title: Chemoenzymatic synthesis of cryptophycin anticancer agents by an ester bond-forming non-ribosomal peptide synthetase module.
abstract: Cryptophycins (Crp) are a group of cyanobacterial depsipeptides with activity against drug-resistant tumors. Although they have been shown to be promising, further efforts are required to return these highly potent compounds to the clinic through a new generation of analogues with improved medicinal properties. Herein, we report a chemosynthetic route relying on the multifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique non-ribosomal peptide synthetase (NRPS) module comprised of condensation-adenylation-ketoreduction-thiolation (C-A-KR-T) domains. We interrogated A-domain 2-keto and 2-hydroxy acid activation and loading, and KR domain activity in the presence of NADPH and NADH. The resulting 2-hydroxy acid was elongated with three synthetic Crp chain elongation intermediate analogues through ester bond formation catalyzed by CrpD-M2 C domain. Finally, the enzyme-bound seco-Crp products were macrolactonized by the Crp thioesterase. Analysis of these sequential steps was enabled through LC-FTICR-MS of enzyme-bound intermediates and products. This novel chemoenzymatic synthesis of Crp involves four sequential catalytic steps leading to the incorporation of a 2-hydroxy acid moiety in the final chain elongation intermediate. The presented work constitutes the first example where a NRPS-embedded KR domain is employed for assembly of a fully elaborated natural product, and serves as a proof-of-principle for chemoenzymatic synthesis of new Crp analogues.
authors.affiliation: Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Yousong
authors.initials: Y
authors.lastname: Ding
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rath
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Bolduc
authors.forename: Kristina
authors.initials: K
authors.lastname: Håkansson
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D047630:Depsipeptides / Q000138:chemical synthesis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D047630:Depsipeptides; C088419:cryptophycin; D010453:Peptide Synthases