Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products

Abstract

Selective access to a targeted isomer is often critical in the synthesis of biologically active molecules. Whereas small-molecule reagents and catalysts often act with anticipated site- and stereoselectivity, this predictability does not extend to enzymes. Further, the lack of access to catalysts that provide complementary selectivity creates a challenge in the application of biocatalysis in synthesis. Here, we report an approach for accessing biocatalysts with complementary selectivity that is orthogonal to protein engineering. Through the use of a sequence similarity network (SSN), a number of sequences were selected, and the corresponding biocatalysts were evaluated for reactivity and selectivity. With a number of biocatalysts identified that operate with complementary site- and stereoselectivity, these catalysts were employed in the stereodivergent, chemoenzymatic synthesis of azaphilone natural products. Specifically, the first syntheses of trichoflectin, deflectin-1a, and lunatoic acid A were achieved. In addition, chemoenzymatic syntheses of these azaphilones supplied enantioenriched material for reassignment of the absolute configuration of trichoflectin and deflectin-1a based on optical rotation, CD spectra, and X-ray crystallography.

Figure 1.

A) Approaches to enabling biocatalytic stereodiversity. B) Biologically active azaphilone natural products feature both the R- and S-configuration at the C7 stereocenter.^,, Biocatalytic oxidative dearomatization gives access to either enantiomer via AzaH or AfoD mediated oxidation.

Figure 2.

A) Sequence similarity network (SSN) of FAD-dependent monooxygenases (Pfam01494) using a sequence alignment score of 110. B) Results of expression, activity with model substrate S9 or S14**, site- and stereoselectivity of enzymes chosen from the SSN in panel A. *FDMO4 demonstrated <10% conversion by UPLC with substrate S9. C) Selected clusters from a more stringent SSN generated with an alignment score of 150 and corresponding analysis of the multisequence alignment of each cluster.

Figure 3.

A) Retrosynthetic analysis of the natural product trichoflectin (17). B) Total synthesis of (S)- and (R)-trichoflectin. C) Calculated and measured CD data for (S)- and (R)-trichoflectin. D) Total synthesis of (S)-deflectin-1a (24). E) Calculated and measured CD data for (S)- and (R)-deflectin-1a. *NADPH recycling system: G6P (2 equiv), NADP(+) (0.4 equiv), G6PDH (1 U/mL).

Figure 4.

A) Retrosynthetic analysis of azaphilone natural product lunatoic acid A (25). B) Total synthesis of lunatoic acid A (25). C) Calculated and measured CD data for lunatoic acid A methyl ester* (28). *NADPH recycling system: G6P (2 equiv), NADP(+) (0.4 equiv), G6PDH (1 U/mL)