66(P-23) 改良Marfey法を用いたペプチド中のチアゾールアミノ酸の絶対配置決定(ポスター発表の部)

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A large number of peptides containing the modified amino acids such as thiazole (Tzl) and oxazole (Ozl) rings have been isolated from cyanobacteria and marine origins. In general, the absolute configuration of such a modified amino acid in a peptide is determined on the basis of that of the corresponding intact amino acid derived from the hydrolysate after chemical treatments. In order to derive intact amino acids from Tzl-amino acids in a peptide, Ireland et al. proposed a method composed of acid hydrolysis combined with ozonolysis in 1983. Since then, although this method has been applied to many peptides containing Tzl-amino acids, no method has been established for the direct detection and the identification of Tzl-amino acids including the absolute configuration. We have established a nonempirical method using LC/MS, the advanced Marfey's method, which includes HPLC with a rational guideline, a sensitive derivatizing reagent, FDLA (1-fluoro-2,4-dinitrophenyl-5-leuciamide), and a racemization procedure using DL-FDLA, for determination of the absolute configuration of constituent amino acids and amines in a peptide. Therefore, we considered that Tzl-amino acids in the hydrolysate can be directly detected and determine the absolute configuration by the "advanced Marfey's method" without treatment of the ozonolysis. For the determination of the absolute configuration of Tzl-amino acids in a peptide, we applied the advanced Marfey's method to the isolated microcyclamide (1) containing two Tzl-amino acids. Tzl-amino acids could be directly detected together with a constituent amino acid in the hydrolysate by this method, although they were racemized under ordinary hydrolysis conditions as expected. According to the proposed separation mechanism of Marfey's method, the elution order of Tzl-amino acids can be basically determine in the same way as amino acids. In order to identify each original peak of Tzl-amino acids, the flash hydrolysis (6M HCl, 110℃, 1 hr) was introduced for the control of the racemization during the acid hydrolysis. Consequently, the absolute configuration of 1 containing Tzl-amino acids was clearly determined. Additionally, this method with the flash hydrolysis was successfully applied to the determination of the absolute configuration of constituent amino acids in two naturally occurring peptides, waiakeamide (2) and gordsporin, possessing Tzl-amino acids. The methodology using LC/MS combined with flash hydrolysis is being further extended for the structural determination of various naturally occurring peptides possessing the modified amino acids and D-amino acids.
天然有機化合物討論会の論文
2001-09-01

66(P-23) 改良Marfey法を用いたペプチド中のチアゾールアミノ酸の絶対配置決定(ポスター発表の部)

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