.
Angewandte
Communications
DOI: 10.1002/anie.201206428
Solid-Phase Chemical Ligation
Towards the Simplification of Protein Synthesis: Iterative Solid-
Supported Ligations with Concomitant Purifications**
Vincent Aucagne,* Ibai E. Valverde, Philippe Marceau, Mathieu Galibert, Nabil Dendane, and
Agnꢀs F. Delmas*
[
9]
Chemical synthesis is a complementary alternative to
recombinant approaches for the production of small (40–80
amino acids) to medium-sized (60–200 amino acids) proteins
and analogues. Protein assembly through chemoselective
coupling of unprotected peptide segments, that is chemical
purification features, as all truncated impurities would be
left unreacted (Figure 1b). This approach has never been
exploited until the present work, despite the fact that a wide
variety of related nonchromatographic purification strategies
[10,11]
were developed during the 1980s and 1990s.
Most of these
[
1]
ligation, has become the state-of-the-art strategy thanks to
strategies rely on the post-elongation chemical tagging of the
target peptide with an N-terminal linker that differentiates it
from the truncated by-products (Figure 1a). After cleavage
from the SPPS resin, the tagged molecules can specifically be
captured on a second, hydrophilic solid support through
[
2]
the pioneering work of Kemp and co-workers, and the
development of native chemical ligation (NCL) by Kent and
[
3,4]
co-workers.
More recently, mechanistically unrelated
[
5]
[6]
amide-forming ligations, and peptidomimetic techniques
have been developed.
[10]
[11]
a chemoselective reaction or noncovalent interactions.
A
The ligation of two peptides is inherently limited to the
synthesis of relatively small proteins. To expand the range of
targets accessible by chemical synthesis to larger and more-
complex proteins, sophisticated, iterative or convergent
synthetic strategies are required. Thus, ligations of more
than three segments, and numerous nontrivial purification
steps, would be required; the handling of the ligation product
intermediates is often tricky, owing to aggregation or limited
solubility.
simple filtration work-up separates the truncated peptides,
and the subsequent cleavage of the tag results in a traceless
release of the purified peptide into solution.
[
10c,d]
A few of these approaches
solid-phase NCLs, but the corresponding studies focus only
have been utilized for
[7]
[12]
[13]
on minimizing the chromatographic and handling steps. All
the examples of sequential solid-phase ligations reported so
far were limited to polypeptides smaller than 100 amino acids.
We thus decided to re-examine the potential of applying
this strategy to develop a self-purifying method by using
contemporary ligation techniques, as we believe that new
reagents or methods that allow for facile and rapid purifica-
tion of the starting materials and intermediates are critical to
the field of protein chemical synthesis. We disclose herein
Peptide segments are typically obtained through solid-
phase peptide synthesis (SPPS), which is generally limited to
[8]
the synthesis of peptides containing up to 40–50 amino acids.
Decades of continuous efforts in optimizing coupling reagents
and protective groups have eliminated most prominent side
reactions, but incomplete amide couplings are still a significant
limitation in Fmoc-based SPPS. Post-coupling acetylations
result in the capping of unreacted amine groups and the
consequent N-truncated peptides constitute most of the by-
products formed in peptide synthesis. We reasoned that the
assembly of a protein by iterative solid-supported ligations of
unpurified peptide segments in the opposite direction to
SPPS, that is the N-to-C direction, should display self-
[14]
a new N-terminal linker and its application to the non-
chromatographic purification of peptides that are difficult to
synthesize by existing methods, as well as to self-purifying N-
to-C ligations. The latter approach is exemplified by an
unprecedented synthesis of a 160-amino-acid triazole-con-
taining analogue of the tandem repeat region of human
[15]
MUC1 protein through successive N-to-C peptidomimetic
[6b]
triazole ligations of four segments.
We designed N -Esoc (2-[2-(2-azido-ethoxy)-ethyl sulfo-
3
nyl]-2-ethoxycarbonyl, Scheme 1) as an N-terminal cleavable
linker functionalized with an azide moiety. The versatility of
this functional group makes it useful for many types of
[
*] Dr. V. Aucagne, Dr. I. E. Valverde, P. Marceau, Dr. M. Galibert,
Dr. N. Dendane, Dr. A. F. Delmas
Centre de Biophysique Molꢀculaire, CNRS UPR 4301
Rue Charles Sadron, 45071 Orlꢀans cedex 2 (France)
E-mail: aucagne@cnrs-orleans.fr
I
[16]
chemoselective couplings such as Cu -catalyzed (this work)
[
17]
and strain-promoted azide/alkyne cycloadditions (CuAAC
[
5a,18]
and SPAAC, respectively) or Staudinger ligations.
These
[
**] The Rꢀgion Centre council is gratefully acknowledged for financial
reactions are superior to the ones employed in previous
capture/release approaches, because of their better chemo-
selectivity, their nonreversible nature, and because of the
chemical inertness of the resulting heterocyclic or amide
linkages. These linkages should be compatible with a wide
range of post-immobilization modifications and thus this
method should extend the range of applications of solid-
support (FibroCat project and PhD fellowship for I.E.V.), as is ANR
(
JCJC08-0138, SynProt project) and la Ligue contre le cancer. We
thank the CBM spectrometric platforms, and in particular Guil-
laume Gabant for recording most of the MALDI-TOF spectra and
Hervꢀ Meudal for the NMR spectra. We also thank Violette Senille,
Dominique Leliꢁvre, and Dr. Chrystelle Derache for peptide syn-
thesis.
[
9,10]
support capture approaches.
The Esoc linker is based on
[
19,10a–c,11d,f]
a alkylsulfonylethoxycarbonyl core,
which is stable
1
1320
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 11320 –11324
Aucagne, Vincent
