Chemical synthesis of lantibiotics and their analogues for structure elucidation and structure-activity relationship (SAR) studies

Lantibiotic peptides, such as lacticin 3147 A1 (11) and A2 (12) represent a promising class of antimicrobial peptides for application in food preservation, veterinary medicine and human therapeutics. The chemical syntheses of two analogues of lacticin 3147 A2 and the attempted total synthesis of lactocin S are first described. In an effort to overcome the oxidative instability of lantibiotics, a carbocyclic-larger ring analogue of lacticin 3147 A2 (43) in which carbon atoms replace the oxidatively unstable sulfur atoms in the lanthionine rings has been successfully synthesized by a combination of solid-phase and solution phase peptide synthesis. During the course of the synthesis of the carbocyclic analogue (43), a highly practical method for the large scale synthesis of the post-translationally modified residues (1→5) of lacticin A2 (45) was also developed. Unfortunately, the carbocyclic analogue did not display any significant antimicrobial activity when tested against the indicator organism Lactococcus lactis HP. In the second project, to study the effect of replacement of the beta-methyllanthionine rings with lanthionine rings, a lanthionine analogue of lacticin A2 (44) has been synthesized on-resin. The key orthogonally protected lanthionine building block (167) was synthesized in solution phase and this fragment along with the previously synthesized residues (1→5) of lacticin A2 were used to assemble the lanthionine analogue of lacticin A2 (44) on solid-support. A preliminary biological evaluation against Lactococcus lactis HP indicates that the lanthionine analogue (44) displays a potent synergistic antimicrobial activity, when tested in combination with lacticin A1. A serial dilution assay showed that the lanthionine analogue is about 100 times less effective than natural lacticin A2 in exhibiting its synergistic activity. A lead compound, residues (6→29) of lanthionine analogue of lacticin A2 with a Fmoc group (173), for potentially simplifying lacticin A2 structure has also been identified. In the third project, attempts towards the total synthesis of lactocin S (48) are described. The two lanthionine rings A and B of lactocin S have been successfully synthesized on solid-support using the preformed orthogonally protected lanthionine building block (167). However, attempts to introduce residues (3→22) of lactocin S, from the bicyclic lanthionine rings, on solid-support did not result in any of the desired product.;LtnJ has been identified as the enzyme responsible for the reduction of dehydroalanines to D-alanines in lacticin 3147 A1 and A2. In order to prepare a substrate for the in vitro reconstitution of LtnJ activity, a 38 amino acid precursor peptide containing a single cysteine (206 ) has been designed and successfully synthesized by solid-phase peptide synthesis. The transfer of the cysteine residue in the precursor peptide to a dehydroalanine by converting the thiol group of the cysteine to a thiocyanate followed by elimination under basic conditions provides the substrate peptide (49) along with secondary cleavage products.;In an effort to make lantibiotics active against Gram-negative bacteria, the hypothesized recognition sequence, residues (75→88) from Gram-negative bacteria active colicin V (50), as well as its analogues with an azide (222) and an alkyne linker (225) have been successfully synthesized on solid support. Efforts towards conjugation of the synthesized peptide to lantibiotics via cycloaddition chemistry are underway.