Cyclobutane carboxamide inhibitors of fungal melanin: biosynthesis and their evaluation as fungicides.

A new fungicide lead has been identified by enzyme screening of a focused combinatorial library. The lead compound 4, a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic activity. The X-ray crystal structure of the enzyme-inhibitor complex and an appreciation for the relationship between physical properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen-methyl group combination was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF3 with methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while significantly increasing systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for outdoor testing in flooded plots which simulate rice paddies.

Synthesis of optically pure chrysobactin and immunoassay development.

Chrysobactin (alpha-N-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of alpha-N-(2,3-dibenzyloxybenzoyl)-epsilon-N-Cbz-D-lysine with L-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10(-8) to 10(-10) mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.

Synthesis of fragments of the peptide component of pseudobactin.

Pseudobactin is a structurally complex and physiologically important siderophore (microbial iron chelator] from Pseudomonas putida-fluorescens. Various fragments of the unusual peptide component of pseudobactin listed below were prepared by solution-phase peptide synthesis. L-Lys.D-threo-beta-OH Asp.L-Ala.D-allo-Thr.L-Ala L-Lys.D-threo-beta OH Asp.L-Ala.D-allo-Thr D-threo-beta-OH Asp.L-Ala.D-allo-Thr.L-Ala.D-N-OH-cycloOrn D-threo-beta-OH-Asp.L-Ala.D-allo-Thr.L-Ala L-Ala.D-allo-Thr.L-Ala.D-N-OH-cycloOrn A class of related peptides named pseudomycins have shown promising antifungal activity. To examine if these peptide fragments above would elicit similar activity, the fragments were tested and found to have no antifungal activity in limited bioassays.