Biomacromolecule-Assisted Screening for Reaction Discovery and Catalyst Optimization.

Reaction discovery and catalyst screening lie at the heart of synthetic organic chemistry. While there are efforts at de novo catalyst design using computation/artificial intelligence, at its core, synthetic chemistry is an experimental science. This review overviews biomacromolecule-assisted screening methods and the follow-on elaboration of chemistry so discovered. All three types of biomacromolecules discussed─enzymes, antibodies, and nucleic acids─have been used as "sensors" to provide a readout on product chirality exploiting their native chirality. Enzymatic sensing methods yield both UV-spectrophotometric and visible, colorimetric readouts. Antibody sensors provide direct fluorescent readout upon analyte binding in some cases or provide for cat-ELISA (Enzyme-Linked ImmunoSorbent Assay)-type readouts. DNA biomacromolecule-assisted screening allows for templation to facilitate reaction discovery, driving bimolecular reactions into a pseudo-unimolecular format. In addition, the ability to use DNA-encoded libraries permits the barcoding of reactants. All three types of biomacromolecule-based screens afford high sensitivity and selectivity. Among the chemical transformations discovered by enzymatic screening methods are the first Ni(0)-mediated asymmetric allylic amination and a new thiocyanopalladation/carbocyclization transformation in which both C-SCN and C-C bonds are fashioned sequentially. Cat-ELISA screening has identified new classes of sydnone-alkyne cycloadditions, and DNA-encoded screening has been exploited to uncover interesting oxidative Pd-mediated amido-alkyne/alkene coupling reactions.

Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis.

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

Rapid Enantioselective and Diastereoconvergent Hybrid Organic/Biocatalytic Entry into the Oseltamivir Core.

A formal synthesis of the antiviral drug (-)-oseltamivir (Tamiflu) has been accomplished starting from m-anisic acid via a dissolving metal or electrochemical Birch reduction. The correct absolute stereochemistry is efficiently set through enzyme-catalyzed carbonyl reduction on the resultant racemic α,ß-unsaturated ketone. A screen of a broad ketoreductase (KRED) library identified several that deliver the desired allylic alcohol with nearly perfect facial selectivity at the new center for each antipodal substrate, indicating that the enzyme also is able to completely override inherent diastereomeric bias in the substrate. Conversion is complete, with d-glucose serving as the terminal hydride donor (glucose dehydrogenase). For each resulting diastereomeric secondary alcohol, O/N-interconversion is then efficiently effected either by synfacial [3,3]-sigmatropic allylic imidate rearrangement or by direct, stereoinverting N-Mitsunobu chemistry. Both stereochemical outcomes have been confirmed crystallographically. The α,ß-unsaturation is then introduced via an α-phenylselenylation/oxidation/pyrolysis sequence to yield the targeted (S)-N-acyl-protected 5-amino-1,3-cyclohexadiene carboxylates, key advanced intermediates for oseltamivir pioneered by Corey (N-Boc) and Trost (N-phthalamido), respectively.

Population dynamics and reproductive biology of Barilius bendelisis (Cyprinidae: Cypriniformes) from river Gaula of Central Indian Himalaya.

The Indian hill trout cyprinid, Barilius bendelisis is a member of family Cyprinidae that dwells in shallow, cold, and clear water. In this study, growth parameters and reproductive biology of Indian hill trout, Barilius bendelisis from river Gaula, Central Himalaya region, India, were studied. The length-frequency data were grouped sex wise and were analyzed to determine the growth and mortality parameters using the computer software programme, FAO-ICLARM Stock Assessment Tool (FISAT II). Altogether, 501 individuals were collected from river Gaula (November 2013-October 2014) and were preserved in formalin for further analysis. The results showed that the female outnumbered the male population. The minimum GSI of females was observed in the month of October (4.93 ± 0.26) and for males in the month of June and July (0.093 ± 0.12), whereas, the maximum value was in the month of April for both females (13.47 ± 0.52) and males (1.21 ± 0.12). Fluctuation in GSI values had a bimodal pattern showing two peaks during March-May and August-September in both the sexes, indicating the common spawning period of fish. The slope of regression showed the negative allometric growth for both males and females (b= 2.65 for male and b= 2.5 for female). A significant relationship between length and weight was observed in the present study (p < 0.05). The ELEFAN-I estimated L∞ and K of the von Bertalanffy growth factor for males (17.33 cm and 0.310 per year), females (17.33 cm and 0.3 per year) and pooled sexes (17.33 cm and 0.240 per year). The results indicated that Barilius bendelisis is a small sized fish having negative allometric growth that spawns twice a year. Thus, the present study on biological traits represents the baseline information for effective production, conservation and restoration planning.

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography.

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Population dynamics and reproductive biology of Barilius bendelisis (Cyprinidae: Cypriniformes) from river Gaula, Central Indian Himalaya / Dinámica poblacional y biología reproductiva de Barilius bendelisis (Cyprinidae: Cypriniformes) en el río Gaula del Himalaya Central, India

Abstract:The Indian hill trout cyprinid, Barilius bendelisis is a member of family Cyprinidae that dwells in shallow, cold, and clear water. In this study, growth parameters and reproductive biology of Indian hill trout, Barilius bendelisis from river Gaula, Central Himalaya region, India, were studied. The length-frequency data were grouped sex wise and were analyzed to determine the growth and mortality parameters using the computer software programme, FAO-ICLARM Stock Assessment Tool (FISAT II). Altogether, 501 individuals were collected from river Gaula (November 2013-October 2014) and were preserved in formalin for further analysis. The results showed that the female outnumbered the male population. The minimum GSI of females was observed in the month of October (4.93 ± 0.26) and for males in the month of June and July (0.093 ± 0.12), whereas, the maximum value was in the month of April for both females (13.47 ± 0.52) and males (1.21 ± 0.12). Fluctuation in GSI values had a bimodal pattern showing two peaks during March-May and August-September in both the sexes, indicating the common spawning period of fish. The slope of regression showed the negative allometric growth for both males and females (b= 2.65 for male and b= 2.5 for female). A significant relationship between length and weight was observed in the present study (p < 0.05). The ELEFAN-I estimated L∞ and K of the von Bertalanffy growth factor for males (17.33 cm and 0.310 per year), females (17.33 cm and 0.3 per year) and pooled sexes (17.33 cm and 0.240 per year). The results indicated that Barilius bendelisis is a small sized fish having negative allometric growth that spawns twice a year. Thus, the present study on biological traits represents the baseline information for effective production, conservation and restoration planning. Rev. Biol. Trop. 64 (3): 1287-1295. Epub 2016 September 01.

ResumenBarilius bendelisis es un miembro de la familia Cyprinidae que habita en agua superficial, fría y clara. En esta investigación se estudiaron los parámetros de crecimiento y la biología reproductiva de Barilius bendelisism en el río Gaula, Himalaya Central, India. Los datos talla-frecuencia fueron agrupados por sexo, y analizados para determinar parámetros de crecimiento y mortalidad usando el programa de computador, FAO-ICLARM Stock Asseessment Toll (FISAT II). En total, 501 individuos fueron recolectados en el río Gaula (Noviembre 2013-Octubre 2014) y fueron preservados en formalina para futuros análisis. Los resultados mostraron que la población de hembras supera en número la población de machos. El GSI mínimo en hembras fue observado en octubre (4.93 ± 0.26) y en machos en Junio y Julio (0.093 ± 0.12), mientras, el valor máximo en abril para ambos, hembras (13.47 ± 0.52) y machos (1.21 ± 0.12). La fluctuación en los valores del GSI tenía un patrón bimodal mostrando dos picos durante Marzo-Macho y Agosto-Septiembre en ambos sexos, indicando el periodo de desove común del pez. La pendiente de regresión muestra crecimiento alométrico negativo tanto para machos como para hembras (b= 2.65 para machos y b= 2.5 para hembras). Se observó una significativa relación entre talla y peso en el presente estudio (p < 0.05). El ELEFAN-I estimó L∞ y K del factor de crecimiento von Bertalanffy para machos (17. 33 cm y 0.310 por año), hembras (17.33 cm y 0.3 por año) y sexos combinados (17.33 cm y 0.240 por año). Los resultados indicaron que Barilius bendelisis es un pez de talla pequeña con un crecimiento alométrico negativo que desova dos veces al año. Además, el presente estudio de rasgos biológicos representa la información de base para la efectiva producción, conservación y planeamiento de la restauración.