Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Molecules. 2010 Sep 14;15(9):6399-410. doi: 10.3390/molecules15096399.
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 T(N) 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(Ac(3)-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-H(2)O (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.
从大鼠附睾胱氨酸丰富的分泌蛋白（Crisp-1）的 N 端制备了 3 种 18 残基肽，序列为 Glp-Asp-Thr-Thr-Asp-Glu-Trp-Asp-Arg-Asp-Leu-Glu-Asn-Leu-Ser-Thr-Thr-Lys，这些肽是亲本序列，加上两个可能的α-O 连接的 T（N）抗原含有糖肽，其中 Thr3 或 Thr4 被 Thr（α-D-GalNAc）取代。在链组装过程中，产生了两个缺失肽[des-Asp2 和 des-Thr（Ac（3）-α-D-GalNAc）]和一个终止肽[N-乙酰化 14 肽]，以及几个在序列的四个可能位置都发生天冬酰胺内酰胺形成的肽。这些副产物约占所需产物的 20%；它们通过 HPLC 和 ESI-MS 识别，并在中间纯化过程中去除。最终产物的总收率为 15-21%，通过 HPLC 纯度和 ESI-MS 进行了表征。所有 3 种纯化肽的圆二色性（CD）谱在纯水中和三氟乙醇-H（2）O（1:1）中记录，表明糖部分的存在不会显著影响采样构象。未来的生物学评价可以阐明 Crisp-1 的糖修饰的性质和位置，并深入了解为什么 Crisp-1 蛋白 E 与精子不可逆结合，而蛋白 D 缺乏 N 端附近的糖，并且仅与精子松散结合。
