2.1 Introduction

Synthetic hydrophilic polymers find promising applications in

pharmacology, biotec:hnology and chemistry. The biocompatibility,

biodegradability and phzumacolo~cal activity of these polymers depend

much on their hydrclphilic nature. Polymer-supported synthesis has

achieved a major place in polypeptide and oligonucleotide synthesis over

1-6 the past three decades. Peptide synthesis has proven indispensable for

the structural elucidation and activity studies of many naturally isolated

products having a peptide structure such as hormones, neuropeptides,

antibiotics and enzymes, which can be isolated only in very small

quantities. Synthetic peptides find application in all areas of biomedical

research including immunology, neurobiology, pharmacology,

enzymology and molecular biology.7 The chemical synthesis of peptides

with the naturally oc~cuning structure is possible; it was used for the

development of artificial vaccines and potent drugs that can

substitute the conventional drugs having various side effects.

Investigation of structure-activity relationship of biologically active

peptides also demand:; the synthesis of many analogues of a given

peptide.

In the beginning of 20th century, Emil Fischer synthesised the

first peptide in solutior~.~ The general chemical requirements for the

synthesis of peptide involve the blocking of carboy1 group of one

amino acid and the a~mino group of the second amino acid. The

activation of the free carboxyl group resulted the formation of

amide bond between the amino acids and the selective removal of

the protecting groups resulted a dipeptide. The method developed by

Fischer was laborious and time-consuming because the intermediate

peptides have to be rernoved, purified and characterised before the

next coupling step. The major limitation of classical solution phase

synthesis of peptides is the low yield and solubility of the

intermediate peptides with increase in chain length. A new

approach was needed jor the synthesis of larger and more complex

peptides with high purity and yield.

Merrifield introduced the concept of solid phase synthesis to

achieve more e6cien.t synthesis of peptides. In SPPS, the peptide chain

was assembled in a ste:pwise manner while the C-terminal end of the

peptide was anchored to an inert cross-linked polymer support and

the peptide was grown from C-terminal to N-terminal residue.

Menifield demonstrated the feasibility of the idea by synthesising a

model tetrapepude ~~leuc~l-~-alanyl-~l~c~l-lvaline.~ Simultaneously

with Memfield, Letsinger and Komet reported the synthesis of a

dipeptide, L-leucyl-glycine on a "Popcorn polymer support" using a

different chemical strategy. ' O The N-terminal amino acid was

anchored to the polymer support and the peptide was grown from

N-terminal to C-terminal. This technique is not so popular

because the cleavage from the support under mild condition is not

possible and always there is a problem of racemisation. Memfield's

invention formed the basis of a new technique of peptide synthesis,

which has been used till now with several methodological

improvements and refin~ements. The design of polymer support, its

chemistry and applications for SPPS have been extensively

reviewed. 3,) 1b16 These refinements and recent developments in

designing solid supports and various factors involving in solid

phase peptide synthesis are reviewed in this chapter.

2.2 Principles of Memfield Peptide Synthesis

SPPS follows the strategy of the stepwise assembly of peptides

by consecutive coupling of amino acids. The stepwise synthesis is carried

17.18 out from the C-terminal to the N-terminal of the target peptide. This

approach can eliminate the possibiity of racemisation. The stepwise

synthesis using low rn.olecular C-terminal protecting group finds less

application because of its sparing solubility in organic polar

solvents does not permit a homogeneous reaction. Carrying out the

synthesis as a heterogeneous reaction is also not very promising

due to poor filterability of the reaction mixture.

Merrifield method employs an insoluble and filterable

polymeric support such as cross-linked polystyrene that function

as the carboxy-protescting group for the C-terminal amino acid of the

peptide. The target peptide sequence was formed in a stepwise

manner by attaching temporary N,-protected C-terminal amino acid

to the chloromethylated PS-DVB resin. After the removal of N,-

protection, the next N,-protected amino acid is coupled and the

process is repeated until the entire desired peptide is assembled on

the polymer support. Clicyclohexyl carbodiide (DCC) is used as the

coupling agent,lg and ,all the reactions are cmied out under non-

aqueous conditions in organic solvents. The target peptide was

deprotected and cleaved from the polymer matrix by acidolysis with HF

or anhydrous TFA in the presence of suitable scavengers.2@21

Even though highly pure peptides can be synthesised by

classical solution phase method, it has the following shortcomings:

i. The method is slow, tedious and laborious. In order to obtain a

peptide with high purity, the constituent amino acids are

incorporated in a stepwise manner starting from the peptide's

C-terminus end. Afiter each completed amino acid addition, the

intermediate peptide is separated from any remaining reactants

before its characteri~zation, leading to lengthy synthesis time.

ii. The increasing insollubility of the growing peptide chain in the

reaction medium causes problems in both purification and in

the next coupling step results in the termination of peptide

chain elongation.

iii. Methods such as; chromatography and crystallization required

during the synthesis results in considerable reduction of the

overall yield of the peptide.

Solid phase peptide synthesis has the following advantages

over the classical so~lution phase method.

i. The peptide is synrhesised while its C-terminus is covalently

attached to an insoluble polymeric support. This permits the

easy separation of t:he growing peptide from any by-products or

excess unused amino acid components.

ii. The reactions are driven to completion by using an excess of

reactants and reage:nts.

iii. No mechanical loss occurs because the growing peptide is retained

on the polymer in a single reaction vessel throughout the

synthesis.

iv. The final peptide is detached from the polymer support by a

single cleavage ster~ at the end of synthesis. The side chain

protecting groups can also be cleaved in the same reaction in

order to simplify the work-up and the isolation of the final

peptide. The cleavage step does not degrade the assembled

peptide.

v. The physical ope:raltions involved in the synthesis are simple,

rapid, and amenable to automation.

vi. The spent resin can be recycled.

In spite of these aldvantages, Merrifield's solid phase method

has a number of limitations and is extensively reviewed. 16,22-24

They are:

i. Non-compatibility of resin and growing peptide chain.

ii. Lack of stability of peptide-resin linkage under the conditions of

synthesis.

iii. Non-equivalence of functional groups attached to the polymer

support.

iv. Formation of errosr peptides due to truncated and failure

sequences.

v. Peptide conformation changes in macroscopic environments

inside the polymer matrix and also due to peptide resin linkage.

A number of modifications have been introduced to overcome

the difficulties associated with Merrifield SPPS which includes:

i. Development of new supports with high swelling properties

permitting improved solvation of both matrix and growing

peptide chain.

ii. Introduction of multi-detachable anchoring groups improving

the flexibility of synthetic strategy.

iii. Development of n'ewer separation method (eg. preparative and

semi-preparative HPLC) and characterization techniques in

peptide synthesis.

Extensive investigation upon reaction rates and kinetic course in

solid phase synthesis revealed that the reaction sites within the

polymeric matrix are chemically and kinetically not equivalent,

resulting in deviations from linear kinetics. Consequently quantitative

reactions are difficult to obtain. The preparation and accessibility of

insoluble polymer reagents appears to limit a more general application

of the compounds. These inherent difficulties in solid phase synthesis

were mostly overcome by the 'Yiquid-phase procedure" proposed by

Bayer and Mutter, 25-30 which makes use of soluble polymeric

groups. These macromolecular groups determine the physical and

chemical properties of low molecular weight components covalently

attached to the polymer. Such a technique allows the effective and

quantitative removal of reagents. All reactions proceed in

homogeneous solutiorl in analogy to the low molecular weight system.

The reduced operatio:nal simplicity and changes in the crystallization

tendency makes the liquid phase peptide synthesis less versatile.

2.3 The Role of Solid Support

The stability of' the solid support under all conditions of

functionalisation an.d synthesis is the prime requirement in SPPS.

It is believed that the peptides show a lower tendency to aggregate

because of limited ir~termolecular interactions when bound to a

polymer than in real solution especially at low substitutional levels.

SPPS could therefore be favoured for solution condensations when

31-33 solubility problems in LPPS arise. The restricted mobility of the

peptide anchored to the polymer could become a disadvantage of

SPPS. The PS-DVB support has been widely used for the synthesis of

peptides using BOC-chemistry.w Polystyrene resin with 1% PS-DVB

showed optimum swelling and stabiity while 0.5% PS-DVB resin is too

fragile and above 2% PS-DVB resin does not swell effe~tivel~.~ The

extent of swelling in polar organic solvents increases as the number of

amino acid residues incorporated to the resin increases.36 This is

due to the lowering of the network free energy based on the

additional solvation of' the growing peptide chain. 33.37,38

Merrifield techniclue has undergone a series of mcdifications and

improvements because of the physicochemical incompatibility of the

growing peptide chain and the rigid hydrophobic macromolecular

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

to optimize the resin structure in SPPS, Sheppard introduced a polar

polydimethyl acrylmnide resin, which is structurally similar to

peptide backbone. 12.39 This helps easy solvation of the peptidyl

resin and thus reduces the steric hindrance during deprotection and

coupling reactions.4043 Cross-linked and funtionalised polydimethyl

acrylamide gel can detained within the pores of fabricated Keiselguhr

which can be used a:s a matrix in continuous flow method. The

support shows effectiv~: swelling in polar solvents but in non-polar

solvents it is very pc~or. The chemical stability of the resin was also

less comparable to that of polystyrene supports.

The mixed PE:G-PS resin, a highly promising class of solid

support, is used successfully in polypeptide synthesis. 44-46

A cross-linked polystyrene-pol~ethyleneglycol graft co-polymer

with a 2-nitrobenzyl ;anchoring group has been used as a solid

support for the stepwise synthesis of peptides.47 Swelling, which is

a sign of good solva1:ion of the resin, is good for polystyrene resins

in non-polar or less polar solvents like DCM, whereas polyacrylamide

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

excellent swelling in common solvents such as THF, acetonitrile and

alcohols.44 The sirnillar polarity of peptides and polyacrylamide

support, both well soluibilised in DMF, makes the support suitable for

SPPS.48 On the basis of solvent-resin interactions, increasing effort

has been made to introduce polyacrylamide where initially

polystyrene supports were used.49

Bis-2-acrylamidoprop- 1 y l polyethyleneglycol cross-linked

dimethyl acrylamide (PEGA) has been introduced as a hydrophilic,

biocompatible and flexible solid flow stable support in peptide

synthesis (1). 50(a,bl

(1)

The first flow stable synthesis resin was obtained by

polymerization of the soft polydimethyl acrylamide gel inside a

solid matrix of sup:porting ~e i se l~uhr . " Small and Sherrington

replaced the irregu1a.r Keiselguhr with more regular rigid 50% cross-

linked polystyrene sponge containing a grafted polydimethyl acrylamide

This technique IwaLs developed for grafting polyethylene glycol

on to 1% cross-linked1 polystyrene,53 which are monodispersed,

spherical and flow stable. Polystyrene grafted to films of

polyethylene has been used for synthesis of peptides under non-

polar conditions (21.~~ Polyhydroxypropyl acrylate coated

polypropylene and coltton has shown some promise as supports

under polar conditions. 55.56 A co-polymer of bis-acrylamido

polyethylene glycol, lV,IV-dimethyl acrylarnide and acryloyl sarcosin

ethyl ester was succe~;sfully employed for the synthesis of peptides. s7(a-b)

The inert polyethy1e:ne glycol cross-linked resins such as

polyoxyethylene-po1yo:rq~propylene (POEPOP) (3) and polyoxyethylene

polystyrene (POEPS) (4) were efficiently used a s flexible and

biocompatible resins in SPPS.~' Cross-linked Ethoxylate Acrylate

Resin (CLEAR)O supp>rf:s developed by Kempe and Barany were also

used successfully m SPFS.~~

Different concepts for multiple peptide synthesis (MPS) or

simultaneous multiple peptide synthesis (SMPS) were developed to

respond the rapidl-y growing demands for a large number of

peptides with conipletely different sequences. The multiple

synthesis methods are mostly applied in hormone and inhibitor

research. For the identification of relevant.

side chains, every am.ino acid of a biologically active peptide can be

systematically substi.tuted, the chain length can be varied, and N-

as well a s C-termini can be rn~dified.~'

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

i as acetylations, biotinylations, succinimidilations or couplings for

/

preparing irnmunogens could be achieved by multiple methods or by

consecutive syntheses. In multiple peptide synthesis it is possible

to simultaneously prepare the same peptide bound to different anchors

and cleave to obtain peptide acid, peptide arnide, alkylated arnide,

hydrazide or a fully protected fragment. The "tea-bag" method proposed

by Houghten belongs )to the oldest strategies of multiple peptide

synthesis." In "tea bag" method, polystyrene in polypropylene mesh

packets were 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.43 Acrylic acid

coated polyethylene rod:; were used as supports in multi-pin synthesis

technology. Valerio el: al. used 2-hydroxy ethyl methacrylate grafted

polyethylene supports in multi-pin peptide synthesis.62 In multi-

column methods Macrosorb-SPR resin was used.63 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.64 Recently cross-linked enzyme crystals (CLECs) of

thermolysin were also used for peptide ~ynthesis .~ '

The polymer matrix has a significant role in SPPS. The

success of SPPS depends on the physicochemical characteristics of

the peptide bearing support. For effective swelling of the resin and

solvation of the peptjde, the polymer should have an optimum

50,52 hydrophobic-hydrophili.~ balance. The structure-reactivity and

structure-property correlations in cross-linked polymeric systems

helped to design new :supports with mechanical stability and optimum

reactivity. Pillai et al. 'developed a series of polymer supports by

introducing polar cro:ss-linking agents such as triethyleneglycol

dirnethacrylate (TEGYDMA), tetraethyleneglycol diacrylate (TTEGDA),

hexanediol diacrylate (HDODA) and 1,4-butanedioldimethacrylak

(BDODMA) to po1ysQm:ne network. - These resins have optimum hydrophobic-hydrophlic balance and swell like a gel in most of the.

organic solvents usedl in peptide synthesis. E f f ~ c i e n of these supports

was demonstrated b y :synthesising large number of peptides in high

purity and yield. The new member of this series described in this

thesis, is the g1yce:rol dirnethacrylate cross-linked polymethyl

methacrylate (GDMPi-F'MMA). This support showed very high swelling

characteristics in various solvents, an effective hydrophobic-

hydrophilic balance and is successfully used for the synthesis of

pep tide^.'^^ The optimum reactivities of these newly developed

resins are due to the greater chain mobility of the cross-linker in

solvents that enable effective interaction between the reactants

and resin bound functional groups.

2.4 Resin-peptide Linkages

Polypeptide synthesis by solid phase technique is more

effective when a specific combination of the linker/handle-resin

was used. These linkers can help the cleavage of the peptide from

the support under specific selected conditions that enable the

peptide free from side reactions. The covalent linkage between the

growing peptide chain and the polymer support is one of the

factors that determine the purity of the peptide.' The design and

development of a :series of bifunctional linkers (handles) have

facilitated and extendled enormously the scope and application of

SPPS approach for Ithe preparation of large peptide through a

convergent strategy7' and also hybrids of peptides with other

72 74 biomolecules containmg labile structures. One end of the bi-

functional spacers is attached to a smoothly cleavable protecting

group and the other end allows coupling to a previously

functionalised support. The linkages are easily formed and stable

to repeated cycles of acylation and deprotection steps.

The initial procedure of SPPS was based on temporary Boc

and permanent Bzl- protection (Boc/Bzl) with benzyl ester linkage of

the peptide to the resin. The HF cleavage of the peptide resulted in

concomitant loss of all protecting groups, thus offering no access to

protected peptide fragments. The protected peptides can be obtained

from Memifield resin by trans-esterification, ammonolysis or

hydrogenation depending on the stability of side chain protection.7541

Recently Fmoc/ t-Bu based protection schemes have become

increasingly popular with the development of a great number of

handles or linker-resiLns and cleavage of the protected peptide from

the resin is achieved under milder conditions by a great variety of

reagents.

Table 2.1

-

No.

-

1

-

2

-.

3

-

4

- -.

5

6

-

7

8

Some common linkers utilised in SPPS -- - - v- I

Structure Cleavage Reagent

0 0 Acetoxybenzyl (HMPAIPBA)lw TFA

0 0

0 0 II Methoxy Dilute

aceto~ybenzyl '~~ T F A

o cH,C Methoxyalkoxy benzyl Dilute R-C-(X ii ( S A S R I N ) ~ ~ ~ TFA

Hydroxymethyl phenyl acetamidomethy l (PAM)'''

0 C I I , l 0 Hydmxymethyl

dimethoxy Dilute phenoxyvaleryl T F A (HAL)"'

TFA

Alkoxy dimethoxy benzhydryl i ink)"^

4-benzyloxy- 4',4" dimethoxy hitylamine (BDMTA)"~

Benzhydrylamine ( B H A ) " ~

Methyl benzhydylamine (MBHA)"'

Methoxybenz hydrylamine (MeOBHA)"'

Dilute TFA

Dilute TFA

Dilute TFA

TFA

Aminomethyl dimethoxy

Dimethoxy b e ~ ~ z h ~ d r y l " ~

Amino xanthyloxy valeryl (XAL)"

Nitro henyl ethyl 1 4

TFA

TFA

Dilute TFA

Dilute TFA

* Cleavage reagent for the peptide

1

2.5 Protecting Groups

Of the various amino protecting groups t-butyloxycarbonyl

(Boc)8l,s2 and fluore:n~~lmethoxycarbonyl ( F m o ~ ) ~ ~ - ~ 5 are widely used.

Boc group can be cleaved under acidic conditions and Fmoc group

under basic conditi.ons, hence they are considered as orthogonal

protecting groups.86 For the removal of Boc group 30% TFA in

DCM us used. Though neat TFA is usually used for reducing the

time required for the synthesis of long peptides, studies show that

incomplete removal of Boc group may happen due to the

22

23

Fluorenyl methy 1"'

~ l l y l e s t e r ~ ~ ~

~ i t r o b e n z ~ l ' ~ '

Nibobenz h y d ~ ~ l a m i n e ' ~ ~

0 II

R g - c - - w w R-C-O(H\ -

I t /

~~~

U II E'

R-C-WH, ,+I= n~-C:-NHctl

~~ --

0

Piperidine

Pd(O)

hv

hv

II

25

-

~-

. ~p

insufficient swelling o:i the resin irk TFA.87 Earlier 4 N HC1-dioxane

was used for the cleprotection.88 However this reagent was not

sufficient to give complete removal of Boc group. TFA in DCM

provides an excellenl: medium for the maximum solvation of

peptidyl resin.8"

Fmoc group can be easily and rapidly removed by using a

secondary arnine (20% piperidine in DMF). Unlike Boc method, the

deprotection step in F~noc strategy avoids the neutralization stage,

thus reducing the time for synthesis. Besides this, Fmoc group

permits the easy monitoring of the progress of the synthesis

spectrophotometrically and hence Fmoc amino acids are widely

used in continuous flow synthesis.90 However, recent studies

reveal the fact that Frnoc group induces P-sheet conformation,

hence in solid phase peptide synthesis, using Fmoc protecting

strategy incomplete removal of Fmoc group is frequently notedg'.

The side chain of Boc amino acids are protected by benzyl based

groups. Boc/Bzl groups are removed during the cleavage step of

peptide from the support. In Fmoc strategy, the side chains are

protected by t-butyl groups which are stable in basic conditions,

but: are very sensitive to acid treatments.

Table 2.2

Commonly used W-protecting groups

Cleavage conditions No Name and Structure

1

2,

3

4

5

6

Benzylo:cycarbonyl (2)Iz9

c)- ,,,- @, ? -

~~

HBdHOAc, HBrITFA, Catalytic hydrogenolysis or Liquid HF

4-rneth0x~t1en.z~lox~carbonyl~~~

H3C f \ CH2-f3C- - ~-

Terr-Butylox)~carbonyl (BOC)"'

FH3 ? CH,-C-0-C-

I CH3

2-(4-Biphenyl)-iso ro oxycarbonyl ( ~ p o c ) P32.83

0

-

RSHITENDCM at 25' C for 5 min.

TFA

-

EIF or TFMSA

,4cidolysis, Catalytic hydrogenation

Dilute TFA

.-

12

13

14

15

16

--- ._.-___~--- 143, 144

Dithiosucc'inoyl ( U ~ S )

4 s. L-nN- 0

~

~- Diphenylphosphinyl ( ~ p p ) ' ~ ~

(2 / ( + + p + O

--

p-~iolueneaulphonyl ( T O S ~ ~ ) ' ~ ~

(-'-Jm2-. CH:,-

- ~~ ~ . -__ - - Allyloxycarbonyl(~lloc)'~~

I? CH2= CH-CH2-0-C--

~

148, 149 4-Methoxybem:yloxycarbonyl (Moz)

f H~cD--( \ CH2-(PC-

1 I I

3 -2 _ . _ ~-

2.6 Coupling Reagents

The formation of amide bond is the key step in peptide

synthesis. The reagent used for coupling should be able to form

the peptide bond in mild conditions, and the reaction should be

fast. They should retain the optical integrity of the peptide during

Thiolysis

Photolysis

Base labile

18

19

.

3-Nitro-2-pyridinesulfenyl (~pys) '~ '

02N

(>s-NH

- - 6-Nitrover:atryloxycarbonyl

(Nvoc)l? I . 1'2

WH3 /

P (XZH; CHZ-0-C-

.. . - - 2-[4-(methyls1llfonyl)phenyl~~lf0nyl]

ethoxycarbonyl ( M ~ C ) ' ~ '

rr,cxo, R {Ss ,,-

synthesis. Some of the recently introduced coupling reagents are

given in Table 2.3.

In spite of all th.ese achievements, dicyclohexyl carbodiirnide

(DCC) is still the widely used coupling reagent.22 The mechanism of

the reaction of DCC with carboxyclic acid via the formation of very

reactive 0-acyl isoure:a intermediate has been proposed by Khorana.g2

DCC when used with excess carboxylic acid make symmetrical

anhydride which art: capable of attacking the nucleophile.93

Recently, DCC active esters94-g6 of amino acids have been

widely used in peptide synthesis. As these are stable at room

temperature, they can, be prepared and stored for long time for the

direct reaction in coupling. Among these, pentaflurophenyl esters

of amino acids are very popular.97

Miyazawa et. al used Cu(I1) chloride for suppressing the

racemisation in I)CC assisted coupling reaction.98 I-Hydroxy

benzotriazole (HOE3t) is used as coupling additive which can

prevent racemisation.99 HOBt is used either in conjunction with

DCC or as active ester or built into a stand alone reagent in the

form of phosphoniurnl00~'01 or u r o n i ~ m . ~ ~ ~ ~ ~ ~ ~ The presence of

tertiary amine, e.g diisopropylethyl amine (DIEA), has been

reported1o4 to increase the reaction rate and product yield and are

comparable with other rapid coupling method using (I-H, 1,2,3-

benzotriaw1)- 1-yloxy tris(dimethy1amino) phosphonium hexafluoro

phosphate (BOP) or 2-.(3,4-dihydro-40x0- 1,2,3-benzotriazin-3yl)-

1,1,3,3-tetramethyl-uror~ium tetrafluoroborate (TDBTU) and no

racemisation is noted. lo4

Comparative stucly of various coupling agents to couple

hindered peptides shours that urethane protected amino acid N-

carboxy anhydride (UNCA) and bromo-tris-(pyrro1idino)-

phosphonium hexafluol-ophosphate (PyBrop) are excellent reagent

compared to 2-(1-13 Benzotriazol- 1- 1-yl) 1,1,3,3-tetramethyl-

uronium hexafluoro-phosphate (HBTU), pentafluorophenyl esters

or mixed anhydrides.10"

Table 2.3

Coupling reagents used in peptide synthesis

Reagent Structure

!

1

2

3

4

6

7

N,N'-Dicyclohexylca~rbodiimide (DCC)"~

- ----

N-Hydroxysuccinimide ( H O S U ) ' ~ ~

--

1-Hydroxybenzotriazole ( H O B ~ ) " ~

(Benzotriazol-l -ylo:~y) tris(dimethy1amino) phosphonium hexafluorophosphate BOP)'^'

-

I-0x0-2-hydrox!idihydrobenzotriazene ( H o ~ h b t ) ' ~ '

- - --

7-Aza-I-hydroxybenzotriazole ( H O A ~ ) ' ~ ~

. . . - __ __--___

4-Am-1-hydroxq.benzotriazole

(~-HoA~) '"

.. - - .. -

0

HO-N?J

0

N I OH

QJN% N' I 0 0 PF, I

\ ,pe / N I N,

N / \

dxxoH aN,,N .

N f OH

02 \ OH

N,N'-Diiso ro ylcarbodiimide k'. 2 (DIPCDI)' ' '

1-Ethyl-3-(3'-dimeth laminopropyl) carbodiimide (EDC) x3,,64

tris dimet thy la mi no:^ phosphonium hexafluorophosphate (AOP)"~

tris (pynolidino) phosphonium hexafluorophosphate ( P ~ A O P ) ' ~ ~

N-methylmethaminiurn hexafluorophosphate N-oxide (HBTU)'~'

0 CI

01 H-N w N = c = N - - /

I

I 0 0 PF, I

\ ,pP / N I N,

N /

1-(1-Pyrrolidinyl-IH-1,2,3-triazolo [4,5-b] pyridin-yl methylene) pyrrolidinium hexaflulorophosphate N-oxide (HAP~u)"'

1,2,3-benzotriazin-3-:{I)-l,1,3,3- tetrameth luroniurn hexafluorophoshate (HDTU)lYZ

Tetramethylfluorofonmamidiniurn hexafluorophospha.te (TFFH)

173.174

Pentatluorophenol ( H O P ~ P ) ' ~ ~

Urethane protected amino acid N- carboxy anydride (UNCA) 177-179

2-(3,4-Dihydro-4-0x0-

1,2,3-benzotriazin-3:yl)-l,I ,3,3- tetramethyluronium tetrafluoroborate ( T D B T U ) ' ~ ~

tetramethyluronium tetrafluoroborate (TPTU)I*I

2-(5-Norborene-2,3-dicarboxamido) 1,1,3,3-tetramethyluronium tetrafluoroborate ( ' T ~ I T U ) ' ~ ~

Fmoc amino acid halide 182-188

Tetrabutylammoniuni hydroxide1 p-toluenesulphonyl chloride189

I -(P-Naphthalene: s l~ l honvloxy)- benzotriazole (NSBt) 790 .

Bromo-tris(dimethy1amine) phosphorium hexduorophosphate (B~oP)"'

--

Bromo-tris-byrrolidino) phosphorium hexafluorophosphal:e ( P ~ B ~ O P ) ' ~ ~

Benzotriazolylox.y-bis-pyrrolidino- carbonium hexafluorophosphate ( B B C ) ' ~ ~

p~ ~

Bib (Boc) amino acid f lu~r ide"~

- - - -- - --

4 N~trophenol (ONp) 197 19X

-- - --- --

Q-I N-N

199-200 N-NHCHC,,

0-(Benzotriazol-1 -o!il)- 38 1 ,I ,3,3-pentameth:yleneuronium

hexafluorophosphate (HBPipU) 202,203

0

0-(7-Azobenzotriaz:oI-1-yl)- [TN'i Q PF6

41 1,3-dimethyl- 1.3..trimethyleneuronium N N'

Me hexafluorophosplhate (HAM'TU)'~'

0

0-(7-Azabenzotriazol- 1 yl)- PF6

F4 ? Me

2-(Benzotr~azol- 1 -yl)oxy- 42 1,3-dimethylimidaz.olidin~um

hexafluorophosphate (901)'"~ M e

- - - - - -- ---

40 1,3-dimethyl-l,3-di~nethyluronium hexafluorophosphate (HAMDU)'~'

00 '

2-(1 H-Benzotriazol-1-yl)-1,1,3,3- tetrameth luronium tetrafluoroborate J (TBTU)' '

2.7 Purification and Characterisation of Peptides

A s a general colasideration, homogeneity and correct covalent

structure are the basic goals of the synthetic endeavour and are the

two factors that pervade any and all consideration of the state of

the synthetic product. Even with optimised SPPS, there will be

typically small amounts of deletion peptides and a family of

peptides with chemical modifications due to the side reactions in

the final deprotection.

The evaluatiorl of the homogeneity and covalent structure of the

peptides are prime :importance and the most commonly used

techniques are analytical H P K , capillaryzone electrophoresis (CE),

amino acid analysis, 1JV spectroscopy, sequence analysis and mass

spectrometry. For the homogeneity and structural determination, each

technique makes its, own specialised contribution to the evaluation

process by providing unique information and generally they all are used

to some extent in a complimentary fashion (Figure 2.6).

Homgenay Covalent Stmcture

\

Amino acid UV

Figure 2. 6 ?he relative contribution of analytical t~ools toward the goal of assessing homogeneity and covalent structure

The developmer~t of HPLC in the late seventies revolutionised the

synthetic peptide chemistq by the adequate, rapid and sensitive

analybcal and punfica.tic)n modes of crude synthetic p r o d u c t s . ~ 5 , ~ It

also flouted one of the veiy early-applied peptide purification method-the

gel filtration method. Because amino acids are the fundamental units of

peptides the chromatographic behaviour of a particular peptide will be

determined by the number and properties (polarity, charge potential) of

the residue side chains it contains. In general the major modes of HPLC

employed in peptide separation take advantage of difference in peptide

size (slze-exclusion HPLC), net charge (ion exchange HPLC) or

hydrophobicity (reve:rse phase HPLC). Within this modes mobile

phase condition may be manipulated to maximise the separation

potential of a particultu :HPLC column. The reverse phase HPLC is the

most important mode of chromatographic technique for synthetic

peptide purification. 'Th.e alkyl derivatised (C8 and C18) silica based

packings are the most frequently employed packing support for

reverse phase HPLC separation of peptides. HPLC has proved

extremely versatile for the purification of a single peptide from the

kind of complex peptide mixtures encountered following solid

phase peptide synthesis, where impurities are closely related to

the peptide of interest: (deletion, terminated, chemically modified

peptides), perhaps missing only one amino acid residue and hence

may be difficult to :separate. The HPLC of very hydrophobic

peptides often pose special problems due to their limited solubility

and tendency to aggr'egate and some times it may be absorbed

irreversibly to some reverse phase sorbents.m7 Despite these, the

contaminants frequemt).y encountered in solid phase, including side

chain protecting groups, coupling reagents, cleavage reagents and

scavengers are often difficult to separate from the desired product

peptide during HPLC on Cs and C18 columns.M8

Either andpcal HPLC or CE is normally done as the frst level

of assessment. Capill,~)r electrophoresis is a high resolution, sensitive,

rapid and quantitative electrophoretic separation technique used for

the characterisatior~ of synthetic peptides. The speed and high

resolution capabilities of CE have found ready application in the area

of synthetic peptide c:hemistry. Its one major role has been to analyse

the products of automated peptide synthesis were both deletion and

incompletely deprotected peptides are commonly present. Separation

of peptides by CE occurs as a result of two interacting forces,

electrophoretic migration and electroosmotic flow. In fact, CE can be

an excellent analytical tool for any synthetic peptide manipulation

that results in a change in the net charge or shape of the molecule.

Amino acid analysis quantifies the amount of peptide and

determines whether the requisite amino acids are present a t

reasonable rati0s.m However it is less reliable for certain amino acids

such as cysteine, methionine and tryptophan and the

compositional accuracy decrease as the peptide gets larger.

Sequence analysis, particularly ,the gas phase sequencing and

analysis of the poly1ne:r bound peptides have made tremendous

opportunity to SPPS. UV spectroscopy monitors the integrity of the

aromatic amino acids particularly tryptophan and is very useful in

peptide purification. But the amount of information that can be

obtained about the covalent state of the peptide is very limited.

The recent developments in the field of mass spectrometry,

such as the introduct~orl of desorption ionisation technique and time

of flight mass measurements have made the ionisation and accurate

mass analysis of peptides and proteins feasible at the precise

juncture. In this c1a.s~ the first emerged one was the fast atom

bombardment mass spectrometry (FAB MS).210-211 The FAB MS has

found a particular niche in the mass analysis of biological

macromolecules up to 4 KDa. A valuable application of FAB MS has

been in the verification of the molecular mass of synthetic peptides

produced by automated synthesis.212 Moreover it has the ability to

determine precisely the molecular mass of the peptide with less

than 1Da. Thus amino acid insertions, deletions or chemical

modifications to the peptides can readily be discerned. FAB

MS/MS allows the elucidation of the primary structure of the

target peptide, even1 tiom a mixture and also permits the rapid

identification of synthetic side products.213 So FAB M S and FAB

MS/MS aid in the unec~uivocal characterisation of synthetic peptides.

The second one, electrospray ionisation mass spectrometry (ESI

MS) is an easy and rapid method for the verification of proper peptide

synthesis and for the identification of most: synthetic peptide

byproducts.214-215 Com~pared to other available mass spectrometric

techniques ESI MS offers the advantage of relatively low cost,

convenient automation and ease of interfacing on-line with liquid

chromatography (LC) or capillary electrophoresis. In addition the

multiple charges on most peptides enhance the abiity of ESI MS to

provide structure elucidation or conformation by fragment analysis. In

this series the most a.dvanced electrospray time of flight instrument will

give better perfomances.216

The matrix assisted laser desorption ionisation mass

spectrometry, an outgrowth of direct laser desorption ionisation

spectroscope (LD-MS)I~''~ pioneered by Hillenkamp and Chait is highly

useful in the characterisation of peptides and protein.218 With the

advent of time of flight mass analyzers, MALDI MS extends from

the low molecular weight range to the very high molecular weight

range upto 200 KDa and greater.21g The least complex instrument,

a typical linear mc~de MALDI TOF M S system is suitable for

monitoring most problems encountered in solid phase peptide

synthesis laboratories.

For a primary in:formation of the conformational state, CD,220

ORD221 a d vibrationsll spectrosCopic techniquesm2w were used

extensively. In the characterisation scenario of synthetic peptides, the

most informative of the analysis array, three dimensional (3D)

structural analysis using one dimensional (ID) and two dimensional

(2D) nuclear magnetic resonance (NMR) spectros~opy,224~~~~ and

X ray crystallography .2"6

2.8 Problems Associated with SPPS

The reactions in heterogeneous media are not like any true

solution type reactions. Hence unless all the different stages in a

multi-step synthetic procedure proceed unequivocally and

quantitatively, we cannot expect pure product at the end of the

synthesis. Even if we consider the chances of other side reactions

such as racemisations during coupling, side reactions associated

with individual ami:no acids, branching of peptide chain from the

side chain of a trifunctional amino acid, and the problems of

detachment of peptilde from the support, the yield of peptide after

10 or 15 residue inco~rporation will be too low.. The accumulation

of significant amount. of deletion peptide are well-documented.41

The inherent probllerns which limit the yield of peptides have

identified and these are attributed to the polymer effect or self

aggregating tendency of the peptide bound to the support via.

P-sheet formation or c:ombination of both.227

It was believed that polymer support does 'not have any

influence on the attached peptide chain. However studies show

that the polymer has a significant effect on the growing peptide. In

SPPS methodology, the growing peptide chain is covalently attached

to the insoluble support. The support imbibes organic solvent, and

comes highly swollen gel. Studies indicate that the interior of

peptide-resin is in a highly mobile environment comparable at a

molecular level to free solution, except the fact that diffusion is

restricted, and the homogenities within the swollen resin are

averaged out in NMR time scale.228

The rate of incorporation of a particular amino acid has been

found to decrease with increasing peptide chain The slow

reactivity has been att.ributed to the steric effect of polymer at

various functional sites. Narita and co-workers studied the

influence of peptide chain length in the coupling efficiencies of

amino acid with terminal amino acid of the oligopeptide bound to

soluble polystyrene, copoly(styrene- 1% DVB), and copoly (styrene-

2% DVB).z31,232 They noted little influence of peptide chain length

upto 10 residue amino acid length in the case of soluble and 1%

crosslinked polystyrente. However coupling efficiencies was lowered

with peptide chain length in the case of 2% crosslinked

polystyrene. This may be due the decreased solvation of polymers

in the higher crosslinked system. Free permeation of reactants and

solvents into the polymer support is an essential condition for

effective gel phase reaction. The decrease in coupling yield in SPPS

is believed to result from the restricted permeation of carboxyl

component into resin matrix. Another reason is that functional groups

that are placed near the crosslinks experience steric effects and are

inaccessible to the incoming reagent. Morawetz carried out extensive

investigations on the reactivity of functional groups attached to

polymer s ~ p p o r t . ~ ~ ~ , ~ ~ ~ He pointed out that introduction of

crosslinks in polymer support changes the local polarity of the

system; however the degree of crosslinking did not exert any

marked influence on reactivity. Ford et al. using NMR pulse-

gradient spin echo (PGSE) method showed that all reactive sites in

polystyrene gels are not equally rea~tive.*~5 The SPPS kinetics reveal

that the reactive sites for the first 90% reaction are effectively

homogeneous, but substantial reduction in rate was observed for the

final 10%. Crosslinkinj; of polymer causes the final stages of the

reaction to be extremely slow.

Solvation studies on PS matrix reveal that polystyrene is

hydrophobic and is compatible with hydrophobic solvents, but as

the peptide chain length is increased the amide bond change the

polarity of the peptidyl r e ~ i n . ~ 3 ~ However Sarin et a1.237 has

demonstrated that polymer and peptide chains mutually enhance

one another's solvation, and this is a supporting evidence for Flor's

view.238

Studies from this laboratory have shown that optimum

hydrophobic-hydroph:ilic balance is required for the polymer

support for effecting the solid phase reactions s u ~ c e s s f u l l ~ ~ - ~ ~ . In

this optimum conditions, the polymer will be compatible with the

peptide chain even if the length of peptide is increased or the

polarity of solvent i:; changed.

Bayerr et a]. introduced PEG as a support in order to avoid

matrix effect and the heterogeneous nature of cross-linked

system.23" PEG ouring to its good solubility in many organic

solvents can solubilise even otherwise insoluble peptides. An

advantage of PEG based liquid phase method is the possibility of

carrying out a better analytical control of the reaction, since

spectroscopic techniques like NMR, IR and CD can routinely be

applied which will help to study the changes in conformation of

peptide during ~ynt.hesis.6~.240 The problems involved in the

process of separation of PEG peptide from reaction media and

insolubility problem in the case of larger peptides bound to PEG

hinder the wide spread use of this technique.

The introduction of PEG grafted crosslinked polystyrene

(PEG-PS) was an attempt to overcome the above problems. There

are two forms of P'EG-PS. The anionic polymerization of ethylene

oxide to attach the PEG chain of controlled length on crosslinked

polystyrene bead containing OH group. This resin is marketed a s

tentagel r e ~ i n . ~ ' . ~ ' Preformed polythyleneglycol has been attached

to polystyrene bead ito obtain PS-PEG. The PEG-PS. in contrast to

PS offer better solvation in protic solvents. In PEG, since not

crosslinked, the reaction sites are highly accessible resulting in

greater reactivity.6' The main limitations of PEG-PS in comparison

to PS are higher cost, reduced loading level, and significant

mechanical instability.

Atherton and Stleppard have investigated on the origin of

the steric hindrance in polymer supported peptide synthesis.241

Based on the concepts of polymer-peptide structures they could

explain the polymer effects on the reactivity in polymer-supported

reactions.

2.10 Aggregation of Peptide

Besides the polymer effect described above, another important

factor which is considered as a general source of problem in peptide

synthesis is the insolubility (in classical peptide synthesis) or

aggregation (in SFJR3) of hydrophobic peptide due to freely

interpenetrating c'oils. Internal aggregation of peptide chains

bound to crosslinked polymer brings extra crosslinking to the

system, reduces the swelling behaviour of polymer support,

eventually leading to the inaccessibility of the reagents and

solvents to the reactive groups in the matrix. The onset of

aggregation in solid phase peptide synthesis can be characterized

by:

> A series of reproducible incomplete amino acylations (0.5-15%).242

P Recoupling or capping provides little or no improvement.235.242

P Occurs irrespective of resin type or ~trategy.242~243

> Difficulq increases with high resin loading.2421 N4

P Aggravated by stericdly hindered amino acid."Z

> Associated with specific sequence^.^^^.^^^

The internal aggregation has been shown to be antiparallel P-

sheet structure by 1TIR.245 Studies by Narita have shown that

si@cant solubility o:f protected hydrophobic peptide was noted when

Aib residues were incorporated into peptide ~ h a i n s . ~ 4 6 - ~ ~ ~ Although

the use of Aib in peptide synthesis finds limited application, the above

studies corroborate the idea that insolubility of protected hydrophobic

peptide segment is due to 0-sheet formation, since a-helix promoting

and 0-sheet disrupting property of Aib is well known.249 The view is

supported by the R,arnan, FTIR and CD studies of protein

aggregates."O-25"

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