Simultaneous detection and determination of the absolute conﬁguration of thiazole-containing amino acids in a peptide Kiyonaga Fujii, a Yukie Yahashi, a Tomoyo Nakano, a Susumu Imanishi, a Susana F. Baldia b and Ken-ichi Harada a, * a Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan b Department of Aquaculture, Southeast Asian Fisheries Development Center, Binangonan Freshwater Station, 1940 Binangonan Rizal, Philippines Received 10 May 2002; accepted 27 June 2002 Abstract—For the simultaneous detection and determination of the absolute conﬁguration of a thiazole-containing (Tzl-) amino acid in a peptide, we have developed a reliable method using the ‘advanced Marfey’s method’, which includes HPLC with a rational guideline, a sensitive derivatizing reagent, 1-ﬂuoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a racemization procedure using DL-FDLA for determination of the absolute conﬁguration of constituent amino acids in a peptide. Tzl-amino acids could be directly detected in the hydrolysate by this method, although they were racemized under ordinary hydrolysis conditions. In order to depress the racemization, the ﬂash hydrolysis was introduced. As a result, the ﬂash hydrolysis for 1 h was sufﬁcient to detect each constituent amino acid, and it was possible to identify the original peak. Consequently, the absolute conﬁguration of microcyclamide (1) possessing Tzl-amino acids was determined by the advanced Marfey’s method combined with ﬂash hydrolysis. Additionally, this method was successfully applied to the simultaneous detection and determination of the absolute conﬁguration of two other naturally occurring peptides, waiakeamide (2) and goadsporin (3). The established method with the ﬂash hydrolysis had an additional advantage in that labile amino acids, such as tryptophan and methionine sulfoxide, during acid hydrolysis can be detected in the intact form. q 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction A large number of naturally occurring peptides containing thiazole, oxazole and methyloxazole rings have been isolated from various origins, and almost all including antibiotics show characteristic biological activities such as cytotoxic, immunosuppressive, antifungal and enzyme inhibitory activities. Thiazole-containing (Tzl-) amino acids in such a peptide arise biosynthetically from the cyclization of cysteine and an adjacent amino acid, and oxazole- (Ozl-) and methyloxazole-containing (mOzl-) amino acids are derived from serine and threonine and their adjacent amino acids, respectively. 1,2 Recently, several cyclic peptides composed of three such cyclically modiﬁed amino acids have been isolated as a group of natural products from cyanobacteria and marine origins, and they have cytotoxic activities in particular. 3–11 In general, the absolute conﬁguration of such a modiﬁed amino acid in a peptide is determined on the basis of that of an amino acid adjacent to the heterocycle. Amino acids adjacent to Ozl- and mOzl-rings can be released from Ozl- and mOzl-amino acids in a peptide by acid hydrolysis. 3–11 However, no adjacent amino acid was obtained from Tzl- amino acid under the usual hydrolysis conditions, and it is difﬁcult to isolate the intact Tzl-amino acid due to its instability. Additionally, acid hydrolysis of a peptide containing Tzl-amino acids causes a serious racemization of liberated Tzl-amino acid. 3,12 In order to obtain amino acids adjacent to Tzl-rings in Tzl-amino acids, Ireland et al. proposed a practical method including ozonolysis followed by acid hydrolysis in 1983. 12 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 identiﬁcation of Tzl-amino acids including the absolute conﬁguration so far. We have carried out the isolation and the characterization of cyanobacterial peptides including toxic peptides for the elucidation of their biosynthesis. 13 – 18 During this study, we have developed the ‘advanced Marfey’s method’ for the rapid and reliable structural determination of isolated cyanobacterial peptides. The advanced Marfey’s method is a nonempirical method for the determination of the absolute conﬁguration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). 19,20 This method consists of Marfey’s method as a chromato- graphic technique for the separation of amino acids into their enantiomers, detection using mass spectrometry, and a 0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0040-4020(02)00748-2 Tetrahedron 58 (2002) 6873–6879 * Corresponding author. Tel.: þ81-52-832-1781ext.333; fax: þ81-52-834- 8780; e-mail: kiharada@ccmfs.meijo-u.ac.jp Keywords: microcyclamide; absolute conﬁguration; advanced Marfey’s method; thiazole-containing (Tzl-) amino acid.