Enzymatic preparation of neomycin C from ribostamycin

The high regio- and stereospecificities of enzymes and their ability to catalyze reactions in the absence of organic solvents make enzymatic synthesis of antibiotics an attractive way to prepare complex natural products. Enzymatic total syntheses of several complex natural products have been recently achieved, which illustrates the power of this synthetic methodology.^{1, 2} Combinations of biosynthetic enzymes and substrates, including unnatural types, can afford structurally diverse natural product-like compounds.³

At first, we prepared 6′′′-deamino-6′′′-hydroxyneomycin C from ribostamycin by incubation with NeoF and NeoD according to earlier methods with minor modifications.⁵ Overexpressed NeoF protein was partially purified by anion exchange chromatography with DEAE Sepharose Fast Flow (GE Healthcare, Chalfont St Giles, UK) and the obtained crude enzyme (17 ml, 22 mg as crude enzyme) was directly used for the glycosylation of ribostamycin (56 mg) with UDP-GlcNAc•2Na (86 mg) at 28 °C for 45 h. Without purification of the glycosylated product, the next deacetylation reaction was carried out by the addition of NeoD (0.3 mg, 0.25 ml) purified by TALON (Clontech, Mountain View, CA, USA) chromatography. After 24 h at 28 °C, the resulting enzymatic solution was heat-treated (100 °C, 2 min) and then applied to a Dowex AG1-X8 (Bio-Rad, Richmond, CA, USA, [OH^–] form, 12 ml) column. The unadsorbed fraction was neutralized with acetic acid and then applied to an Amberlite CG50 (Rohm and Haas Co, Philadelphia, PA, USA, [NH₄⁺] form, 8 ml) column. A stepwise gradient of aqueous NH₄OH (0.04 M and then 2 M) eluted 6′′′-deamino-6′′′-hydroxyneomycin C, which was further passed through Dowex AG1-X8 ([SO₄²⁻] form, 8 ml) to obtain 6′′′-deamino-6′′′-hydroxyneomycin C sulfate (45 mg, 2 steps 45% yield).

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