Transaminase-Catalyzed Synthesis of ß-Branched Noncanonical Amino Acids Driven by a Lysine Amine Donor.

Transaminases are choice biocatalysts for the synthesis of chiral primary amines, including amino acids bearing contiguous stereocenters. In this study, we employ lysine as a "smart" amine donor in transaminase-catalyzed dynamic kinetic resolution reactions to access ß-branched noncanonical arylalanines. Our mechanistic investigation demonstrates that, upon transamination, the lysine-derived ketone byproduct readily cyclizes to a six-membered imine, driving the equilibrium in the desired direction and thus alleviating the need to load superstoichiometric quantities of the amine donor or deploy a multienzyme cascade. Lysine also shows good overall compatibility with a panel of wild-type transaminases, a promising hint of its application as a smart donor more broadly. Indeed, by this approach, we furnished a broad scope of ß-branched arylalanines, including some bearing hitherto intractable cyclopropyl and isopropyl substituents, with high yields and excellent selectivities.

Formation, Selective Encapsulation, and Tautomerization Control of Isoindolone Utilizing Guanidinium Sulfonate Frameworks.

Herein we report the use of tetrakis (guanidinium) pyrenetetrasulfonate (G4PYR) and bis (guanidinium) 1,5-napthalene disulfonate (G2NDS) to catalyze the cyclization of 2-cyanobenzamide (1) to isoindolone (2). Moreover, we demonstrate the remarkable selectivity of these guanidinium organosulfonate hosts in encapsulating 2 over 1. By thoroughly investigating the intramolecular cyclization reaction, we determined that guanidinium and the organosulfonate moiety acts as the catalyst in this process. Additionally, 2 is selectively encapsulated, even in mixtures of other structurally similar heterocycles like indole. Furthermore, the tautomeric state of 2 (amino isoindolone (2-A) and imino isoindolinone forms (2-I)) can be controlled by utilizing different guanidinium organosulfonate frameworks.

General Synthesis of Conformationally Constrained Noncanonical Amino Acids with C(sp3)-Rich Benzene Bioisosteres.

Recent years have seen novel modalities emerge for the treatment of human diseases resulting in an increase in beyond rule of 5 (bRo5) chemical matter. As a result, synthetic innovations aiming to enable rapid access to complex bRo5 molecular entities have become increasingly valuable for medicinal chemists' toolkits. Herein, we report the general synthesis of a new class of noncanonical amino acids (ncAA) with a cyclopropyl backbone to achieve conformational constraint and bearing C(sp3)-rich benzene bioisosteres. We also demonstrate preliminary studies toward utilities of these ncAA as building blocks for medicinal chemistry research.

From Powders to Single Crystals: A Crystallographer's Toolbox for Small-Molecule Structure Determination.

Although the crystal structures of small-molecule compounds are often determined from single-crystal X-ray diffraction (scXRD), recent advances in three-dimensional electron diffraction (3DED) and crystal structure prediction (CSP) methods promise to expand the structure elucidation toolbox available to the crystallographer. Herein, a comparative assessment of scXRD, 3DED, and CSP in combination with powder X-ray diffraction is carried out on two former drug candidate compounds and a multicomponent crystal of a key building block in the synthesis of gefapixant citrate.

Electrochemical Synthesis of Hindered Primary and Secondary Amines via Proton-Coupled Electron Transfer.

Accessing hindered amines, particularly primary amines α to a fully substituted carbon center, is synthetically challenging. We report an electrochemical method to access such hindered amines starting from benchtop-stable iminium salts and cyanoheteroarenes. A wide variety of substituted heterocycles (pyridine, pyrimidine, pyrazine, purine, azaindole) can be utilized in the cross-coupling reaction, including those substituted with a halide, trifluoromethyl, ester, amide, or ether group, a heterocycle, or an unprotected alcohol or alkyne. Mechanistic insight based on DFT data, as well as cyclic voltammetry and NMR spectroscopy, suggests that a proton-coupled electron-transfer mechanism is operational as part of a hetero-biradical cross-coupling of α-amino radicals and radicals derived from cyanoheteroarenes.

Five-Step Enantioselective Synthesis of Islatravir via Asymmetric Ketone Alkynylation and an Ozonolysis Cascade.

A 5-step enantioselective synthesis of the potent anti-HIV nucleoside islatravir is reported. The highly efficient route was enabled by a novel enantioselective alkynylation of an α,ß-unsaturated ketone, a unique ozonolysis-dealkylation cascade in water, and an enzymatic aldol-glycosylation cascade.

Imaging local electric fields produced upon synchrotron X-ray exposure.

Electron-hole separation following hard X-ray absorption during diffraction analysis of soft materials under cryogenic conditions produces substantial local electric fields visualizable by second harmonic generation (SHG) microscopy. Monte Carlo simulations of X-ray photoelectron trajectories suggest the formation of substantial local electric fields in the regions adjacent to those exposed to X-rays, indicating a possible electric-field-induced SHG (EFISH) mechanism for generating the observed signal. In studies of amorphous vitreous solvents, analysis of the SHG spatial profiles following X-ray microbeam exposure was consistent with an EFISH mechanism. Within protein crystals, exposure to 12-keV (1.033-Å) X-rays resulted in increased SHG in the region extending ∼ 3 µm beyond the borders of the X-ray beam. Moderate X-ray exposures typical of those used for crystal centering by raster scanning through an X-ray beam were sufficient to produce static electric fields easily detectable by SHG. The X-ray-induced SHG activity was observed with no measurable loss for longer than 2 wk while maintained under cryogenic conditions, but disappeared if annealed to room temperature for a few seconds. These results provide direct experimental observables capable of validating simulations of X-ray-induced damage within soft materials. In addition, X-ray-induced local fields may potentially impact diffraction resolution through localized piezoelectric distortions of the lattice.

Dynamic X-ray diffraction sampling for protein crystal positioning.

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by limiting the dose and area exposed for image reconstruction and crystal positioning using data collection hardware present in most macromolecular crystallography end-stations.

Unified Theory for Polarization Analysis in Second Harmonic and Sum Frequency Microscopy.

A unified theoretical framework for the recovery of second-order nonlinear susceptibility tensors and sample orientations from polarization-dependent second harmonic generation and sum frequency generation microscopy was developed. Jones formalism was extended to nonlinear optics and was used to bridge the experimental observables and the local-frame tensor elements. Four commonly used experimental architectures were explicitly explored, including polarization rotation with no postsample optics, polarization-in polarization-out measurement, and polarization modulation with and without postsample optics. Polarization-dependent second harmonic generation measurement was performed on Z-cut quartz and the local-frame tensor elements were calculated. The recovered tensor elements agree with the expected values dictated by symmetry.

Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging.

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s(-1)). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-fold improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.

Nonlinear Optical Characterization of Membrane Protein Microcrystals and Nanocrystals.

Nonlinear optical methods such as second harmonic generation (SHG) and two-photon excited UV fluorescence (TPE-UVF) imaging are promising approaches to address bottlenecks in the membrane protein structure determination pipeline. The general principles of SHG and TPE-UVF are discussed here along with instrument design considerations. Comparisons to conventional methods in high throughput crystallization condition screening and crystal quality assessment prior to X-ray diffraction are also discussed.

Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals.

The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy.

Parts per Million Powder X-ray Diffraction.

Here we demonstrate the use of second harmonic generation (SHG) microscopy-guided synchrotron powder X-ray diffraction (PXRD) for the detection of trace crystalline active pharmaceutical ingredients in a common polymer blend. The combined instrument is capable of detecting 100 ppm crystalline ritonavir in an amorphous hydroxypropyl methylcellulose matrix with a high signal-to-noise ratio (>5000). The high spatial resolution afforded by SHG microscopy allows for the use of a minibeam collimator to reduce the total volume of material probed by synchrotron PXRD. The reduction in probed volume results in reduced background from amorphous material. The ability to detect low crystalline loading has the potential to improve measurements in the formulation pipeline for pharmaceutical solid dispersions, for which even trace quantities of crystalline active ingredients can negatively impact the stability and bioavailability of the final drug product.

High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories.

A simple beam-scanning optical design based on Lissajous trajectory imaging is described for achieving up to kHz frame-rate optical imaging on multiple simultaneous data acquisition channels. In brief, two fast-scan resonant mirrors direct the optical beam on a circuitous trajectory through the field of view, with the trajectory repeat-time given by the least common multiplier of the mirror periods. Dicing the raw time-domain data into sub-trajectories combined with model-based image reconstruction (MBIR) 3D in-painting algorithms allows for effective frame-rates much higher than the repeat time of the Lissajous trajectory. Since sub-trajectory and full-trajectory imaging are simply different methods of analyzing the same data, both high-frame rate images with relatively low resolution and low frame rate images with high resolution are simultaneously acquired. The optical hardware required to perform Lissajous imaging represents only a minor modification to established beam-scanning hardware, combined with additional control and data acquisition electronics. Preliminary studies based on laser transmittance imaging and polarization-dependent second harmonic generation microscopy support the viability of the approach both for detection of subtle changes in large signals and for trace-light detection of transient fluctuations.

Benchmarking Guanidinium Organosulfonate Hydrogen-Bonded Frameworks for Structure Determination of Encapsulated Guests.

Single crystal X-ray diffraction (SCXRD) is arguably the most definitive method for molecular structure determination, but it is often challenged by compounds that are liquids or oils at room temperature or do not form crystals adequate for analysis. Our laboratory previously reported a simple, cost-effective, single-step crystallization method based on guanidinium organosulfonate (GS) hydrogen bonded frameworks for structure determination of a wide range of encapsulated guest molecules, including assignment of the absolute configuration of chiral centers. Herein, we expand on those results and report a head-to-head comparison of the GS method with adamantoid "molecular chaperones", which have been reported to be useful hosts for structure determination. Inclusion compounds limited to only two GS hosts are characterized by low R1 values and Flack parameters, infrequent disorder of the host and guest, and manageable disorder when it does exist. The structures of some target molecules that were not included or resolved using the adamantoid chaperones were successfully included and resolved by the GS hosts, and vice versa. Of the 32 guests attempted by the GS method, 31 inclusion compounds afforded successful guest structure solutions, a 97% success rate. The GS hosts and adamantoid chaperones are complementary with respect to guest inclusion, arguing that both should be employed in the arsenal of methods for structure determination. Furthermore, the low cost of organosulfonate host components promises an accessible route to molecular structure determination for a wide range of users.

Supramolecular Mille-Feuille: Adaptive Guest Inclusion in a New Aliphatic Guanidinium Monosulfonate Hydrogen-Bonded Framework.

During the past three decades, the ability of guanidinium arenesulfonate host frameworks to encapsulate a wide range of guests has been amply demonstrated, with more than 700 inclusion compounds realized. Herein, we report crystalline inclusion compounds based on a new aliphatic host, guanidinium cyclohexanemonosulfonate, which surprisingly exhibits four heretofore unobserved architectures, as described by the projection topologies of the organosulfonate residues above and below hydrogen-bonded guanidinium sulfonate sheets. The inclusion compounds adopt a layer motif of guanidinium sulfonate sheets interleaved with guest molecules, resembling a mille-feuille pastry. The aliphatic character of this remarkably simple host, combined with access to greater architectural diversity and adaptability, enables the host framework to accommodate a wide range of guests and promises to expand the utility of guanidinium organosulfonate hosts.

Electrosynthesis of iminophosphoranes and applications in nickel catalysis.

P(v) iminophosphorane compounds are accessed via electrochemical oxidation of commercially available P(iii) phosphines, including mono-, di- and tri-dentate phosphines, as well as chiral phosphines. The reaction uses inexpensive bis(trimethylsilyl)carbodiimide as an efficient and safe aminating reagent. DFT calculations, cyclic voltammetry, and NMR studies provide insight into the reaction mechanism. The proposed mechanism reveals a special case of sequential paired electrolysis. DFT calculations of the frontier orbitals of an iminophosphorane are compared with those of the analogous phosphines and phosphine oxides. X-ray crystallographic studies of the ligands as well as a Ni-coordination complex provide structural insight for these ligands. The utility of these iminophosphoranes as ligands is demonstrated in nickel-catalyzed cross-electrophile couplings including C(sp2)-C(sp3) and C(sp2)-C(sp2) couplings, an electrochemically driven C-N cross-coupling, and a photochemical arylative C(sp3)-H functionalization. In some cases, these new ligands provide improved performance over commonly used sp2-N-based ligands (e.g. 4,4'-di-tert-butyl-2,2'-bipyridine).

Fluorinated Isoindolinone-Based Glucosylceramide Synthase Inhibitors with Low Human Dose Projections.

Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼10(3)-10(4)-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

Synchrotron X-Ray Diffraction Dynamic Sampling for Protein Crystal Centering.

A supervised learning approach for dynamic sampling (SLADS) was developed to reduce X-ray exposure prior to data collection in protein structure determination. Implementation of this algorithm allowed reduction of the X-ray dose to the central core of the crystal by up to 20-fold compared to current raster scanning approaches. This dose reduction corresponds directly to a reduction on X-ray damage to the protein crystals prior to data collection for structure determination. Implementation at a beamline at Argonne National Laboratory suggests promise for the use of the SLADS approach to aid in the analysis of X-ray labile crystals. The potential benefits match a growing need for improvements in automated approaches for microcrystal positioning.