Gut microbiome and metabolome profiling in Framingham heart study reveals cholesterol-metabolizing bacteria.

Accumulating evidence suggests that cardiovascular disease (CVD) is associated with an altered gut microbiome. Our understanding of the underlying mechanisms has been hindered by lack of matched multi-omic data with diagnostic biomarkers. To comprehensively profile gut microbiome contributions to CVD, we generated stool metagenomics and metabolomics from 1,429 Framingham Heart Study participants. We identified blood lipids and cardiovascular health measurements associated with microbiome and metabolome composition. Integrated analysis revealed microbial pathways implicated in CVD, including flavonoid, γ-butyrobetaine, and cholesterol metabolism. Species from the Oscillibacter genus were associated with decreased fecal and plasma cholesterol levels. Using functional prediction and in vitro characterization of multiple representative human gut Oscillibacter isolates, we uncovered conserved cholesterol-metabolizing capabilities, including glycosylation and dehydrogenation. These findings suggest that cholesterol metabolism is a broad property of phylogenetically diverse Oscillibacter spp., with potential benefits for lipid homeostasis and cardiovascular health.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.

Origin of high stereocontrol in olefin cyclopropanation catalyzed by an engineered carbene transferase.

Recent advances in metalloprotein engineering have led to the development of a myoglobin-based catalyst, Mb(H64V,V68A), capable of promoting the cyclopropanation of vinylarenes with high efficiency and high diastereo- and enantioselectivity. Whereas many enzymes evolved in nature often exhibit catalytic proficiency and exquisite stereoselectivity, how these features are achieved for a non-natural reaction has remained unclear. In this work, the structural determinants responsible for chiral induction and high stereocontrol in Mb(H64V,V68A)-catalyzed cyclopropanation were investigated via a combination of crystallographic, computational (DFT), and structure-activity analyses. Our results show the importance of steric complementarity and non-covalent interactions involving first-sphere active site residues, heme-carbene, and the olefin substrate, in dictating the stereochemical outcome of the cyclopropanation reaction. High stereocontrol is achieved through two major mechanisms. First, by enforcing a specific conformation of the heme-bound carbene within the active site. Second, by controlling the geometry of attack of the olefin on the carbene via steric occlusion, attractive van der Waals forces and protein-mediated π-π interactions with the olefin substrate. These insights could be leveraged to expand the substrate scope of the myoglobin-based cyclopropanation catalyst toward non-activated olefins and to increase its cyclopropanation activity in the presence of a bulky α-diazo-ester. This work sheds first light into the origin of enzyme-catalyzed enantioselective cyclopropanation, furnishing a mechanistic framework for both understanding the reactivity of current systems and guiding the future development of biological catalysts for this class of synthetically important, abiotic transformations.

Myoglobin-Catalyzed C-H Functionalization of Unprotected Indoles.

Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal-catalyzed carbene transfer has provided an attractive route to afford C3-functionalized indoles, these protocols are viable only in the presence of N-protected indoles, owing to competition from the more facile N-H insertion reaction. Herein, a biocatalytic strategy for enabling the direct C-H functionalization of unprotected indoles is reported. Engineered variants of myoglobin provide efficient biocatalysts for this reaction, which has no precedents in the biological world, enabling the transformation of a broad range of indoles in the presence of ethyl α-diazoacetate to give the corresponding C3-functionalized derivatives in high conversion yields and excellent chemoselectivity. This strategy could be exploited to develop a concise chemoenzymatic route to afford the nonsteroidal anti-inflammatory drug indomethacin.

Cyclopropanations via Heme Carbenes: Basic Mechanism and Effects of Carbene Substituent, Protein Axial Ligand, and Porphyrin Substitution.

Catalytic carbene transfer to olefins is a useful approach to synthesize cyclopropanes, which are key structural motifs in many drugs and biologically active natural products. While catalytic methods for olefin cyclopropanation have largely relied on rare transition-metal-based catalysts, recent studies have demonstrated the promise and synthetic value of iron-based heme-containing proteins for promoting these reactions with excellent catalytic activity and selectivity. Despite this progress, the mechanism of iron-porphyrin and hemoprotein-catalyzed olefin cyclopropanation has remained largely unknown. Using a combination of quantum chemical calculations and experimental mechanistic analyses, the present study shows for the first time that the increasingly useful C═C functionalizations mediated by heme carbenes feature an FeII-based, nonradical, concerted nonsynchronous mechanism, with early transition state character. This mechanism differs from the FeIV-based, radical, stepwise mechanism of heme-dependent monooxygenases. Furthermore, the effects of the carbene substituent, metal coordinating axial ligand, and porphyrin substituent on the reactivity of the heme carbenes was systematically investigated, providing a basis for explaining experimental reactivity results and defining strategies for future catalyst development. Our results especially suggest the potential value of electron-deficient porphyrin ligands for increasing the electrophilicity and thus the reactivity of the heme carbene. Metal-free reactions were also studied to reveal temperature and carbene substituent effects on catalytic vs noncatalytic reactions. This study sheds new light into the mechanism of iron-porphyrin and hemoprotein-catalyzed cyclopropanation reactions and it is expected to facilitate future efforts toward sustainable carbene transfer catalysis using these systems.

Highly Diastereo- and Enantioselective Synthesis of Trifluoromethyl-Substituted Cyclopropanes via Myoglobin-Catalyzed Transfer of Trifluoromethylcarbene.

We report an efficient strategy for the asymmetric synthesis of trifluoromethyl-substituted cyclopropanes by means of myoglobin-catalyzed olefin cyclopropanation reactions in the presence of 2-diazo-1,1,1-trifluoroethane (CF3CHN2) as the carbene donor. These transformations were realized using a two-compartment setup in which ex situ generated gaseous CF3CHN2 is processed by engineered myoglobin catalysts expressed in bacterial cells. This approach was successfully applied to afford a variety of trans-1-trifluoromethyl-2-arylcyclopropanes in high yields (61-99%) and excellent diastereo- and enantioselectivity (97-99.9% de and ee). Furthermore, mirror-image forms of these products could be obtained using myoglobin variants featuring stereodivergent selectivity. These reactions provide a convenient and effective biocatalytic route to the stereoselective synthesis of key fluorinated building blocks of high value for medicinal chemistry and drug discovery. This work expands the range of carbene-mediated transformations accessible via metalloprotein catalysts and introduces a potentially general strategy for exploiting gaseous and/or hard-to-handle carbene donor reagents in biocatalytic carbene transfer reactions.

Biocatalytic Synthesis of Allylic and Allenyl Sulfides through a Myoglobin-Catalyzed Doyle-Kirmse Reaction.

The first example of a biocatalytic [2,3]-sigmatropic rearrangement reaction involving allylic sulfides and diazo reagents (Doyle-Kirmse reaction) is reported. Engineered variants of sperm whale myoglobin catalyze this synthetically valuable C-C bond-forming transformation with high efficiency and product conversions across a variety of sulfide substrates (e.g., aryl-, benzyl-, and alkyl-substituted allylic sulfides) and α-diazo esters. Moreover, the scope of this myoglobin-mediated transformation could be extended to the conversion of propargylic sulfides to give substituted allenes. Active-site mutations proved effective in enhancing the catalytic efficiency of the hemoprotein in these reactions as well as modulating the enantioselectivity, resulting in the identification of the myoglobin variant Mb(L29S,H64V,V68F), which is capable of mediating asymmetric Doyle-Kirmse reactions with an enantiomeric excess up to 71 %. This work extends the toolbox of currently available biocatalytic strategies for the asymmetric formation of carbon-carbon bonds.

Low-temperature synthesis of homogeneous solid solutions of scheelite-structured Ca1-xSrxWO4 and Sr1-xBaxWO4 nanocrystals.

A series of compositionally complex scheelite-structured nanocrystals of the formula A1-xAWO4 (A = Ca, Sr, Ba) have been prepared under benign synthesis conditions using the vapor diffusion sol-gel method. Discrete nanocrystals with sub-20 nm mean diameters were obtained after kinetically controlled hydrolysis and polycondensation at room temperature, followed by composition-dependent thermal aging at or below 60 °C. Rietveld analysis of X-ray diffraction data and Raman spectroscopy verified the synthesis of continuous and phase-pure nanocrystal solid solutions across the entire composition space for A1-xAWO4, where 0 ≤ x ≤ 1. Elemental analysis by X-ray photoelectron and inductively coupled plasma-atomic emission spectroscopies demonstrated excellent agreement between the nominal and experimentally determined elemental stoichiometries, while energy dispersive X-ray spectroscopy illustrated good spatial elemental homogeneity within these nanocrystals synthesized under benign conditions.

Timpanismo e empiema de bolsa gutural conseqüente a garrotilho / Timpany and empyema of guttural pouch associated with strangles

Garrotilho é uma doença infecto-contagiosa do trato respiratório superior dos eqüídeos, freqüente e importante, que causa pirexia, descarga nasal e abscedação dos linfonodos adjacentes. Acomete basicamente equídeos jovens, e é causada pelo Streptococcus equi â-hemolítico. Ocasionalmente, o garrotilho pode deixar seqüelas como sinusites, empiema das bolsas guturais e formação de abcessos à distância, principalmente no mesentério. O objetivo deste estudo é avaliar o timpanismo e empiema de bolsa gutural, clínico e cirúrgico, conseqüente a garrrotilho, ocorrido num eqüino jovem.