Synthesis of ramariolide natural products and discovery of their targets in mycobacteria

Abstract

Ramariolides A-D are natural products with antibacterial activity. To exploit their cellular mechanism, we here devise the first total synthesis and prepare a photoprobe for target identification. Antibacterial testing against several pathogenic strains including Mycobacterium tuberculosis revealed the highest potency for ramariolide A. Chemical proteomics unraveled binding to essential proteins for amino acid anabolism.

Fig. 1. Molecular structures of ramariolides A–D.

Scheme 1. Consecutive synthesis of ramariolides A–D. (a) (COCl)₂, DMF, CH₂Cl₂, rt, 3 h; (b) TMS-CHN₂, ACN, 0 °C, 16 h; (c) HBr, Et₂O, 60 °C, 3 h; (d) PPh₃, CHCl₃, rt, 16 h; (e) KOH, MeOH : H₂O = 4 : 1, rt, 3 h; (f) maleic anhydride, PhMe, 110 °C; (g) NaBH₃CN, THF, rt, 1 h then add HCl for 1 h; (h) Ti(Oi-Pr)₄, (+)-DIPT, t-BuO₂H, –32 °C, 16 h; (i) (COCl)₂, DMF, CH₂Cl₂, rt, 2 h, then add 1a and pyridine, 2 h; (j) m-CPBA, CH₂Cl₂, 3 h; (k) DMP, CH₂Cl₂, rt, 2 h; (l) Zn(BH₄)₂, THF, 0 °C, 2 h, then add HCl; (m) (±)-CSA, CH₂Cl₂, rt, 3 h. DMF = N,N-dimethylformamide, TMS = trimethylsilyl-, THF = tetrahydrofuran, DIPT = diisopropyl tartrate, m-CPBA = meta-chloroperoxybenzoic acid, DMP = Dess–Martin periodinane, CSA = camphorsulfonic acid.

Fig. 2. (A) Scatter plot of gel-free ABPP dimethyl labeling experiments with 30 μM 8 vs. DMSO in M. smegmatis mc²155 soluble fraction after UV-irradiation (criteria: enrichment ≥2 and p-value ≤0.05). (B) Fluorescence scan of gel-based ABPP experiments with recombinant expressed mycobacterial Ask labeled with 8 in E. coli. “+” indicates induction of protein expression by addition of IPTG; “–“ represents absence of inducer. (C) Scatter plot of gel-free ABPP dimethyl labeling experiment with 30 μM 8 vs. DMSO in M. smegmatis mc²155 soluble fraction without UV-irradiation. (criteria: enrichment ≥3 and p-value ≤0.05) (D) Scatter plot of M. smegmatis mc²155 soluble fraction preincubated with 100 μM 1a followed by treatment with 30 μM 8 (criteria: enrichment ≥3 and p-value ≤0.05). Red dots indicate proteins essential for bacterial growth. Green dots depict non-essential protein hits. DMSO = dimethylsulfoxide, IPTG = isopropyl-β-d-thiogalactopyranosid.