Proceedings book download - 5Z.com

acids were incorporated using the Fmoc/tBu strategy with three equivalents of amino acidand three equivalents of N,N’-dicyclohexyl-carbodiimide in DMF for 3 hours. The couplingefficiency was monitored with the Kaiser test [3]. Fmoc-deprotection was made with 20%piperidine in DMF. Before coupling of Boc-protected glycosylated aspartic acidderivatives, the resin was pre-washed with dry DCM and then the resin was treated twicewith 0.2 M SnCl 4 in dry DCM for 10 minutes, giving the resin a red colour due to complexformation with SnCl 4 . Coupling of the last two amino acid was performed with the standardFmoc-amino acids and N,N’-dicyclohexyl-carbodiimide. Cleavage of the peptides from theresin was performed by addition of a mixture containing TFA:H 2 O (8:2) for 3 hours. Aftercleavage the peptides were precipitated onto the resin in ice cold diethyl ether andlyophilized after solubilization in H 2 O. The synthesis strategy of H-Gly-Val-Glu-Asp-Ile-[Xil(β1-O)]Ser-Gly-Leu-Pro-Ser(Bzl)-Gly-NH 2 was the same as in case of N-glycopeptideswith the exception that in this case the peptide contains a glycosylated serine derivativewith three unprotected hydroxyl functional groups and several trifunctional amino acids,which side-chains must be protected during the synthesis. Based on our earlier research theSer-Bzl, Ser(Xil)-Bz, Glu-, Asp-ODmab protecting group combination was suitable for thepreparation of this glycopeptide. Dmab-deprotection was made with 2% hydrazinehydrate/DMF,10 min, on resin. Bz-deprotection was made in solution-phase with 20%hydrazine-hydrate/MeOH, 1h.In summary we found a covenient solid-phase synthesis strategy for the preparation ofthe above glycosylated-hexapeptide conjugates. We have developed a strategy thatcombines the Fmoc and Boc SPPS approaches for the preparation of N-andO-glycopeptides in high purity and resonable yield, by making use of SnCl 4 for Bocdeprotection, which leaves the acid sensitive glycosidic bonds intact (Figure 2).1000tg100111cso #18-41 RT: 0.73-1.68 AV: 24 NL: 4.81E5T: + c ESI ms [ 49.99-2500.01]576.861009590750858075Abs.500250Relative Abundance706560555045400353025587.76-2505 10 15Time (min)20151050300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/zFig. 2. Analytical HPLC (220 nm) chromatogram and ESI-MS spectrum of the purifiedpeptide Leu-Lys-[Man(β14)GlcNAc(β1-4)GlcNAc(β1-N)]Asn-Gly-Gly-Pro-NH 2 .598.67768.691152.66393.84 776.101174.80599.50788.691175.68267.45 394.74496.07 731.60 878.44 1012.42 1151.58 1304.80 1441.14 1551.54 1608.79 1728.79 1827.71 1943.71AcknowledgmentsWe are grateful for the grant from the Hungarian National Science Foundation (OTKA-71753) for thefinancial support.References1. Freeman, N.S., Gilon, C.H. Synlett 13, 2097-2100 (2009).2. Hudáky, P., Stráner, P., Farkas, V., Váradi, G.Y., Tóth, G., Perczel, A. Biochemistry 47, 1007-1016(2008).3. Kaiser, E., Colescott, R.L., Bossinger, C.D., Cook, P.I. Analytical Biochemistry 34, 595 (1970).99