POLYMER- SUPPORTED

SOLID PHASE REACTIONS: AN OVERVIEW

During 1980's research in synthetic organic chemistry was dominated

by asymmetric synthesis. The significant progress made by the synthetic

organic chemist is the development of chiral auxiliaries for asymmetric

carbonyl group transformation and reagent for enantioselective

chemocatalysts.'" \Nhile intensive research in asymmetric synthesis

continues and its importance remains, this decade has seen its popularity

equaled and position as vogue subject in synthetic organic ~ h e m i s t r y ~ , ~

challenged by solid phase peptide synthesis.

Merrifield pioneered solid phase synthesis back in 1963 and his

primary focus was on the solid phase peptide synthesis5. The breadth of

synthetic organic chemistry applied to solid phase peptide synthesis has

been widened owing to the emergence of combinatorial ~ h e m i s t r y , ~ , ~ ~

pharma~eutical'.~ and agrochemical research.' Moreover the interest was

focused in creating structurally diverse analogous on resins using

multicomponent condensation reaction." The preparation of small

molecules and drug like compounds on solid phase has undergone

exhaustive study. Specific target molecules for drug discovery programme

10 Chapter 2

such as hydroxamic acids," polyamines,12 biotinylated probes,13

peptid~mimetics'~ and benzodiazepines15 have been prepared.

Here, an attempt is made to discuss the new trends in polymer

supported reactions, its rriorphology related aspects and an overview of

solid phase peptide synthesis (SPPS).

2.1 Polymer support

A reactive functional group attached to a polymer has quite different

reactivity due to the so called 'polymer effects'. The origin may be physical

(viscous, diffusion effect, steric effect, site isolation, local concentration effect)

or chemical (micro environment interaction, coordination interaction).'"."

The reactivity of the polymeric reagent is influenced by nature of

the solvents and reagent:; to which the polymer is likely to be subjected

during the course of its f~~nctionalisation and subsequent application as a

reagent. It depends also on the chemical behaviour of the polymer support

which in turn depends upon the physical form, crosslink density, flexibility

of the chain segments and the degree of subst i t~t ion. '~~'~ Appropriate

choice of the support as well as reaction condition can overcome the

problem of low reaction rate and product y~eld compared to homogeneous

reactions.20

%+- S u p p o r t e d S o ~ u i ~ e Reactwm : An Ouewiew 11

2.2 Nature of the support

A polymeric support not only binds the reactive species but also

play an important role in determining the reactivity of the attached

~pecies.~'~~"he major requirements of the polymer support are:

1. ease of preparation with controlled degree of crosslinks to

give spherical porous beads.

2. compatibility with most organic solvents,

3. chemicial and mechanical stability,

4. inertness of the backbone towards reactants and reagents,

5. low cost and commercial availability of the monomers, and

6. cost of functionalisation.

A wide variety of supports was developed giving due consideration

to the above mentioned requirements. Several types of natural and

synthetic polymers have been chemically functionalised for use as reactive

supports.

2.3 Linear and crosslinked supports

Linear and c:rosslinked organic macromolecular species have found

application as synthetic reagents, catalysts and substrate carriers. Each

type possesses its own desired advantages and disadvantages depending

on the final utilisation. The advantage associated with the use of linear

polymers include the fact that the reaction can be carried out in a

12 Chapter 2

homogeneous medium without much diffusional problems and with equal

accessibility to all the funclional groups of the polymer. This is important in a

reaction which is known tc~ be sluggish, for substrates having large size and

which cannot penetrate the pores of crosslinked polymer matrix. But here

separation of the polymer from the low molecular weight material is difficult

and it can be achieved ol ly by u~trafiltration.~~ When the functional groups

were attached to the crosslinked sites, they were not available to the

reactants due to steric and other microenvironment factors

Linear N-chlorinated nylon polymers with high chlorine capacity were

used to oxidise primary and secondary alcohols to aldehydes and k e t ~ n e s . ~ ~

The halogenation of Nylcin 66 requires 3 hours at 1 5 ' ~ and converted 94%

of N-H bonds in the or~ginal polyamide into N-Cl bond. In addition, the

polyamide which was originally insoluble became readily soluble after

chlorination in chloroform. This may be due to the reduced intramolecular H-

bonding. The possibility of the side reactions producing unwanted crosslinks

and the difficulty in the recovery of support after the completion of the

reaction are the two major problems when linear polymers are used as

supports. The insolubility of a low molecular weight species in the

precipitating medium prevents the complete removal of impurities from the

precipitated polymer. Gel formation is also another problem associated with

linear polymer.

P05mer- SupgortedSoliilPhnse @actions :An Overview 13

In contrast, crosslinked polymers, being insoluble in all solvents,

offer great ease of processing and they can be prepared in the form of

spherical beads. Lclw molecular weight contaminants can be removed from

the insoluble crosslinked polymer by simple filtration and washing with

various solvents. In suitable solvents, beads with low degree of crosslinking,

swells extensively exposing the inner reactive groups to the soluble

reagent^.^"

Macroporo~rs or gel type resins are generally prepared by

suspension polymerisation2' using a mixture of vinyl monomer and small

amounts of crosslinking agents containing no additional solvents.

Macroporous and macroreticular resins are also prepared by suspension

polymerisation with higher amount of crosslinking agents and inclusion of an

inert solvent. Small particles are obtained by increasing the waterlmonomer

ratio or diluting the organic phase with a solvent for the polymer to be

produced. Increasing the amount of crosslinking agent has opposite effect.

Temperature also has a significant influence on the morphology of

crosslinked polymer bead. However, the most important factors are the

choice of dispersing agent and stirring rate.

Crosslinked polymers are also prepared by p o p ~ o r n ' ~ . ~ ~

polymerisation, by gently warming a mixture of vinyl monomer and a small

amount of crosslinking agent (0.1-0.5%) in the absence of initiators or

solvents which results in a white glassy opaque granular material fully

insoluble and porous with low density. In such cases the growing polymer is

propagated as a helix in the presence of high concentration of monomer. In

styrene-DVB copolymers, the monomers act as good solvents for the

growing polymer.

Particle with narrow distribution are easy to obtain in the submiron

range by emulsion polymerisation with the help of an emulsifier.'' A simple

process has been described involving the dispersion polymerisation of

styrene in a non solvating medium containing the dissolved cellulose

polymer. The particle thus obtained has a bead size around 10-20 microns

The beads with 100-600 microns are preferred as a polymer support

for functional group in organic synthesis or catalysts. The simplest way to

get beads with a given particle size is by sieving process.29

Gel type resins are highly crosslinked and have no permanent

porosity. They are found to be less reactive than linear polymers as reaction

will be limited by diffusion of the reagent within the resin pores. These resins

are referred to as microporous and less sensitive to the choice of solvents.30

2.4 Problems faced in polymer supported solid phase reactions

A proper choice of the polymer matrix is an important factor for

successful utilisation of the polymeric reagents. The industrial applications

of the polymer supported reagents are not frequent except for Merrifield

synthesis. This is due to certain limitations of the polymeric system.

Stability is one of the majcr limitations of polymer supported catalysts.3' The

B@r- ~ u p g o r t e d ~ o T i P h e @xctionr :An Ovewiew 15

hydroformylation of olefins with supported rhodium catalyst has been

extensively studied but not developed commercially. Another limitation

especially of the stoichiometric reaction is the resin capacity which is

controlled by the skeleton of the support itself.32 The third limitation is the

non accessibility or non availability of active site to low molecular weight

substrate. Most of them are not on the solid surface as it is in the case of

classical heterogeneous catalysts. They are buried deep in the highly

viscous crosslinked polymer matrix swollen with solvent system.

The support interacts with the surrounding medium. It may or may

not swell depending on its thermodynamic affinity with the medium and its

method of synthesis. It may selectively absorb one of the reactants or

products as a result of preferential solvation. The macromolecules can be a

linear species capable of forming a molecular solution in a suitable solvent.

Alternatively crosslinked species, the so called resin, which though ideally

being solvated by a suitable solvent remain macroscopically insoluble.33

The ease of chemical modification of a resin and success of its

application as a reagent or as a catalyst depend substantially on the

physical properties of the resin.34 Functionalised polymer support must

possess a structure which permits enough diffusion of the reagents into the

reactive site. The kinetics of chemical reaction is controlled by the diffusion

into the microparticles upto the point where there are enough functional

groups on the pore surface.

While considering the reactivity of the polymer supported

reagents, one important factor which is relevant is the diffusion and

distribution of the substrate between the support and the bulk reaction

medium. Entropy of mixing also contributes to the reactivity. Favourable

interaction between the support and the substrate may enhance the rate

of the reaction. But if the support and the substrate are mutually

incompatible, the effective concentration of the substrate in the support

volume will be diminished considerably which may lead to decreased

reaction rate. The concept of diffusion controlled transport of the

substrate has given importance to the consideration of the size of the

substrate as well as the particle size of the support.

A soluble low molecular weight species when attached to a

crosslinked polymer acquires the latter's property of complete insolubility in

all common solvents. Moreover, if the polymer is highly porous, the bound

molecules will remain fully accessible to the solvent and solute molecules.

So, it does not loss much of its reactivity which they exhibit in a

homogeneous medium The polymer matrix contributes a special

microenvironment for carrying out the reaction. Polymer matrix will

generally impose a restriction to molecular diffusion which is controlled by

pore parameters, chemical structure of the substituents on the polymer

backbone, and the distance between the attached molecule and the

polymer backbone.35

Pobmer- ~u~portedso&fa& %actions :An Overviav 17

For polymer supported solid phase reactions, the reagent moieties

are attached to linear as well as crosslinked polystyrene, polyacrylamide,

vinylheterocyclic polymers, polyvinylpyridine and polyacrylic acid supports.

These resins provide functionalities for the direct attachment of the reagent

species to it.

One of the major disadvantages cited for polymeric reagents was

slowness with which a functional group conversion takes place compared to

the homogeneous reactions. Polymer aided heterogeneous reactions are

comparatively very slow and requires vigorous conditions. This decrease in

reactivity is due to the close proximity of the macromolecular matrix and the

functional group. 'This is more pertinent in the case of crosslinked polymers

where the active groups are either flanked by crosslinks or buried in the

interior of the polymer. Such groups are not readily accessible to the

reagent or substrate in the continuous phase. It has been observed that the

reactivity of the immobilised fur~ctional groups can be considerably

increased if the active site is separated from the polymer backbone. Such

enhancement in property is observed when the functional group is

separated from the polymer backbone by flexible arm. This makes the active

function protrude away from the matrix into the solution phase where they

are accessible to low molecular weight species and so~vents.~"~'

The hydrogenation of carbonyl compounds catalysed by

poly(benzimidazole)~d38 catalyst demonstrated the enhancement through

spacers. Better results are obtained when the catalytic function is removed

from the benzene ring by a three carbon spacer. When the active site is

close to the polymer backbone, a higher activation energy is required for

effective molecular collision between the low molecular weight substrate and

polymeric reagent resulting in the retardation of the reaction rate. Such

limitations are encountered with polymeric t-butyl hydroperoxides also. To

overcome these limitations, studies are carried out to determine the

efficiency of polymeric t-butyl hydroperoxide reagents in which the reactive

sites are well separated from the polymer backbone.

2.5 Diffusion of solvents into the polymer matrix

Penetration of the solvent into the polymer matrix brings the

polymer to a state of complete solvation and this allows easy diffusion of

low molecular weight s~ibstrates into the polymer matrix. The degree of

penetration of the solvent will determine the effective pore size and

molecular weight exclusion limit for the diffusion of substrate molecules. It

is possible to cause a reaction to occur at a fraction of the available sites

by the control of the swelling properties of the polymer. Reaction in

swollen 1-2% crossli~iked polymer bead resemble to those in

homogeneous solution. Highly crosslinked beads are less reactive due to

hindered diffusion of the substrate into the polymer. If the amount of

crosslinks in the polymer is comparatively small, network chains are fairly

long and the molecules of low molecular weight substrate can easily

PO+- .Su~edSoTulPtia.se @actions : J4n Overview 19

penetrate into the solid matrix. When the network links are short, the

polymer completely losses its ability to swell. Even though a crosslinked

network of polymer cannot dissolve, the individual segments can solvate to

give a swollen gel. The maximum swelling occurs, when the dilution

accompanying the swelling equals the contraction force of rubber like

elasticity of the polymer chain. That is, when the solubility parameters of

the solvent and the polymer are substantially equal. It is believed that

crosslinked matrix ~ehaves like an osmotic membrane.

The swelling and diffusion characteristics of a polymer bead

depends on its porous structure, which in turn depends on the conditions of

polymerisation such as rnonomer to diluent ratio and temperature. The

influence of diluent on the pore parameters of the crosslinked bead was

studied by Coutinho et They used scanning electron microscopy for

observing the variation of the matrix structure with the change of diluent type

and amount, when :styrene and DVB were copolymerised using toluene and

heptane as diluent mixtures. The increase of thermodynamic affinity of the

diluent mixture promoted the formation of the beads with a smooth surface.

The copolymer syntnesised with toluenelheptane in the ratio (45155) had the

characteristics of a smooth surface with small channels regularly distributed.

The beads obtained by using large contents of the non solvent had a rough

surface. In these cases, the channels were bigger and more irregularly

20 Chapter 2

distributed. Evidently the polymer domains separated by voids were bigger

when large content of heptane was employed in the copolymer synthesis.

The physical parameters of a solid support can be characterised in

terms of total surface area, total pore volume and average pore diameter.

The reactivity of a polymer bead can be changed by the variation in

structural parameters like porosity and surface area. In the swollen state, the

size and shape of the polymer networks continuously change with the

solvating effect of a good solvent and hence the mobility of the polymer

segments.

Gel type supports having relatively small pore diameter and a large

effective surface area under certain circumstances give rise to high binding

capabilities upto approximately 10mmollg. The macroporous or

macroreticular supports have large pore diameters but relatively small

surface area. Chemical modification occurs largely on the pore surface.

Highly crosslinked and entangled polymer chains within the matrix are not

readily available for func:tionalisation. A resin of high surface area can be

prepared by using a good solvent as porogenic agent. The total pore

volume and pore size distribution depends upon the type and relative

volume of the diluent, degree of crosslinking and reaction conditions. The

high porosity of the matrix has two desirable effects. It leads to good flow

properties and does not hinder the penetration of the molecules of high

molecular weight substrete.

c%+- SupportedSolii Phase Qactionr : pn Owrvierv 21

The swelling characteristics of a crosslinked polymer largely

depends upon the nature and amount of crosslinking agent. The crosslink

ratio controls the behaviour of a resln in contact with a solvent and the

swelling is found to be inversely proportional to the crosslink density of the

polymer backbone.'''

At low cross1 nk density the solvent swollen polymer may resemble a

homogeneous solution. Here, the gel network consists largely of the solvent

with only a small fraction of the total mass being the polymer backbone. But

with increasing crosslink density, the tendency of the polymer backbone to

expand in a good solvent is reduced and penetration of the reagent into the

interior may become impaired. In the presence of solvents which are not

able to swell the polymer network, the movement of the reagents within such

a network become diffusion controlled. At higher concentration of the

crosslinking agent, during suspension copolymerisation, in addition to

crosslinking, considerable chain entanglements also occur. This reduces the

extent of swelling in the presence of a good solvent. Polymers with large

pores, macroporous and macroreticular resins also absorb reasonable

amount of solvent :simply by filling the available voids. Good solvent may

penetrate and solvate highly entangled areas of the polymer. Solvent

compatibility of the support can be adjusted by incorporating suitable

monomer unit in the polymer chain by copolymerisation. In the case of

highly swollen gel type resin, pools of solvent appear within the polymer

matrix through which molecules can diffuse quickly without the requirement

of molecular motion of the polymer ba~kbone.~'

In the case of crosslinked polymer networks, the distribution of the

functional group on the polymer backbone is non homogeneous and there is

some extent of non equivalence of the functional group. It might be expected

that groups placed in the vicinity of the crosslink points are less accessible

to reagents and solvents than groups situated away from the crosslinks. In

the solvolysis of p-nitrophenyl acetate catalysed by Poly 2-acrylamido

pyridine, the reaction rate was increased upto a certain crosslink ratio and

then decreased. At very high crosslink density, due to decreased swelling of

the gel, the accessibility of the pyridine group is less. In the bromination of

polyacrylamide the molecular character and extent of crosslinking was found to

influence the functionalisation and reactivity. According to Pillai, V.N.R et al..

reactivities of brominated 5% and 10% NNMBA crosslinked polymer are

higher than those of the linear ones4'. But with higher amount of

crosslinking, the polymer network becomes rigid and the penetration of the

solvent and substrate molecules into the active site becomes difficult. The

initial delayed activity of hear polymeric reagent was due to the conversion

of the linear chains into blocks during the course of the reaction. The

crosslinked reagents appear as small particles and upon wetting they swell

into small beads and the extent of swelling decreases with increase in

c r o s ~ l i n k i n g . ~ ~ ~ ~

PO+- ~upported~otidPhase @actions : ,8n C~J& 23

One of the major areas in polymer supported solid phase reaction

that requires modernisation is in the design of polymer supports since

synthetic peptides comprising several amino acids have emerged as a

powerful tool in biological research. The study of the mechanism of hormone

action, enzyme-substrate, antigen-antibody and protein-DNA interaction is

possible.45-47 Chenlical synthesis of peptides can confirm the naturally

occurring structure!;. It can be made available in greater quantities for further

investigations to delineate antigenic determinants, to allow preparation of

artificial vaccines, and potent new drugs by judicious chemical substitutions

that change functional groups and conformation of the parent peptide.

Chemical synthesis is the practical way of providing useful quantities of

material. In addition, it allows systematic variation of the structure necessary

for developing peptides for therapeutic use. The main challenge in peptide

synthesis is to establish synthetic routes to homogeneous products of

defined covalent structure. The use of polymeric resins as a solid support for

the synthesis of peptides have become popular ever since divinylbenzene

crosslinked polystyrene (PS-DVB) was introduced by Merrifield in 1 9 6 3 . ~ ~

Cumulative advances have made it possible to synthesise large peptides by

stepwise synthesis using different supports. 45.46

Chemical assembly of amino acids to form peptides is achieved

either by the 'solution phase' or by the 'solid phase' method. The classical

solution phase neth hod for the synthesis of polypeptide began with Fischer

24 Chapter 2

at the beginning of the 20th century when he synthesised the first peptide

and coined the word '~eptide'.~' Chemical synthesis of peptide involves the

blocking of the carboxyl group of one amino acid and the amino groups of

the second amino acid. Then by activating the free carboxylic group of the

second amino acid the peptide bond was formed. The protected peptide

was then precipitated. C-terminal and N-terminal protections were

selectively removed and the resulting dipeptide was purified before

proceeding to the incorporation of the third amino acid. This method of

peptide synthesis is laborious and time consuming because intermediate

peptides have to be removed, purified and characterised before proceeding

to the next coupling step.48

In 1963, Merrifield introduced the concept of solid phase peptide

synthesis. The feasibility of this method was demonstrated by synthesising

a tetrapeptide L-leucyl-L-alanyl-glycyl-L-va~ine.~ In the same year Letsinger

and Kornet reported the synthesis of a dipeptide L-leucyl-glycine on a

'Popcorn polymer suppolt' using a different chemical approach. Since then

several methodological ilnprovements and refinements have been effected

in the synthesis of peptide. The design of polymer support, its chemistry and

application of the solid phase peptide synthesis have been well

documented.""' Here, an updated overview of solid phase peptide

synthesis emphasising new polymeric supports is given.

T o e - SupportedSohf T k Ractbns :%I - 25 2.6 Principle and strategy of solid phase peptide synthesis

The basic idea of solid phase approach involves the covalent

attachment (anchoring) of the C-terminal amino acid of the target peptide

chain to an insoluble polymeric support in all stages of the synthesis. The

target peptide sec!uence is formed in a stepwise manner in the C+N

direction using activated Nu-protected amino acids. After the incorporation of

the N-terminal amino acid N-terminal Boc protection is removed and the

target peptide is cleaved from the support and purified. The general steps

involved in the Merrifield solid phase peptide synthesis using

chloromethylated d~vinyl benzene crosslinked polystyrene support is outlined

in Scheme 2.1.

In Merrifielcl's solid phase peptide synthesis, the C-terminal amino

acid with a ternporary Nu-blocking function like t-butyloxy carbonyl (Boc) was

attached to the chloromethyl resin by a benzyl ester linkage. The ternporary

amino protecting group Boc can be removed with 1M HCI in glacial acetic

acid, 4N HCI in dioxane or with 30% TFA in D C M . ~ ~ " The resulting amine

salt was neutralisecl with a tertiary amine like triethyl amine.

l deprlecllan 30 ",TFA In DCM

I Neotrallsatlon 5 O . DlEA in NMP

1 Elongation of peptide chain

I Cleavage TFA l scavenge,

Scheme 2.1. Merrifield solid phase peptide synthesis, using chloromethylated divinyl benzene-crosslinked polystyrene support

fibmer- SupportedsofidiirPhare Qacrrbm : fin O v e d 27

The free amino group of resin bound amino acid was then coupled

to the next Boc-amino acid using DCC as a coupling agent.6' Another

important feature cmf this technique is that all the coupling and deprotection

steps can be monitored using ninhydrin. All the above reactions are carried

out under non aqueous conditions in organic solvents like DCM, DMF, and

NMP. Finally, the completed peptide was deprotected and cleaved from

the support with HF,~' or neat TFA in presence of scavenger^.^^

The classical solution phase peptide synthesis produced peptides

of high purity, but it still suffers from the following drawbacks:

Peptide synthesis is slow, tedious and laborious. To obtain

peptides with high purity, intermediate peptides has to be purified

and characterised. This leads to longer synthesis time,

S Insolubility of the intermediate peptide in solvents that are used

during the synthesis causes problems in purification and in the

next coupling reaction it results in abrupt termination of chain

elongation, and

Large nl~mber of manipulations result in considerable reduction in

overall yield of the peptide.

Solid phase peptide synthesis has a number of advantages over

the classical solution phase peptide synthesis. They are:

28 Chapter 2

S The peptide can be synthesised while its C-terminus is covalently

attached to an insoluble polymeric support. This permits easy

separation of the by-products or constituent amino acids from the

growing peptide,

z All the reaction steps can be driven to completion by using an

excess of reactants and reagents

H No loss occurs because the growing peptide is retained by the

polymer in a single reaction vessel throughout the synthesis,

d Due to the speed and simplicity of the repetitive steps, physical

operations involved in the solid phase procedure are amenable to

automation, and

6 The spent resin can be recycled

Merrifield's solid phase method possesses several limitations and

have been critically discussed by several a~ thors .~ ,~"he major limitations

of solid phase peptide synthesis are:

Y Non compatibility of the resin and the growing peptide chain,

Stability of pef>t.de - resin linkage under the conditions of peptide

synthesis,

Formation of error peptides due to deletion and truncated sequences,

and

PO+- ~ u ~ d ~ o f i d r p h a . s e %actions : An Overview 29

u Changes ;n the peptide conformation occur in macroscopic

environment within the polymer matrix due to peptide-resin linkage.

Solid support plays an important role in determining the purity and

homogeneity of the peptide. In order to achieve this, the reagents and

solvents must be freely accessible to the growing resin bound peptide

chain. Divinylbenzene crosslinked polystyrene (DVB-PS) support has been

widely used in solid phase peptide synthesis with considerable success.

However, low penetration of the reagents, difficulty in selecting a good

solvent for both polymer and growing peptide, and low rates of acylation

and deprotection are the major limitations of using the support. 48

Swelling characteristic and mechanical stability of the support also

play an importanl role in solid phase peptide synthesis5. Though PS-DVB

resin was rigid and showed h~gh mechanical strength, its swelling

characteristics in non polar solvents was poor due to the hydrophobic

macromolecular environment of the polymer.50 Since the growing peptide

is hydrophilic, it gets aggregated in non polar solvents, which will affect the

purity, and the homogeneity of the synthetic peptides.5',52

The solid support polystyrene crosslinked with divinyl benzene (1)

introduced by klerrifield, has been widely used for the peptide synthesis

using Boc c h e r n i ~ t r ~ . ~ ~ . ~ '

30 Chapter 2

The ideal resin \ ~ i t h optimum swelling and stability was 1%

crosslinked polystyrene. 0.5% crosslinked resin was found to be too fragile

while above 2% crosslinked polystyrene does not swell sufficiently in

DCM.~' AS the peptide grows within the resin beads, increased swelling of

the resin was observed.69 This increased swelling is due to the change in

the chemical potential of a swollen network. It has been attributed to

lowering of the network free energy based on the additional solvation of

the growing peptide

Merrifield's technique has undergone a series of modifications and

improvements because of the physico-chemical incompatibility of the

growing peptide chain and the rigid hydrophobic macromolecular

environment created by tile PS-DVB network of the support. In order to

@ ' o h - SupportzdSolidPhe Qactionr :An Oumview 31

optimise the resin structure in SPPS, Sheppard introduced a polar

polydimethyl acrylamide resin, which is structurally similar to peptide

ba~kbone.'~.'~ Th~s helps easy solvation of the peptidyl resin and thus

reduces the steric hindrance during deprotection and coupling rea~t ions . '~ .~~

Crosslinked and functionalised polydimethyl acrylamide gel can be

detained within the pores of fabricated Keiselguhr which can be used as a

matrix in continuous flow method. The support shows effective swelling in

polar solvents but in non polar solvents it is very poor. The chemical

stability of the resin is also less comparable to that of polystyrene supports.

in multi-pin synthesis technology (PIN) acrylic acid coated

polyethylene rods are used as support. In simultaneous multiple peptide

synthesis (tea bag method) polystyrene in polypropylene mesh packets are

used as supp0r1.~' In multicolumn methods Macrosorb-SPR resin was

used.82 The mixec PEG-PS resin, a highly promising class of solid support, is

used successfully in polypeptide s y n t h e ~ i s . ' ~ ~ A crosslinked polystyrene-

polyethylene glycol graft copolymer with a 2-nitrobenzyl anchoring group has

been used as a solid support for the stepwise synthesis of peptides.'"

Swelling, which is a sign of good solvation of the resin, is good for

polystyrene resins in non polar solvents like DCM, whereas polyacrylamide

resins swell mucn better in DMF. Mixed PEG-PS polymers show excellent

swelling in common solvents such as THF, acetonitrile and alcohol^.'^ The

similar polarity of peptides and polyacrylamide support, both well

32 Chapter 2

solubilised in DMF, makes the support suitable for SPPS.'' On the basis of

solvent-resin interactions, increasing effort has been made to introduce

polyacrylarnide supports where initially polystyrene supports were used."

Bis-2-acrylarnidoprc~p-1-ylpolyethyleneglycol crosslinked dirnethyl

acrylarnide (PEGA) has been introduced as a hydrophilic, biocornpatible and

flexible solid flow stable support in peptide synthesis (2).89

(2) The first flow stable synthesis resin was obtained by polymerisation of

the soft polydirnethyl acrylarnide gel inside a solid matrix of supporting

~eiselguhr.'' Small and Sherrington replaced the irregular Keiselguhr with

more regular rigid 50% crosslinked polystyrene sponge containing a

grafted polydimethyl acrylamide gel." This technique was developed for

grafting polyethylene glycol on to 1% crosslinked po~ystyrene,'~ which

were rnonodispersed, spherical and flow stable. Polystyrene grafted to

%+- SuprtedSold P h e Xgactzbns : A n O v e r u k 33

films of polyethylene has been used for synthesis of peptides under non

polar conditions (3).93 Polyhydroxypropyl acrylate coated polypropylene

and cotton has shown some promise as supports under polar condition^.^^,^^

A copolymer of bis-acrylamido polyethylene glycol, N, N'-dimethyl acrylamide

and acryloyl sarcosin ethyl ester was successfully employed for the

synthesis of peptide!;.g6

(3)

The inert polyethylene glycol crosslinked resins such as

polyoxyethylene-polyoxypropylene (POEPOP) (4) and polyoxyethylene

polystyrene (POEPS) (5) were efficiently used as flexible and biocompatible

resins in SPPS." Crosslinked Ethoxylate Acrylate Resin (CLEAR) supports

developed by Kempe and Barany were also used successfully in SPPS."

34 Chapter 2

Different concepts for multiple peptide synthesis (MPS) or

simultaneous multiple peptide synthesis (SMPS) were developed to respond

to the rapidly growing demands for a large number of peptides with

completely different sequences. The various methods can be distinguished by

the differences in the polytneric supports employed, the number of peptides

possible and the amount of products obtained. The multiple synthesis methods

are mostly applied in hormone and inhibitor research.

For the identification of relevant side chains, every amino acid of a

biologically active pepticle can be systematically substituted, the chain

length can be varied, ana N-as well as C-tennini can be modified."

Po&ner- SupportedSo&f 'Phase @actions :Art Overview 35

In the initial work of SPPS, the various N-terminal modifications

such as acetylations, biotinylations succinimidilations or couplings for

preparing immunogens could be achieved by multiple methods or by

consecutive synthesis. In multiple peptide synthesis it is possible to

prepare simultaneously the same peptide bound to different anchors and

cleaved to obtain peptide acid, peptide amide, alkylated arnide, hydrazide

or a fully protected fragment. The 'tea-bag' method proposed by Houghten

belongs to the oldest strategies of multiple peptide syn thes i~ . ' ~~ In 'tea

bag' method, polystyrene in polypropylene mesh packets was used as

supports. Geysen et al. developed the concept of multi-pin synthesis

technology (PIN) and several hundred peptides can be simultaneously

prepared using this procedure.lq2 Acrylic acid coated polyethylene rods were

used as supports in multi-pin synthesis technology. Valerio et al. used 2-

hydroxy ethyl methacrylate grafted polyethylene supports in multi-pin

peptide synthesis 101,102 In rnulticolurnn methods Macrosorb-SPR resin

63 was used. Frank et al. proposed an inexpensive procedure for the

preparation of polymer bound peptides in which the first amino acid was

coupled to a sheet of cellulose paper.103 Crosslinked enzyme crystals

(CLECs) of thermolysin are also used for peptide synthesis.'04

For effective swelling of the resin and solvation of the peptide, the

polymer should have optimum hydrophobic-hydrophilic b a l a n ~ e . ' ~ ~ . ' ~ ~ The

structure-reactivity correlations in crosslinked polymeric systems helped

Pillai, V.N.R et al. to design new series of supports with optimum

reactivity, mechanical stability and other essential requirements of a

polymeric support. Compared to other supports, styrene based polymer

supports showed high mechanical and chemical stability towards various

reagents and solvents that were used for polypeptide synthesis. The

problems associated with PS-DVB resin could be overcome to some

extent by the judicious choice of the crosslinker that can provide optimum

hydrophobic-hydrophilic balance to the resin. This increases the swelling of

the polymer in various o'rganic solvents and thus enhances the coupling

and deprotection rate. These new resins are highly solvated in various

solvents. This results in fast and quantitat~ve reactions. Their polarity must

also be compatible with those of reagents and solvents used.

The tetraethyleneglycol diacrylate crosslinked polystyrene (PS-

TTEGDA) (6) was developed by introducing hydrophilic flexible TTEGDA

crosslinker to polystyrene. The polymer was synthesised by aqueous

suspension polymerisation of styrene with TTEGDA using benzoyl

peroxide as radical initiator, toluene as the diluent and 1% polyvinyl

alcohol (MW - 75,000) as suspension stabiliser. Polymer was obtained in spherical uniform beads of 200-400 mesh size

Polymerisation in diluents like hexane and cyclohexane results in

the formation of polymer in fine powdered form. The physico-chemical

property of the polymer was found to be determined by the chemical

nature of rnonomer and the mole percentage of the crosslinker.

Reproducible results are obtained by adjusting the monomer to diluent

ratio, amount of the stabiliser, geometry of the vessel and the stirring rate.

Compared to various crosslinking density a 4% PS-TTEGDA resin showed

optimum hydrophobic-hydrophilic balance, mechanical stability, excellent

swelling properties and performance i r~ the stepwise synthesis of medium to

large peptides in high yield and purity.

A second polymer developed by introducing hexanediol

diacrylate as crosslinker to polystyrene network was proved to be a very

38 Chapter 2

good candidate for the solid phase polypeptide synthesis. This polymer

was synthesised by the aqueous suspension polymerisation of the

respective monomers using toluene as diluent and benzoyl peroxide as

radical initiator.

A third support has been developed by the copolymerisation of

styrene and butanediol dimethacrylate (BDODMA) for the solid phase

synthesis of peptides (7) The resin support was synthesised with varying

crosslinking densities (l'', 2%, 3%, 4%, 6%, 8% and 10%) by the free

radical aqueous suspension polymerisation using toluene as diluent and

benzoyl peroxide as initi~ator. The insoluble polymer support was obtained

as spherical uniform beads. The beads were sieved and the main fractions

obtained were of 100-200 mesh size. Reproducible results were obtained

in the preparation of beads of 100-200 mesh size by adjusting the amount

of stabiliser PVA, geometry of the vessel and stirrer, and stirring rate.

These supports were stable under all peptide synthetic conditions.

The ester crosslinkage of the resin was stable enough to withstand strong

acidic and alkaline conditions. The optimum hydrophobic-hydrophilic

balance of the resln results in high swelling in different polar and non polar

solvents. So, the range of chemistry that could be conducted on the

support makes it an efficient one for different organic reactions. The ease

of preparation, functionalisation and workup procedure are the major

advantages of these new polymers over conventional polymer supports.

The enhanced coupling rate during peptide bond formation, high sensitivity

in monitoring the coupling reactions, economical use of reagents, reactants

and solvents, and the yield and purity of the peptides are the other

advantages of these new resin supports. The physico-chemical

compatibility of the macr~~molecular support and the growing peptide chain

helps to synthesise peptides of very high purity and homogeneity.

2.7 Recent techniques in solid phase peptide synthesis

2.7.1 The resin linkages

In solid phase rnethod the covalent attachment of the growing

peptide chain to the polymeric support has been found to be one of the

critical problems of efficient peptide synthesis.56 A chemical linkage of

peptide to the support b'y using a suitable 'handle' unit, allows the whole

synthesis under precise conditions. Handles are bifunctional spacers

incorporating the featuraes of an easily cleavable protecting group on one

end and the other end allows coupling to a previously functionalised

support. Their linkages have to be easily formed, stable to repeated cycles

of acylation and deprotection and yet easily cleaved at the end of the

synthesis without damage to newly formed peptide bond. These handles

also help to cleave the polypeptides as free acids, side chain protected

acid fragments, amides, hydrazides and as carboxyl derivatives

An orthogonal protection involves classes of groups that are

removed by different chemical mechanisms. It can be removed in any

order using appropriate reagents. For the synthesis of peptide acids a

p-alkoxybenzyl alcohol resin ( l ) was developed by ~ a n g . ' ~ ' . ' ~ ~ Here, the

p-alkoxy substituent of the benzyl alcohol moiety enhances the acid

sensitivity between the peptide and the resin. So, this anchoring bond can

be cleaved with TFA. The support (I) was used for the peptide synthesis

-l*e !@Utio?ls : % Overvipw 41

using Fmoc amino acids in which Nu-protection was removed by an

organic base. 109,110

From the support (1) Nu-protection Boc was removed with 30% TFA in

DCM. It can also result in a minute loss of peptide from the resin. This problem

was serious when large peptides were synthesised. So, a new support,

containing oxymethylphenylacetamidomethyl handle (Pam-resin) (2) was used

for the solid phase peptide synthesis. 111-113 The electron withdrawing linker

makes the ester bond hundred times more acid stable than the simple

alkoxybenzyl alcohol resin.

4-Hydroxylphenylthiomethyl resin (3) can be used for the solid

phase peptide synthesis using Boc amino acid^.^^^^"^ The peptide can be

cleaved from the support either by using H2O2IAcOH or by nucleophiles.

pNitrobenzophenoneoxime-resin (4; was prepared from the

corresponding ketoresin by the reaction with hydroxylaminehydrochloride and

pyridine in refluxing ethanol. The finished peptide can be cleaved from

functionalised support by using a range of nucleophiles like aqueous or

alcoholic hydroxide, thiophenoxide in DMF to give peptide acid. Alcohols in

presence of tertiary amine gives peptide ester, ammonia gives peptide

amide and hydrazine gives peptide hydrazide.'l""g

Benzhydrylamine-4-oxycarbonyl methyl-resin (5) can be used for

the solid phase synthesis of peptide. Cleavage of the resin bound peptide

with HF gives peptide amide

%+- SupportedSofifPhase a(eactiom : An O v m 43

Supports like 4-hydroxymethylbenzarnidomethyl re sir^^'^'^' (6)

4-hydroxy-methylphenoxyacetarnidornethyl resinqz0 (7) or 3-methoxy-4-

hydroxymethyl-phenoxyacetarnidomethyl resin"' (8) are used in solid

phase peptide synthesis. The peptide acids can be cleaved from the

support by using TFA under various conditions.

Peptide arnides can be synthesised by stepwise addition of Fmoc amino

acid to a 442 ',4 ' dirnethoxyphenylaminomethyl) phenoxymethyl resin (9). 122,123

The peptide arnide can be cleaved from the support using AcOH or dilute

TFA.

Peptide arnide can also be synthesised using a 4,4 ' methoxybenzhydril phenoxyacetamidornethyl resin (10). Cleavage of the peptide arnide from the

support was effected using TFA."~

10 Acid labile 9-xanthenyl resin (11) prepared from 3-hydroxyxanthone

can also be used for the synthesis of peptide arnides by Frnoc rneth~d."~

2.7.2 Multidetachable supports in solid phase peptide synthesis

Multidetachable anchoring groups in solid phase peptide synthesis

gives maximum flexibility in the methods used for removing the peptide

ToIjmer- S u p p o t t e d S o l i d ~ T(pctions : .51 OyeryiprU 45

from the resin^.""'^' It provides greater chemoselectivity between the

protecting groups of the a-amine side chain and the anchoring bond which

connect the peptide with the solid support. The multidetachable resin

supports can be cieaved at different positions with acids, nucleophiles or

by pho t~ l ys i s . ' ~~ After the synthesis, the final peptide can be obtained as a

free acid, as a protected peptide which is suitable for segment

condensation or in a form which contains the removable spacer which can

be again attached 1:o suitable resin support for further e longat i~n. '~~

Based on the above concept Tarn et al. have designed two resins

2-(4-hydroxymethyl) phenylacetoxypropionyl resin (12) and 4-[4-(hydroxymethyl)

phenylacetoxyrnethyl]-3-nitrobenzamidomethy resin (13) which can be cleaved

selectively by acidolysis, at position A or by photolysis at position B . ' ~ ' , ' ~ ~

Benzhydrylamine-4-oxycarbonylmethyl-resin (14) can also be used

as a multidetachable support which can be cleaved with acid at position A

and with a variety of nucleophiles at position B.

14

2.7.3 Fmoc protection in solid phase peptide synthesis

Merrifield's solid phase peptide synthesis requires the use of

protecting groups of differential stability towards acidolytic cleavage. The

temporary Na-protecting Boc group is cleavable by mild acid treatment and

anchor bond is cleavable by strong acid. These repeated acid treatments

may result in partial loss of side chain protecting group.'30 Nucleophilic

side chain of Tyr, Trp, Met and His can undergo trifluoroacetylation during

TFA treatment.'"'

Reactions on solid support are influenced by the dissimilar

solvation properties of the polymer and growing peptide chains. The

dierence in the solvation characteristics of polystyrene bound polymer support

and peptide chain causes the decreased rate of peptide bond forming reaction.

and deprotection. Fmoc polyamide solid phase synthesis was designed by

Pofjmer- SupportedSo&fllse ~ a c t w n s : pn Oventiew 47

Sheppard et a/. to overcome some of the problems experienced with

Merrifield's solid phase peptide synthesis using Boc chemistry. These supports

are well solvated in polar, aprotic solvents like DMF and N M P . ' ~ ' ~ ' ~ ~ In this

strategy, Fmoc pmtection was used for amino blocking of the respective

amino acid. Fmoc group can be easily removed by using non hydrolytic

bases like piperidine. Fmoc protection was used with t-butyl side chain

protection. p-Benzyloxy benzyl ester resin anchorage offers a novel

approach to solid phase peptide synthesis without repetitive a c i d o ~ ~ s i s . ' ~ ~

The target peptide from this Fmoc polyamide solid phase peptide synthesis

was exposed only once to acidolysis with TFA at the final stage.

Fmoc removal occurs rapidly in polar medium like DMF than non

polar medium like DCM. Fmoc group was found to be completely stable to

acids like TFA, HHrlHOAc or HBrlnitromethane.

A number of model peptides and protein sequences were prepared

using these polar polyamide supports. Continuous flow Frnoc solid phase

peptide synthesis has been demonstrated for resins encapsulated inside a

low density, highly permeable inorganic matrix that can withstand

continuous flow pressure. The inorganic support Kieselguhr was

successfully used as a continuous flow matrix with polyamide resin for

Fmoc solid phase peptide ~yn thes is . '~~

48 Chapter 2

2.7.4 Attachment of C-terminal amino acid

The usual procedure for the synthesis of peptide acid is to attach the

C-terminal amino acid to the polymeric support via an ester bond. In Merrifield

method esterification was carried out by heating TEA salt of Boc-amino acid in

EtOH w~th chloromethyl resin. This reaction was slow and complete

esterification was usually not possible while there was a possibility to form

quaternary ammonium group on the polymer. If esterification was not

complete, then quarternisation can occur at every neutralisation step

during the synthesis (Scheme 2.2). By converting the Boc amino acid to

tetraethyl ammonium salt or cesium salt, esterification reaction can be

driven to completion.

Scheme 2.2. Quarternisation of chloromethyl resin by trimethylamine.

Amino acids containing easily alkylatable functional groups like His,

Cys and Met can cause difficulty during esterification reactions. If EtOH

was used as solvent for esterification reaction, some ester interchange may

occur with Asp and Glu. All these problems can be avoided by using hydroxy

methyl resin or resins with suitable linkers containing the hydroxy group.

BoclFmoc amino acids were attached to the resin in a variety of methods.

Esterification with prefonned symmetric anhydride of Boc or Fmoc amino acid

%5ner- SttpportedSolidPhare @utionr : An Owrview 49

was the most popular method. 4-(Dimethy1amino)pyridine (DMAP) was used

as the catalyst for esterification reaction.13' This catalyst was able to

promote hydrolysi:; of activated Fmoc amino acid as well as reaction with

resin bound hydroxyl group. So, less than one equivalent (relative to resin

functionality) of DMAP was sufficient. The resin and the solvents used for

the first attachment should be perfectly dried. DMAP also significantly

promote racemisation of activated urethane protected amino acids.13' It

can also cleave Fmoc protecting group leading to the formation of

dipeptide on the resin. These side reactions could be avoided by

repeating the esterification with fresh reagents after 50 minutes, if the

esterification reaction was not complete in 50 minutes.

2.8 Sequential attachment of amino acids and deprotection

The factors that depend on the efficiency of coupling reactions

include the nature of the acylating agent and the activated species of

protected amino acids. The solvation of the resin-bound growing peptide

chain can also have a profound influence in the coupling steps.

Subsequent amino acids must be added to the growing peptide-resin in a

highly activated form to ensure rapid and quantitative coupling. The common

activating species ~ ~ s e d in solid phase synthesis are the amino acid symmetrical

anhydride and the amino acid active ester with HOBt. The symmetrical

anhydride method using DCC (1) provides the derivatives of greatest reactivity.

But their instability and the formation of insoluble DCU during acylation are its

50 Chapter 2

drawbacks. Diisopropyl carbodiimide (DIPCDI, 2) and t-butyl ethyl carbodiimide

(3) are the other carbodiimdes used in SPPS. ' ~~ , ' ~~

The symmetrical anhydride method suffers from a number of

disadvantages. Some of them are:

i) they must be prepared just before use because of their instability,

ii) amino acids are wasted during the anhydride formation as two

'moles of amino acids are required for each mole of symmetrical

anhydride produced,

iii) the most efficient solvent (DCM) for rapid symmetric anhydride

formation is not the solvent of choice (DMFINMP) for peptide

synthesis, and

ToIjmer- SupportedSolidTbe S&ctionc :an avnviav 51

iv) the possibility of racernisation.

EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) (4) have

also been used for the coupling of successive amino acids in SPPS.'~'

The limitations of symmetrical anhydride method can be overcome by

using the pentafluorophenyl (Pfp) (5), HOBt (6) and 3-hydroxy-2, 3-dihydro-4-

0x0-benzotriazine (oDhBt) (7) esters of amino acid^.'^','^^ These esters

suppress the racernisation during coupling reaction

52 Chapter 2

Benzotriazol-l-yl-oxy-tris(dimethy1 amino) phosphonium hexafluoro

phosphate (8) along with DIEA is one of the advancing acylating agents in

144 peptide synthesis.

The synthesis of hydrophobic peptides are difficult because of internal

aggregation of constituent amino acids via p-sheet formation or by association

between protected chain with the synthetic support matrix. The use of 2-(IH-

benzo triazol-l-yl) 1,1,3,3-tetramethyl uroniumhexafluorophosphate (HBTU)

(9) or 2-(1 H- benzotriazol-l -yl) 1 ,l ,3,3-tetramethyl uroniurntetrafluoroborate

145.147 (TBTU) (10) during the cc~upling reaction can avoid these problems. The

coupling reaction proceecls rapidly at low level of racemisation. HOBt can act

as a catalyst for this coupling reaction and the base like DIEA can activate the

1-Hydroxy-7-azabenzotriazol(HOAt~11) and its uroniurn and

phosphoniurn salt derivatives such as o-(7-azabenzotriazol-l-yl>l,1,3, 3-

tetrarnethyl uroniurnhexa-fluorophosphate (HATU) (12), N7-azabenzotriazol-

l-yl)-l,l,3.3-bis(tetramethy1ene)uroniurn hexafluorophosphate (HAPyU)

(13), 7-azobenzotriazol-l-yl oxytris-dirnethyl amino-phosphoniurn

hexafluorophosphate (AOP) (14), and 7-azobenzotriazol-l-yloxytris-

pyrrolidino-phosphoniurn hexafluorophosphate (PyAOP) (15) in

presence of DlEA can be used as acylating agents in acylation

reactions.14'

(15)

The reduction in product loss, minimisation of failure sequences,

greater control over coupling steps, increased reaction efficiency,

decreased racemisation and reduced coupling time are the advantages of

using HOAt and it's derivatives, especially in the preparation of peptides

containing hindered amino acids.I4'

2.9 Cleavage

Solid phase peptide synthesis was primarily designed for acidolysis

of peptide-linker bond by TFA with suitable scavengers. Time required for

cleavage of the peptide from the resin depends upon the type of linker that

is used between the peptide and the resin. During cleavage all acid labile

side chain protection is removed. Peptides can also be cleaved from the

resin in fully protected form to produce a unique peptide carboxyl

termination by using photolysis, fluoride ion, alkali or hydrogenation.

Trifluoromethane sulphonic acid (TFMSA) and HF have both been used for the

final cleavage and side chain depr~tection.'~ PAM linker can be readily removed

by TFMSA.'~ Trimethyl silyltrifluoromethane sulphonate (TMSOTf) in the

presence of thioanisol can be used as a cleaving agent.15' It can also remove

several side chain protections.

Cleavage of peptide from benzyl ester type linkers by 2-dimethylamino

ethanol or N,N-diethyl hydroxylamine yield side chain protected peptide

acidsg3. Protected peptide alkylamide may be generated by alkylamine

cleavage and side chain protected pept~de acids by catalytic transfer

hydrogenation. Silyl containing linkers have been designed for cleavage of

protected peptide by fluoride ion. Tetrabutyl ammonium fluoride was used

to generate fluoride ion.

Potjnrer- ~ u p p o r t e d S o L i d i @actions :pn Ovnview 57

REFERENCES

Chen, H.; Lurll, J.P.; Garaby, C. Tetrahedron Lett. 2005,46,3319.

Boxendale, 1.3; Davidson, T.D.; Ley, S.V.; Perni, R.H. Heterocycles.

2003,60,2707.

Huang, H.Q.; Li, S.C.;Qin,Z.H; Cao, S.L.;Yao,Y.; Liu,Y.S.; Li, H.Y.;

Shi, Y.E. Bioorganic and Medicinal Chemistry Lett.2005, 15, 2415.

Akelah. A., Synthesis, 1981, 6, 13.

Merrifield, R.B., JI. of Am. Chem. Soc. 1963, 85, 3045.

Dolle, R. E. JI. of Combinatorial Chem.2004, 6, 623.

Lakshmi, S.; Nirmala R. J.; Nisha, V. S.; Jayakrishnan, A. JI. of

applied polylner science. 2003, 88, 2580.

Carrigan, C.N.; Imperiali, B. Analytical Biochemistry. 2005, 341, 290.

Parlow J.J.; Norrnon Cell. J.E. Mol. Diversity, 1996, 1, 266.

Armstrong. R W.; Combs A.P.; 'Tempest. P.A.; Brown S.D.; Keating T.A.

Acc. Chem. Res.1996,29.123.

Floyd C.D.; Lewis C.N.; Whittaker. M. Chem. Brit. 1996,31.

Nash I.A.; Byc:roft B.W.; Chanwc, Tetrahedron Lett. 1996,37,2635

Kumar P.; Bhatia. D.; Garg B.S.; Gupta K.C. Bioorganic and Medicinal

Chemistry Lelt.1996, 4, 1761.

Castex, C.; Lalanne, C.; Mouchet, P.; Lemaire, M,; Lahana, R

Tetrahedron Lett. 2005.61, 803.

Bunin B. A.; Ellman J.A., JI. of Am. Chem. Soc. 1992,114, 10997

Donati, D.; Morelli, C.; Porcheddu, A. Taddei, M. JI. of Organic

Chem. 2004 69, 9316.

Sreedhar, K.M. Ph.11. Thesis, Mahatma Gandhi University. 2004.

Nair, V. A.; Suni, M.M.; Sreekurnar K. Designed Monorners and

Polymers. 2003, 6, 81.

Wojaczynska. M.; Kolarz. B N. JI. of Chromatography. 1986, 358, 129

Valeur, E.; Bradley, Fvl. Chemical Cornm. 2005, 9, 1164

Chacko, A.; Mathew B. Jl. of Appl. Poly. Sci. 2003, 90,3708.

Aiswaryakurnari, K.; Sreekumar, K. Polymer International. 1998, 45, 255.

Chettiyar, K.; Sreekurnar. K. Indian JI. of Chern.Tech.2003, 11, 59.

Akelah. A.. Moet. A. 'Functionalised Polymers and Their Application'

Chapmann & Hall, London. 1990.

Schuttenberg. H.; Klurnp G.; Kacmar. U,; Turner S.R.; Schulz. R. C. J.

Macrornol. Sci. Che~n. 1973, A7, 1085

Kurnar, G.S.V.; Mathew, B. JI. of Macrornol. Sci.: Pure Appl. Chern. 2004,

A41,1037.

Chacko, A.; Mathew. B. JI. of Macrornol. Sci.: Pure and Appl. Chem.

2003, A40, 1035.

Hodge, P. Current Opinion in Chemical Biology. 2003, 7, 362.

Tornoi, M.; Ford, M.P.; Tsyurupa Die. Angewandte Makrornoleculare

Chemie. 1980, 91, 127.

John. M,; Jose, J . Mathew, B. JI. of Macrornol. Sci.: Pure Appl. Chern.

2004, A4 1. 85.

Lee, B.S.; Mahajan, S.; Janda, K.D. Tetrahedron. 2005, 61, 3081.

Chettiyar, K.; Sreekumar, K. Indian JI. of Chern.Tech.2004, 11, 59.

Borchard, W.; Steinbrecht , W. Colloid Polyrn. Sci., 269, 1991, 269.

Boris Sket, Marko Zupan and Pavle Zupet, Tetrahedron, 40,1984, 1603.

A.E. Poomanadhan, P. Rajalingam and Ganga Radhakrishnan Polymer,

1993,34, 1485.

Herforth, C.; Heidler, P,; Franke. S.; Link, A. Biorganic and Medicinal

Chem. 2004, 12,2895.

Sreekumar K.; Rajasekharan Pillai. V.N. Proceedings of Indian Academy

of Sci. (Chem. Sci.). 1989, 101, 335.

Jomol. M.; Korir~.; Kakuchi. H. Makromol. Chem. 1986,187, 2753.

Coutinho. F.M.13.; Rabelo. D. Euro. Polym. JI. 1992, 28, 1553

Okay. 0.; Guron C, JI. of Appl. Polym. Sci. Part A Polym. Chem. 1990,

28, 2585.

Greig, J.A.; Sherrington, D.C. Polymer. 1978,19,163

George, B.K.; Pillai, V.N. R. JI. of Polym. Sci. Part A Polym. Chem.. 28.

1990,2585.

Roice, M.; Pillai, V.N.R. JI. of Appl. Polymer. Sci. 2003, 88, 2897

Arunan, C.; Pillai, V.N.R. JI. of Appl. Polymer. Sci. 2002, 87, 1290

Zatsepin, T.S.; Stetsenko, D.A.; Gait, M.J.; Oretskaya, T.S. Tetrahedron

Lett. 2005,46,3191.

Karidi. K.; Garoufis, A.; Hadjiliadis, N.; Reedijik, J. Dalton Transactions.

2005,4,728.

Galpin, I. J. "Amino Acids, Peptides and Proteins", Vo1.16, The Royal

Society of Chemistry, Burlington House, London. 1985 pp.272-341.

Fischer, E.; Fourneau, E. Ber. Dtsch. Chem. Ges.1901, 34,2868.

Kumar, K.A.; Mathew, B. JI. of Appl. Polymer Sci. 2002, 86, 1717.

Letsinger, R. L.; Komet M. J. JI. of Am. Chem. Soc.1963, 85, 3045

51) F'flaum, 2.: Rucman. R. Acta Chimica Slovenica. 2005, 52, 34.

52) Kurnar, K.A.; Mathew, B. Lett. Peptide Sci. 2002, 8,339

53) Kumar, K.A.; Mathew, B. Lett. Peptide Sci. 2002, 7, 317.

54) Kumar, K.A.; Mathen., B. Eur. Polyrn. JI. 2002, 38, 183.

55) Pillai, V. N. R.; Mutter, M. Topic Curr. Chem. 1982,106,119.

Barany G. and Merrifield R. B. "The Peptides: Analysis, Synthesis and

Biology", Vol. II (Eds, Gross E. and Meienhofer J.) Academic Press, New

York, 1979 pp. 1-284.

Barany, N.; Cordonier, K.; Mullen, D.G. Ind. JI of Pept. Protein. Res. 1987

30, 705.

Merrifield, R.B. JI. of Am. Chern. Soc. 1964246, 1922.

Stewart, J.M.; Wool!{, D.W. Nature (London), 1965,206,619.

Varkey, J.T.; Pillai, \I.N.R. JI. of Peptide Sci. 1999, 5, 577.

Arunan, C.; Pillai, V.N.R.; Protein and Peptide Lett.1999,6,391.

Sheehan, J. C.; Hess G. P. JI. of Am. Chern. Soc. 77,1955, 1067

Lenard, J.; Robinscn. JI. ofArn. Chem. Soc. 1967, 89, 181.

Wunsch, E. Angw. Chern. Int. Ed. 1971,10,786.

Erickson, B.W.; Nlerrifield, R.B. The Proteins, 3rd Edn., (Eds. Neurth

H., Hill R.L. and Boeder C.L.) Academic Press, New York. 1976.

Stewart, J.M.; Macromol. Sci. Chem. 1976, 10, 254.

Menifield, R.B., Science. 1986, 232, :%l.

Birr, C., "Aspect!; of Merrifield Peptide Synthesis", Springer-Verlag,

Heidelberg 1978.

Hancock, W.S.; Prescott, D.J.; Vagelos, P.R.; Marshall, G.R. JI. of Org.

Chem. 38 1973. 774.

Flory. P.J. Macromolecule.1974, 12, 199

Tsay, H.M.; Ullrnan R. Macromolecule. 1988, 21, 2963

Sarin, V.K.; Ken, S.B.H.; Merrifield, R.B. JI. of Am. Chem. Soc. 1980, 102,

5463.

Atherton E., Gait M.J., Sheppard R. C., William B. JI. of Bio. Org. Che.

1979, 8, 351.

Sheppard R.C. in Peptides (1971), Proc. of the ll* Europ. Pept. Symp.

Nesvadba H. E.ds. North Holland, Amsterdam.1973, 11 1.

Sheppard, R.C. Sci. Tools. 1986, 33,9

Kents Clark, L.J. Synthetic Peptides in Biology and Medicine, Alitalok.

Partanenm P,; Veheri, A Eds. Elsevier Science Publishers.

Amsterdam.1985, 29-57.

Kumar, K.A.; hrlathew, B. JI. of Peptide Sci.2002, 86, 1717.

Geyseri, H.M.; Meloen, R.H.; Barteling. S.J. Proc. Natl Acad. Sci. USA,

1984, 81, 399i3.

Houghten, R. A.; DeGraw, S.T.; Bray, M.K.; Hoffman, S.R.; Frizzel, N.D.

Bio Technique. 1986, 4, 522.

Holm A. and Meldal M. "Peptide 1988" (Eds. Jung. G. and Bayer E.)

Walter de Gn~yter and Company, Berlin, 1989, pp.208-210.

Barany, G.; Abericio, F. in Peptides 1990, Proc. of the 21'' Europ. Pept.

Symp. Giralt 13. Andrew D. (Eds) ESCOM, Leiden, 1991, 139.

Bayer, E.; Dengler, M.; Hernmassi, B. Int. JI. of Peptide Protein Res.

1985, 25, 178.

62 Chapter 2

Barany, G.; Albericio, F.; Sole, N.A.; Griff~n, G.W.; Kates, S.A.; Hudson, B.

In Peptides, 192 Proc. 22nd Europ. Pept. Syrnp. Schneider C.H. Eberle,

A.W. Eds. ESCOM, Leiden 1993,267.

Pillai, V.N.R.; Renil, M,; Haridasan, V.K. Indian JI. of Chem. 1991,

308, 205.

Atherton, E.; Carneron, L. R.; Sheppard, R.C. Tetrahedron. 1988, 44,

843.

Findeis, M.A.; Kciser, E.T. JI, of Org. Chern. 1989, 54, 3478

Meldal, M. Tetrahedron Lett., 1992, 33, :3077

Auzanneau, F.I.; Meldal, M,; Bock, K. JI. of Peptide Sci. 1995, 1, 31

Atherton, E.; Brown, E.; Sheppard, R.C.; Rosever, A.J., Chern. Soc.

Chern. Cornrnun. 1981,1151

Small, P.W.; Sherrington, D.C. JI. of Chern. Soc. Chern. Cornrnun. 1989,

1589.

Rapp, W.; Zhang. L.; Aabich, R.; Bayer, E. in Peptides Proc. of 20th Eur.

Pept. Syrnp. Jung. G. (Ed) Waker de Gruyter and Co., Berlin. 1989,195.

Berg, R.; Arndal, K.; Pedersen, W.B.; Holm, A.; Tarn, J.P.; Merrifield. R.B.

JI. of Am. Chern. Soci. 1989,111, 824.

Daniels. S.B.; Bernatowiez. M.S.; Coulh, J.M.; Koster, H. Tetrahedron

Lett. 1989, 30, 4345.

Eichler, J.; Beinert, A.; Stiernadora, A.; I-ebel, M. Peptide Research. 1991,

4,296.

Renil, M.; Meldal, M. Tetrahedron Lett. 1996, 36, 4647.

Renil, M.; Ferreras. M.; Delaisse. J.M.; Foged, N.T.; Meldal, M.J. Peptide

Sci. 1998, 4, 195.

98) Renil, M,; Melclal, M. Tetrahedron Lett. 1996, 34,6185.

99) Kempe, M.; Barany, G. JI. of Am. Chem. Soc. 1996,118,7083

100) Beck Sickingel-, A.G.; Jung, G.; Gaida, W.; Koppen, H.; Schorrenberg, G.;

Lang, R. Eur. ,ll. of Biochem. 1990, 176,458.

101) Houghten, R. A. Proc. Natl. Acad Sci., U.S.A. 1985, 182,5131

102) Valerio, R.M.; Bary, A. M.; Campbell, R.A.; Dipasquale, A.; Margellis, C.;

Rodda, S.J.; Geysen, H.M.; Maeji, W.J. Int. JI. of Peptide Protein Res.

1993,42,1.

103) Holm, A.; Medal, M., Peptide 1988, Jung G. Bayer E., Eds. Walter de

Gmyter and Cc)., Berlin. 1989, 208.

104) Frank R.; Gulctr, S.; Kamse, S.; Lindenmaire, W.in Peptides 1990, Proc.

21st Eur. Pept. Symp. Giralt E., Andreau D. Eds ESCOM., Leiden 1991.

105) Renil, M.; Pillai, V.N.R. Tetrahedron. 1994,50, 6681

106) f'flauni, Z.; Rucman, R. Acta Chimica Slovenica.2005, 52, 34.

107) Meldal, M. Tetrahedron Len.1992, 33, 3077.

108) Wang. S.S. JI. of Org. Chem. 1973, 95, 1328.

109) Wang, S. S. JI. of Org. Chem. 1975, 40, 1235.

110) Chang, C.D.; tvleienhofer J. Int. JI. of Pept. Protein Res. 1978, l l , 246.

11 1) Atherton. E.; Lc~gan, C.J.; Sheppard, R.C. JI. of Chem. Soc. Perkin Trans.

1981.1, 538.

112) Mitchel, A.R.; Erickson, B.W; Ryabtsev, M.N.; Hodges, R.S.;

Merrifield, R.B. JI. of Am. Chem. Soc. 1976, 98,7357.

113) Mitchel, A.R.; Kent, S.B.H.; Erickson, B.W.; Merrifield, R.B. Tetrahedron

Lett. 1976,17,3i195.

64 Chapter 2

114) Mitchel, A.R.; Kent, S.B.H.; Engelhard, M,; Menifield, R.B. Jl. of Org.

Chem. 1978,43,2845.

115) Marshall, D.L.; Liener, I.E. JI. of Org. Chem. 1970, 35, 867.

116) Flanigan, E.; Marshall, G.R. Tetrahedron Lett, 1970, 2403.

117) DeGrado, W.F.; Kaiser, E.T. JI. of Org. Chem. 1980, 45, 1295.

118) DeGrado, W.F.; Kaiser, E.T. JI. of Org. Chem.1982, 47, 3258,

119) Nakagawa. S.H.; Lau, H.S.; Kezdy, F.J.; Kaiser, E.T. JI. of Am. Chem.

Soc. 1985,107,7087

120) Tarn, J.P. Jl. of Org. Chem. 1985,50, 5291

121) Colrnbo, R..; Atherton, E.; Sheppard, R.C.; Wooley, V. Int. JI. of Pept.

Protein. Res. 1983, 21, 118.

122) Sheppard, R.C.; William, B. J. Int. JI. of Pept. Protein. Res. 1982,20,451.

123) Rink, H.; Sieber, P. "Peptide 1988, (Eds., Jung. G. and Bayer E.) Walter

de Gruyter and Company, Berlin 1989, pp. 139-141.

124) Rink, H.Tetrahedron Lett. 1987, 28, 3787.

125) Breipohl, G.; Knolle, J.; Stuber, W. Tetrahedron Lett, 1987, 28, 5651.

126) Sieber, P. Tetrahedr'm Lett. 1987,8, 2107.

127) Tarn J.P., Tjoeng F.S. and Menifield R.B., Tetrahedron Lett.1979, 20,

4935.

128) Tarn, J.P.; Tjoeng, F.S.; Menifield, R.B. JI. of Am. Chern. Soc. 1980, 102,

6117.

129) Sparrow, J.P. JI. of Clrg. Chem. 1976,41, 1350

130) Wang, S.S.JI. of Org. Chem. 1976,41,3258.

131) Yaron, A; Schlossmann, S.F. Biochemistry. 1968, 7, 2673

132) Kent, S.B.H.; Mitchell, A.R.; Engelhard, M.; Menifield, R.B. Proc. Natl.

Acad. Sci. US,\. 1979, 76, 2180.

133) Atherton, E.; Clive. D.L.J.; Sheppard, R.C. JI. of Am. Chem. Soc. 1975,

97, 6584.

134) Arshady, R.; Atherton, E.; Gait, M.J.; Lee K.; Sheppard, R.C. JI. of Chem.

Soc. Chem. Commun.l979,423.

135) Arshady, R.; Atherton, E.; Clive, D.L.J.; Sheppard, R.C. JI. of Chem. Soc.

Perkin Trans.'l981, 1, 538.

136) Arshady, R.; Atherton, E.; Sheppard, R. C. Tetrahedron Lett. 1979, 20,

1521.

137) Atherton, E.; Sheppard, R.C.; Ward P. JI. of Chem. Soc. Perkin Trans.

1986, 1, 2065.

138) Hofle, G.; Sl:eglids, W.; Volbrt~ggen, H. Angew. Chem. Int. Ed. Eng.

1978, 17, 569.

139) Atherton, E.; Gait, M.J.; Sheppard. R.C.; William, B. JI. of Bioorganic

Chem. 1979,8,351. .

140) Izdebski, J.; I3ondaruk, J.; Gurnalka, S.W.; Krzaschi, P. Int. JI. of Peptide

and Protein f7es. 1989,33, 77.

141) Izdebski, J.; Pelka, J., Peptide 1984, Ranganarsson. U,, Eds. Almquist

and Wickshell International Stockholm, 1984, 11 3-1 16.

142) Sipos, F.; Gaston, D.W., Peptide Proc. 11th Eur. Pept. Symp., North

Holland Publ. Amsterdam 1973, 165.

143) Koning, W.; Geiger, R. Chem. Ber. 1970, 103, 775,

144) Kisfaludy, L.; Schon, I. Synthesis. 1983, 325

145) Castro, B.; Ilormong, J.R.; Evin, J.G.; Selve, C. Tetrahedron Lett. 1975,

14. 214.

66 Chapter 2

146) Knorr, R.; Trzeeiak, .A.; BannWarth, W.; Gillessen, D. Tetrahedron Lett.

1989,30,1927.

147) Fischer, P.M.; Cosms, A.; Howden, M E. JI. of lrnmunol. Method. 1989,

118, 119.

148) Posnett, D.N.; McGralh, H.; Tom, J.P. JI. of Biol. Chem. 1988,263, 1719.

149) Jeremic, T.; Linden, A.; Moehle, K.; Heimgartner, H. Tetrahedron. 2005,

61, 1871.

150) Beilan, H.; Noble R.L.; Ashton, C.P.; Hadfield, D.P. "Peptide, Chemistry

and Biology" (Ed., h'larshall G.R.) Escom, Leiden. 1988 pp.198-201.

151) Ni, F.; Knoishi, Y.; FI-azier. R.B.; Scheraga, H.A.; Lord, S.T. Biochemistry.

1989,28,3082.