Angewandte
Chemie
DOI: 10.1002/anie.200700546
Glycopeptide Synthesis
Second-Generation Sugar-Assisted Ligation: A Method for the
Synthesis of Cysteine-Containing Glycopeptides**
Simon Ficht, Richard J. Payne, Ashraf Brik, and Chi-Huey Wong*
Glycosylation is a very common post- or co-translational
modification of proteins that has extensive biological signifi-
cance.[1] Indeed, it is estimated that over fifty percent of all
human proteins are glycosylated.[2] Protein glycosylation
plays an important role for a variety of biological recognition
events such as cell adhesion, cell differentiation, and cell
growth.[3,4] Additionally, some parasites use heavily glycosy-
lated membrane-bound proteins as port of entry.[5] Aberrant
glycosylation of proteins often modifies intracellular recog-
nition and is linked with several serious illnesses including
autoimmune diseases, infectious diseases, and cancer.[6] In
order to understand the role of the glycosylation at a
molecular level, it is important to have access to homoge-
neous glycopeptides and glycoproteins. The glycosylation
pattern of a given glycoprotein, unlike the protein element, is
not under the control of a coding template, but rather is
dictated by the relative activities of the constituent enzymes.
The use of biological expression systems for production and
study of glycoproteins has proved difficult and is hampered by
the heterogeneity of the resulting products.
The necessity for homogeneous glycoproteins can be met
by chemical and chemoenzymatic intervention.[7–10] In partic-
ular, a number of ligation methods have recently gained
significant attention as techniques to facilitate the synthesis of
such targets. Native chemical ligation (NCL), a chemoselec-
tive condensation reaction between a peptide thioester and a
peptide bearing an N-terminal cysteine has proven very useful
in this regard.[11–13] This method has been successfully
implemented in the synthesis of hundreds of proteins to
date.[13] The success of this method for peptide and protein
synthesis has inspired many laboratories to employ this
technology for studies towards the synthesis of glycopeptides
and glycoproteins.[14–17] Although NCL has proved to be
extremely powerful, certain limitations still exist with this
method. The obvious limitation of NCL is the requirement for
a cysteine residue at the ligation junction.[18] Cysteine has a
relatively lowabundance in nature (ca. 1.7%), and as such,
there is a high probability that the target does not have a
cysteine at a synthetically useful position. This led to the
development of a number of cysteine-free ligation techni-
ques,[19,20] some of which have been successfully implemented
in the synthesis of glycopeptides.[21,22] However, the use of
these methods, which rely upon the incorporation of an N-
terminal auxiliary, is restricted to ligation sites containing
amino acids of lowsteric bulk.
Our laboratory has recently reported a ligation method
for the synthesis of cysteine-free O- and N-linked glycopep-
tides.[23,24] This method, dubbed sugar-assisted ligation (SAL),
utilizes a glycopeptide in which the carbohydrate (N-acetyl
glucosamine) is derivatized with a mercaptoacetate auxiliary
at the 2-position. In the presence of a peptide thioester and
under suitable ligation conditions, thioester exchange is
followed by an S!N acyl transfer affording a ligated product
with a native peptide backbone. The reaction cascade showed
high sequence tolerance at the ligation junction, therefore
expanding the number of potential targets accessible by this
method. Additionally, the reaction was shown to be chemo-
selective even in the presence of nucleophilic amino acid side
chains such as lysine.[23] This means that ligations could be
conducted on glycopeptides free of protecting groups. For
these reasons, SAL has recently gained the spotlight as a
feasible method for the total synthesis of glycoproteins.[25] The
major pitfall of this method, however, is the incompatibility of
the conditions used for the removal of the auxiliary, which
requires hydrogenation, with other thiol-containing residues.
As mentioned above, the abundance of cysteine is low;
however, a large proportion of naturally occurring glycopro-
teins contain this residue in their sequence. In addition many
glycoproteins contain cysteine residues in nonstrategic posi-
tions, and as such, NCL cannot be implemented.
[*] S. Ficht,[+] R. J. Payne,[+] Prof. C.-H. Wong
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+1)858-748-2409
E-mail: wong@scripps.edu
and
Academia Sinica
Taipei (Taiwan)
To circumvent this problem of chemoselective auxiliary
removal, we embarked on the development of a second-
generation SAL. This method relies on the incorporation of
an auxiliary which can be removed in the presence of other
cysteine residues after ligation. An acid-labile auxiliary
cannot be used, as strongly acidic conditions are used in the
solid-phase glycopeptide synthesis. In contrast, a base-labile
auxiliary, in this case a mercaptoacetic acid moiety bound
through an ester on the 3-position of the bridgehead sugar,
would fulfill the required orthogonality. The mechanism of
the proposed second-generation SAL is depicted in Scheme 1.
Dr. A. Brik
Department of Chemistry
Ben Gurion University
BeerSheva (Israel)
[+] S. Ficht and R. J. Payne contributed equally.
[**] This work was supported by the NIH and the Skaggs Institute for
Chemical Biology. S.F. is grateful to the Deutsche Akademische
Austauschdienst (DAAD) fora postdoctoral fellowship. R.J.P. is
grateful for funding provided by the Lindemann Trust Fellowship.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2007, 46, 5975 –5979
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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