Biocatalytic cascade process of islatravir: Analytical and regulatory control strategy of minor enantiomer.

Commercial process of islatravir (MK-8591, EFdA) utilizes biocatalytic cascade reactions to construct the ribose moiety of the molecule which bears three chiral centers. However, this biocatalytic process also brought analytical challenges where all stereoisomers and process related compounds are controlled in one isolated intermediate, the final drug substance. A chiral LC method was developed to resolve all those compounds from islatravir and its minor enantiomer by thorough column screening and careful optimization. Detail of designing key method validation components such as method linearity, precision and robustness is discussed, and their results were presented. The method was successfully validated to fulfill various expectation from each individual health authority including FDA, EMA, PMDA, and ANVISA.

Development and validation of ion-pairing HPLC-CAD chromatography for measurement of Islatravir's phosphorylated intermediates.

Biocatalytic processes have become more prevalent in the pharmaceutical industry, leading to analytical challenges not faced when characterizing more traditional synthetic routes. A novel one-pot biocatalytic process has been established for Islatravir, an HIV reverse transcriptase translocation inhibitor for the treatment and prevention of HIV-1. As a one-pot reaction, the Islatravir chemistry contains multiple intermediates that are not isolated. Additionally, these unisolated intermediates have no chromophores, making traditional LC-UV techniques ineffective for characterization. A hydrophilic interaction chromatography (HILIC) method with a charged aerosol detector (CAD) was initially developed, however numerous inorganic species present in the one-pot reaction were retained; this led to co-elution of compounds and poor peak shapes. An innovative ion-pairing LC method was developed in order to resolve inorganic species, intermediates, and the API, for use during in-process control of the Islatravir biocatalytic reaction. Aided by a volatile ion-pairing reagent compatible with the CAD, this method successfully retains and resolves the highly polar intermediates of interest and Islatravir API. This novel method was successfully validated and has allowed the Islatravir biocatalytic process to be fully characterized from the early intermediates through the final API within the one-pot reaction without the need for isolations. This novel ion-pairing HPLC-CAD technique lays the groundwork for method development on current and future biocatalytic-produced drug substances.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

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.

Ultrafast Chiral Chromatography as the Second Dimension in Two-Dimensional Liquid Chromatography Experiments.

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.

Online sensing of palladium in flowing streams.

Rapid palladium (Pd) catalyzed deallylation of an uncoloured reagent within a flowing stream affords a dose dependent colour formation that can be used for convenient online analysis of trace levels of Pd contamination using a modified HPLC instrument. An application to the online sensing of Pd breakthrough from a flow through Pd adsorption cartridge is described. An alternative configuration of the instrumentation allows the rapid (<1 min) and accurate measurement of Pd levels within samples injected via a conventional HPLC autosampler.

Conformationally Distorted π-Conjugation for Reaction-Based Detection of Nickel: Fluorescence Turn-on by Twist-and-Fragment.

A conformationally twisted N-arylbenzotriazole was designed as a fluorescence turn-on molecular probe. Under ambient conditions, metal-catalyzed deallylation reactions restore an intense blue emission. This reaction scheme is applicable exclusively to Group 10 transition metal ions and optimized, in particular, for nickel to allow sub-micromolar detection with no competition from other first-row transition-metal ions.

Predictors of Successful Smoking Cessation after Inpatient Intervention for Stroke Patients.

BACKGROUND: Smoking is a well-known risk factor of cancer, chronic disease, and cerebrovascular disease. Hospital admission is a good time to quit smoking but patients have little opportunity to take part in an intensive smoking cessation intervention. The purpose of this study was to identify the factors of successful smoking cessation among stroke patients who undergo an intensive cessation intervention during the hospitalization period. METHODS: Thirty-nine male smokers who were admitted with stroke were enrolled in the study. They participated in a smoking cessation intervention during hospitalization. Smoking status was followed up by telephone 3 months later. Nicotine dependence, sociodemographic factors, and other clinical characteristics were assessed. RESULTS: After 3 months post-intervention, the number of patients who stopped smoking was 27 (69.2%). In addition, there was no significant difference in nicotine dependence, sociodemographic factors, and clinical characteristics. Only the stages of readiness for smoking cessation were a significant predictor (odds ratio, 18.86; 95% confidence interval, 1.59-223.22). CONCLUSION: This study shows that a patient's willingness to quit is the most significant predictor of stopping smoking after Inpatient cessation Intervention for stroke Patients.

A competitive and reversible deactivation approach to catalysis-based quantitative assays.

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.

High-fidelity self-assembly of crystalline and parallel-oriented organic thin films by π-π stacking from a metal surface.

Organic semiconductor applications will significantly benefit from atomically precise, cofacial stacking of extended π-conjugated molecular systems for efficient charge transport. Surface-assisted self-assembly of poly(hetero)cyclic molecules via donor-acceptor type π-π stacking is a promising strategy to organize functional, many-layered architectures. We have employed tris(N-phenyltriazole) as a model system to achieve molecular-level structural ordering through more than 20 molecular layers from its own metal-templated monolayer. Effective charge transport through such layers enabled molecular-resolution imaging by scanning tunneling microscopy. The structure and chemical composition of the films, grown on Ag(111) or Au(100), were further analyzed by noncontact atomic force microscopy and X-ray photoelectron spectroscopy, revealing a cofacial stacking geometry of the molecular layers. Scanning tunneling spectroscopy measurements show a decrease of the band gap with increasing film thickness, consistent with π-π stacking and electron delocalization. The present study provides new strategies for the fabrication of normally inaccessible structural motifs, atomic precision in organic films, and the effective conduction of electrons through multiple organic molecular stacks.

Stereodynamics of metal-ligand assembly: what lies beneath the "simple" spectral signatures of C2-symmetric chiral chelates.

A series of C2-symmetric chiral tetra-dentate ligands were prepared by using [4,5]- or [5,6]-pinene-fused 2,2'-bipyridyl units that are supported across a rigid arylene-ethynylene backbone. These conformationally pre-organised chelates support stable 1:1 metal complexes, which were fully characterised by UV/Vis, fluorescence, circular dichroism (CD), and (1)H NMR spectroscopy. A careful inspection of the exciton-coupled circular dichroism (ECCD) and (1)H NMR spectra of the reaction mixture in solution, however, revealed the evolution and decay of intermediate species en route to the final 1:1 metal-ligand adduct. Consistent with this model, mass spectrometric analysis revealed the presence of multiple metal complexes in solution at high ligand-to-metal ratios, which were essentially unobservable by UV/Vis or fluorescence spectroscopic techniques. Comparative studies with a bi-dentate model system have fully established the functional role of the π-conjugated ligand skeleton that dramatically enhances the thermodynamic stability of the 1:1 complex. In addition to serving as a useful spectroscopic handle to understand the otherwise "invisible" solution dynamics of this metal-ligand assembly process, temperature-dependent changes in the proton resonances associated with the chiral ligands allowed us to determine the activation barrier (ΔG(≠)) for the chirality switching between the thermodynamically stable but kinetically labile (P)- and (M)-stereoisomers.

Interdigitated hydrogen bonds: electrophile activation for covalent capture and fluorescence turn-on detection of cyanide.

Hydrogen-bonding promoted covalent modifications are finding useful applications in small-molecule chemical synthesis and detection. We have designed a xanthene-based fluorescent probe 1, in which tightly held acylguanidine and aldehyde groups engage in multiple intramolecular hydrogen bonds within the concave side of the molecule. Such an interdigitated hydrogen bond donor-acceptor (HBD-HBA) array imposes significant energy barriers (ΔG(‡) = 10-16 kcal mol(-1)) for internal bond rotations to assist structural preorganization and effectively polarizes the electrophilic carbonyl group toward a nucleophilic attack by CN(-) in aqueous environment. This covalent modification redirects the de-excitation pathways of the cyanohydrin adduct 2 to elicit a large (>7-fold) enhancement in the fluorescence intensity at λmax = 440 nm. A remarkably faster (> 60-fold) response kinetics of 1, relative to its N-substituted (and therefore "loosely held") analogue 9, provided compelling experimental evidence for the functional role of HBD-HBA interactions in the "remote" control of chemical reactivity, the electronic and steric origins of which were investigated by DFT computational and X-ray crystallographic studies.

Reactivity-based detection of copper(II) ion in water: oxidative cyclization of azoaromatics as fluorescence turn-on signaling mechanism.

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.

Fluorescence switching of imidazo[1,5-a]pyridinium ions: pH-sensors with dual emission pathways.

Imidazo[1,5-a]pyridinium ions are identified as highly emissive and water-soluble fluorophores accessed by an efficient three-component coupling reaction. Synthetic modifications of groups conjugated to the polyheterocyclic core are shown to profoundly impact the emission properties of these molecules. Notably, two structural isomers of functionalized imidazo[1,5-a]pyridinium ions were found to exhibit distinct de-excitation pathways, which are responsible for either a fluorescence turn-on or ratiometric response to pH change.

Conformationally dynamic π-conjugation: probing structure-property relationships of fluorescent tris(N-salicylideneaniline)s.

We recently reported the design and synthesis of a series of conformationally dynamic chromophores that are built on the C(3)-symmetric tris(N-salicylideneaniline) platform. This system utilizes cooperative structural folding-unfolding motions for fluorescence switching, which is driven by the assembly and disassembly of hydrogen bonds between the rigid core and rotatable peripheral part of the molecule. Here, we report detailed time-resolved spectroscopic studies to investigate the structure-property relationships of a series of functionalized tris(N-salicylideneaniline)s. Time-resolved fluorescence decay spectroscopy was applied to determine the main relaxation mechanisms of these π-extended fluorophores, and to address the effects of hydrogen bonding, steric constraints, and extension of the π-conjugation on their relaxation dynamics. Our results agree well with the conformational switching model that was previously suggested from steady-state experiments. Notably, extension of the π-conjugation from peripheral aryl groups resulted in the stabilization of the excited states, as evidenced by longer lifetimes and lower nonradiative decay constants. As a consequence, an increase in the fluorescence quantum yields was observed, which could be explained by the suppression of the torsional motions about the C-N bonds from an overall increase in the quinoid character of the excited states. A combination of time-resolved and steady-state techniques also revealed intermolecular interactions through π-π stacking at higher concentrations, which provide additional de-excitation pathways that become more pronounced in solid samples.

Ratiometric detection of mercury ions in water: accelerated response kinetics of azo chromophores having ethynyl ligand tethers.

The metal-induced intramolecular cyclization reaction of an azo dye was exploited for the colorimetric detection of mercury ions in water. The molecular probe 3 responds selectively to nM-level Hg(II) ions in neutral aqueous solutions.

Turn-on fluorescence detection of cyanide in water: activation of latent fluorophores through remote hydrogen bonds that mimic peptide beta-turn motif.

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(-).