Annual-Report-2019

DEVELOPMENT OF NOVEL CHEMOSELECTIVE
LIGATION TECHNIQUES FOR PROTEIN SYNTHESIS
Dr Thimmalapura
Marulappa Vishwanatha
LE STUDIUM / Marie Skłodowska-Curie
Research Fellow
Smart Loire Valley General Programme
From: University Medical College Groningen - NL
In residence at: Center for Molecular
Biophysics (CBM) - Orléans
Nationality: Indian
Dates: February 2019 to May 2020
I have joined the Prof. Sureshbabu’s laboratory
(Bangalore University, India) in July 2009 for a
PhD programme. The main objectives of my
research work were the design and synthesis of
a novel class of peptidomimetics. After finished
my PhD in 2014, Alexander Dömling (University
of Groningen, The Netherlands) offered a postdoc
position to work on multicomponent reactions.
I am very fortunate to have had an additional
experience to work on radiochemistry laboratory
at the medical college under the guidance of Prof.
Elsinga. I have committed for the opportunity to
expand my ideas and past research activities
for the synthesis of complex structures such as
peptides and proteins. I worked as a postdoc
in Dr Aucagne’s group on drug discovery and
organosulfur-based peptidomimetic synthesis.
I received several award grants such as CSIR
senior research fellowship, travel grants to attend
the 2018 International Symposium on Chemical
Biology in Switzerland and 2019 American Peptide
symposium in the USA.
Dr Vincent Aucagne
Host scientist
Vincent Aucagne received his PhD from the
University of Orléans (2002), working with
Patrick Rollin on the development of synthetic
methodologies to elaborate carbohydrate mimics.
Following post-doctoral research with Prof. David
Leigh at the University of Edinburgh (2003-2006) in
the field of mechanically-interlocked architectures
and molecular machines, he returned to Orléans
to join the CNRS Center for Molecular Biophysics
(CBM), as a CNRS Chargé de Recherche (2006)
in the group of Dr Agnès Delmas. He currently
holds a Director de Recherche position, leads the
“Synthetic Proteins and Biorthogonal Chemistry”
research group, and is the coordinator of the
“Molecular, Structural and Chemical Biology”
team. His current research interests focus on
the development of synthetic methodologies for
the chemical synthesis of proteins for application
to the deciphering of biological processes at the
molecular level.
The production of proteins by chemical synthesis is a very promising
alternative to biotechnological techniques for applications to the
deciphering of biological mechanisms at the molecular level, drug discovery
and synthetic biology. It is particularly useful for accessing site-specifically
modified proteins. Current technologies focus on the modular assembly
of unprotected peptide fragments through highly chemoselective reactions
called “chemical ligations”.
This approach revolutionised the field about thirty years ago, but there is still
only a very few reactions compatible with this purpose available to date. The
overall goal of the project is to develop novel ligation reactions for chemical
protein synthesis. In particular, the chemical ligation of peptide thioacids
with N-activated peptides (imidazolyl ureas) has been investigated, with the
goal to transform a known non-chemoselective reaction (carboxylic acid/
imidazoylurea coupling develloped by Campagne) into a chemo and regioselective
reaction compatible with aqueous environments typically used for
the ligation of unprotected peptides.
The rational behind the idea to replace carboxylic acids by thioacids is
that, under acidic conditions, thioacids are reactive whereas side chain
functional groups in the peptides such as amines and carboxylic acids are
expected to be unreactives.
The ligation reaction between peptide thioacids and imidazolyl urea
peptides involves three key challenges:
1. synthesis of unprotected peptide thioacids
2. synthesis of imidazolyl urea peptides and
3. ligation under aqueous conditions.
First, I investigated the synthesis of unprotected peptide thioacids
which are notably difficult to prepare, and rather unstable. A model
peptide was synthesized having a C-terminal hydroxy-benzyl cysteine
group at the C-terminus (so called crypto-thioester) as described
previously in the host laboratory. After the elongation on solid support,
the peptide was released in solution and purified through HPLC. This
crypto-thioester peptide was treated with trimethoxy benzyl thiol
(Tmob-SH) leading to the formation of trimethoxy benzyl thioester
peptide which was purified by HPLC. Very conveniently, trimethoxy
benzyl thioester peptide is efficiently converted into peptide thioacid
by a simple acidic treatment prior to use in ligation reactions. The
second challenge was the synthesis of imidazolyl urea peptides.
For this, activation of amine was carried out on solid support by the
treatment with carbonyl diimidazole.
The imidazolyl urea activated peptide was then released from resin. I
systematically studied the stability of model imidazolyl urea peptide
in aqueous conditions. It was found that, the imidazolyl urea activated
peptides are reasonably stable in water at acidic pH, thus compatible
with the reaction with peptide thioacids.
Next step will be to carry out ligation reaction between unprotected
peptide thioacids and unprotected Imidazolyl urea peptide under
acidic pH based on a previously synthesized dipeptide Boc-Phe-Ala-
OMe as a solid proof of concept.
NH 2
Peptide 1
COOH
OH
O
Peptide thioacid
Novel
Novel
synthesis
synthesis
developed
developed
+
SH N
N
O
N
H
NH 2
Peptide 2
COOH
OH
imidazolyl urea peptides
solvents
Stabilty Stabilty were
in in a investigated
range range of of
solvents were investigated
H 2 O
NH 2
Peptide 1
COOH
OH NH
O
2
N
H
Ligation
Peptide 2
COOH
OH
Life & Health Sciences 2019
Chemoselectivity and and
is to be investigated
reaction reaction in in water water
is to be investigated 51