Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C—H Functionalization

Abstract

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

Chemoenzymatic Route to Oxyfunctionalized Cembranoids Facilitated by Substrate and Protein Engineering

“…Reported chemoenzymatic total syntheses of complex natural compounds relying on selectiveo xidations often involve P450 enzymes from biosynthetic pathways. [45] However, high substrate specificity and narrow substrates pectrumo fs uch P450s limits their application in the oxidation of synthetic substrates. This limitation can be overcome either by using engineered P450 enzymes [46] or via substrate engineering.…”

In Vitro Type I Modular Polyketide Synthase Catalysis for New Antibiotics

“…Recently, a chemoenzymatic PKS catalysis approach was developed by Professor David Sherman's group, by which the surrogate two domains required for biosynthesis of tylactone from juvenimicin BGC (JuvEIV and JuvEV) were identified and overexpressed in Escherichia coli cells, and used for final step condensation of two ketide units to produce tylactone from synthetic intermediate (Scheme ) …”

“…The method significantly reduced the number of steps required for the total chemical synthesis from 21 to 15 steps. Some of the newly synthesized derivatives exhibited enhanced antibacterial activity against Gram‐positive and Gram‐negative bacteria …”

P450 Monooxygenases Enable Rapid Late‐Stage Diversification of Natural Products via C−H Bond Activation

“…The Sherman group had already studied several biocatalytic syntheses of macrolactone glycosides in the past . Recently, they examined the interconnection of pharmaceutically valued macrolides within this compound family via P450 diversification, thus giving an example of the application of this enzyme class in actual drug discovery . Following up on its first biocatalytic total synthesis, antibiotic M‐4365 G 1 ( 20 ) was successively functionalised to a set of juvenimicin and rosamicin macrolides possessing the same core structure.…”

“… Late‐stage diversification of natural product M‐4365 G 1 ( 20 ) with three natural P450s to obtain the improved antibiotic forms ( 21 – 28 ) . The functional groups introduced bare the same colour to the respectively catalysing P450 enzyme.…”