Chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides

Article abstract of DOI:10.1039/c0cc00828a

MUC1 glycopeptide was efficiently coupled to glycosylphosphatidylinositol (GPI) derivatives by sortase A (SrtA), verifying that SrtA can accept sterically hindered glycopeptide as substrate for ligation with GPIs. This work has established a practical method for the chemoenzymatic synthesis of GPI-linked glycopeptides.

Full text of DOI:10.1039/c0cc00828a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored
glycopeptidesw
Zhimeng Wu, Xueqing Guo and Zhongwu Guo*
Received 8th April 2010, Accepted 26th May 2010
DOI: 10.1039/c0cc00828a
MUC1 glycopeptide was efficiently coupled to glycosylphospha-
tidylinositol (GPI) derivatives by sortase A (SrtA), verifying
that SrtA can accept sterically hindered glycopeptide as
substrate for ligation with GPIs. This work has established
a practical method for the chemoenzymatic synthesis of
GPI-linked glycopeptides.
Glycopeptide FmocNH-GGVT(aGalNAc)S(aGalNAc)ADP-
T(aGalNAc)LPKTGGS-OH (1) containing a partial sequence
of MUC1, three O-glycans and the signal peptide LPKTG was
used as the model in this research. MUC1 was targeted as
it is densely glycosylated. Moreover, mucins have been found
GPI-anchored.19 The presence of several O-glycans in the struc-
ture of 1, especially the attachment of a glycan to Thr imme-
diately next to the signal peptide, makes 1 sterically hindered.
Thus, SrtA acceptance of 1 as a substrate can be strongly
indicative that SrtA may accept larger proteins/glycoproteins.
In the structure of 1, an additional Gly residue was installed at
the N-terminus. Therefore, after the GPI-anchored glycopeptides
are prepared, they may be used as SrtA substrates for further
elongation of the glycopeptide chain to obtain more complex
GPI-anchored glycopeptides.
Many surface proteins and glycoproteins are associated to the
cell membrane by glycosylphosphatidylinositol (GPI) anchors,
a class of naturally occurring glycolipids sharing a conserved
core structure Mana1,2Mana1,6Mana1,4GlcNH2a1,6Inositol-
1-(P)-lipid.1 The C-terminus of proteins/glycoproteins always
links to the 6-O-position of the GPI nonreducing end mannose
through a phosphoethanolamine bridge. GPI-anchored proteins/
glycoproteins play a pivotal role in many biological events.1–5
To study the biological functions of GPI-anchored proteins/
glycoproteins in detail, currently a major hindrance is access to
these molecules in homogeneous form and sufficient quantity.
Cell expression has the microheterogeneity problem, while the
chemical synthesis of these very complex molecules represents
a significant challenge.6–10 To address this issue, we have
recently developed a chemoenzymatic strategy for GPI-anchored
peptide/protein synthesis, which is based on sortase A (SrtA)-
mediated GPI-peptide ligation.11,12
The synthesis of glycopeptide 1 was a main challenge, which
was achieved by solid-phase peptide assembly starting from a
pre-loaded Wang resin 2 (Scheme 1), using commercial Fmoc
amino acids and synthetic Fmoc-Ser(Ac3-aGalNAc)-OH 3
and Fmoc-Thr(Ac3-aGalNAc)-OH 420 as building blocks.
Peptide assembly was essentially performed with an automatic
peptide synthesizer by standard Fmoc chemistry, while
the coupling reactions of 3 and 4 were carried out manually
using HBTU as the condensation reagent. In this case, only
2.5 equivalents of the precious 3 and 4 were utilized to guarantee
high coupling efficiencies. After the desired glycopeptide was
assembled, it was released from resin with 95% TFA, which
deprotected the amino acid side chains concomitantly. Finally,
the glycans were deprotected with NaOMe to afford 1, which
was purified by HPLC and characterized with MS.
SrtA is a transpeptidase of Staphylococcus aureus origin. It
recognizes a LPKTG peptide sequence near the C-terminus of
substrate proteins, cleaves the peptide bond between Thr and
Gly to form a reactive thioester between the acyl group of Thr
and SrtA Cys184 thiol group, and finally transfers the Thr acyl
group to the amino group of a Gly residue at the N-terminus
of a peptioglycan, which is the other substrate of SrtA.13 SrtA
has been disclosed to be substrate-promiscuous and has been
broadly used as a ligation tool for a variety of synthetic and
biological applications.14–18 We have recently shown that SrtA
can efficiently catalyze peptide ligation with GPI derivatives
with one or two Gly units linked to the phosphoethanolamine
group at the glycan nonreducing end of the GPI core.11,12
Because this construct of GPI-anchored proteins/glycoproteins
is highly conserved, the new synthetic strategy can be generally
useful. We further envisioned that this strategy may be employed
to prepare GPI-anchored glycopeptides/glycoproteins. To verify
the hypothesis, we investigated herein the first SrtA-mediated
GPI-glycopeptide ligation.
With 1 in hand, we then explored its reactions with GPIs in
the presence of SrtA (Scheme 2). The GPI derivatives used in
this research were 6–8 that were formerly employed to explore
SrtA-catalyzed GPI-peptide ligation.12 Reactions were carried
out at 37 oC in 0.3 M Tris-HCl buffer (pH 7.5) containing
0.15 M NaCl, 5 mM CaCl2, and 0.2 mM mercaptoethanol,
and the concentrations of 1, 6–8, and SrtA were 0.5 mM,
2.5 mM, and 12.6 mM, respectively. The reaction progress was
monitored by HPLC and MALDI-TOF MS. After ca. 24 h of
incubation, reactions were quenched by adding the same
Department of Chemistry, Wayne State University,
5101 Cass Avenue, Detroit, MI 48202, USA.
E-mail: zwguo@chem.wayne.edu; Fax: +1 313-577-8822;
Tel: +1 313-577-2557
w Electronic supplementary information (ESI) available: Experimental
procedures and data; NMR and MS spectra of the intermediates and
final products. See DOI: 10.1039/c0cc00828a
Scheme 1 Solid-phase synthesis of glycopeptide 1.
Chem. Commun., 2010, 46, 5773–5774 | 5773
ꢀc
This journal is The Royal Society of Chemistry 2010

View Article Online
Scheme 3 SrtA-catalyzed further elaboration of 9.
derivatives, and it has established a practical method for
GPI-anchored glycopeptide synthesis. Moreover, because
SrtA can tolerate very sterically hindered substrates, including
both the peptide donor, such as 1, and the peptide acceptor,
such as 7, 8 and 9, we anticipate that SrtA will be able to
handle more complex protein/glycoprotein and GPI substrates
and that the method may be generally useful. Our laboratory
is currently studying the application of this synthetic method
to full-size GPI-anchored proteins/glycoproteins.
This work was supported by Wayne State University and in
part by NSF (CHE-0554777). Z. Wu thanks Mr Benjamin
M. Swarts for his help with the automatic peptide synthesizer.
Notes and references
1 M. A. J. Ferguson and A. F. Williams, Annu. Rev. Biochem., 1988,
57, 285–320.
2 K. Hwa, Adv. Exp. Med. Biol., 2001, 491, 207–214.
3 V. Eckert, P. Gerold and R. T. Schwarz, in Glycosciences, ed. H.-J.
Gabius and S. Gabius, Chapman & Hall, Weinheim, Germany,
1997, pp. 223–243.
4 R. N. Cole and G. W. Hart, in New Comprehensive Biochemistry,
ed. J. Montreuil, J. F. G. Vliegenthart and H. Schachter, Elsevier,
Amsterdam, 1997, vol. 29, pp. 69–88.
5 J. R. Thomas, R. A. Dwek and T. W. Rademacher, Biochemistry,
1990, 29, 5413–5422.
6 Y. Tanaka, Y. Nakahara, H. Hojo and Y. Nakahara, Tetrahedron,
2003, 59, 4059–4067.
Scheme 2 SrtA-catalyzed GPI-glycopeptide ligation.
7 J. Xue, N. Shao and Z. Guo, J. Org. Chem., 2003, 68, 4020–4029.
8 N. Shao, J. Xue and Z. Guo, Angew. Chem., Int. Ed., 2004, 43,
1569–1573.
9 M. G. Paulick, A. R. Wise, M. B. Forstner, J. T. Groves and
C. R. Bertozzi, J. Am. Chem. Soc., 2007, 129, 11543–11550.
10 C. F. W. Becker, X. Liu, D. Olschewski, R. Castelli, R. Seidel and
P. H. Seeberger, Angew. Chem., Int. Ed., 2008, 47, 8215–8219.
11 X. Guo, Q. Wang, B. M. Swarts and Z. Guo, J. Am. Chem. Soc.,
2009, 131, 9878–9879.
12 Z. Wu, X. Guo, Q. Wang, B. M. Swarts and Z. Guo, J. Am. Chem.
Soc., 2010, 132, 1567–1571.
13 L. A. Marraffini, A. C. DeDent and O. Schneewind, Microbiol.
Mol. Biol. Rev., 2006, 70, 192–221.
14 S. Pritz, O. Kraetke, A. Klose, J. Klose, S. Rothemund,
K. Fechner, M. Bienert and M. Beyermann, Angew. Chem., Int.
Ed., 2008, 47, 3642–3645.
15 S. Samantaray, U. Marathe, S. Dasgupta, V. K. Nandicoori and
R. P. Roy, J. Am. Chem. Soc., 2008, 130, 2132–2133.
16 S. Pritz, Y. Wolf, O. Kraetke, J. Klose, M. Bienert and
M. Beyermann, J. Org. Chem., 2007, 72, 3909–3912.
17 R. Parthasarathy, S. Subramanian and E. T. Boder, Bioconjugate
Chem., 2007, 18, 469–476.
18 H. Mao, S. A. Hart, A. Schink and B. A. Pollok, J. Am. Chem.
Soc., 2004, 126, 2670–2671.
19 E. S. Nakayasu, D. V. Yashunsky, L. L. Nohara, A. C. T.
Torrecilhas, A. V. Nikolaev and I. C. Almeida, Mol. Syst. Biol.,
2009, 5, 261.
volume of 0.1% TFA, and reaction yields were finally deter-
mined with HPLC. The reactions of 1 with 6–8 were found to
proceed smoothly to afford ligation products 9 [MS (m/z):
calcd 2430, found 2431 (M+H)+], 10 [MS (m/z): calcd 2845,
found 2890 (M+2Na–H)+], and 11 [MS (m/z): calcd 2902,
found 2903 (M+H)+] in 76%, 75%, and 84% yields, respec-
tively. The products were further purified by HPLC and
characterized with MS. These results indicated that SrtA could
accept 1 as a substrate and effectively couple it to GPI
analogues that were modified to carry one or two Gly residues.
Encouraged by the above results, we then studied the SrtA-
catalyzed further elongation of the glycopeptide chain of 9
using 1 as the glycopeptide donor (Scheme 3). In this context,
9 was first treated with DBU to disclose its N-terminal Gly
residue. Thereafter, the resultant product reacted with 1 in the
presence of SrtA under the same conditions described above.
HPLC analysis showed the formation of 12 in a 59% yield, as
well as the N-terminal deprotected product 13 in a 10% yield,
both of which were purified by HPLC and characterized with
MALDI-TOF MS.
In summary, this work has verified that SrtA could accept
complex glycopeptides as substrates for the ligation with GPI
20 G. Arsequell and G. Valencia, Tetrahedron: Asymmetry, 1997, 8,
2839–2876.
ꢀc
This journal is The Royal Society of Chemistry 2010
5774 | Chem. Commun., 2010, 46, 5773–5774