Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Circular Dichroism Sensing: Strategies and Applications.

The analysis of the absolute configuration, enantiomeric composition, and concentration of chiral compounds are frequently encountered tasks across the chemical and health sciences. Chiroptical sensing methods can streamline this work and allow high-throughput screening with remarkable reduction of operational time and cost. During the last few years, significant methodological advances with innovative chirality sensing systems, the use of computer-generated calibration curves, machine learning assistance, and chemometric data processing, to name a few, have emerged and are now matched with commercially available multi-well plate CD readers. These developments have reframed the chirality sensing space and provide new opportunities that are of interest to a large group of chemists. This review will discuss chirality sensing strategies and applications with representative small-molecule CD sensors. Emphasis will be given to important milestones and recent advances that accelerate chiral compound analysis by outperforming traditional methods, conquer new directions, and pioneering efforts that lie at the forefront of chiroptical high-throughput screening developments. The goal is to provide the reader with a thorough understanding of the current state and a perspective of future directions of this rapidly emerging field.

Determination of Enantiomeric Excess and Diastereomeric Excess via Optical Methods. Application to α-methyl-ß-hydroxy-carboxylic acids.

Characterization of chiral molecules in solution is paramount for measuring reaction success. However, techniques to distinguish between chiral molecules containing more than one stereocenter through the use of optical techniques remains a challenge. Herein, we report a techique using a series of circular dichroism spectra to train multivariate regression models that are capable of predicting the complete speciation of 3-hydroxy-2-methylbutanoic acid stereoisomers. From this, it is possible to rapidly and accurately determine the enantiomeric excess and diastereomeric excess of the solution without the need for chiral chromatography.

11B NMR Spectroscopy: Structural Analysis of the Acidity and Reactivity of Phenyl Boronic Acid-Diol Condensations.

Phenyl boronic acids are valuable for medical diagnostics and biochemistry studies due to their ability to readily bind with carbohydrates in water. Incorporated in carbohydrates are 1,2-diols, which react with boronic acids through a reversible covalent condensation pathway. A wide variety of boronic acids have been employed for diol binding with differing substitution of the phenyl ring, with the goals of simplifying their synthesis and altering their thermodynamics of complexation. One method for monitoring their pKa's and binding is 11B NMR spectroscopy. Herein, we report a comprehensive study employing 11B NMR spectroscopy to determine the pKa of the most commonly used phenyl boronic acids and their binding with catechol or d,l-hydrobenzoin as prototypical diols. The chemical shift of the boronic acid transforming into the boronate ester was monitored at pHs ranging from 2 to 10. With each boronic acid, the results confirm (1) the necessity to use pHs above their pKa's to induce complexation, (2) that the pKa's change in the presence of diols, and (3) that 11B NMR spectroscopy is a particularly convenient tool for monitoring these interconnected acidity and binding phenomena.

Data-Driven Prediction of Circular Dichroism-Based Calibration Curves for the Rapid Screening of Chiral Primary Amine Enantiomeric Excess Values.

Here, we describe the prediction of the circular dichroism (CD) response of a three-component chiroptical sensor for enantiomeric excess (ee) determination of chiral amines using a multivariate fit to electronic and steric parameters. These computationally derived parameters can be computed for nearly any amine and correlate well with the CD response of the 12 amines comprising the training set. The resulting model was used to accurately predict the CD response of a test set of chiral amines. Theoretical calibration curves were then created and used to determine the ee of solutions of unknown ee. Using this method, the error in ee determination differed by less than 10% compared to experimentally generated calibration curves.

High-throughput screening of α-chiral-primary amines to determine yield and enantiomeric excess.

A novel screening protocol was developed using a combination of a fluorescent indicator displacement assay and a circular dichroism (CD) active Fe(II) complex to determine concentration and enantiomeric excess (ee) of α-chiral amines, respectively. The analyte concentration is quantified with a pre-formed non-fluorescent imine, where transimination with the chiral amine results in displacement of the fluorophore 2-naphthylamine. After discerning the concentration of amine via fluorescence in a wellplate reader, the analyte is then incorporated into a three-component octahedral Fe(II) assembly for ee determination using an EKKO CD plate-reader. With these two assays, both the ee and yield of asymmetric transformations of 192 samples could be determined with acceptable errors in under fifteen minutes (not counting the preparation time). This combined speed and accuracy provides an attractive solution to overcoming analytical bottlenecks when creating α-chiral amines.

A Colorimetric Method for Quantifying Cis and Trans Alkenes Using an Indicator Displacement Assay.

A colorimetric indicator displacement assay (IDA) amenable to high-throughput experimentation was developed to determine the percentage of cis and trans alkenes. Using 96-well plates two steps are performed: a reaction plate for dihydroxylation of the alkenes followed by an IDA screening plate consisting of an indicator and a boronic acid. The dihydroxylation generates either erythro or threo vicinal diols from cis or trans alkenes, depending upon their syn- or anti-addition mechanisms. Threo diols preferentially associate with the boronic acid due to the creation of more stable boronate esters, thus displacing the indicator to a greater extent. The generality of the protocol was demonstrated using seven sets of cis and trans alkenes. Blind mixtures of cis and trans alkenes were made, resulting in an average error of ±2 % in the percentage of cis or trans alkenes, and implementing E2 and Wittig reactions gave errors of ±3 %. Furthermore, we developed variants of the IDA for which the color may be tuned to optimize the response for the human eye.

The reliability of skin biopsy with measurement of intraepidermal nerve fiber density.

Intraepidermal nerve fiber density (IENFD) is a sensitive measure of small fiber injury, and holds promise as a clinical trial endpoint measure. A total of 48 punch biopsies were obtained from 22 patients. Tissue was sectioned and stained with PGP9.5. The relative intertrial variability (RIV) of IENFD measurements for each section and punch made by two different observers was determined (interobserver variability). Intraobserver variability (same observer measuring twice) was determined for 50% of the sections and punches. Sections from 12 punch biopsies were also stained at a second laboratory. The effect of the number of sections counted and processing site on reproducibility was investigated. A total of 223 sections were analyzed. The mean IENFD was 6.7 fibers/mm. Mean (+/-standard deviation) interobserver variability was 9.6%+/-9.4 for each biopsy site and 10.2%+/-11.9 for individual sections. Mean intraobserver variability was 9.6%+/-8.9 for biopsies, and 8.8%+/-9.0 for sections. There was no significant difference in IENFD for tissue stained at different laboratories. Intraclass correlation coefficients were greater than 0.98 for each comparison. There was no relationship between absolute IENFD and reproducibility. Reproducibility was highest when four sections were counted. IENFD measurement is highly reproducible. At least four sections should be analyzed. Reliability does not vary with severity of disease. These findings suggest IENFD may be a useful endpoint measure in future neuropathy treatment trials.

Degraded cardiomyocytes and dysfunctional energy metabolism; their relationship with canine cardiomyopathies.

Tissue sections from hearts of dogs suffering from cardiomyopathies were studied histologically and ultrastructurally. Two types of mitochondrial changes were defined and quantitated. Mitochondrial hypertrophy occurred in cardiomyocytes of middle-aged and older dogs. Significant numbers of degraded cardiomyocytes related to hydropic degeneration of hypertrophied mitochondria associated with autolysis of contractile elements and loss of cellular physiochemical functions were seen in hearts of old dogs with cardiomyopathy. Dogs with cardiac disease that were treated to enhance dysfunctional energy production early in the course of disease had cellular changes similar to those seen in old dogs but they survived heart disease and ultimately died of other causes.

A double dissociation within striatum between serial reaction time and radial maze delayed nonmatching performance in rats.

Lesions involving the intralaminar thalamic nuclei have been associated with impairments in working memory and intentional motor function in human clinical cases and animal models of amnesia. The intralaminar nuclei have afferent and efferent connections related to striatum. To test whether disruption of striatal function can account for impairments produced by intralaminar lesions, we investigated the effects of striatal lesions on two tasks known to be impaired by intralaminar damage in the rat: radial maze delayed nonmatching (DNM), a measure of spatial working memory, and self-paced serial reaction time (SRT), a measure of intentional response speed. We compared the effects of lesions in four sites: the medial and lateral caudate putamen, nucleus accumbens, and olfactory tubercle. We found that lesions of the medial, accumbens, or tubercle sites impaired DNM performance, and that lesions of the lateral caudate putamen increased choice response time for the SRT task. There was a double dissociation between the effects of the ventral and the lateral lesions on these two tasks. For both tasks, the effects of striatal lesions were qualitatively similar and at least as large as intralaminar lesions in previous studies. These results provide evidence that striatal dysfunction can account for the DNM and SRT impairments produced by intralaminar lesions. The dissociation of functional impairments suggests that lateral sensorimotor areas of caudate putamen are important for responding based on external sensory stimuli and limbic-related areas in ventral striatum are important for responding based on information held in working memory.