Angewandte
Chemie
DOI: 10.1002/anie.200903710
Amide Activation
Fmoc Solid-Phase Synthesis of C-Terminal Peptide Thioesters by
Formation of a Backbone Pyroglutamyl Imide Moiety**
A. Pernille Tofteng, Kasper K. Sørensen, Kilian W. Conde-Frieboes, Thomas Hoeg-Jensen,* and
Knud J. Jensen*
The use of C-terminal peptide thioesters in synthetic protein
chemistry has inspired the search for optimal solid-phase
peptide synthesis (SPPS) strategies for protein assembly.[1,2]
However, peptide thioesters are not directly accessible by
Fmoc-SPPS (Fmoc = 9-fluorenylme-
backbone pyroglutamyl imide, which, after displacement by a
thiol, provides the peptide thioester (Scheme 1). General
Fmoc-SPPS protocols were applied for the synthesis of
peptides prior to activation.
thoxycarbonyl) owing to the nucleo-
philicity of the secondary amine
required for Fmoc removal. Peptide
thioesters can be accessed by Boc-
SPPS
(Boc = tert-butoxycarbonyl),
however, the conditions are incompat-
ible with many posttranslational modi-
fications.[3] The development of meth-
ods for Fmoc-SPPS of peptide thioest-
ers has therefore been a major chal-
lenge over the past decade, and several
approaches have been reported,[2] for
example, Kennerꢀs safety-catch linker
system,[4] as well as other methods.[5–7]
Very recently, thiolysis of peptides
with a C-terminal peptide N-acylurea
unit was described.[8] Peptide thioest-
ers with a C-terminal achiral glycine
unit can be conveniently synthesized.[9]
However, new, more general methods
for such peptides are also of interest.
We reasoned that the activation of
a peptide backbone amide bond by
increasing its nucleofugality could
Scheme 1. The strategy for the synthesis of peptide thioesters through a backbone amide
activation. Pg=protecting group, TES=triethylsilane, TFA=trifluoroacetic acid, R1 =amino acid
side-chain.
ꢀ
render the C N bond susceptible to
thiolysis and provide peptide thioest-
ꢀ
ers. Cleavage of the C N bonds in a backbone amide to
provide a thioester can occur biologically in intein-mediated
protein splicing.[10]
The synthesis of C-terminal peptide thioesters by this
strategy entails anchoring of a C-terminal glutamic acid
residue with a selectively removable side-chain protecting
group to a solid support (Scheme 1). Upon peptide chain
assembly, the glutamic acid side-chain would be selectively
deprotected. In the first key step, strong activation of the
deprotected carboxylic acid should result in the on-resin
formation of the pyroglutamyl (pGlu) imide moiety. The
novel formation of the backbone pGlu imide involves the
acylation of an amide nitrogen atom, which is a very poor
nucleophile, although the reaction is likely to be entropically
favored. In the second key step, nucleophilic displacement by
treatment with a thiol was expected to release the protected
peptide thioester from the solid support, which then would be
deprotected in solution.
Herein, we describe an unprecedented method for the
activation of a backbone amide in a peptide by formation of a
[*] Dr. K. W. Conde-Frieboes, Dr. T. Hoeg-Jensen
Diabetes Protein and Peptide Chemistry
Novo Nordisk Park D6.1.142, 2760 Maaloev (Denmark)
E-mail: tshj@novonordisk.com
Dr. A. P. Tofteng, K. K. Sørensen, Prof. Dr. K. J. Jensen
IGM, Faculty of Life Sciences, University of Copenhagen
Thorvaldsensvej 40, 1871 Frederiksberg (Denmark)
Fax: (+45)35-33-23-98
E-mail: kjj@life.ku.dk
[**] A grant from the Danish Council for Strategic Research to K.J.J. is
To implement this strategy for the synthesis of thioesters,
a heptamer peptide related to the enkephalins was assembled
on-resin to provide the Boc-FY(tBu)GGFAE(PhiPr)-Rink-
gratefully acknowledged. Fmoc=9-fluorenylmethoxycarbonyl.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2009, 48, 7411 –7414
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7411