (0.21 m l , 1.5 mmol) was s t i r r e d with a suspension of NEPIS (l?) (0.383 g , 1.51 mmol) in a c e t o n i t r i l e (7 ml) at 0 ° C , and added a f t e r 1 hour to a suspension of hydra laz ine HCI (0.299 g , 1.52 mmol) in DMF (8 ml) conta in ing t r i e thy lamine (0.21 m l , 1.5 mmol). Almost a l l s o l i d d i s s o l v e d a f t e r the mixture was s t i r r e d f o r 18 hours at room temperature. Solvent was removed from the s o l u t i o n under reduced p r e s s u r e , and the s t i c k y res idue sonicated with 0.5N HCI (40 ml) to give an amorphous s o l i d . However, a f t e r a per iod of hours , a gas was evolved and most of the s o l i d d i s s o l v e d . L i t t l e TAP product could be ext racted from the aqueous s o l u t i o n with C H ^ C ^ or ethyl ace ta te . A f t e r the pH of the aqueous s o l u t i o n was adjusted to 7 .5 , TAP was extracted in to CH 2 CI ' 2 . TLC of the C H 2 C 1 2 s o l u t i o n showed predomi- nantly 3-NH2CH2-TAP and only a small amount of 3 - ( N - B 0 C - g l y ) - T A P . 193 6.7 SOLID-PHASE STUDIES H y d r o x y m e t h y l - R e s i n , 1 1 6 P $ -CH 2 0H Chloromethyl -poly (s tyrene-co-1% div inylbenzene) (1.34 mmol of C l / g , 200-400 mesh, Bio-Beads S - X l ; 29.3 g , 39.2 mmol) was suspended in Methyl C e l l o s o l v e (200 ml) and s t i r r e d with potassium acetate (10.Og, 102 mmol) at 135°C f o r 70 hours. The r e s i n was c o l l e c t e d and washed s u c c e s s i v e l y with water and methanol. The product was s t i r r e d with f r e s h l y prepared 0.51N NaOH s o l u t i o n (200 m l , 101 mmol) f o r 72 hours, then f i l t e r e d , washed with water and methanol, and vacuum d r i e d . The r e s i n was used immediately f o r the preparat ion of methylchloroformyl r e s i n . IR (HCB m u l l ) : v (0 -H) , 3575 and 3385 cm" 1 ( l i t . 3610 and 3448 c m " 1 ) . 1 0 7 0 M e t h y l c h l o r o f o r m y l - R e s i n , 1 1 6 P -CH 20C-C1 Hydroxymethyl r e s i n (27.9 g) was t reated with 12.5% phosgene in benzene s o l u t i o n (200 ml ) . The s l u r r y was d i l u t e d f u r t h e r with benzene (50 ml) and s t i r r e d f o r 6 hours, fo l lowed by f i l t r a t i o n and washing with benzene and e ther . The r e s i n thus obtained was dr ied in vacuo and s tored in a vacuum d e s i c c a t o r over D r i e r i t e . Volhard t i t r a t i o n f o r c h l o r i d e ind ica ted a r e s i n capac i ty of 1.36 mmol of c h l o r i d e / g of r e s i n . IR (HCB m u l l ) : v(C=0),' 1772 cm" 1 ( l i t . 1779 c m " 1 ) . 1 0 7 0 0 R e s i n - A l a n i n e , P -Q-LOCNHCHCOEt s c | C H 3 DL-Alanine ethyl es te r hydrochlor ide (1.37 g , 8.94 mmol) and t r ie thy lamine (2.0 m l , 36 mmol) were s t i r r e d fo r 23 hours at room temperature with chloroformylmethyl r e s i n (3.09 g , 4.20 mmol) suspended in dry chloroform (50 ml ) . The r e s i n was f i l t e r e d , washed with ch loro form, then resuspended in 194 dry chloroform (50 ml) and reacted with diethylamide (0.50 m l , 4.9 mmol) f o r 4 hours. The r e s i n was washed with a ch loroform-ether mixture (concentrat ion gradient from 0 to 100% ether) and d r ied in vacuo. The r e s i n - a l a n i n e ethyl es te r was sapon i f i ed with 0.5N K0H (50 ml) in methanol-acetone (1:1) f o r 21 hours, f i l t e r e d , a c i d i f i e d with d i l u t e H C l , and washed with a methanol- ether mixture with a concentrat ion gradient of ether varying from 0 to 100%. The r e s i n was dr ied in vacuo f o r 18 hours. The carboxylate content by t i t r a t i o n with NaOH i s 0.63 mmol/g r e s i n . 0 0 R e s i n - g l y c i n e , PS-CH20C.NHCH2C0H Glyc ine ethyl es ter hydrochlor ide (1.60 g , 11.5 mmol) and t r i e t h y l - amine (5.0 m l , 36 mmol) were s t i r r e d f o r 21 hours at room temperature with chloroformylmethyl r e s i n (8.49 g , 11.5 mmol) suspended in dry chloroform (100 ml ) . The r e s i n was f i l t e r e d , washed with ch loroform, then resuspended in chloroform and s t i r r e d with diethylamide (1.0 m l , 9.7 mmol) f o r 5 hours to block unreacted ac id c h l o r i d e groups. The product was washed - i t h a mixture of chloroform and methanol whose composit ion was var ied from pure chloroform to pure methanol. The r e s i n was sapon i f i ed with 0.5N K0H (100 ml) in methanol-acetone (1:1) f o r 18 hours. A f t e r washing with a methanol- ch loroform-ether mixture (mixture composit ion var ied from 100% CH^OH to 100% E t 2 0 ) , the r e s i n was suspended in 75 ml of a methanol-acetone mixture (2 :1) , and s t i r r e d with 12N HCl (1 ml) f o r 1 h o u r . . The r e s i n - g l y c i n e was washed with copious amounts of water, ethanol and e t h e r , then dr ied in vacuo. The carboxylate content by t i t r a t i o n with NaOH is 0.62 mmol/g r e s i n . 195 0 0 R e s i n - g l y c y l g l y c i n e , P -CH0C0NHCH2C0NHCH2C0H G l y c y l g l y c i n e ethyl es ter hydrochlor ide (1.89 g , 9.63 mmol) and t r ie thy lamine (5.0 ml , 36 mmol) were s t i r r e d f o r 23 hours with c h l o r o f o r m y l - methyl r e s i n (3.50 g , 4.75 mmol) suspended in dry chloroform, (100 ml ) . The res in was f i l t e r e d , washed with chloroform and e t h e r , and d r ied in vacuo. Tiie product was suspended in chloroform (75 ml) and reacted with diethylamine (0.50 ml , 4.9 mmol) f o r 5 hours to block unreacted ac id c h l o r i d e groups. A f t e r washing with chloroform and e t h e r , the r e s i n - g l y g l y O E t was d r ied by pumping on a vacuum l i n e . The r e s i n was sapon i f i ed with 0.5N NaOH (50 ml) in methanol-acetone (1:1) f o r 17 hours, then washed with water , methanol, and ether p r i o r to dry ing in vacuo. A n a l y s i s fo r c a r b o x y l i c ac id by t i t r a t i o n with NaOH gave a value of 0.19 mmol/g. The low value probably r e f l e c t s the g l y c y l g l y c i n e subs t i tuen t being present p r i m a r i l y as the sodium s a l t , and not n e c e s s a r i l y from low s u b s t i t u t i o n of d ipept ide onto the r e s i n . Coupling of R e s i n - g l y c i n e with Hydralazine using Iso-buty l Chloroformate Isobutyl chloroformate (5 g , 37 mmol) was s t i r r e d at - 8 ° C with a suspension of r e s i n - g l y c i n e (1.86 g , 0.689 mmol) in dry chloroform (25 ml) and t r i e thy lamine (5.0 ml ) . A f t e r one hour, the r e s i n was f i l t e r e d and washed with dry ch loroform. A co ld mixture of hydra laz ine hydrochlor ide (1.96 g , 10.0 mmol) in chloroform (100 ml) conta in ing t r i e thy lamine (1.56 m l , 11.2 mmol) was added. The suspension was s t i r r e d fo r 1 hour at - 8 °C and f o r 19 hours at room temperature, f i l t e r e d , and s t i r r e d with 75 ml water f o r 2 hours to remove any undissolved hydra laz ine h y d r o c h l o r i d e . The r e s i n was f i l t e r e d , v/ashed with water, acetone, ch loro form, and e the r , and d r i e d under reduced pressure . 196 Cleavage from the Res in: 2 -Aminomethy l -s -Tr iazo lo [3 ,4 -a ]phtha laz ine from the Coupling Reaction with Isobutyl Chloroformate as Carboxyl A c t i v a t i n g Agent fo r R e s i n - g l y c i n e Hydrogen bromide was introduced f o r 3 1/2 hours in to a suspension of r e s i n - 3 - g l y c y l - T A P i n ^ t r i f l u o r o a c e t i c ac id (5 ml) and the suspension was allowed to stand f o r 15 hours. The res in was removed by f i l t r a t i o n and washed with t r i f l u o r o a c e t i c a c i d . Evaporat ion of the f i l t r a t e under reduced pressure gave a l i g h t orange syrup. P u r i f i c a t i o n of the res idue by preparat ive sca le TLC on s i l i c a gel with CHC1 3 /CH 30H (10:1) gave a main f l u o r e s c e n t band at low R f which showed UV spectra c h a r a c t e r i s t i c of TAP, and a ye l low non- f luorescent band at high R f which showed a prominent band at 278 nm in the UV spectrum reminiscent of decomposed hydra laz ine (c f . Chapter 5) . The f l u o r e s c e n t band was extracted with methanol and p u r i f i e d two more times by preparat ive sca le TLC. A ye l low impurity was removed by washing the product with ch loroform. Comparison of UV and mass spect ra with those of authent ic 3-NH 2CH 2~TAP confirms the i d e n t i t y of the product . UV (MeOH), A m a v (nm): 235 s h , 241, 248, 265, 274, 285 s h . Mass spectrum, m/e (Re l . I n t e n s i t y ) : Parent and Base at 199; 199 (100), 198 (59), 184 (15), 183 (15), 171 (48), 149 (13), 145 (15), 129 (24), 117 (11), 115 (14). Coupling of R e s i n - g l y c i n e with Hydralazine using EEDQ l - E t h o x y c a r b o n y l - 2 - e t h o x y - l , 2 - d i h y d r o q u i n o l i n e (27, EEDQ) (0.249 g , 1.01 mmol) was s t i r r e d at room temperature with r e s i n - g l y c i n e (1.61 g , 1.0 mmol) suspended in dry te t rahydrofuran (25 ml ) . Tr ie thy lamine (0.40 ml) was added to a suspension of hydra laz ine .HCl (1.00 g , 5.09 mmol) in d r y , perox ide- f ree THF (25 ml) under a n i t rogen atmosphere and the suspension f i l t e r e d . 197 A f te r the r e s i n - g l y c i n e was a c t i v a t e d f o r 45 minutes, the hydra laz ine s o l u t i o n was added under a n i t rogen atmosphere, and the mixture s t i r r e d f o r 21 hours. From the weight of hydralazine.HCI recovered , f ree -base hydra laz ine in THF s o l u t i o n was estimated to be 0.10 g (0.51 mmol). The r e s i n was f i l t e r e d , and washed with THF, methanol, and e ther . Cleavage from the Resin: 3 -Aminomethy l -s -Tr iazo lo [3 ,4 -a ]phtha laz ine from the Coupling Reaction with EEDQ as Carboxyl A c t i v a t i n g Agent fo r R e s i n - g l y c i n e Hydrogen bromide was bubbled f o r 4 hours in to a suspension of r e s i n - 3 -g lycy l -TAP in g l a c i a l a c e t i c ac id and the suspension was l e f t standing f o r 24 hours. The r e s i n was removed by suct ion f i l t r a t i o n and washed with a c e t i c a c i d . Evaporat ion of the f i l t r a t e under reduced pressure gave a l i g h t orange syrup which showed r e l a t i v e l y c lean UV spectra in methanol s o l u t i o n charac- t e r i s t i c of the TAP chromophore: A m a x (nm), 233 s h , 238, 245, 261, 271, 281 s h . Coupling of R e s i n - g l y c i n e with Hydralazine using D icyc lohexy lcarbodi imide (51) Hydralazine hydrochlor ide (1.02 g , 5.19 mmol) was suspended in dry chloroform (50 ml) under a ni t rogen atmosphere, and s t i r r e d with t r ie thy lamine (0.72 m l , 5.2 mmol) f o r 1 hour. The undissolved hydralazine.HCI was removed by f i l t r a t i o n under N 2 with Schlenk g lass -ware , and the hydra laz ine s o l u t i o n , a f t e r coo l ing to 0°C was added to r e s i n - g l y c i n e (1.60 g , 1.01 mmol) with d icyc lohexy lcarbod i im ide (0.286 g , 1.39 mmol). The mixture was s t i r r e d at 0°C f o r 2 hours, and at room temperature f o r 15 hours. UV spectra of the s o l u t i o n showed cons iderab le hydra laz ine decomposit ion evidenced by a strong band at 278 nm a f t e r overnight r e a c t i o n . Ref luxing the reac t ion mixture fo r 2 hours showed no change in the UV spectra of the s o l u t i o n . The r e s i n was f i l t e r e d , washed with ch loroform, methanol, and e the r , and dr ied in vacuo f o r 4 hours. 198 Cleavage from the Res in : 3-Aminomethyl -s-Tr iazo1o[3,4-a]phtha1azine from the Coupling Reaction with DCC as Carboxyl A c t i v a t i n g Agent f o r R e s i n - g l y c i n e Hydrogen bromide was bubbled fo r 3 1/2 hours in to a suspension of r e s i n - 3 - g l y c y l - T A P (1.63 g) in g l a c i a l a c e t i c ac id (10 ml ) . The suspension was allowed to stand f o r 16 hours, f i l t e r e d , and washed with a c e t i c a c i d . Evaporat ion of the s o l u t i o n in vacuo gave an orange sy rup , which on t r i t u r a t - ing with ch loro form, c r y s t a l l i z e d to a white s o l i d . UV spect ra of the s o l i d in methanol s o l u t i o n showed bands c h a r a c t e r i s t i c s of the c lean TAP chromo- phore. Polyacrylamide Ac id Ch lor ide Carboxypolyacrylamide (Bio-Gel CM-2; 100-200 mesh, 5 meq/g, Na + form) (50 g , 250 meq) was suspended in Dioxane/2N NaOH (3:1) (250 ml) f o r 2 hours, and in Dioxane/2N HCI (3:1) f o r 2 hours. The polymer was washed with water , acetone, and e ther , and d r ied in vacuo f o r 19 hours. The polymer thus p u r i f i e d was s t i r r e d f o r 3 1/2 hours in f r e s h l y d i s t i l l e d th iony l c h l o r i d e (conta in ing 5% pyr id ine ) (100 ml ) . The d e r i v a t i z e d product was f i l t e r e d , washed with dry benzene and e the r , and d r ied in vacuo fo r 18 hours. The polyacrylamide ac id c h l o r i d e was stored over d r i e r i t e . Ch lor ide A n a l y s i s of the Chioroformylmethyl Resin by the modif ied Volhard Method 1 9 2 The chloroformylmethyl r e s i n (^200 mg) was hydrolyzed in pyr id ine (3 ml) f o r 3 hours at 100°C. The s o l u t i o n was t r a n s f e r r e d q u a n t i t a t i v e l y to a 125 ml Erlenmeyer f l a s k with 50% aqueous a c e t i c ac id (30 m l ) , and a c i d i f i e d with cone, n i t r i c ac id (5 ml ) . The c h l o r i d e was p r e c i p i t a t e d with 0.100N AgNOo (5.00 ml ) . The AgCI formed was coated with toluene (about a 1/4 inch 199 layer of toluene on the water sur face) and the excess AgNO^ b a c k - t i t r a t e d with standard 0.100N NH^SCN s o l u t i o n , using a saturated f e r r i c alum s o l u t i o n as an i n d i c a t o r . A red c o l o r , due to the formation of FelSCN)^, i nd ica ted that the end point has been reached. Ana lys is fo r c h l o r i d e content in P - ^ O C O C l was done in d u p l i c a t e , and agreed wi th in + 0.01 mmol C l / g r e s i n . Hydro lys is of the r e s i n was i n e f f i c i e n t with IN NaOH s ince the r e s i n f l o a t e d in th is medium, and tended to r i s e up the s ide of the con ta ine r . In a d d i t i o n , the r e s i n swel led poor ly in IN NaOH s o l u t i o n . A n a l y s i s of the Resin-Amino Ac id (peptide) by T i t r a t i o n with Hydroch lor ic Ac id The r e s i n (^500 mg) was suspended in 95% ethanol (25 ml) and standard 0.100N NaOH s o l u t i o n (25.00 ml) added. The mixture was heated to r e f l u x f o r 10 minutes, and cooled to room temperature. The excess base was b a c k - t i t r a t e d with 0.100N H C l , using phenolphthalein as i n d i c a t o r . A l l analyses were done in d u p l i c a t e , and u s u a l l y agreed wi th in + 0.02 mmol/g r e s i n . The t i t r a t i o n f o r ac id content i s i n d i r e c t l y a measure of the amino a c i d s u b s t i t u t i o n on the r e s i n . 200 6.8 TRANSITION METAL COMPLEXES AND HYDROLYSIS STUDIES Sodium T r i s c a r b o n a t o c o b a l t a t e ( l I I ) T r i h y d r a t e , 1 6 8 Na[Co(C03)3].3H20 A s t i r r e d s l u r r y of sodium bicarbonate (42.0 g , 0.50 mol) in 50 ml of H 20 was cooled to 0°C and a s o l u t i o n of C o C l 2 . 6 H 2 0 (23.8 g , 0.10 mol) and 30% H 2 0 2 (10 ml) in 50 ml of H 20 was added dropwise over a per iod of 20 mins. (Note: vigorous e f f e r v e s c e n c e ) . The o l i v e - g r e e n s l u r r y was al lowed to stand at 0°C f o r 1 hour with continuous s t i r r i n g . The product was f i l t e r e d and washed with three 10 ml por t ions of co ld H 2 0 . The complex was u s u a l l y used immediately f o r the preparat ion of c i s - g - [ C o ( t r i e n ) 0 H ( H 2 0 ) ] ( C 1 0 4 ) 2 , however, i t may be stored f o r fu ture use i f protected from moisture . g is -g -Carbonato t r ie thy lenete t raminecoba l t (111 ) Perchlorate Monohydrate [ C o ( t r i e n ) C 0 3 ] ( C 1 0 4 ) 2 . H 2 0 Concentrated (60%) p e r c h l o r i c a c i d (15.2 ml) was slowly added to an i c e - c o l d s o l u t i o n of t r i e thy lenete t ramine (12.5 ml) in 75 ml of H 2 0 . The complete batch of f r e s h l y prepared N a 3 [ C o ( C 0 3 ) 3 ] . 3 H 2 0 was added and the mixture s t i r r e d for 30 minutes at 0 ° C , (Note: vigorous ef fervescence) and then warmed to 60°C fo r 30 minutes. A l i g h t purple s o l i d was removed from the hot s o l u t i o n by suct ion f i l t r a t i o n with a coarse , s i n t e r e d - g l a s s f u n n e l . The s o l u t i o n was concentrated to ^75 ml by ro tary evapora t ion , and then placed in the cold f o r 1 hour to complete c r y s t a l l i z a t i o n of the complex. The product was c o l l e c t e d by suct ion f i l t r a t i o n and washed with 95% methanol. The red s o l i d was d i s s o l v e d in hot water (35 ml ) , and sodium perch lora te (8 g) and methanol (15 ml) were added. The product r e c r y s t a l l i z e d on coo l ing and was f i l t e r e d and washed with 95% ethanol u n t i l the washing was c o l o r l e s s . One f u r t h e r r e c r y s t a l l i z a t i o n from water gave a n a l y t i c a l l y pure m a t e r i a l . Absorpt ion spectrum, A (nm): 506, 359. 201 T e t r a a q u o b i s ( s - t r i a z o l o [ 3 , 4 - a ] p h t h a 1 a z i n e ) n i c k e l ( I I ) Perch lora te [ N i ( 3 - H - T A P ) 2 ( H 2 0 ) 4 ] ( C 1 0 4 ) o On s t i r r i n g n icke l (II) perch lora te hexahydrate (1.101 g , 3.01 mmol) with 10 ml 2,2-dimethoxypropane a dense immiscible o i l y layer of the metal s a l t was formed. Within an hour, the c o l o r changed from green to ye l low . A f t e r 1 1/2 hours , a s o l u t i o n 3-H-TAP (0.881 g, 5.18 mmol) in methanol was added to the mixture . A pale blue s o l i d p r e c i p i t a t e d slowly as a f i n e powder. The reac t ion mixture was s t i r r e d overn igh t , f i l t e r e d , a n d the s o l i d washed with methanol ( s l i g h t l y s o l u b l e ) . The product was d r ied in vacuo a t room temperature fo r 13 hours over D r i e r i t e . A n a l . C a l c d . f o r [ N i ( 3 - H - T A P ) 2 ( H 2 0 ) 4 ] - ( C 1 0 4 ) 2 : C, 32 .3; H, 3 .0; N, 16.7. Found C, 32.2; H, 3 .0; N, 16.7. D i a q u o t e t r a k i s ( s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e ) coba l t ( I I ) Perch lora te Mono- h ^ d r a t e [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 Cobal t ( I I ) perch lora te hexahydrate (0.391 g , 1.07 mmol) was s t i r r e d with 20 ml 2,2-dimethoxypropane under a n i t rogen atmosphere f o r 2 hours. s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (0.726 g , 4.27 mmol) was d i s s o l v e d in 20 ml warm methanol and added to the s o l u t i o n of coba l t p e r c h l o r a t e . A tangerine p r e c i p i t a t e appeared slowly which was f i l t e r e d a f t e r two hours, washed with dry methanol and e t h e r , and dr ied under vacuum at room temperature f o r 18 hours. The complex d id not show any d e f i n i t e melt ing p o i n t , but turned v i o l e t c a . 224°C . A n a l . C a l c d . fo r [ C o ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 : C, 43 .6; H, 3 .1 ; N, 22.6. Found: C, 43 .7 ; H, 3 .0 ; N, 22.6. 202 D i a q u o t e t r a k i s ( s - t r i a z o l o [ 3 , 4 - a j p h t h a l a z i n e ) n i c k e l ( I I ) Perch lora te l ^ ^ ^ l ? [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 N icke l ( I I ) perch lora te hexahydrate (0.156 g , 0.455 mmol) was s t i r r e d in 5 ml 2,2-dimethoxypropane f o r one hour, dur ing which time the suspended s o l i d changed from a pale green c o l o r to orange. Methanol (1.5 ml) was added <• to d i s s o l v e a l l the s o l i d , and the deep orange s o l u t i o n was s t i r r e d fo r a fu r ther per iod of one hour. s - T r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (0.308 g , 1.81 mmol) was d i s s o l v e d in 15 ml warm methanol and added to the s o l u t i o n of n icke l p e r c h l o r a t e . The c o l o r of the s o l u t i o n l ightened u n t i l i t was almost c o l o r l e s s , and a pale blue s o l i d g radua l ly p r e c i p i t a t e d from s o l u t i o n . A f t e r two hours, the s o l i d was f i l t e r e d , washed with dry methanol (the complex i s moderately s o l u b l e ) , and e ther , and d r ied in vacuo a t room temperature fo r 18 hours. A n a l . C a l c d . f o r [ N i ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 . H 2 0 : C, 43 .6; H, 3 .1 ; N, 22.6. Found C, 43 .8; H, 3 .0 ; N, 22.5 . D i a q u o t e t r a k i s ( s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e ) c o p p e r ( I I ) Perch lora te [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 Copper(I I ) perch lora te hexahydrate (0.245 g , 0.662 mmol) was s t i r r e d with 5 ml 2,2-dimethoxypropane f o r two hours. s - T r i a z o l o [ 3 , 4 - a ] - phthalaz ine (0.449 g , 2.64 mmol) was d i s s o l v e d in 15 ml warm methanol and added to the blue o i l of copper p e r c h l o r a t e . A l i g h t blue p r e c i p i t a t e was formed which immediately changed to purp le . The p r e c i p i t a t e was f i l t e r e d , and on washing with methanol (the complex i s s l i g h t l y s o l u b l e ) , the blue co lo r re turned. On drying in vacuo a t room temperature fo r 43 hours, the product was obtained as a purple amorphous s o l i d . On exposure to moist a i r or s o l v e n t s , the co lour changed to l i g h t b lue . The in te rconvers ion between the purple and blue forms appear to be r e a d i l y r e v e r s i b l e . A n a l . C a l c d . f o r [ C u ( 3 - H - T A P ) 4 ( H 2 0 ) 2 ] ( C 1 0 4 ) 2 ' ( p u r p l e form): C, 44 .2; H, 2 .9 ; N, 22.9 . Found: C, 44 .0; H, 2 .8 ; N, 22.9 . 203 H e x a k i s ( s - t n ' a z o 1 o [ 3 , 4 - a ] p h t h a l a z i n e ) c o b a l t ( H I ) Perchlorate Monohydrate [ C o ( 3 - H - T A P ) g ] ( C I 0 4 ) 3 . H 2 0 Sodium t r is (carbonato )coba1ta te ( I I I ) (0.749 g , 1.55 mmol) and ' s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e (1.582 g , 9.30 mmol) were suspended in 75 nil 95% ethanol and 10 ml 2M HCIO^. The suspension v/as heated to r e f l u x f o r 3 hours, during which time the c o l o r of the s o l i d changed from dark green to a l i g h t brown. Reaction was continued fo r an hour at room temperature. The s o l i d was f i l t e r e d , washed with 95% ethanol and e t h e r , and d r ied in vacuo at room temperature f o r 18 hours. A n a l . C a l c d . fo r [Co(3-H-TAP)gj - ( C 1 0 4 ) 3 . H 2 0 : C, ,46.5; H, 2 .7 ; N, 24 .1 . Found: C , 46 .5 ; H, 2 .9 ; N, 24.3. c - is -g -2 -an inomethy lpyr id ine ( t r ie thy lenete t ramine)coba l t (111 ) Perchlorate [Co( t r i en ) (2 -AMPy) ] (C10 4 ) 3 Two equiva lents of 2M HC10 4 (2.3 ml) were added to 8 - [ C o ( t r i e n ) C 0 3 ] - C10 4 .Hp0 (1.045 g , 2.731 mmol) and the s o l u t i o n warmed at 70°C u n t i l C 0 o ceased to be l i b e r a t e d (10 ni i n ) . On c o o l i n g , the pH of the s o l u t i o n was adjusted to about 4 with IM NaOH, and 2-aminomethylpyridine (2-AMPy) (0.296 g , 2.74 mmol) added. A f t e r heating the s o l u t i o n to 70°C f o r 15 minutes, the s o l u t i o n was quenched to pH 4 with 2M HCIO^, and a purple p r e c i p i t a t e f i l t e r e d o f f . A f t e r the s o l u t i o n was concentrated by rotary evapora t ion , the product was loaded onto the top of a chromatography column conta in ing Sephadex CM-25 ca t ion exchange r e s i n , and e luted with sodium perch lora te s o l u t i o n (0 .1-2.0M). If a s u f f i c i e n t l y long column i s used, two isomers of the product can be obtained as separate orange bands. Sodium perch lora te impuri ty was removed by concentrat ing the e luents conta in ing the complexes and c r y s t a l l i z i n g the NaClO^ from s o l u t i o n whi le rep lac ing the water with methanol. The s o l i d thus obtained always contained some i laCl0^- impur i ty , however. Absorpt ion spectrum, X m a x (nm): 1st isomer e l u t e d , 342 and 471. 2nd isomer e l u t e d , 339 and 467. 204 (3-Acetimi d o m e t h y l - s - t r i a z o l o [ 3 , 4 - a ] p h t h a l a z i n e ) t r i e t h y l e n e t e t r a m i n e - coba l t ( I I I ) Perch lora te _ . . . . [ C o ( t r i e n ) ( 3 - ( N - A c - g l y ) - T A P ) J ( C 1 0 4 ) 2 B- [Co( t r i e r . )C0 3 ]C10 4 .H z 0 (0.126 g , 0.329 mmol) was converted to 3 - [ C o ( t r i e n ) ( 0 H 2 ) 2 ] 3 + with two equiva lents of 2.0M HC10 4 (0.33 ml ) . A f te r the evo lu t ion of C 0 2 ceased, the s o l u t i o n was heated to 60°C f o r 10 minutes. On c o o l i n g , the pH of the s o l u t i o n was adjusted to about 4 by a d d i t i o n of 1M NaOH, and 3 - (N -Ac-g ly ) -TAP (0.080 g , 0.33 mmol) was added. The pH of the s o l u t i o n was ra ised to 8.0 with NaOH s o l u t i o n . A f t e r heating the s o l u t i o n to 6 0 ° C , methanol was added to d i s s o l v e 3 - (N-Ac-g ly ) -TAP complete ly , and the reac t ion continued f o r 1 hour at 60°C and 18 hours at room temperature. 3 - ( N - A c - g l y ) - TAP tended to p r e c i p i t a t e from s o l u t i o n , so s u f f i c i e n t methanol was added to prevent t h i s from o c c u r r i n g . The s o l u t i o n was concentrated by ro ta ry evapora- t i o n , and the components of the s o l u t i o n were separated by ion-exchange chromatography on Sephadex CM-25 using sodium perch lora te (0.1M - 0.5M) f o r e l u t i o n . Two orange bands which appear to be isomers of [ C o ( t r i e n ) ( 3 - ( N - A c - - 2+ g ly ) -TAP) ] were e luted with 0.2 - 0.3M NaClO^. The products were contami- nated with NaC10 4 , and p u r i f i e d by f r a c t i o n a l r e c r y s t a l l i z a t i o n from methanol s o l u t i o n , complexes, . However, i t was d i f f i c u l t to remove a l l t races of NaC10 4 from the Absorpt ion spectrum, A (nm): 1st isomer e luted ^355 - 360 s h , max and 472. 2nd isomer e luted ^355 sh and 475. 2+ Hydro lys is Study on 3 - (N-Ac-g ly ) -TAP in the Presence of Co A s o l u t i o n conta in ing equimolar amounts of 3 - (N -Ac-g ly ) -TAP and C o C l 2 . 6 H 2 0 (0.0125 M) in 1.00 M HCI, and a cont ro l s o l u t i o n conta in ing only 3 - (N-Ac-g ly ) -TAP at the same concentra t ion (0.0125 M) in 1.00 M HCI were incubated at 44°C . The progress of the h y d r o l y s i s reac t ions was monitored by TLC on s i l i c a gel GF a f t e r n e u t r a l i z a t i o n of a sample of the s o l u t i o n 205 with d i l u t e NaOH. The concentrat ions of the spots on the TLC p la tes were estimated v i s u a l l y . A f t e r 3 hours the degree of hydro lys is was s m a l l , and there was no s i g n i f i c a n t d i f f e r e n c e in the concent ra t ion of hydrolyzed product 2+ conta in ing C6 ions from the contro l s o l u t i o n . Hydro lys is study on 3 - (N-Ac-g ly ) -TAP in the Presence of Cu*1 ' An aqueous s o l u t i o n conta in ing equimolar amounts of 3 - (N -Ac-g ly ) -TAP and CuSO^.SH^O was adjusted to pH 10 with concentrated NaOH (Cu p r e c i p i t a t e s as C u t O H ^ ) , and then to pH 0.8 with 5N HCl . A cont ro l s o l u t i o n conta in ing only 3 - (N -Ac-g ly ) -TAP at the same concentra t ion (0.0125 M) was t reated s i m i l a r l y with base and a c i d . Both s o l u t i o n s were incubated at 5 0 ° C . The rate of h y d r o l y s i s of the acetamidomethyl group was monitored by TLC with s i l i c a gel on a n e u t r a l i z e d sample. A f t e r 5 hours, TLC a n a l y s i s showed a r e l a t i v e l y low concentra t ion of hydrolyzed product , which however, was s l i g h t l y 2+ higher in the s o l u t i o n conta in ing Cu i o n s . Attempted Hydro lys is of 3- (N-Ac-g1y)-TAP with a Cation-exchange Resin An aqueous s o l u t i o n of 3 - (N-Ac-g ly ) -TAP was e luted through a chromatography column conta in ing cat ion-exchange r e s i n (B io -Rad , Dowex 50W-X8; 200-400 mesh, 5.1 meq/g, H + form) (2.335 g , 11.91 meq) with water. The e luent was monitored by UV spectroscopy and TLC on s i l i c a gel 3F. No UV-absorbing mater ia l passed through the column. The b.ound TAP was e lu ted from the column with saturated NaCl s o l u t i o n . UV spectra were c o n s i s t e n t with protonated 3 - (M-Ac-g ly ) -TAP. There was no evidence of hydrolyzed product in th in layer chromatograms. 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