| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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