L-Alanyl-L-prolyl-L-aspartyl-3-O-[(5-acetamido-3,5-dideoxy-D-
glycero-a-D-galacto-2-nonulopyranosylonic acid)-(2 → 3)-O-b-D-
galactopyranosyl-(1 → 3)-O-(2-acetamido-2-deoxy-a-D-galacto-
pyranosyl)]-L-threonyl-L-arginyl-L-prolyl-L-alanine amide (7).
Synthesis, purification on Waters Sep-Pak C18 solid-phase extrac-
tion column with gradient elution 0 → 50% MeOH in H2O and
futher by preparative HPLC (gradient 0 → 10% CH3CN in H2O
during 30 min, then 10 → 100% during 30 min) gave 7 (3 mg, 60%
yield). MS (ES): calcd for C55H91N13O28 m/z (M + H)+ 1382.618,
observed 1382.617.
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L-Alanyl-L-prolyl-L-aspartyl-3-O-[(5-acetamido-3,5-dideoxy-D-
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prolyl-L-alanine amide (8). Sialylation of 3 was essentially
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added to purified ST6GalNAc-I (12 m units)53 in 20 mM Bis-Tris
buffer (pH 6.5) containing CMP-b-D-sialic acid disodium salt
donor (2 mM), EDTA (20 mM), and dithiothreitol (1 mM). The
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◦
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solution was then incubated at 37 C for 6 h, and monitoring of
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combination with MALDI-TOF mass spectrometry. Purification
by preparative HPLC (gradient 0 → 80% CH3CN in H2O in
60 min) gave 8 (2.37 mg, 60% yield). MS (ES): calcd for
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General procedure for the lactonization
The sialylated benzyl glycoside 6 and glycopeptides 7 and 8 were
dissolved in CD3COOD and incubated at ambient temperature.
The reactions were monitored using mainly 1H NMR spectroscopy
over a period of 14 days in order to verify completion. The product
mixture 10a and 10b was freeze-dried several times from acetic
acid, dissolved in DMSO-d6 and the set of NMR experiments
were recorded, which allowed us to elucidate their structures.
Hydrolysis of mixture of lactones 10a and 10b
The lactones 10a and 10b were freeze-dried several times from
DMSO and dissolved in D2O. Hydrolysis was performed in NMR
tube at ambient temperature and several NMR experiments were
run every second day during 30 days in order to verify the reaction
progress. The NMR spectra in DMSO-d6 were also recorded after
multiple lyophilisation from water.
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Acknowledgements
35 K. C. Nicolaou, C. W. Hummel, N. J. Bockovich and C.-H. Wong,
J. Chem. Soc., Chem. Commun., 1991, 870–872.
This work was funded by the EU Prime Boost grant (QLRT-
2001-02010). Celso A. Reis was supported by FCT (POCTI/
CBO/44598/02) and the Association for International Cancer
Research (Grant 05–088).
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