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

人们坚信，只有L氨基酸才能参与高等动物的功能。然而，从蛙皮、非洲巨螺的神经节和漏斗网蜘蛛的毒液中发现了该序列中含有D-氨基酸(S)的几个肽(如德莫芬、Deltorphin、achatin I和omega-agatoxin-TK)。近年来，随着药物制剂技术的进步，胰岛素、生长激素等生物活性多肽药物已被用于治疗。然而，在某些情况下，由于药物制剂的稳定性不足和不良反应，野生型多肽不能作为药物使用。为了解决这些缺点，序列中的L-氨基酸(S)被转化为D-对映体(S)。在这些方面，开发一种方法来描述多肽序列中每个氨基酸的构型是非常必要的。我们开发了光学活性的Edman型荧光试剂，即4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2，1，3-benzoxadiazole的S(+)和R(-)对映体(NbD-PYNCS)和4-(3-异硫氰基吡咯烷基-1-基)-7-(N，N-二甲氨基磺酰基)-2，1，3-苯并恶二唑(DBD-PYNCS)。这些试剂已应用于氨基酸对映体的拆分。该试剂还能在温和的条件下与多肽中的N-末端氨基反应。在三氟乙酸四氮唑酮的作用下，硫代甲酰基衍生物被转化为荧光硫代海因(激发波长约为460 nm，发射波长约为550 nm)。以乙腈/磷酸盐缓冲液(pH 6.8)为淋洗液，用反相色谱分离硫代海因，可用于D/L氨基酸的鉴定。用[D-Ala2]-亮氨酸脑啡肽和Deltorphin II验证了该方法在多肽序列分析中的适用性。

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.