Chapter 2 Solid Phase Peptide Synthesis: An Overview

Chapter 2

Solid Phase Peptide Synthesis: An Overview

O v e r the last two decades, there has been a rapid progress in the chemistry

of large peptides and

Peptide synthesis has proven as an

indispensable tool for the structural elucidation of many recently isolated

natural products having a peptide structure such as hormones, neuropeptides

and antibiotics, which often could be isolated only in minute quantities.

Recent developments in the biotechnology of new proteins as well as

advances in immunology and the development of pharmaceuticals based on

inhibitors and antagonists have led to immense demands for synthetic

peptides. The fields of research in modem peptide chemistry include

synthesis and analysis, isolation and structure determination, conformation

investigations and molecular modeling.

The advances in chemical peptide synthesis over the last fifty years have made the synthesis of large peptides and proteins a realistic possibility. Chemical synthesis is probably the most practical way of providing usehl quantities of material, and in addition, allows the systen~aticvariation of structure necessary for the development of peptides for therapeutic use.2 Analogs of the peptides and modified structures containing specifically

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labeled amino acids or non-DNA-encoded amino acids and also peptide mimetics, are more efficiently made by chemical synthesis. Reports on the studies connected with the synthetic peptides revealed that they are used to raise anti-peptide antibodies, to study enzyme substrates and the binding properties of viral proteins to identify and locate gene products and in NMR studies of peptide structure.

The earliest modes of peptide bond formation pioneered by

~ ~ r t i aunds i~sh~her'^ at the turn of this century yielded impressive but not

yet practical results. Introduction of the amino protecting benzyloxycabonyl

led to a new era of peptide synthesis. Improvements in the method

of pcptide bond formation, particularly the invention of carbonic acid mixed

anhydridez8 gave a new impetus to peptide synthesis and in 1953, the

methodology reached a degree of sophistication which allowed the

synthesis of a peptide hormone, Oxytocin, by Du Vigneaud and his

associates. From here on, synthesis of peptides progressed by leaps and

bounds. Introduction of di~~clohex~lcarbodiimiades~t~ill unsurpassed

coupling reagent had a major impact on the methodology of peptide bond

formation and further refinement was brought about by the development of

active esters.30 Equally important improvements could be noted in the

methods of protection: acid labile blocking groups built on the stability and

thus ready formation of the tertiary butyl m at ion,^' the

tertiarybutyloxycarbonyl group particularly, remain among the tools of

unchallenged importance even after the introduction of base sensitive

blocking in the form of the 9-Fmoc

Yet, the most conspicuous

milestone in the history of peptide synthesis is the invention of solid phasc

peptide synthesis by R. B. Merrifield in 1963.~ Through painstaking

meticulous research, Merrifield determined the best conditions for his solid

%

phase synthesis. Since his 1963 article appeared, thousands of peptides and -- -

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other biological polymers including carbohydrates and nucleic acids have been synthesized using this method both by him and by others. In 1965, together with John Stewart, he automated the process, and today commercial models with microcomputer controls are available. In recognition for Merrifield's development of methodology for chemical synthesis on a solid support, he was awarded the 1984 Nobel prize in chemistry.

The synthesis of peptides is achieved either by the solution phase or by the solid phase methods. The solution phase method of peptide synthesis is laborious and time consuming as the intermediate products have to be removed, purified and characterized before proceeding to the next coupling step. Insolubility of the intermediate peptide in solvents used for the synthesis and mechanical losses are other problems associated with this method. Therefore, a new approach was needed if large amounts of peptides were required or if larger and more complex peptides were to be made.

The advances made in peptide chemistry and biology would not have been possible without the availability of the new methods of peptide synthesis. The feasibility of this technique was first shown by the synthesis of the crystalline tetrapeptide, L-leucyl-Lalanyl-glycyl-L-valine. Numerous developments have been made which widened the scope of the method.33 There is a greater demand for new strategies, faster synthesis,34 better coupling reagents, protecting groups and especially methods for simultaneous preparation and analysis of very large number of peptides in a short time. Stepwise peptide synthesis on polymer supports is regaining importance due to the recent developments made in protecting group strategy,35,36 anchoring techniques37-39 and support properties. 40,41

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2. 1. Principles of Solid Phase Peptide Synthesis

Solid phase peptide synthesis follows the stepwise assembly of peptides by consecutive coupling of amino acids. Memfield employed an

insoluble and f< il-tera~~b-le polymer support such as chloromethylated, 1% \ ~~

DVB-crosslinked polystyrene which functions as the carboxy protecting group for the C-terminal amino acid. Ailer incorporation of the first amino acid to the polymer through a benzyl ester linkage, the terminal amino group is deprotected under conditions which do not cleave the resin-amino acid ester bond. Then, another Na protected amino acid is coupled to the amino group of the polymer bound substrate using DCC ester coupling. The W deblocking and coupling steps are desired sequence is assembled on the polymer support.

After completion of the synthesis, the peptide is support. Memfield's strategy used strong acids like TFA, HBr-AcOH for

the cleavage of peptides fiom the polymer. This results in simultaneous Na-

deblocking and deprotection of most of the side chain functionalitiesto give the e e e peptide which is then purified by suitable procedures. Owing to standardization of the steps involved, solid phase synthesis can be automated. The chemical steps involved in the Memfield's synthesis using chloromethylated polystyrene are outlined in Scheme 2 . 1.

The Essential Advantages Associated with SPPS The reactions can be driven to completion using excess soluble low

molecular weight reagents and final products are obtained in good yield.

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The excess reagents can be easily separated from the polymer-bound peptide by simple filtration. As a result, the laborious and cumbersome

purification of intermediate peptide is avoided and this results in a

tremendous saving of time. Since it is possible to cany out all reactions in a

single reaction vessel, manipulations and attendant losses involved in the

repeated transfer of materials can be avoided. After synthesis, the spent

resin can be recycled as such or with some chemical modifications. So the

process is economical. The polymeric support should be insoluble, rigid

and capable of funtionalization to a relatively high degree. The functional

groups should undergo a straight forward reaction with reagents and must

be free of any side reactions. The support should swell in suitable solvents

and should be physicochemically compatible with the bound substrate,

reagents and solvents used, for effective reactions to occur. There are no

solubility problems encountered when adding one amino acid per cycle. In '

this respect, solid phase peptide method appears-to be more suitable for i.,nuo.,

protein synthesis.

1/ I .

The Limitations Associated with the Solid Phase Synthesis

Physicochemical incompatibility of the growing peptide chain with the polymer support, non-equivalence of functional groups attached to the polymer support, racemisation leading to optically impure products and formation of error peptides from deletion and truncated sequences.

(a) First amino acid attachment

(b) Deprotection & Neutralisation

I

Cesium salt method

RI Boc HN~coo-CHI

H

(i) TFA 30% (ii) DlEA 5%

2 H HN T C O O - C H 2 a

(c)Coupling (Active ester)

DCC/HOBt

I

R2 BOC H N ~ C O O H

H

R2

R1

Boc H N ~ C O - H N t C 0 0 - C H *

H

H

(d) Elongation of c h a i n

Repeat steps (b) a n d (c) n times

Rn-I R2 i

Boo N H V O - ( N H t C O . J t C O - H N r C O O - C H 2 a

H

H H

H

(e) Cleavage

I

TFA/thioanisole

Scheme 2. 1. General protocol for the assembly of amino acids by solid phase peptide synthesis.

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2. 2. Insoluble Supports and Anchoring Linkages in SPPS

Most recent work on the chemical modification of polymers has centered on the introduction and modification of various functionalities on polystyrene. Usually, 1-2 % DVB-crosslinked polystyrene has been used successfully in SPPS. The ideal resin with optimum swelling and stability was found to be 1% crosslinked polystyrene. Polystyrene, chloromethylated and ring lithiated polystyrene are used in the chemical modification of styrene polymers as they provide a method of attaching a wide variety of both electrophilic and nucleophilic species. In addition, a number of other supports incorporating functional groups like phenacyl, hydrazyl, acylsulfonyl, benzhydryl, aminomethyl, etc. have been used in S P P S . ~ ~

Newer resins have been developed with different aims such as improving resin-peptide bond stability, solvent-resin product compatibility, support loading, coupling efficiency, cleavage of finished resin-peptide bond and synthesis of protected peptide fragments including peptide esters, amides or hydrazides. Functionalized resins incorporating safety catch

device^,^' pellicularised resins based on silica,44 polyoxyethylene-

polystyrene graft copolymeric support (POE-PS);~ polyacrylate-DVB copolymer,46polyamide-kieselguhr support:7 isocyano resin:' Rink resin,4y 5[4 (9-Fmoc) amino methyl 3,5-dimethoxylphenoxy] valeric acid (PAL) resin,jO2-chloro trityl chloro resin," carboxylamide terminal (CAT) resin," tertiary alcohol re~in,'~2-methoxy-4-benzyloxy benzylalcohol resin,544nitrobenzophenone resin,55 4-[2, 4-dimethoxy phenyl (amino) methyl] methyl resins6and acid labile 9-Xanthenyl resin57have been developed for SPPS.

Recently, more supports were developed for multiple peptide synthesis. In Houghten's tea bag method, PS-DVB (1%) in polypropylene mesh packets were used as supports.58 In multi-pin synthesis technology, amino Iimctionalized polyoxyethylene rods are employed as supports.59 An inexpensive procedure recently developed by Frank et al uses a sheet of cellulose paper as support.60 In multicolurnn methods, macrosorb-SPR resin was used?' Multiple peptide synthesis on acid-labile handle derivatised polyethylene supports has been developed.62 Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports have been reported?' Multiple column peptide synthesis employing Fmoc-amino acid -0-Dhbt or -P@ esters in continuos flow version of the polyamide method on small packed columns of Kieselguhr supported resin in a reaction block of Teflon has been reported.64 An automated multiple peptide synthesis method to synthesis, cleave and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods.65

Fmoc SPPS using ~erloza" beaded cellulose has been reported. Fmoc-amino acids were anchored to amino propyl Perloza beaded cellulose via the TFA labile Coxymethyl phenoxyacetyl (HMPA) linker. Using Fmoc-aminoacyl-4-oxymethylphenoxy acetyl-2,4-dichlorophenylesters, all 20 amino acids were anchored at substitution levels ranging from 0.37-0.65 r n r n ~ l l ~ mC.o~nt~inuos flow synthesis of peptides using a polyacrylamide gel resin ( ~ x ~ a n s ihnas~b)een proved to be ~onvenient.~T' he hydrophilic support beaded cellulose (Perloza) can be used for peptide synthesis with modified Fmoc and Boc protocols.68 Beaded, hydrophilic, crosslinked aminoallcjl polydimethyl acrylamide supports have been used for the

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