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~atic variation of

structure necessary for the development of peptides for therapeutic use.2

Analogs of the peptides and modified structures containing specifically

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 u s ~ ~ and ish 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~lcarbodiimide~~ a still 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 - - -

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 35,36 37-39 strategy, anchoring techniques and support properties. 40,41

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 filterable 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 functionalities to give

the eee 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.

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 I Cesium salt method

RI Boc HN~coo-CHI

H

(b) Deprotection & Neutralisation (i) TFA 30% (ii) DlEA 5%

2 H N T C O O - C H 2 a H

(c) Coupling (Active ester) DCC/HOBt

I R2

BOC H N ~ C O O H H

R2 R1 Boc HN~CO-HNtC00-CH*

H H

(d) Elongation of chain Repeat steps (b) and (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.

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,46 polyamide-kieselguhr support:7 isocyano resin:' Rink resin,4y

5[4 (9-Fmoc) amino methyl 3,5-dimethoxylphenoxy] valeric acid (PAL)

resin,jO 2-chloro trityl chloro resin," carboxylamide terminal (CAT) resin,"

tertiary alcohol re~in, '~ 2-methoxy-4-benzyloxy benzylalcohol resin,54 4-

nitrobenzophenone resin,55 4-[2, 4-dimethoxy phenyl (amino) methyl]

methyl resins6 and acid labile 9-Xanthenyl resin57 have 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 '*K"7

Fmoc-aminoacyl-4-oxymethyl phenoxy acetyl-2,4-dichlorophenyl esters, all

20 amino acids were anchored at substitution levels ranging from 0.37-0.65

r n r n ~ l l ~ m . ~ ~ Continuos flow synthesis of peptides using a polyacrylamide

gel resin ( ~ x ~ a n s i n ~ ) has been proved to be ~onvenient.~' The 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

assembly of peptides using standard Boc or Fmoc chemistry in automated

equipment. Manual SPPS on resins with high loading capacity requiring

small volumes of solvents have been described.69 NHS-activated Pharmacia

HiTrap Sepharose was modified with 1,3-diaminopropane to give an amino

hctionalized support for S P P S . ~ ~

Some of the recent developments in the field of polymer-supported

peptide synthesis are new hydrophilic matrices for the synthesis of small

peptides by either batch or continuos flow methods7' and [2-(2-nitrophenyl

ethyl)] (NPE) resin7' for the synthesis of protected peptides and

oligonucleotides. Bis-2-acrylamidoprop-1-yl polyethyleneglycol crosslinked

dimethylacrylamide (PEGA) has been introduced as a hydrophilic,

incompatible and flexible support in peptide synthesis.73 Recently, a new

method for preparation of high capacity PEGA resins with well defined

loading of functional groups has been described for continuos flow

synthesis by Meldal and co- worker^.'^

Although the earlier solid phase chemistry was very usehl for

making small peptides and even small proteins, it was clear that there was a

need for improvement in several areas. Several tailor made anchoring

linkages have also been introduced between the first amino acid and the 75,76 polymer support to improve the synthetic procedures of SPPS. These

include phenylacetamido (PAM), p-methyl benzhydrylamide (MBHA), p-

alkoxy benzylester systems, etc. The introduction of multidetachable

anchors in SPPS making use of chemoselectively removable protecting

groups provide maximum flexibility and adaptability to the methods used

for removing the peptide from the polymer supports.77

By introducing a photolabile anchoring group between the resin

and the growing peptide chain, the finished peptide can be cleaved under

mild conditions by photolysis.78 A photolabile o-nitro benzhydrylamino

polystyrene support (NBHA-resin) has been used for solid phase synthesis

and C-terminal amidation of peptides.79 1-chloromethyl-2-nitro TTEGDA-

crosslinked polystyrene resin has been used as a photosensitive solid

support for preparation of fully protected peptides.80 Development of new

photolabile protecting groups like Menpoc (a-methyl nitropiperonyl

oxycarbonyl) and Menvoc (a-methyl nitro veratryloxycarbonyl) were

reported in the 13" American peptide symposium.8' Polymer-supported

solid phase synthetic procedures have been reported for the synthesis of C-

terminal peptide arnides using a new photolytically cleavable a-methyl

phenyl arnido anchoring linkage between the support and growing peptide.82

In Fmoc methodology, all resins are substituted with a linker such

as 4-hydroxymethyl phenoxy acetic acid (HMF'A) requiring attachment of

the first amino acid as an ester. This step can lead to low substitution,

racemisation or dipeptide formation. An efficient catalyst is DMAP. A

good alternative is the use of 2,6-dichloro benzoylchloride in DMF.'~ This

method can be used for the attachment of the first amino acid onto

hydroxymethylated resins. 5, 9-(9-Fmoc amino xanthen-2-oxy) valeric acid

(XAL) has been introduced as an acid labile handle for Fmoc-based peptide

amide synthesis.84 Other acid labile anchoring linkages used in SPPS are

dimethoxyacido labile linker ( D A L ) ~ ~ and 3-methoxy-4-hydroxymethyl

phenoxyacetic acid.86 Barany and Merrifield have reviewed on the recent q

developments on handles and supports for S P P S ~ An oxidation labile [ -- < C '

phenyl hydrazide group has been recently reported as a linker for solid

phase peptide synthesis.88 PAL handle is usehl for the synthesis of

dipeptide C-terminal amides using Fmoc/t-Bu strategy.50 Mc.lnnes and co-

workers have developed an efficient linker for SPPS based on

dibenzocyclohepta-l,4-diene system.89

Barter and co-workers have demonstrated the synthesis and

application of a novel silicon linker offering highly efficient cleavage to

small molecule libraries bearing no memory of resin atta~hment.~' 2-(NM

tert.butyloxycarbonyI-5-methyl-imidazol-4yl)-2-hydroxyacetic acid, a

safety catch linker recently developed by Hoffman and Frank allows direct

release of peptide acids into aqueous buffer after Fmoc solid phase

synthesis.9' A new procedure using dihydropyran-2-carboxylic acid as a

bifunctional linker for the synthesis of peptide alcohols has been

de~cribed.~' Linkers based on the Dde [I-(4,4-Dimethyl-2,6-

dioxocyclohexylidene) ethyl] primary mine protecting strategy have been

developed and their utility demonstrated in the solid phase synthesis of a

naturally occurring spider toxin. The linkers are stable to both acid and

base conditions and cleaved either by 2% vlv hydrazine hydrate or by

transamination with a volatile primary alkylamine in a variety of organic

solvents.

Some new polymeric supports based on polyacrylamide and

polystyrene have been developed for solid phase peptide synthesis.

Polyacrylamide supports include N,N'-methylene - bis - acrylarnide

(NNMBA), tetraethyleneglycol diacrylate (TTEGDA), triethyleneglycol

dimethacrylate (TEGDMA) and divinylbenzene (DVB)-crosslinked

polystyrene supports.'3s93 Polystyrene supports include TTEGDA,

TEGDMA and 1,6-hexanediol diacrylate (HD0DA)-crosslinked supports.93

These new resins were found superior to PS-DVB in terms of stability and

solvation properties. This resulted in the increased use of these resins in

solid phase peptide synthesis. Here, we have used polystyrene crosslinked

with tetraethyleneglycol diacrylate (IITEGDA) as the polymer support,

which can be easily prepared and functionalized. The polymer has high

mechanical stability, good solvation properties and suitable reactivity

characteristics, thus favoring speed and completion of all reactions during

synthesis.

2. 3. Use of Fmoc Groups in Peptide Chemistry

Carpino and Han described the use of Fmoc group for the

protection of amino function, which provides a simple, rapid and efficient

alternative to the common use of Boc-amino acids.32 It is also an

exceptionally mild procedure, avoiding both the repetitive trifluoroacetic

acid treatment in each cycle and the harsh liquid HF cleavage of the product

from the solid support. It offers the following advantages:

quantitative and rapid p-protective group cleavable by mild

nonhydrolytic base treatment.

use of tertiary butyl type side chain protection, which is known to be

completely stable to base.

cleavage of completed target peptide from resin by mild acidolysis . facile UV monitoring of both coupling and Nu-deprotection steps, thus

opening up the possibility of spectroscopically monitoring the peptide

synthesis.

Frnoc chemistry can be enhanced by combining it with HBTU

activation, a combination known as Fast Moc chemistry.94 Fmoc polyamide

solid phase synthesis was designed by Sheppard et a1 to overcome some of I > * , , A

4 the problems associated with SPPS using Boc chemistry. The new

polyamide support has a polarity similar to peptides and swells much better

than polystyrene in the solvents commonly used in peptide synthesis, eg:

NMP, DMF etc. A recent development in the polyamide method has been

the introduction of continuos flow synthesis which has advantages over the

batch synthesis recommended by Merrifield. Bayer et al described the first

continuos flow synthesis on chemically modified silica gel supports.95

Frank and Gausepohl employed loosely packed cartridges of different C'

polymeric gels for the continuos flow synthesis.96 Atherton and Sheppard

et al polymerised polydimethyl acrylamide gels on Kieselguhr and

successfully used the material obtained for continuos flow synthesis using 97 an automated synthesizer.-

2.4. Solvents in SPPS

Numerous developments in the area of SPPS have widened the

scope of the method, but one problem remained substantially unresolved,

i.e., the occurrence of aggregation within the peptide-resin matrix.98 It was

suggested that the incomplete solvation of peptide-resin complex might be a

source of difficulty in solid phase synthesis.99 Such incomplete solvation

was thought to encourage inter or intra chain association leading to more

compact structures within the complex compared with the freely solvated

state.

Dimethylsulfoxide has proved to be a superior reaction medium for

SPPS in aggregating systems and a solution to the difficult sequence

problem.100 The influence of resin swelling was recognized at the very

beginning of SPPS. HCI-dioxane, a powerful swelling solvent was found to

give good deprotection of Boc groups whereas HCI-AcOH, which did not / c

swell the resin, did not."' - Mixed solvent systems may optimize peptide- -- - 7

resin solvation by combining relatively polar and non-polar solvents.'02

Several mixed solvent systems used successfully in solid phase peptide

synthesis include trifluoroethanol (TFE)-DCM,"' dimethylsulfoxide

(DMSO)-DCM,"~ p-dioxane-~MF,'05 urea-DM~,"~ DMSO-NMP,'" and

1, I, 1,3,3,3-hexafluoro-2-propanol (HFIP)-DCM.'" An improved solid

phase synthesis of a difficult-sequence peptide using hexafluoro-2-propanol

has been suggested.

Recently, theory incorporating solvent electron donor and acceptor

numbers have been used to create mixed-solvent systems that minimize the

intermolecular 0-sheet formation.'09 Strong electron donor solvents such as

hexamethylphosphonic triamide (HMPA) or DMSO are mixed with DMA,

DMF or NMP."~ The partial substitution or complete replacement of tBu-

based side chain protecting groups for carboxyl, hydroxyl and amino side

chains by more polar groups would also aid peptide-resin so~vation."~ The

use of solvent mixtures containing both a polar and non-polar component,

such as 35% 1'HF-NMP or 20% TFE-DCM is recommended to alleviate the

problem of side chain-induced resin collapse.'"

Chaotropic salts have been shown to inhibit interchain P-sheet

aggregates and hence improve peptide-resin solvation and coupling

efficiencies. ' I 2 0.4 M NaC104, KSCN or LiBr was helpful for several

couplings in DCM-DMF (1:l) during Boc SPPS of Rnase I-13-MBHA-

PS."' The use of LiBr in anhydrous THF was reported to be extremely

effective in disrupting P-sheet structure in resin bound peptides."4 The use

of hexafluoro acetone trihydrate as a structure stabilizer for peptides have

been reported."5

2. 5. New Methods of Protection and Deprotection in SPPS

N-2-(2,4-dinitro phenyl) ethoxy carbonyl, 2-chloro-3-indenyl

methyloxycarbonyl (CLIMOC) and Benz (F) inden-3-yl methyloxy

carbonyl (BIMOC) are some of the base labile protecting groups similar to

9-fluorenyl methyloxy carbonyl group suitable for solid phase synthesis."6

Another newly introduced base labile a-amino protecting group is 2-(4-nitro

phenyl) sulphonyl ethoxy carbonyl (Nsc)."~

Several modifications to the widely used tertiarybutyloxycarbonyl

(Boc) group have been made. 118,119 4-methyl sulphenyl benzyloxycarbonyl

(Msz) group is a new protecting group removed by reductive acidolysis.

Acid labile monomethoxy trityl and dimethoxy trityl were used as amino

protecting groups in SPPS. Several new side chain protecting groups for

amino acids such as S-phenylacetamidomethyl (Phacm) group for

cysteine,120 2-(4-acetyl-2-nitrophenyl) ethyl group for the y carboxyl of

aspartic acid,I2' Nm -cyclohexyloxycarbonyl group (HOC) '~~ and N" -

allyloxycarbonyl group (Aloc) for tryptophan,'23 2,4-dinitrophenyl group

(Dnp) for hydroxyl function of t y r ~ s i n e , ' ~ ~ p-(methyl sulphinyl) benzyl

group for serine,12' 2,2,4,6,7-pentamethyl dihydrobenzofuran-5-sulfonyl

group (Pbf) for ~ ~ i n i n e ' ~ ~ and 2-adamantyl oxycarbonyl group (2-Adoc)

for &-amino group of sine'^^ have been introduced. For imidazole group

of histidine, W (1-adamantyloxy methyl) group (W-1-Adom) removed by

TFA, W-t-butoxymethyl group removed by mild acidolysis and W-

benzyloxymethyl group cleaved rapidly and cleanly by HBr in TFA or by

catalytic hydrogenolysis has been reported. 128-130 The new mild acid labile

protecting groups for the guanidino function of N-Fmoc-L-Arginine, 10,ll-

dihydro-5H dibenzo [a, dl cyclohepten-5-yl, 2-methoxy-l0,l I-dihydro-5H-

dibenzo [a, dl cyclohepten-5-yl and 5 H-dibenzo [a, dl cyclohepten-5-yl

groups in SPPS has been reported. 13'

New protecting groups like 2-(1-adamanty1)-propanol-2-esters

(Adp) removable under mild acid and 2-bromoethyl and 2-

iodoethyl esters deprotected by samarium diiodide found application in

SPPS . '~~ The use of 9-fluorenyl methylesters for protection of carboxyl

group has been proposed.'34 The advantage is selective deprotection under

mild conditions using secondary mines without racemization. The use of

2-phenyl isopropyl esters as carboxyl terminus protecting groups have been I .

reported in the fast synthesis of peptide fragments."' Robles and co-

workers have reportkd a new base labile carboxyl protecting group [2-(4-

acetyl-2-nitrophenyl) ethyl] ( A n ~ e ) . ' ~ ~

Tetrafluoroboric acid has been employed as a useful deprotecting

reagent in Fmoc-based SPPS.'~' A newly developed reagent for the

deprotection of t-butyloxycarbonyl and No-benzyloxycarbonyl

is iodotrichlorosilane. A report from the 22"d European peptide

symposium (1992) is about the optimized deprotection procedure for

peptides containing Arg (Mtr), Cys (Acm), Trp and Met residues.'40 A new

stepwise deprotection method using reductive acidolysis followed by

fluoide - ion in SPPS has been found to minimize aspartimide formation in

peptides containing an Asp-Gly sequence.I4' Sodium borohydride in

presence of palladium (0) catalyst could be used for effective removal of N-

ailyl oxycarbonyl (Alloc) protecting Recently, Wensbo has

described the selective removal of N-Boc protecting group using silica gel 143 at low pressure. The microwave assisted silica gel deprotection of N-Boc

derivatives have been reported by Vaquero and c o - ~ o r k e r s . ' ~ ~

The use of Fmoc-N-(2-hydroxy-4-methoxybenzyl) amino acids in

peptide synthesis has been reported.'45 The use of 2-methoxyethylester as a

protecting group in peptide and glycopeptide synthesis has been reported.

The selective removal of ME esters by lipases was achieved under mild

conditions (pH 7, 37OC) leaving all other linkages including peptide bonds

and other ester protecting groups ~naf fec ted . '~~

2. 6. Coupling Reagents in SPPS

The nature of acylating agent, protected amino acid activated

species and solvation of the growing peptide chain decide the efficiency of

coupling reactions. The introduction of DCC as a reagent for peptide bond

formation was a major event in the history of peptide synthesis. Other

carbodiimides utilized in SPPS are diisopropyl carbodiimide (DIPCDI) and

di-t-butyl ethylcarbodiimide. 147,148 Recently, benzotriazol-1-yl-oxy-tris

(dimethylamino) phosphonium hexafluoro phosphate (BOP) found

application in the synthesis of fairly complex pep tide^.'^^ Novel activating

agents like benzotriazolyloxy tri (pyrro1idine)-phosphonium

hexafluorophosphate (P~BOP),"' Bis[4-(2,2-dimethyl 1,3-dioxolyl)

methyl]-carbodiimide (BDDC),'" bromo tris (pyrro1idino)-phosphonium

hexa fluoro phosphate (P~B~oP)"* have been introduced.

Spccific sequences which contain dificult couplings during SPPS

can bc drivcn to completion using 2-(1H-bcnzotriazol- I -yl) 1,1,3,3-

tetramethyl ammonium hexafluoro phosphate (HBTU) or 2-(1H-

benzotriazol-1 -yl) 1,1,3,3-tetramethyl uronium tetrafluoroborate (TBTU)

mediated coupling. 153-155 Enhancement of peptide couplings was

recommended by a combination of 4-dimethylaminopyridine (DMAF')-

dicyclohexyl carbodiimide (DCC) in the case of hindered amino acid

residues.Is6 Simultaneous use of 1-HOBt and copper (11) chloride as

additives for racemisation free and efficient peptide synthesis by the

carbodiirnide method has been reported. Recently, l-Hydroxy-7-

azabenzotriazole (HOAt) has been described as a superior peptide coupling

additive, which enhances couplings yields in solution by about 6-32

times.Is7 The uronium and phosphonium salts of HOAt is also used. 3-

Dimethyl phosphinothioyl-2(H)-oxazolone (MPTO) was introduced as a

promising new reagent for racemisation eee couplings.'s8 Some of the

other newly introduced coupling reagents are N-cyclohexyl-N-isopropyl

carbodiimide (cIc),"~ 2-(benzotriazol-1 -yl) oxy- 1,3-dimethyl

imidazolidinium hexafluoro phosphate (BOI),'~' Tris (pyrrolidino)

phosphonium reagent,l6I Toppip U [2-(2-0x0-l(2H)pyridyl)-1,1,3,3-

bispentamethylene uronium tetrafluorob~rate]'~~ and FDP

(pentafluorophenyl diphenyl phosphate).163 Some of the newly developed

activating agents in peptide synthesis are 1-a-Naphthalene sulfonyloxy

benzotriazole ( N S B ~ ) , ' ~ 6-Nitro-1-a-naphthalene sulfonyloxy

benz~triazole,'~~ di-ter-butyl pyrocarbonate in presence of pyridine and

ammonium hydrogen ~arbona te , '~~ and tetramethylfluoroformadinium

hexafluorophosphate for solution and solid phase synthesis.167 Coupling

methods for the safe incorporation of cysteine with minimal racemisation in

9-Fmoc SPPS include BOP (or HBTU or HATU)/HOBt (or H0At)EMP

(4:4:4) without preactivation in CH2C12-DMF (1:1), DIPCDI/HOBt (or

HOAt) (4:4) with Smin. preactivation and preformed pentafluoro

phenylesters in CH2CI2-DMF (1 : 1).'6s A novel algorithm for the coupling

control in SPPS has been described. The control scheme relies on a feed-

forward artificial neutral network algorithm which can predict the final

yield of the reaction within its initial 5 min. by analyzing the conductivity

signal profile.169

Goodman and co-workers have described the utility of a-aminoacid

N-carboxyanhydrides protected with the three urethane protecting groups

most commonly used for peptide synthesis, 9-Fmoc, Boc and Cbz, as highly

effective reagents in peptide synthesis in both solid phase and in solution.'70

Fmoc-amino acid fluorides, whether as the stable isolated species or as

intermediates generated in situ, represent convenient inexpensive reagents

for peptide coupling.17' Rapid coupling occurs even for sterically hindered

amino acids. In the case of preformed acid fluorides, coupling does not

require the presence of base, thus precluding the incursion of any base-

catalyzed side reactions, including loss of configuration at the carboxyl

group undergoing reaction. The coupling of Fmoc-amino acid chlorides can

be mediated by the potassium salt of 1-HOBt and 1-HOAt. 172.173 Coupling

is fast and racemisation free.

2. 7. Cleavage of the Peptide-Resin Bond

On completion of the chemical synthesis of the peptide chain, the

final step requires removal from the solid phase support and liberation of

the protected side-chains of the trihctional amino acids. The most popular

reagent for cleavage of peptides from Boc-based resins is anhydrous HF.

But, this necessitates the use of expensive HF resistant fumehoods and

cleavage apparatus.

More recently, an improvement over the conventional strong acid

SN1 deprotection process has been developed by Tam and co -~orke r s . ' ~~

This is a two step procedure that incorporates an SN2 process as an initial

step followed by an SN1 step to remove the more resistant pretecting

groups. This two step procedure has been named the low-high HF

deprotection procedure. Over the past few years, the use of other strong

acids such as TFMSA (trifluoromethanesulfonic acid) or TMSOTf

(trimethyl silyl trifluoroacetate) have begun to appear in the literature as

alternatives to HF cleavage of PAM and MBHA resins. Dilute HBr in TFA

containing pentamethylbenzene and thioanisole was used in the cleavage

and deprotection of peptides on MBHA-resin.

A new two-step deprotection/cleavage procedure for Boc-based

SPPS is reported.'75 First, the protective groups are removed from 4-

(oxymethy1)-phenylacetamidomethyl (PAM) resin attached peptide with the

weak hard acid, trimethylsilylbromide-thioanisole~TFA. In the second step,

the peptide is cleaved flom the resin with a stronger hard acid such as

trimethylsilyl trifluoromethane sulfonate in TFA or with HF. The hard acid

deprotection with 1M trirnethylsilyl trifluoromethanesulfonate in TFA has

been applied for the porcine peptide w."~

In addition to acid cleavage, several resin types such as oxime can

be cleaved using a variety of different methods to yield peptide hydrazides

and analogs of protected fragments. Some resins like Br-Wang and the Br-

PPOA resins are even cleavable by light. Cleavage of peptide from

benzylester type linkers by 2-dimethyl arninoethanol or N,N-diethyl

hydroxylamine yield sidechain protected peptide acids.177 Reductive

cleavage of peptide-resin ester bond in classical Memfield resins give

peptide-alcohols when treated with lithium borohydride in THF.'~*

2. 8. Purification and Characterization of Cleaved Peptides

Since Memfield's breakthrough in peptide chemistry, advances in

instrumentation, chemistry and computer technology have simplified

peptide synthesis and have made purification and characterization easier.

Automated peptide synthesizers eliminate time consuming manual

operations and minimize contact with caustic chemicals.

Solid phase peptide synthesis has proved to be an established

procedure only after the introduction of efficient HPLC. Semipreparative or

preparative HPLC, usually on reverse phase is necessary to achieve purities

of > 95%.179 Depending on the peptide size and hydrophobicity, packings

of C18, C8 or C4 are recommended. Newer techniques such as reverse

phase flash chromatography and perfusion chromatography may become

very important in the future. 180,181

Mass spectroscopy, especially in the FAB mode is a very usehl

technique for the structure elucidation of peptide The

introduction of electrospray ionization (ESI) and matrix assisted laser

desorption ionization (MALDI) has enabled the intact ionization of large

biomolecules for mass spectral analysis. 183,184 High resolution 2D NMR

spectroscopy has become one of the important tools in the elucidation of

three dimensional solution structure of fair sized biomolecules. Some recent

reviews put an insight into structure determination of proteins by three- and

four-dimensional NMR spectroscopy. 185,186 Bandekar studied the IR and

Raman spectroscopic results on amide bands in proteins, peptides and

polypeptides. Copenhagen has described the use of near-1R.Fourier

Transform Rarnan spectroscopy as a new method for monitoring the

secondary structure of the peptide chain during solid phase peptide

synthesis. 187,188 Some more examples of conformational analysis of

peptides come kom the laboratories of Throntan, Narita, Baldwin and

Ponnuswamy. 189-192 The use of molar ellipticity at 222 nm and

deconvolution of the experimental CD curves for secondary structure

estimation is well established. 193*194 Among the interesting new additions to

the technique of solid phase synthesis, the incorporation of internal

standards such as norleucine seem to have a lasting value, as it allows

monitoring of the progress of a synthesis by simple means. 195,196

2. 9. S S Bridging in Peptides

Disulfide bridges formed between cysteine residues constitute an

important structural determinant in peptide and protein s t ruct~res . '~~ One

need only mention the role of Actinomycin D in the study of protein

biosynthesis and of Valinomycin in the study of membranes. The artificial

introduction of extra disulfide bridges into peptides or proteins allows the

creation of conformational constraints that can improve biological activity

or confer therm~stability.'~~

Starting with the pioneering work of du Vigneaud on Oxytocin, the

challenge to reproduce and engineer increasingly complex arrays of

disulfide bridges as found in natural peptides and proteins has stimulated

the efforts and ingenuities of many peptide chemists.199 Within the peptide

field, there are a number of native molecules containing disulfide bonds

including Somatostatin, Endothelin and Calcitonin. Many of the peptide

toxins from snakes and scorpions possess multiple disulfides. Following

these examples, disulfide cystine linkages have been incorporated into a

large number of synthetic peptide sequences with the aim of reducing the

conformational freedom of the native molecules. For example, cysteine

residues have been incorporated in place of non-essential residues in the

peptide Opiate enkephalins, RGD sequences and Somatostatin. 200,201 In

1982, Schiller and co-workers used a D-C~S', L-C~S' cyclization to

incorporate conformational constraint into Leu-Enkephalin and

~ ~ n o r p h i n . ~ ~ ~

A number of model peptides containing disulfide linkages have

been examined using NMR and X-ray dimaction. These studies have

mainly focused on the ability to induce p-turns and P-sheets?'' P-hairpin

mimics constrained by disulfide bridges have been explored in solution?04

Cystine containing cyclic and acyclic bicystine peptides have been reported

to represent excellent models of antiparallel P-sheets. The use of disulfide

linkages across helices to stabilize helical bundles is reported by several 205,206 groups. In TASP approach of protein designing, disulfides have been

used as conformational constraints of templates.207 More recently, the

importance of small disulfide loops in the functional active site in the

redox-proteins, thioredoxins and glutaredoxins has been recognized?08

Disulfide bridges are important structural motifs in natural and

engineered peptides and proteins. The preparation of disulfide-containing

peptides hinges on reliable chemistry to form disulfide bonds. Usually, a

linear sequence is assembled by solid phase method and then protecting

groups as well as the anchoring linkage is cleaved. There follows oxidation

in dilute solution to minimize unwanted dimerisation and oligomerisation.

The alternative of carrying out deprotection and oxidation of the cysteines

while the peptide chain remains anchored to the polymer support is of

obvious interest and has received considerable recent attraction. 209,210 Such

an approach takes advantage of pseudo-dilution, which is a kinetic

phenomena expected to favor facile intramolecular processes.2'l

The disulfide bond forming reaction is a key step in the synthesis of

cystine-containing peptides. Usually, air oxidation or iodine oxidation has

been employed for this reaction.212 However, the former is time-consuming

under highly diluted conditions and the latter needs particularly controlled

conditions. Methyl trichlorosilane or tetrachlorosilane in trifluoroacetic

acid (TFA) in the presence of sulfoxide, can cleave various S-protecting

groups of cysteine to form cystine directly?'3 A mild and highly efficient

method for intramolecular disulfide bond formation in peptides mediated by

charcoal has been developed. Completion of charcoal-assisted catalysis of

disulfide bond formation took less than 6 h, testing a series of peptides with

ring sizes varying from 2 to 17 amino

Cyclic peptides have become powerful tools in the hands of

biochemists and have served as the means whereby great

made in the study of a number of biochemical processes. .$ ,. . .

2.10. Synthesis of Hydrophobic Peptides

In 1959, K a m a n n suggested that

defined as the tendency of non-polar side chains to pack together in the

interior of the protein molecule where they can avoid contact with water,

might be important for the stabilization of the native structure of

It is now widely assumed that hydrophobicity is the major factor in

maintaining the specific structure of native The hydrophilic-

hydrophobic balance can be estimated theoretically from the calculated

hydrophobicity values and experimentally £rom the retention times in

reverse phase high performance chromatography.

The hydrophobicities of the individual amino acid side chains have

been measured experimentally in a variety of ways, using the free amino

acids, amino acids with the amino and carboxyl groups blocked and side

chain analogues with the backbone replaced by a hydrogen atom and using

a variety of non-polar solvents including ethanol, octanol, cyclohexane and

di~xane.~" Synthesis of hydrophobic peptides is a difficult process because

of the non-polar side chains and due to the coiling tendency of the

peptides.2's Peptides with substantial hydrophobic character also tend to

aggregate with increasing concentrati~n.~'~

Hydrophobic proteins are the vital components of every living cell.

Often associated with cell membranes, their functions vary from ion or

molecule transport to cell recognition to signal transduction. Owing to their

limited solubility in aqueous solvents, structural analysis by conventional

techniques has been problematic. Mass spectrometry has become a

valuable tool for the peptide and protein analysis. The development of mass

spectroscopy for the analysis of hydrophobic peptides and proteins provide

an extremely valuable tool in structural studies of this important class of

proteins.