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 themultifunctional enzyme CrpD-M2 that incorporates a 2-hydroxy acid moiety (unit D) into Crp analogues. CrpD-M2 is a unique nonribosomal 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
