Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2239–2241
Synthesis of a thioester linker precursor for a general preparation
of peptide C-terminal thioacids
Hubert Gaertner,a Matteo Villain,a,* Paolo Bottia and Lynne Canneb,
aGeneprot, 2, Rue Prꢀe de la Fontaine, PO Box 125, CH-1217 Meyrin 2, Switzerland
bGryphon Therapeutics, 600 Gateway Blvd., South San Francisco, CA 94080, USA
Received 15 October 2003;revised 8 December 2003;accepted 18 December 2003
Abstract—A general procedure to prepare peptide thioacids by solid-phase peptide synthesis is presented. The method involves the
synthesis of 4-[a-(S-acetyl)mercaptobenzyl]phenoxyacetic acid as general precursor. This reagent once attached to a solid support is
derivatized with the Boc-amino acid of choice after deprotection of the thiol.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Peptide a-thioacids are key intermediates in the syn-
thesis of proteins by chemical ligation of unprotected
peptides.1–3 The key feature of the thioacid moiety lies in
its ability to act as a nucleophile at pH 3–6, in a unique
window where all nucleophiles present in peptides and
proteins are basically unreactive. Thus, a peptide-
aCOSH can be easily converted into a peptide thioester
by reaction in aqueous acidic conditions with either an
alkyl bromide or with an activated symmetrical disul-
fide. A case in point is the coupling of a peptide-aCOSH
with the alkyl bromide at the N-terminus of another
segment generating a single polypeptide chain with an
artificial thioester bond at the ligation site.2 Further-
more due to its unique reactivity this functionality has
also been exploited in (i) the convergent chemical liga-
tion of multiple unprotected peptide segments (when the
protein multiple sub-domains are synthesized in parallel
by ligation of two fragments with a normal thioester
strategy, and the final protein is generated by ligation of
the sub-domains after activation of the thioacid)3 and
more recently (ii) in extended chemical ligation involv-
ing a removable auxiliary group4 when a highly reactive
thioester is required.5
Kent.6 This procedure however has some disadvantages6
since it requires the preparation in solution of the Boc-
aminoacyl-S-linker-COOH for each amino acid before
attachment to the amino-methyl resin. Therefore the
development of a unique resin precursor that allows the
loading of the amino acid or the reagent of choice is
crucial to offer a more attractive route to thioacid pep-
tides in a straightforward manner.
In this paper we report the synthesis of 4-[a-(S-ace-
tyl)mercaptobenzyl]phenoxyacetic acid in a four-step
route. This reagent is used to generate a general resin for
thioacid peptide synthesis, which can be functionalized
with the Boc-amino acid of choice. The preparation of
this precursor is described in Scheme 1.
The 4-(a-hydroxybenzyl)phenoxyacetic acid was easily
synthesized in three steps from the corresponding
4-hydroxybenzophenone using the reported synthetic
pathway.7 The problems outlined by Goldstein8 could
be overcome with a supplementary purification step of
the 4-benzoylphenoxyacetic acid ethyl ester by ethyl
acetate extraction, washing and crystallization before
the saponification step. The 4-(a-hydroxybenzyl)phen-
oxyacetic acid was isolated as the DCHA salt in a 55%
yield. The corresponding free acid was then reacted with
concentrated HCl and the resulting 4-(a-chlorobenz-
yl)phenoxyacetic acid crystallized upon addition of
petroleum ether and was isolated in a 79% yield.9
Reaction of CH3COSH in a 1.1 M excess over the
chloro-substituted linker in the presence of equimolar
amounts of DIEA in DMF was almost quantitative
after 20 min incubation at room temperature, generating
Thioacid peptides are generally synthesized on the
appropriate Boc-aminoacyl-S-resin as described by
Keywords: Thioacids;Thioesters;C-terminal modified peptides.
* Corresponding author. Tel.: +41-22-7198996;fax: +41-22-7198999;
Present address: Exelixis, Inc., 170 Harbor Way, South San
Francisco, CA 94083-0511, USA.
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.12.160