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ACTIVATION OF CARBOXYL GROUPS IN PEPTIDE SYNTHESIS 2. - 2.1 A REVIEW Introduction Over the last two decades there has been a rapid growth in interest in the chemistry of peptides and proteins. 1-2 The study of the mechanisms of hormone action and the enzyme-substrate, antigen-antibody and protein-DNA inter- actions have now moved to the forefront of contemporary chemistry. One of the m a i n d i f f i c u l t i e s f a c i n g the researchers in this field is the isolation of sufficient amount of the peptide or protein in a pure form. The isolation of protein from natural sources is laborious and often provides only tiny quantity of material. Among the contemporary methods for peptide and protein synthesis biotechnological techniques are currently used, out of which the most useful method is the cloning of genes.3 - 4 They are powerful techniques but their drawbacks still create problems. Although, such methods can be used to provide modified protein structure,5 they are really inappropriate for developing analogous systems which are routinely necessary for investigating the structure-activity relationships. The advances in peptide synthesis over the last five decades have made the synthesis of larger peptides and proteins a realistic possibility. Chemical synthesis is probably the most practical way o f providing sufficient amount of material, and in addition, allows the systematic variation of structure necessary for the development of peptides for various biochemical studies. Analogous to peptides, specifically modified structures containing labelled amino acids and also peptide mimetics are more efficiently made by chemical synthesis. However, for larger peptide molecules 6 , the synthesis of chemically homogeneous m a t e r i a l i s a c h a l l e n g i n g field but success in this endeavour cannot necessarily be guaranteed. Organic chemists since Emil p is her' have noticed the problems that chemical syntheses of proteins entail. syntheses of insulin8 , ribonuclease ' A analogues1' and also of lysozyme are milestones in this field. larger p e p t i d e s in The The synthesis of s o l u t i o n , i n which the various intermediate compounds are purified and characterised is, in p r i n c i p l e , t h e c o r r e c t w a y of obtaining homogeneous material. ''-I4 However, this synthetic approach of larger peptides is not without problems such as insolubility o f peptides in various solvents, epimerisation at the activated C-terminal end, lack of powerful methods of purification etc. In spite of these, synthesis in solution is one of the important methods of peptide synthesis and continues to find various applications. The large scale peptides with unusual amino acids of shorter , peptides16 and the semisynthesis of all often carried out in solution Synthesis of peptide with a amino acid residues is a fairly com sequence of ess. The crux of peptide synthesis lies in the 'activation of carboxyl component' followed by aminolysis for the formation of peptide bond. To convert carboxylic acids into activated form. their hydroxyl group must be replaced by an electron withdrawing substituent ( X ) to enhance the polarisation ot the carboxyl group and thereby the electrophilicity at the carbon centre. This facilitates the nucleophi lic attack by the amino and alcoholic groups. The following are the important conventional methods used for carboxyl activation. 2.2 Acid chlorides The first attempt on the activation process was made by Emil ish her^ peptide at the cradle of this century itself and bonds w e r e method "ll'. formed using this acyl chloride Here the chloride ion is used as the electron withdrawing moiety. Acyl chlorides 1 were formed by using phosphorous pentachloride or thionyl chloride. 2.3 Acid azides Parallel to the acyl chloride method, acid azide method was developed by curtius'', which remains even today as a p o w e r f u l and practical approach for the synthesis o f peptides. T h e ' o r i g i n a l steps o f activation involves h y d r a z i n o l y s i s o f a l k y l esters and conversion of the hydrazides to acid azides 2 with the help of nitrous acid. In recent years direct conversion of carboxylic acids to acid azides with the help of diphenyl phosphoryl azide (3) or hydrazide21,22 serves as an attractive alternative. The reagent obtained from diphenyl phosphoryl chloride and sodium azide, proved to be very effective for the synthesis of peptides. Even in solid phase peptide synthesis the azide method was implemented by Felix and Merrifield. 2 3 H e r e , stepwise synthesis o f peptides by the initial attachment of an amino acid, t-butyloxycarbonyl hydrazide through its oC -amino group to a polystyrene resin was investigated. N,N3-Bis(2-oxo-3-oxazolidinyl) phosphorodiamide (BOPA, 4) is another useful coupling agent. 2 4 azide The peptides prepared by this method are of high purity and the extent of recemization is same as that of DCC/HOBt method. 2.4 Anhydrides A very simple and most efficient method of activation' is the treatment of amines with anhydrides of carboxylic acids 5. Symmetrical anhydrides are nowadays prepared b y treating carboxylic acid with phosgene. The most successful generation of mixed anhydrides is by treating carboxylic acids with alkyl chlorocarbonates. The resulting mixed 25-27 anhydrides are readily used in the synthesis of peptides. A particular advantage of alkyl carbonic acid mixed anhydrides is that the by-products of the acylation reaction can easily be removed from the reaction mixture. A very interesting development of the method of mixed anhydrides is the application of 1,2-dihydroquinoline derivatives in the s y n t h e s i s of peptides. 28-30 N- Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6 ) which can be readily o b t a i n e d f r o m ethyl chloroformate and quinoline, give carboxylic acids. rise to mixed anhydrides on reaction with Various peptides have been synthesised in good yields by this procedure. A polymer modification of EEDQ has been developed and various peptides have been prepared using this31 reagent. A closely related reagent N - isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIOQ, 713' is used nowadays for peptide synthesis which is powerful than EEDQ. 2.5 Esters Another well established mt.rhod to prepare amides of blocked amino acids or peptides is by treating their alkyl esters with ammonia. Electron withdrawing inductive effect in the alcoholic component of the ester renders the carbon atom of the carboxyl group more electrophilic and more ready to attack nucleophiles. T h u s , methyl e s t e r s could be further activated by ~ ~ S s t i t u t i owith n strongly electron withdrawing groups. Aminolysis of cyanomethyl esters 3 3 was found suitable for the preparation of peptides. Aryl esters were proved to be more promising and it was observed that reactivity would be increased using proper electron withdrawing substitutions. nitrophenyl esters (8 & Thus, eand para- 9) were well studied as the tools for peptide synthesis.3 4 - 3 7 Along with the nitrophenyl esters, chloro and fluor0 phenyl esters (10. 11 L peptide synthesis.3 8 - L C 12) were found to be very useful for N-Hydroxysuccinimide and N-hydroxyphthalimide esters ( 1 3 , 1 4 ) used more f r e q u e n t l y than p - n i t r o p h e n y l and chlorophenyl esters for p e p t i d e s y n t h e s i s . 4 1 - 4 3 T h e synthesis of N-hydroxysuccinimide e s t e r s p r e s e n t no difficulties and they provide completely racemization free peptides . Esters derived from aldoximes ( 1 5 ) an& ketoximes ( 1 6 ) w e r e a l s o s t u d i e d in peptide s y n t h e s i s . 4 4 Esters o f k e t o x i m e s m a d e it p o s s i h l e to s y n t h e s i z e s e r i n e and threonine peptides without protecting the hydroxyl group Interesting results have been obtained from esters derived from pyridine-4-aldoximes. Much attention has been devoted to activated esters whose reactivity is explained by i n t r a m o l e c u l a r base catalysis. Prominent among these was the ester derived from 8-hydroxyquinoline (17).4 5 The effect o f the intramole- cular base catalysis is manifested to a greater extent in esters based on 2-hydroxypyridine (la), 2-mercaptopyridine (19) and lLhydroxypiperidine(20). 46-40 Esters of l-hydroxybenzotriazole ( H O B t ) ( 2 1 ) and indole-3-acetic acid agents.4 9 7 5 0 (22) arc extremely potent acylating Enhanced reactivity of the former group is attributed to the anchimeric assistance. 2.6 Coupling Reagents Introduction of carbodiimides, especially dicyclohexyl carbodiimide (23) as reagent for the formation of peptide bond w a s a turning point synthesis.5 1 in the history of peptide These compounds can be directly added to a mixture o f carboxylic acid and amine s o that 1~ sit2 activation and coupling occurs.5 2 Eventhough, activation and coupling in a single step make this process so-popular, they providr pcptide scgments with loss of optical purity. Also, the reaction leads to thc formation of unreactive by-products such as N-acyl ureas (24). Both these difficulties to a possible extent can be overcome by the addition of auxiliary nucleophiles such as 1-hydroxybenzotriazole and N-hydroxysuccinimide (25) to the reaction system.53.51, 4 e0 O!-!-~"G 24 25 It should be noted that since HOBt is regenerated during acylation, its concentration remains almost constant during coupling. A more efficient coupling agent, 1-hydroxy 7-azobenzotriazole (HOAt, 2 6 ) is used nowadays for the effective coupling of peptides. 55 The success achieved in using DCC stimulated the search for better coupling agents. Among them, carbonyl diimidazole (27) was found to be an effective one which mediates coupling intermediate. 5 6 9 5 7 through a reactive N-acyl Since rigorous anhydrous conditions are needed both in the preparation of the reagent and in bond formation, it appears to be too demanding for general use Castro BOP reagent a.introduced a little more ideal reagent, [benzotriazol-1-yl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate] (28) for peptide synthesis in solution phase. 5 8 , 5 9 The rate of coupling using BOP was greater than DCC and other methods. The BOP coupling using the solid phase procedures 6 0 - 6 2 proceeded more rapidly and to a greater d e g r e e of completion f o r p e p t i d e bond formations. hexafluoro phosphate (HBTU, 29) was found to be an efficient c o u p l i n g a g e n t w h i c h p r o v i d e s peptides w i t h m i n i m a l racemization and side reactions.6 3 Just like BOP reagent, this is highly useful for rapid activation in solid phase peptide synthesis. Since 1940, N-carboxyanhydrides (NCA) have been used in the formation of peptide bonds and were extensively applied to the synthesis of poly ( A - a m i n o acids). 64-66 Eventhough, several small peptides were prepared using this unprotected NCAs, they are not suitable for larger ones.6 7 Urethane protected NCAs (UNCA) should result in readily controlled acylation reactions. Among them (9-fluorenylmethoxy) carbonyl protected NCAs (F-moc NCAs) have been used in the successful synthesis of a decapeptide in a flow reactor.68 Similarly g-butyloxycarbonyl (Boc) protected NCAs gave excellent results and were comparable with BOP and HBTU. 69 2.7. Photochemical Activation In the conventional a c t i v a t i o n m e t h o d o l o g y , the carboxyl functional group is usually converted into the corresponding acyl h a l i d e , mixed a n h y d r i d e or active ester. 1 8 * 2 5 9 3 3 In these c a s e s , t h e c a r b o x y l function possesses enhanced reactivity towards nucleophilic attack. All these approaches require somewhat drastic conditions which involve the use of a base. Sometimes the substrate may be sensitive to these conditions and c a n create serious difficulties. Therefore, the use of activating groups which could be accomplished u n d e r n o n - d e s t r u c t i v e neutral conditions, a v o i d i n g the need for r i g o r o u s chemical treatment is a suitable alternative. Photochemical reactions could provide an ideal way of activation of the carboxyl groups under mild, neutral conditions. photochemical activation procedures find These promising applications i n the field of p e p t i d e a n d macrolide synthesis.7 0 - 7 3 A photochemical activating group contains a chromphore which is sensitive to light, but relatively stable to most of the w i d e encountered. variety of chemical reagents commonly The wavelen~th of the radiation is absorbed only by the activating group and i t should not affect other parts of the molecule. Morcover, the photochemical reaction of the activating chromophore should no way affect the substrate molccule and the photoproduct should be readily separable. In the photochemical activation approach, the functional group is derivatized with a light sensitive chrornophore, which can serve as a latent activator of the functional group.7 4 On irradiation with light of suitable wavelength, the functional group is converted to an active form and the light sensitive chromophore is removed. The active species in which the functional group possesses enhanced reactivity towards the desired effective synthesis of peptides,7 5 , 7 6 reaction permits macrolides 7 7 , 7 8 and carbohydrates 7 9 3 8 0 under m i l d , neutral photochemical conditions. Photolabile groups have been used frequently in peptide synthesis as protecting and or activating groups and as handles of polymeric support. The photolysis reaction is very selective and highly efficient, especially when it is carried out under homogeneous conditions and even when the group is attached to a polymer. It has been shown that photolysis at 350 nm causes no damage towards the most light sensitive amino acids such as Trp and Tyr.8 4 The following review gives an insight into the photolabile carboxyl activating groups, touching upon their applications in this field. Amit et al.8 5 , 8 6 found that N-acyl derivatives of 5 - bromo-7-nitroindoline (Bni, 3 0 ) and aromatic or simple aliphatic acids undergo efficient photosolvolysis to yield acids, esters or amides depending on the nucleophile present during irradiation. Photosolvolysis of the 5-bromo-7-nitro- 1-indolino group was obseryed even using light above 400 nm. When were N-acyl derivatives of 5-bromo-7-nitroindoline irradiated in the presence of nuclrophiles such as amines, alcohols etc. the respective products were isolated. This reaction has been developed for the synthesis of peptides. 87 Goissis et a1.88 could synthcsitc protected peptide acids and esters by photosolvolysis of l-peptidyl-5bromo-7-nitroindolines. ii) 2-Thionothiazolidines - Burton and whiteBg has reported the photochemical activation of carboxyl thionothiazolidines (31). group via N-acyl 2- Photolysis of 31 in the presence of alcohols resulted in the formation of the corresponding csters in very good yield. The N-acyl derivatives are prepared by the treatment of the sodium salt of 2 - thionothiazolidine with the corresponding acyl chlorides in benzene. It was explained that the kinetic product formed in this reaction is the S-acyl derivative, a facile S - - > which undergoes N thermal rearrangement to yield the N- acylated product (Scheme2.1), 90,91 0 II ___* 0J-R R-C-OR' Scheme 2.1 A mechanism involving \(-hydrogen abstraction by the sulphur atom is postulated for this activation process (Scheme 2.2). This mechanism which involves a ketene intermediate is analogous to the mechanistic pathway for the photolysis of g-phenylethyl Barton and coworkers.9 2 thiobenzoates investigated by This activation approach using 2- thionothiazolidines has been extended to the synthesis of amides and peptides.93-95 Scheme 2 . 2 iii) 5-Azido-1.3.4-oxadiazo- A conceptually novel approach to the synthesis of peptides based on the photochemistry of 2-substituted involves incorporation of the 5-azido-1,3,4-oxadiazole carboxyl group of a suitably protected amino acid in the heterocyclic system.96797 oxadiazoles afford On irradiation, these 1 , 3 , 4 - the activated acyl cyanide corresponding to the carboxylic acid, with the extrusion of two molecules of gaseous nitrogen (Scheme 2.3). irradiation of 2-substituted When the 5-azido-1,3,4-oxadiazoles was carried out in the presence of nucleophiles such as amines and alcohols, the corresponding products result with an overall yield of 80-100%. N-N % -N Scheme 2.3 iv) O x ~ ~ z o ltri.amide erearrangement In order to illustrate the generation of activated carboxyllc species, Wasserman a &. sensitized of oxazoles to triamides. photooxygenation developed the dye- Under mild conditions the oxazoles convert to triamides, eventually each of the carbon atoms in the oxazole ring transforms to a carboxylate derivative (Scheme 2.4).98,99 Scheme 2.4 By proper choice of the substituents R1, R 2 and R 3 it was possible to limit the reaction of the triamide with nucleophiles to one of the three carboxyl groups. l o o This selective reaction has been utilised by Wasserman the synthesis of macrolide lactones of peptides. a.for and in the synthesis In peptide condensation, the carboxyl group of an N-acylated amino acid was protected by conversion to an oxazole derivative, which on photooxygenation regenerated the carboxyl group in the activated triamide form.7 8 ACTIVATION OF CARBOXYL GROUPS USING 2-MERCAPTOBENZOTHIAZOLE