2. ACTIVATION OF CARBOXYL GROUPS IN PEPTIDE SYNTHESIS

- A REVIEW

2.1 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 main difficulties facing 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 relation-

ships.

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 of 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, modified structures containing specifically

labelled amino acids and also peptide mimetics are more

efficiently made by chemical synthesis. However, for larger

6 peptide molecules , the synthesis of chemically homogeneous

material is a challenging 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. The

8 syntheses of insulin , ribonuclease A' and also of lysozyme

analogues1' are milestones in this field. The synthesis of

larger peptides in solution, in which the various

intermediate compounds are purified and characterised is, in

principle, the correct way of obtaining homogeneous

material. ''-I4 However, this synthetic approach of larger

peptides is not without problems such as insolubility of

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 of shorter

peptides with unusual amino acids ,

peptides16 and the semisynthesis of

all often carried out in solution

Synthesis of peptide with a sequence of

amino acid residues is a fairly com 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^ at the cradle of this century itself and

peptide bonds were formed using this acyl chloride

method "ll'. 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

powerful and practical approach for the synthesis of

peptides. The'original steps of activation involves

hydrazinolysis of alkyl 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 hydrazide 21,22 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

Here, stepwise synthesis of 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 azide

(BOPA, 4) is another useful coupling agent. 2 4 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 by

treating carboxylic acid with phosgene. The most successful

generation of mixed anhydrides is by treating carboxylic

acids with alkyl chlorocarbonates. The resulting mixed 2 5 - 2 7

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 synthesis of peptides. 28-30 N -

Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6 ) which

can be readily obtained from ethyl chloroformate and

quinoline, give rise to mixed anhydrides on reaction with

carboxylic acids. 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. Thus, methyl esters could be further

activated by ~~Sstitution with 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. Thus, e- and para-

nitrophenyl esters (8 & 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 12) were found to be very useful for

peptide synthesis. 3 8 - L C

N-Hydroxysuccinimide and N-hydroxyphthalimide esters

(13,14) used more frequently than p-nitrophenyl and

chlorophenyl esters for peptide synthesis. 41-43 T h e

synthesis of N-hydroxysuccinimide esters present no

difficulties and they provide completely racemization free

peptides .

Esters derived from aldoximes (15) an& ketoximes (16)

were also studied in peptide synthesis.44 Esters o f

ketoximes made it possihle to synthesize serine 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 intramolecular base

catalysis. Prominent among these was the ester derived from

8-hydroxyquinoline (17). 4 5 The effect of 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 (HOBt) (21) and

indole-3-acetic acid (22) arc extremely potent acylating

agents. 4 9 7 5 0 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 was a turning point in the history of peptide

synthesis. 51 These compounds can be directly added to a

mixture of carboxylic acid and amine so 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

( 2 4 ) . 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 2 4 2 5

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, 26) is used nowadays for the

effective coupling of peptides. 5 5

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 through a reactive N-acyl

intermediate. 5 6 9 5 7 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 a. introduced a little more ideal reagent, BOP reagent [benzotriazol-1-yl-oxy-tris-(dimethylamino)-

phosphonium hexafluorophosphate] (28) for peptide synthesis

in solution phase. 58 ,59 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 degree of completion for peptide bond

formations.

hexafluoro phosphate (HBTU, 29) was found to be an efficient

coupling agent which provides peptides with minimal

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-amino acids). 64-66 Eventhough,

several small peptides were prepared using this unprotected

NCAs, they are not suitable for larger ones. 67 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. 6 8

Similarly g-butyloxycarbonyl (Boc) protected NCAs gave

excellent results and were comparable with BOP and HBTU. 69

2 . 7 . Photochemical Activation

In the conventional activation methodology, the

carboxyl functional group is usually converted into the

corresponding acyl halide, mixed anhydride or active

ester. 18*25933 In these cases, the carboxyl 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 can create serious

difficulties. Therefore, the use of activating groups which

could be accomplished under non-destructive neutral

conditions, avoiding the need for rigorous chemical

treatment is a suitable alternative. Photochemical

reactions could provide an ideal way of activation of the

carboxyl groups under mild, neutral conditions. These

photochemical activation procedures find promising

applications in the field of peptide and macrolide

synthesis. 70-73

A photochemical activating group contains a chromphore

which is sensitive to light, but relatively stable to most

of the wide variety of chemical reagents commonly

encountered. The wavelen~th of the radiation is absorbed

only by the activating group and it 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 reaction permits

effective synthesis of peptides, 7 5 , 7 6 macrolides 7 7 , 7 8 and

carbohydrates 7 9 3 8 0 under mild, 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 N-acyl derivatives of 5-bromo-7-nitroindoline

were 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-5-

bromo-7-nitroindolines.

ii) 2-Thionothiazolidines

- Burton and whiteBg has reported the photochemical

activation of carboxyl group via N-acyl 2 -

thionothiazolidines (31). 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, which undergoes

a facile S - -> N thermal rearrangement to yield the N -

acylated product (Scheme 2.1), 90,91

0 II

J-R ___* R-C-OR'

0

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 thiobenzoates investigated by

Barton and coworkers. 9 2 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

5-azido-1,3,4-oxadiazole involves incorporation of the

carboxyl group of a suitably protected amino acid in the

heterocyclic system. 96797 On irradiation, these 1,3,4-

oxadiazoles afford the activated acyl cyanide

corresponding to the carboxylic acid, with the extrusion of

two molecules of gaseous nitrogen (Scheme 2.3). When the

irradiation of 2-substituted 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) Ox~~zole- tri.amide rearrangement

In order to illustrate the generation of activated

carboxyllc species, Wasserman a &. developed the dye-

sensitized photooxygenation of oxazoles to triamides.

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, R2 and R3 it

was possible to limit the reaction of the triamide with

nucleophiles to one of the three carboxyl groups. loo This

selective reaction has been utilised by Wasserman a. for the synthesis of macrolide lactones and in the synthesis

of peptides. 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. 78

ACTIVATION OF CARBOXYL GROUPS USING 2-MERCAPTOBENZOTHIAZOLE