Nilsson M R, Nguyen L L, Raleigh D P
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
Anal Biochem. 2001 Jan 1;288(1):76-82. doi: 10.1006/abio.2000.4887.
The polypeptide hormone amylin forms amyloid deposits in patients with type 2 diabetes mellitus. Amyloid-forming peptides are often very difficult to synthesize and purify. Amylin and fragments of amylin are no exception. In this paper we describe the efficient synthesis and purification of two amyloidogenic fragments of human amylin. One fragment corresponds to residues 17 to 37 of the full-length hormone and the other corresponds to residues 24 to 37. These fragments have previously been identified in vivo and have been shown to form amyloid in vitro. The strategy used to elucidate appropriate conditions for the synthesis and purification of these peptides is generally applicable to other peptides that are difficult to synthesize. These peptides were prepared using solid-phase peptide synthesis with Fmoc alpha-amino protection. The effects of varying the solvent, side-chain-protecting group and choice of cleavage conditions were examined. The use of NMP as the main solvent and cleavage with trifluoroacetic acid, phenol, ethanedithiol, thioanisole, and water proved to be optimal. 1,1,1,3, 3,3-Hexafluoro-2-propanol (HFIP) was found to be the best solvent for solubilizing the crude peptides. A wide range of HPLC conditions for the purification of the peptides were examined and an acetonitrile-based solvent system with HCl as the ion pairing agent provided efficient purification.
多肽激素胰淀素在2型糖尿病患者体内形成淀粉样沉积物。形成淀粉样的肽通常很难合成和纯化。胰淀素及其片段也不例外。在本文中，我们描述了人胰淀素两个淀粉样生成片段的高效合成与纯化。一个片段对应全长激素的第17至37位氨基酸残基，另一个对应第24至37位氨基酸残基。这些片段此前已在体内被鉴定出来，并已证明在体外可形成淀粉样物质。用于阐明这些肽合成和纯化适宜条件的策略通常适用于其他难以合成的肽。这些肽采用具有Fmoc α-氨基保护的固相肽合成法制备。研究了改变溶剂、侧链保护基团和切割条件选择的影响。结果表明，以NMP作为主要溶剂，并用三氟乙酸、苯酚、乙二硫醇、硫代苯甲醚和水进行切割最为适宜。发现1,1,1,3,3,3-六氟-2-丙醇（HFIP）是溶解粗肽的最佳溶剂。研究了多种用于纯化这些肽的HPLC条件，以HCl作为离子配对剂的基于乙腈的溶剂系统提供了高效的纯化效果。
