Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Photoexcitation is a common strategy for initiating radical reactions in chemical synthesis. We found that photoexcitation of flavin-dependent “ene”-reductases changes their catalytic function, enabling these enzymes to promote an asymmetric radical cyclization. This reactivity enables the construction of five-, six-, seven-, and eight-membered lactams with stereochemical preference conferred by the enzyme active site. After formation of a prochiral radical, the enzyme guides the delivery of a hydrogen atom from flavin—a challenging feat for small-molecule chemical reagents. The initial electron transfer occurs through direct excitation of an electron donor-acceptor complex that forms between the substrate and the reduced flavin cofactor within the enzyme active site. Photoexcitation of promiscuous flavoenzymes has thus furnished a previously unknown biocatalytic reaction.

This Perspective highlights the advances of optical methods for asymmetric reaction discovery. Optical analysis allows for the determination of absolute configuration, enantiomeric excess and reaction yield that is amenable to high-throughput experimentation. Thus, the synthetic organic community is encouraged to incorporate the methods discussed to expedite the development of high-yielding, enantioselective transformations.

We present here a new design motif for strained, conjugated macrocycles that incorporates two different aromatics into the cycle with an -A-B-A-B- pattern. In this study, we demonstrate the concept by alternating electron donors and acceptors in a conjugated cycle. The donor is a bithiophene, and the acceptor is a perylene diimide derivative. The macrocycle formed has a persistent elliptiform cavity that is lined with the sulfur atoms of the thiophenes and the π-faces of the perylene diimide. Due to the linkage of the perylene diimide subunits, the macrocycles exist in both chiral and achiral forms. We separate the three stereoisomers using chiral high-performance liquid chromatography and study their interconversion. The mechanism for interconversion involves an "intramolecular somersault" in which one of the PDIs rotates around its transverse axis, thereby moving one of its diimide heads through the plane of the cavity. These unusual macrocycles are black in color with an absorption spectrum that spans the visible range. Density functional theory calculations reveal a photoinduced electron transfer from the bithiophene to the perylene diimide.

The association between an achiral copper(II) host (1) and chiral carboxylate guests was studied using exciton-coupled circular dichroism (ECCD). Enantiomeric complexes were created upon binding of the enantiomers of the carboxylate guests to the host, and the sign of the resultant CD signal allowed for determination of the configuration of the studied guest. The difference in magnitudes and shapes of the CD signals, in conjunction with linear discriminant analysis (LDA), allowed for the identity of the guest to be determined successfully. A model was created for the host:guest complexes which successfully predicts the sign of the observed CD signal. Further, Taft parameters were used in the model, leading to rationalization of the observed magnitudes of the CD signals. Finally, the enantiomeric excess (ee) of unknown samples of three chiral carboxylic acid guests was determined with an average absolute error of ± 3.0%.

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.

Cryptospirolepine is the most structurally complex alkaloid discovered and characterized thus far from any Cryptolepis specie. Characterization of several degradants of the original, sealed NMR sample a decade after the initial report called the validity of the originally proposed structure in question. We now report the development of improved, homodecoupled variants of the 1,1- and 1,n-ADEQUATE (HD-ADEQUATE) NMR experiments; utilization of these techniques was critical to successfully resolving long-standing structural questions associated with crytospirolepine.

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