PMC

Activities of (−)-fragin (1) and (+)-fragin (11). a Structure of (−)-fragin (1). b Disc diffusion assays of synthetic (−)- and (+)-fragin to determine the antifungal activity. Representative pictures of an antifungal spray assay against the fungus F. solani are shown. Scale bar indicates 10 mm

Proposed model of fragin and valdiazen biosynthesis. The amino group of l-valine, which is bound to HamD, is transformed into a diazeniumdiolate group by the tailoring enzymes HamA, HamC, and HamE. The resulting intermediate molecule is then released in a two-step process. The first 2-electron reduction step catalyzed by the HamC R-domain results in an aldehyde intermediate, which can undergo a second 2-electron reduction to yield valdiazen. Alternatively, the aldehyde intermediate can be transaminated by HamG, resulting in an amino derivative, which can be acylated by HamF to yield fragin. A: adenylation domain, T thiolation domain, R reductase domain; e⁻ electron

Enantioselective total synthesis of (−)-fragin (1) and (+)-fragin (11), and X-ray crystal structure of isolated (−)-(R)-fragin (1). Color code: blue = nitrogen; gray = carbon; red = oxygen; white = hydrogen. a Isobutyl chloroformiate, NaBH₄, DME, 1.5 h, –15 °C; b PPh₃, DEAD, DPPA, THF, 0 °C to RT, 10 h; c Pd/C Et₃SiH, MeOH/CHCl₃, RT, 14 h; d octanoyl chloride, DIPEA, DMAP, 0 °C to RT, 35 h; e AlCl₃, toluene, RT, 4 h; f dibenzoylperoxide, K₂HPO₄, THF, RT, 21.5 h; g N₂H₄ x H₂O, EtOH, RT, 2.5 h; h isopentyl nitrite, NH_3(g), EtOH, RT, 0.5 h

A subset of the ham genes synthesize the signal molecule valdiazen. a Cross-streaks of all seven ham gene mutants against the reporter strain (H111 ΔhamD/PhamA-lacZ) show that a subset of ham genes produces a diffusible signal molecule, which regulates ham promoter activity. The genes hamB, hamF, and hamG are dispensable for the production of the signal molecule. Scale bar indicates 5 mm. b Structure of valdiazen (2). c The promoter activity of the hamABCDE operon (PhamA-lacZ) was comparable in the wild-type strain (H111 wt) and the hamF mutant strain (H111 ΔhamF). The hamD mutant strain (H111 ΔhamD) showed only very low promoter activity, which could be activitated by the addition of synthetic valdiazen. Results are presented as mean and error bars represent SD. n = 6 (H111 ΔhamF, H111 ΔhamD 10 μM and 50 μM valdiazen), n = 8 (H111 wt, H111 ΔhamD 100 μM valdiazen), n = 9 (H111 ΔhamD 10 μM and 50 μM fragin)