Shenoy N R, Shively J E, Bailey J M
Beckman Research Institute of the City of Hope, Division of Immunology, Duarte, California 91010.
J Protein Chem. 1993 Apr;12(2):195-205. doi: 10.1007/BF01026041.
In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Bailey et al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50 degrees C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.
在之前旨在从羧基末端对肽和蛋白质进行测序的研究中，我们使用乙酸酐和三甲基硅基异硫氰酸酯（TMS - ITC）将C末端衍生化为硫代乙内酰脲，随后将其水解以形成能够进一步降解的缩短肽和可通过反相高效液相色谱鉴定的氨基酸硫代乙内酰脲。这种化学方法目前的局限性包括无法衍生化脯氨酸以及天冬酰胺和天冬氨酸残基的产率较低（贝利等人，1992年）。为了解决其中一些问题，我们研究了使用除乙酸酐之外的试剂来活化C末端羧酸。这些试剂包括对甲苯磺酸2 - 氟 - 1 - 甲基吡啶鎓、碘化2 - 氯 - 1 - 甲基吡啶鎓和乙酰氯。将TMS - ITC添加到由2 - 卤代吡啶鎓盐活化的肽中形成了预期的肽基硫代乙内酰脲，但此外还形成了在C末端化学修饰的肽，该肽被阻断进行C末端序列分析。当使用乙酸酐或乙酰氯进行活化时，未获得这种衍生物。发现形成这种衍生物除了卤代吡啶鎓盐外还需要异硫氰酸酯试剂的存在。硫氰酸钠、TMS - ITC和一种用于硫代乙内酰脲合成的新试剂三丁基锡异硫氰酸酯（TBSn - ITC）都被发现能够形成这种类似物。对C末端修饰氨基酸的结构解析表明它是一种2 - 亚氨基吡啶鎓类似物。通过使用2 - 氯吡啶鎓试剂而非2 - 氟试剂，并在50℃或更低温度下进行反应，可以使这种C末端被阻断的肽的形成最小化。与使用乙酸酐活化C末端羧酸相比，2 - 卤代吡啶鎓试剂具有潜在优势。这些优势包括：反应条件更温和、反应时间更快以及能够对C末端天冬氨酸进行测序。发现TBSn - ITC试剂在形成肽基硫代乙内酰脲方面与TMS - ITC相当。
