Structure of Subtilosin A, a Cyclic Antimicrobial Peptide from Bacillus subtilis with Unusual Sulfur to α-Carbon Cross-Links: Formation and Reduction....

The complete primary and three-dimensional solution structures of subtilosin A (1), a bacteriocin from Bacillus subtilis, were determined by multidimensional NMR studies on peptide produced using isotopically labeled [13C, 15N]medium derived from Anabaena sp. grown on sodium [13C]bicarbonate and [15N]nitrate. Additional samples of 1 were also generated by separate incorporations of [U-13C,15N]-L- phenylalanine and [U-13C,15N]-L-threonine using otherwise unlabeled media. The results demonstrate that in addition to having a cyclized peptide backbone (amide between N and C termini), three cross-links are formed between the sulfurs of Cys 13, Cys7, and Cys4 and the a-positions of Phe22, Thr28, and Phe31, respectively. The stereochemistry of all residues in 1 except for the three modified ones was confirmed to be L by complete desulfurization with nickel boride, acid hydrolysis to the constituent amino acids, and conversion of these to the corresponding pentafluoropropanamide isopropyl esters for chiral GC MS analysis. The stereochemistry at the modified residues was determined by subjecting each of the eight possible stereoisomers of 1 to eight rounds of ARIA structure calculations, starting with the same NMR peak files and assignments. The stereoisomer with the L stereochemistry at Phe22 (α-R) and D stereochemistry at Thr28 (α-S) and Phe31 (α-S) (LDD isomer) fit the NMR data, giving the lowest energy family of structures with the best rmsd. Thus, biochemical formation of the unusual thio links proceeds with net retention of configuration at Phe22, and inversion at Thr28 and Phe3 1. Model amino acid derivatives bearing a sulfide moiety at the α-carbon were synthesized by reaction of the corresponding α-alkoxy compounds with benzyl thiol and SnCl4. [ABSTRACT FROM AUTHOR]