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.

An oxidative cyclization reaction transforms nonemissive azoanilines into highly fluorescent benzotriazoles. We have found that introduction of multiple electron-donating amino groups onto a simple o-(phenylazo)aniline platform dramatically accelerates its conversion to the emissive polycyclic product. Notably, this chemistry can be effected by μM-level concentrations of copper(II) ion in water (pH = 6-8) at room temperature to elicit >80-fold enhancement in the green emission at λ(em) = 530 nm. Comparative kinetic and electrochemical studies on a series of structural analogues have established that the accelerated reaction rates correlate directly with a systematic cathodic shift in the oxidation onset potential of the azo precursors. In addition, single-crystal X-ray crystallographic analysis on the most reactive derivative revealed the presence of a five-membered ring intramolecular hydrogen-bonding network. An enhanced contribution of the quinoid-type resonance in such conformation apparently facilitates the mechanistically required proton transfer step, which, in conjunction with electron transfer at lower oxidation potential, contributes to a rapid cyclization reaction triggered by copper(II) ion in water.

Chromatographic separation and analysis of complex mixtures of closely related species is one of the most challenging tasks in modern pharmaceutical analysis. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. However, the relatively slow speed of chiral separations has limited the use of chiral stationary phases (CSPs) as the second dimension in 2D-LC, especially in the comprehensive mode. Realizing that the recent revolution in the field of ultrafast enantioselective chromatography could now provide significantly faster separations, we herein report an investigation into the use of ultrafast chiral chromatography as a second dimension for 2D chromatographic separations. In this study, excellent selectivity, peak shape, and repeatability were achieved by combining achiral and chiral narrow-bore columns (2.1 mm × 100 mm and 2.1 mm × 150 mm, sub-2 and 3 μm) in the first dimension with 4.6 mm × 30 mm and 4.6 mm × 50 mm columns packed with highly efficient chiral selectors (sub-2 μm fully porous and 2.7 μm fused-core particles) in the second dimension, together with the use of 0.1% phosphoric acid/acetonitrile eluents in both dimensions. Multiple achiral × chiral and chiral × chiral 2D-LC examples (single and multiple heart-cutting, high-resolution sampling, and comprehensive) using ultrafast chiral chromatography in the second dimension are successfully applied to the separation and analysis of complex mixtures of closely related pharmaceuticals and synthetic intermediates, including chiral and achiral drugs and metabolites, constitutional isomers, stereoisomers, and organohalogenated species.

Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation.

A molecular probe was prepared that selectively responds to cyanide in aqueous solutions by fluorescence enhancement. Using the peptide beta-turn as a structural template, we designed a series of diphenylacetylene derivatives in which the pi-conjugated backbone was functionalized with an aldehyde group to render the molecule nonfluorescent. The N-H...O hydrogen bond across the 2,2'-functionalized diphenylacetylene turn motif activates the carbonyl group toward nucleophilic attack, and chemical transformation of this internal quencher site by reaction with CN(-) elicits a rapid (k = 72 M(-1) s(-1)) enhancement in the emission at lambda(max) = 375 nm. Tethering of an ammonium group to the hydrogen bond donor fragment significantly increased both the response kinetics and the intensity of the fluorescence signal. In addition to providing electrostatic attraction toward the CN(-) ion, this positively charged R-NH(3)(+) fragment can engage in a secondary hydrogen bond to facilitate the formation of the cyanohydrin adduct responsible for the signaling event. The structurally optimized molecular probe 3 responds exclusively to microM-level cyanide in neutral aqueous solutions, with no interference from other common anions including F(-) and AcO(-).