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, 133 (37), 14492-5

Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-Ribosomal Peptide Synthetase Module

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Chemoenzymatic Synthesis of Cryptophycin Anticancer Agents by an Ester Bond-Forming Non-Ribosomal Peptide Synthetase Module

Yousong Ding et al. J Am Chem Soc.

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.

Figures

Figure 1
Figure 1
CrpD-M2 biosynthetic scheme. (A) Enzymes used in this study, CrpD-M2 and Crp TE. Domains within the CrpD-M2 polypeptide are noted with squares, and the phosphopantetheinyl arm is denoted by the linked SH group. (B) Reactions catalyzed by CrpD-M2 and Crp TE mediated cyclization. (B) SNAC-ABC analogs utilized (3–5). (D) Cyclic cryptophycins (6–8), and linear seco-cryptophycins (9–11) generated by CrpD-M2 and Crp TE. formula image, formula image, formula image, and formula image.
Figure 2
Figure 2
Examination of CrpD-M2 A domain substrate specificity using the radio-PPi exchange assay. (A) Relative activity of the A-domain normalized to 2. (B) Extender units investigated in this assay.
Figure 3
Figure 3
CrpD-M2 T domain active-site bound with extender unit intermediates monitored by LC FTICR-MS. A zoomed mass spectrum averaged over the elution window is presented. The deconvoluted monoisotopic mass, and observed charge state are shown. Reactions of CrpD-M2 with ATP and: (A) formula image (2), (B) formula image (20), (C) formula image (1), (D) formula image, (E) formula image. Further data are provided such as LC IT-MS/MS25 (Table S4–5 and Figures S26–34).
Figure 4
Figure 4
FTICR-MS analysis of cryptophycin products from the reaction of unit C monomethyl chain elongation intermediate (3-amino-2(R)-methylpropionyl, 3) with l/d-2HIC, ATP, CrpD-M2, and Crp TE. EICs are presented at +/− 15 ppm as time versus absolute signal. Inset mass spectra are time averaged over the 1 minute elution window corresponding to the asterisk in the extracted ion chromatogram. Inset mass spectra are presented as m/z versus absolute signal. Monoisotopic mass MH+ and the experimental mass error in ppm are reported. Reactions with l-2HIC and d-2HIC monitor the formation of cyclic (A and C) and linear products (B and D). No enzyme control reactions for cyclic (E) and linear product formation (F) are provided.

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