Record Details

Solid phase peptide synthesis using glycerol based cross linked polymer supports

Shodhganga@INFLIBNET

View Archive Info
 
 
Field Value
 
Title Solid phase peptide synthesis using glycerol based cross linked polymer supports
 
Contributor Pillai, V N Rajasekharan
 
Subject Polimers
Peptide Synthesis
 
Description An important objective of the modem biochemical research is to
understand the molecular basis of the numerous and intricate biological
activities of peptides and proteins and therefore able to predict and control
these activities. The impc~rtance dramatically increased today because of the
explosive success ofthe gtnome-sequencing project, which revealed hundreds
of thousands of new proteins, but only as predicted sequence data. The
chemical synthesis and elucidation of the biological function of a predicted
protein molecule is thus a challenge of great significance. The introduction of
unnatural chemical group; into the covalent structure of a peptide or protein
molecule can provide valuable insight into questions about peptidelprotein
functions and enzyme activity. The most efficient way of introducing these
unnatural amino acids into peptides and small proteins through the
complementary approach of total chemical synthesis is by the stepwise solid
phase peptide syn1:hesis. The yield and the purity of these products are
depending upon various factors of which the most important one is the choice
of solid support, its mechanical and chemical stability, swellability and
compatibility with a range of solvents with different polarity. For a successful
synthesis, the reaction should go to completion in each step at a higher rate.
The physicochemic:al incompatibility of the support with the growing peptide
chain is considered as another major factor that affects the performance of the
resin in polypeptide synthesis. The design and synthesis of an efficient
polymer support depends on the judicious selection of monomers, their
structure and polarity and the net hydrophobic/l~ydrophilic balance of the
polymer. The work. presented in the thesis describes the development of a new
TRPGGDA cross-linked polystyrene support with a view to approaching the
above mentioned problems.
A brief ov':rviea of the key characteristics of various solid supports
and their modific:ation with suitable linkers, protection strategies, carboxyl
activation reagent:; and the difficulties encountered in SPPS are described in
the first and second chapters. This review aims to give a comprehensive
outline of the recent developments and refinements in the field of polymer
Summary 188
supported polypepticle synthesis and various factors ~nvolved in optimising the
purity and the yield of the synthetic peptide.
The third chapter describes the synthesis of the new polymer support,
PS-TRPGGDA. This has been designed to increase the flexibility of the
polymer backbone allowicg better diffusion of reagents through the matrix.
The polymer is synthe5ised by the aqueous free radical suspension
polymerisation of siyrene and TRPGGDA. The resultant polymers have
propyleneglycol-like character, which provides a spacer effect from the rigid
polystyrene core, and the glycerol moiety present in the cross-linker serves as
the growth site for the polypeptide synthesis. The hydrophillicity of the crosslinker
and its content in the resin determines the hydrophobic/hydrophilic
balance of the new polymzr. It also results in the very high swelling of the
polymer in a wide range of solvents. The support was stable to the various
chemistries employed during the synthesis. The support was characterised by
CP-MAS I3c NMR and F?'-IR techniques. The shape, size and morphological
features of the cross-ilinked polymer beads were analysed by scanning electron
microscopy. SEM analysis of the polymer showed that they are uniform
spherical beads. This chapter also describes the functional interconversion of
the hydroxy resin to various other derivatives like chloro and amino resins.
The support was a1:io fur~ctionalised with various commercially available
linkers like HMPA, HMPB and Rink amide so that the peptide or its derivative
can be cleaved from the support in short time.
The fourth chapter describes the optimisation of various chemical
reaction conditions encountered during polypeptide synthesis when the new
resin is used as the support for the synthesis. A systematic study of the nature
and the percentage of the cross-linker present in the resin on its swelling
behaviour in various solvents were carried out. This chapter also critically
evaluates the various para:neters like optimum reaction conditions for the
C-terminal amino acid incorporation, Nu-deprotection, coupling reaction and
final cleavage of the target peptide from the support. A kinetic comparison of
Summary 189
the amide bond fonnation was carried out on the new support with Merrifield
resin. The quantificatior of these closely related parameters was found
necessary for the judicious selection of the optim~sed reaction conditions.
The fifth chapter discusses a comparative synthetic study of some
difficult peptide sequen-es, which are considered as the test peptides to
evaluate the capability of a new support with commercially available
Merrifield, Sheppard and Pam resins. Various techniques like HPLC, amino
acid analysis and MALIII-TOF-MS were employed to analyse the purity of
the synthetic peptides. T"1e high yield and purity of the peptides synthesised on
the new support reveal t'ne ability of the resin to assist solvation and break the
aggregation of peptide by direct amphipathic interaction between the polymer
and the peptide chain. This study revealed the superiority of the new support
over various commercially available supports.
The sixth chapter discusses the utility of the new resin in the synthesis
of a number of d~fferent biologically active peptides in good yield and purity
using either by Boc- or by Fmoc chemistry. PS-TRPGGDA supports with
various cross-linking densities were utilised for the synthesis. These sequences
include the NR2B and NR2A peptide substrates and their mutations of
~a~+/calmodulin bindig protein and various peptides from the non-structural
regions of hepatitis C ~ ~ i r a l polyprotein and nuclear export signals. The purity
of these synthetic peptides were analysed by RP-HPLC using C18 column and
further confirmed by amino acid analysis and MALDI-TOF-MS. Circular
dichroism measurements were also carried out on selected peptides.
The work presented in this thesis introduces a new class of polymer
support for solid phase peptide synthesis. This new highly flexible and
chemically inert PS-TIWGGDA support, combining with the high mechanical
stability of the hydrophobic polystyrene matrix, makes it a better polymer
support than most of ,:he commercially available supports. The oxypropylene
chains together with 1:ster functionality's and hydroxyl groups of the crosslinker
confer a hydrophilic character to the polymer. By selecting the
Summary 190
percentage of cross-linE:er in the polymer an optimum hydrophobic1
hydrophilic character can be imparted to the resin. The high swelling
characteristics of the resin in various solvents employed in the polypeptide
synthesis can result in an effective interaction between the resin bound
functionality and the rez.ctive species in the solvent. Since the secondary
hydroxyl group in the resln is selected as the peptide growth point, the drastic
in~tial functionalisation steps involved in polystyrene type polymers with
various reagents like ch'oromethymethyl ether can be avoided in the new
resin. The degree of functional groups available for C-terminal amino acid
incorporation can be easiiy controlled by selecting the degree of cross-linking
in the resin. This makes the resin more economical when compared to most of
the other commercially available resins. The performance of PS-TRPGGDA
resin in polypeptide synthesis, especially in the case of sterically hindered
peptide sequences and thase which tends to form a secondary structure during
the synthesis, makes it 3 better choice when compared to various styrenebased
supports.
Abbreviation, Abstract included
 
Date 2010-07-29T06:50:38Z
2010-07-29T06:50:38Z
2010-07-29
August 1997
December 2001
31/01/2003
 
Type Ph.D.
 
Identifier http://hdl.handle.net/10603/302
 
Language English
 
Rights university
 
Format 190p.
CD
 
Publisher Kottayam
Mahatma Gandhi University
School Of Chemical Sciences
 
Source INFLIBNET