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Solid-Phase Synthesis and Evaluation of Glycopeptide Fragments from Rat Epididymal Cysteine-Rich Secretory Protein-1 (Crisp-1) ‡
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA
This work is dedicated to Professor Gary R. Gray on the occasion of his retirement after 38 years of teaching and research on carbohydrates as a member of the Chemistry faculty at the University of Minnesota - Twin Cities campus
Current address: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
* Author to whom correspondence should be addressed.
Received: 17 May 2010; in revised form: 24 August 2010 / Accepted: 6 September 2010 / Published: 14 September 2010
Abstract: Three 18-residue peptides with the sequence Glp-Asp-Thr-Thr-Asp-Glu-Trp-Asp-Arg-Asp-Leu-Glu-Asn-Leu-Ser-Thr-Thr-Lys, taken from the N-terminus of the rat epididymal cysteine-rich secretory protein (Crisp-1) that is important in the fertilization process, were prepared by Fmoc solid-phase synthesis using a convergent strategy. These peptides were the parent sequence, plus two possible α-O-linked TN antigen-containing glycopeptides with a Thr(α-D-GalNAc) residue in place of either Thr3 or Thr4. During chain assembly, two deletion peptides [des-Asp2 and des-Thr(Ac3-α-D-GalNAc)] and one terminated peptide [N-acetylated 14-mer] arose, as did several peptides in which aspartimide formation had occurred at each of the four possible positions in the sequence. These by-products totaled ~20% of the desired product; they were recognized by HPLC and ESI-MS and removed during the intermediate purifications. Final products, obtained in 15-21% overall yields, were characterized by HPLC purities and ESI-MS. Circular dichroism (CD) spectra for all three purified peptides, recorded in pure water and in trifluoroethanol-H2O (1:1), revealed that the presence of a sugar moiety does not significantly impact the sampled conformations. Future biological evaluation could elucidate the nature and locus of sugar modification of Crisp-1, and provide insight as to why Crisp-1 protein E binds sperm irreversibly, in contrast to protein D that lacks a sugar near the N-terminus and only binds sperm loosely.
Keywords: glycopeptides; solid-phase synthesis; cysteine-rich secretory protein (Crisp-1); circular dichroism; TN antigen
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Liu, M.; Hamilton, D.W.; Barany, G. Solid-Phase Synthesis and Evaluation of Glycopeptide Fragments from Rat Epididymal Cysteine-Rich Secretory Protein-1 (Crisp-1) ‡. Molecules 2010, 15, 6399-6410.
Liu M, Hamilton DW, Barany G. Solid-Phase Synthesis and Evaluation of Glycopeptide Fragments from Rat Epididymal Cysteine-Rich Secretory Protein-1 (Crisp-1) ‡. Molecules. 2010; 15(9):6399-6410.
Liu, Mian; Hamilton, David W.; Barany, George. 2010. "Solid-Phase Synthesis and Evaluation of Glycopeptide Fragments from Rat Epididymal Cysteine-Rich Secretory Protein-1 (Crisp-1) ‡." Molecules 15, no. 9: 6399-6410.