Generation of reduced macrolide analogs by regio-specific biotransformation

In this study, an attempt was made to expand the applicability of the unique bio-hydrogenation activity of S. venezuelae toward other unsaturated macrolide antibiotics (natural oligomycin A and semi-synthetic tilmicosin) that possess the above mentioned identical catalytic scaffold, and the structure and antibiotic potential of two previously uncharacterized macrolides, 2,3-dihydro-oligomycin A (1) and 10,11-dihydro-tilmicosin (2), isolated from a S. venezuelae culture supplemented with oligomycin A and tilmicosin, respectively, were evaluated.

A recombinant strain of S. venezuelae YJ028,⁶ which had both the pikromycin polyketide synthase-encoding gene and desosamine biosynthetic genes deleted, was provided with oligomycin A or tilmicosin. The organism was cultivated in SCM media (1.5% soluble starch, 2.0% soytone, 0.01% CaCl₂, 0.15% yeast extract, and 1.0% MOPS) in baffled Erlenmeyer flasks. After 2 days growth at 30 °C, the cultures were supplemented with either macrolides oligomycin A (Sigma, St Louis, MO, USA) and tilmicosin (Sigma) at a final concentration of 5 μg ml⁻¹, and then incubated for additional 3 days. The whole cultures were extracted and partitioned using EtOAc, and then the organic extracts were concentrated under vacuum. The dried residues were dissolved in 200 μl of MeOH and a portion of the solvent was subjected to HPLC-ESI-MS (Waters, Milford, MA, USA) analysis.⁶ Consequently, supplementation of each non-native macrolide led to ∼67 and 30% conversion yields for the corresponding reduced macrolides 1 and 2, respectively (see Supplementary Figure S1). To isolate and structurally characterize both converted macrolides 1 and 2, crude extracts containing 1 were prepared from 150 ml (three batch cultivations) of the fermentation broth from YJ028 strain fed with oligomycin A (total 0.75 mg), whereas crude extracts containing 2 were from 250 ml (five batches) culture broth from the same strain supplemented with tilmicosin (1.25 mg). These new macolides in the crude extracts were purified by chromatographic isolation using a preparative reversed-phase HPLC on a Watchers 120 ODS-BP (250 × 10.0 mm, 5.0 μm, Daiso, Osaka, Japan)⁶ and subsequent ESI-MS analyses of each fraction (5 ml per fraction) collected. Compound 1 was obtained as a white powder (0.41 mg), which analyzed for the molecular formula C₄₅H₇₆O₁₁ by the HR-ESI-MS ([M+H]⁺ m/z 793.5409, calcd. 793.5421) using LCT-premier XE mass spectrometer (Waters; see Supplementary Figure S2). The MS/MS spectrum of 1 at m/z 793, which represents its protonated molecular ion, showed the main fragment ion at m/z 447, that is identical to that of oligomycin A,⁷ suggesting that the structural modification is likely to occur in the polyketide chain spanning from C1 to C12 position (see Supplementary Figure S3). On the other hand, HR-ESI-MS of 2 (light yellow powder; 0.30 mg) gave an m/z of 871.5835 as [M+H]⁺, which corresponds to the calculated molecular formula for C₄₆H₈₂N₂O₁₃ (871.5850; see Supplementary Figure S2). The MS/MS spectrum of tilmicosin at m/z 869 indicated two fragment ions at m/z 174 and 696, which arise from the cleavage of a mycaminose moiety attached at C5 position, whereas MS/MS spectrum of 2 showed a similar fragment at m/z 174 as in case of tilmicosin, but the characteristic product ion at m/z 698 that is different by 2 Da from a fragment ion at m/z 696 typical to tilmocosin,⁸ implying the disappearance of a double bond in the polyketide scaffold (see Supplementary Figure S3). Their chemical structures were further elucidated by ¹H- and ¹³C-NMR spectroscopic analyses. The NMR samples were prepared by dissolving each macrolide in 200 μl of CDCl₃ in a 5 mm Shigemi advanced NMR microtube (Sigma) matched to the solvent. Their ¹H- and ¹³C-NMR spectra were acquired using a Varian INOVA 500 NMR spectrometer (Varian, Inc., Palo Alto, CA, USA) at 298 K. The chemical shifts of each converted macrolide were assigned by a comparison with those of the parent macrolides oligomycin A and tilmicosin. Compounds 1 and 2 were identified as 2,3-dihydro-oligomycin A and 10,11-dihydro-tilmicosin, respectively (see Supplementary Tables S1 and S2).

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