Eurystatin A

The structures of eurystatins A and B, new prolyl endopeptidase inhibitors, have been elucidated by chemical degradation and spectral studies. They have in common a unique 13-membered cyclic peptide core composed of L-leucine, L-ornithine and (S)-3-amino-2-oxobutyric acid, and differ from each other in the alpha,beta-unsaturated fatty acid attached to the alpha-amino moiety of the ornithine.

Accurate and precise component analysis of eurystatin analogs in fermentation broth was devised by HPLC methods with and without 2,4-dinitrophenylhydrazonation. Detailed optimization of fermentation conditions and strain improvement by HPLC analysis significantly increased the eurystatin productivity of Streptomyces eurythermus. Chemically defined fermentation media which produced eurystatins A and B at fermentation yields comparable to complex media were elaborated for radio-isotope fermentation studies and controlled biosynthesis. Radio-isotope incorporation study using 14C-labeled amino acids in chemically defined medium demonstrated that L-leucine and L-ornithine were the direct precursors for the L-leucine and L-ornithine moieties of eurystatins A and B, respectively. Based on this finding, L-valine and L-isoleucine were supplemented to the growing culture of S. eurythermus in chemically defined medium, which resulted in the controlled biosynthesis of new eurystatin analogs named eurystatins C, D, E and F.

Eurystatins A and B, were isolated from the cultured broth of Streptomyces eurythermus R353-21. They showed specific and potent inhibitory activity against prolyl endopeptidase and did not show antimicrobial activity. No lethal toxicity was observed for the two compounds after ip administration in mice at 200 mg/kg.