G. Kragol, L. Otvos, Jr/ Tetrahedron 57 (2001) 957±966
965
resin) in a glass vial for 4 h. After precipitation into cold
ether, the peptides were centrifuged, ®ltered and puri®ed
using preparative RP-HPLC (gradient 1). The integrity of
the peptides was veri®ed by MALDI-MS. Overall yields
were 7 mg for G-12/Ser and 0.55 mg for G-11/Ser.
support with a mixture (2.5 ml per 0.1 g of resin) of
TFA:water:thioanisole (90:5:5, v/v/v) for 4 h, precipitated
in cold ether, centrifuged and ®ltered. Existence of the
protected glycopeptides G-12/Man and G-11/Man was
checked using analytical HPLC (gradient 1).
4.1.7. Construct G-12/Man. Successful synthesis of
construct G-12/Man was achieved by manual coupling of
two molar excess (0.16 g, 0.24 mmol, calculated to free
amino groups on the resin) of N-Fmoc-O-(2,3,4,6-tetra-O-
acetyl-a-d-mannopyranosyl)-l-serine onto the side chains
of backbone lysines in construct A (made on 0.2 g of resin,
Acknowledgements
The authors thank Dr W. Gerhard for his critical reading of
the manuscript. This work was supported by a research grant
(GM45011) from the National Institutes of Health.
2
1
0.15 mmol g ). The amino acid was activated with an
equivalent amount of HATU (calculated to the glycoamino
acid) in the presence of 1 equiv. of diisopropylethylamine in
References
1.5 ml of DMF. The mixture was shaken for 3 days in a
glass vial. After coupling and extensive washing with
DMF, the ®nal glycopeptide was cleaved off the resin
with a mixture (5 ml) of TFA:water:thioanisole (90:5:5,
v/v/v) for 4 h and precipitated into cold ether. Puri®cation
using preparative RP-HPLC (gradient 1) yielded 4.8 mg of
the protected glycopeptide. MALDI-MS was used for the
analysis of the protected product. Deacetylation and Fmoc
removal were accomplished with a 30 min treatment
employing 0.1M NaOH (1 ml per ,0.5 mg peptide). After
neutralization with 0.2M HCl and further puri®cation on
preparative HPLC (gradient 1) 3.35 mg of deprotected
glycopeptide G-12/Man was obtained. MALDI-MS was
used for the ®nal characterization.
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4
.1.8. Construct G-11/Man. Construct G-11/Man was
successfully synthesized on a PS3 batch mixing synthesizer
Rainin Technologies, Woburn, MA) using double
7. Hsu, S. C.; Chargelegue, D.; Obeid, O. E.; Steward, M. W.
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(
couplings (2£6 h) with N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-
8. Meyer, D.; Torres, J. V. Mol. Immunol. 1999, 36, 631±637.
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a-d-mannopyranosyl)-l-serine onto construct B (made on
2
1
0
.12 g of resin, 0.15 mmol g ) and HATU activation.
While the ®rst coupling employed a four molar excess
0.19 g, 0.29 mmol calculated to the number of free amino
10. Fitzmaurice, C. J.; Brown, L. E.; Kronin, K.; Jackson, D. C.
Int. Immunol. 2000, 12, 527±535.
(
groups) of N-Fmoc-O-(2,3,4,6-tetra-O-acetyl-a-d-manno-
pyranosyl)-l-serine, the second used only 2 equiv.
11. Cruz, L. J.; Quintana, D.; Iglesias, E.; Garcia, Y.; Huerta, V.;
Garay, H. E.; Duarte, C.; Reyes, O. J. Pept. Sci. 2000, 6, 217±
224.
(
0.095 g, 0.145 mmol). The glycopeptide was cleaved off
the resin with a mixture of TFA:water:thioanisole (90:5:5,
v/v/v) for 4 h and precipitated into cold ether and centri-
fuged. Because the very hydrophobic nature of the protected
peptide prevented the RP-HPLC puri®cation of the product
12. Sallusto, F.; Cella, M.; Danieli, C.; Lanzavecchia, A. J. Exp.
Med. 1995, 182, 389±400.
13. Tan, M. C.; Mommaas, A. M.; Drijfhout;, J. W.; Jordens, R.;
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(
gradient 1), the Fmoc and acetyl protecting groups were
removed from the crude glycopeptide with 0.1M NaOH as is
described for G-12/Man. After neutralization with 0.2M
HCl, and puri®cation on preparative RP-HPLC (gradient
1
) 0.03 mg of ®nal glycopeptide G-11/Man was obtained.
15. Palladino, G.; Modzanowska, K.; Washko, G.; Gerhard, W.
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MALDI-MS was used for the characterization of the ®nal
product.
1
7. Frace, A. M.; Klimov, A. J.; Rowe, T.; Black, R. A.; Katz,
J. M. Vaccine 1999, 17, 2237±2244.
4.1.9. Conditions for manual coupling of Fmoc-Ser-
(
structs A (made on 0.30 g of resin, 0.15 mmol g ) and B
Ac -Man)-OPfp. Mannosylations of resin-bound con-
18. Neirynck, S.; Deroo, T.; Saelens, X.; Vanlandschoot, P.; Jou,
W. M.; Fiers, W. Nature Med. 1999, 5, 1157±1163.
19. Brumeanu, T. D.; Kohanski, R.; Bona, C. A.; Zaghouani, H.
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4
2
1
2
1
made on 0.12 g of resin, 0.15 mmol g ) were attempted
(
with 2 equiv. of Fmoc-Ser(Ac -Man)-OPfp (0.29 g,
4
0
.35 mmol for construct A and 0.125 g, 0.15 mmol for
20. Gerhard, W.; Haberman, A. M.; Scherle, P. A.; Taylor, A. H.;
Palladino, G.; Caton, A. J. J. Virol. 1991, 65, 364±372.
21. Liblau, R. S.; Tisch, R.; Shokat, K.; Yang, X.; Dumont, N.;
Goodnow, C. C.; McDevitt, H. O. Proc. Natl. Acad. Sci. USA
1996, 93, 3031±3036.
construct B in 2 and 1 ml of DMF, respectively) in the
presence of 1 equiv. of diisopropylethylamine (and 1
equiv. of HOBt in the case of construct B). The mixtures
were shaken for 2 days. Peptides were cleaved off the solid