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Development of Chiral Edman Degradation Method and Sequential Analysis of D/L-Amino Acid (s) in Peptide

肽中 D/L-氨基酸的手性 Edman 降解方法和序列分析的开发

基本信息

批准号：
07672321
负责人：
TOYO-OKA Toshimasa
金额：
$ 1.47万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672321/
关键词：
Chiral derivatization reagent D L-Amino Acid Chiral Edman degradation Peptide Diastereomer Fluorescence analysis Liquid chromatography D,L-アミノ酸配列分析

项目摘要

It was firmly believed that only L-amino acid participates in the functioning of a higher animal. However, a few peptides comprising D-amino acid (s) in the sequence (e.g.dermorphin, deltorphin, achatin I and omega-agatoxin-TK) have been discovered from frog skin, the ganglia of African giant snail and the venom of the funnel web spider. With recent advance of drug formulation technique, many drugs involving bioactive peptides such as insulin and growth hormon have been used for therapy. In some cases, however, wild type peptides can not be apply as the drug due to insufficient stability for drug formulation and undesirable side-effects. To solve these disadvantages, L-amino acid (s) in the sequence are converted to D-enantiomer (s). In these aspect, it is highly desirable to develop a method to discrimate the configulation of each amino acid in peptide sequence. We have developed optically active fluorescent Edman-type reagents, i.e., S(+) and R(-) enantiomers of 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-PyNCS) and 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-PyNCS). These reagents have been applied to the resolving of amino acid enantiomers. The reagent also reacted with N-terminal amino functional group in the peptide under mild conditions. The resulting thiocarbamoyl derivative was converted to fluorescent thiohydantin (excitation at around 460 nm and emission at around 550 nm) via thiazolinone in trifluoroacetic acid. The separation of the thiohydantoins by reversed-phase chromatography with acetonitrile/phosphate buffer (pH6.8) as the eluent was good enough for the identification of D/L-amino acid. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala2]-leucine-enkephalin and deltorphin II.

人们坚信，只有l -氨基酸参与高等动物的功能。然而，从青蛙皮肤、非洲巨蜗牛的神经节和漏斗网蜘蛛的毒液中已经发现了一些含有d -氨基酸序列的肽（如dermorphin、deltorphin、achatin I和omega-agatoxin-TK）。近年来，随着药物配方技术的进步，胰岛素、生长激素等含有生物活性肽的药物已被广泛用于治疗。然而，在某些情况下，由于药物配方稳定性不足和不良副作用，野生型肽不能作为药物应用。为了解决这些缺点，序列中的l -氨基酸被转化为d -对映体。在这些方面，非常需要开发一种方法来区分肽序列中每个氨基酸的构型。我们研制了具有光学活性的edman型荧光试剂，即4-（3-异硫氰酸托吡咯烷-1-基）-7-硝基-2,1,3-苯并恶二唑（NBD-PyNCS）和4-（3-异硫氰酸托吡咯烷-1-基）-7-（N，N-二甲氨基磺酰基）-2,1,3-苯并恶二唑（DBD-PyNCS）的S（+）和R（-）对映体。这些试剂已应用于氨基酸对映体的拆分。该试剂还能在温和条件下与肽中的n端氨基官能团发生反应。得到的硫氨基甲酰衍生物经噻唑啉酮在三氟乙酸中转化为荧光硫酰肼（激发波长约460 nm，发射波长约550 nm）。以乙腈/磷酸缓冲液（pH6.8）为洗脱液，反相色谱法分离硫脲类化合物，可用于D/ l -氨基酸的鉴定。用[D-Ala2]-leucine-enkephalin和deltorphin II证明了该方法对肽序列分析的适用性。