R.B. Leveson-Gower

Germylenes with two ylide-substituents are usually highly nucleophilic species. Herein, we demonstrate that introduction of a cyano moiety into the ylide backbone decreases the LUMO energy and thus significantly enhances the electrophilic nature of these species. This is reflected in the reactivity but also in the molecular structure of the diylidylgermylene.

Abstract

Owing to the strong electron-donating ability of ylide substituents, diylidyltetrylenes are usually highly nucleophilic species with strong donor capacities. Here, we demonstrate that their electronic properties are in fact highly flexible and can be effectively tuned through variation of the substituent in the ylide backbone. Initial density functional theory studies showed that cyano groups are particularly capable in lowering the LUMO energy of diylidyl germylenes thus turning these usually highly nucleophilic species into electrophilic compounds. This was confirmed by experimental studies. Attempts to synthesize the germylene (Y_CN)₂Ge [with Y_CN=Ph₃P-(C)-CN] from the corresponding metalated ylide Y_CNK selectively led to germanide [(Y_CN)₃Ge)K]₂ thus reflecting the electrophilic nature of the intermediate formed germylene. XRD analysis of single crystals of (Y_CN)₂Ge – serendipitously obtained through protonative cleavage of one ylide from the germanide – revealed a monomeric structure with rather long Ge-ylide linkages, which corroborates well with a pure single bond and no stabilization of the empty p_π orbital at germanium through π bonding. The germanide exhibits methanide-like reactivity towards chalcogens but a likewise weak Ge−C bond as demonstrated by the insertion of carbon dioxide.

The conjugation of Au complexes with proteins and enzymes, generating new types of artificial metalloenzymes, has proven to be interesting and effective to obtain materials with improved properties such as higher stability, catalytic activity and selectivity. In this work, a novel method has been developed for the synthesis and design of artificial gold metalloenzymes at 50ºC in aqueous media, using two genetically modified variants of the alkalophilic lipase Geobacillus thermocatenulatus (GTL). The only difference between these two enzymatic variants is the possible coordination of the Au via active site (GTL-114) or Lid site (GTL-193). TEM analysis of the metalloenzymes revealed the formation of Au (0) nanoparticles with different structures (nanowires, nanorods, nanoshells, nanoclusters) and sizes depending on the mutant and the pH used during the synthesis. Characterisation by fluorescence spectroscopy demonstrated that conjugation of the enzyme to Au altered the tertiary structure of the protein. On the other hand, all metalloenzymes showed excellent reductase like activity. Finally, the selectivity of the enzyme-Au bioconjugates was tested in the asymmetric reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. The protein environment played a key role in the reactivity and selectivity of the metalloenzymes, obtaining chiral nanoparticles with an enantiomeric excess of up to 39% towards (R)-1-phenylethanol after two hours of reaction using GTL-114 pH 10 as catalyst.

By using Rosetta enzyme design and MD simulations, the active site of a thermostable ketoreductase was redesigned to invert the stereoselectivity. Simultaneous introduction of 6–8 mutations without laboratory screening of mutant libraries gave active enzymes catalyzing asymmetric synthesis of 1-phenylethanols with high enantiomeric excess.

Abstract

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6–8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of k_cat /K _M values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

We report a homogeneous photocatalytic system based on cobalt porphyrin substituted myoglobin (CoMb) for CO₂ to CO conversion in water. By optimizing the reaction conditions, our catalysts achieved up to 2000 TON(CO) at low enzyme concentrations, and a product selectivity of 80 % with an increased enzyme loading. We show that the efficiencies of CO generation and overall TON(CO) can be improved by introducing positively charged residues near the active site of CoMb.

Abstract

While native CO₂-reducing enzymes display remarkable catalytic efficiency and product selectivity, few artificial biocatalysts have been engineered to allow understanding how the native enzymes work. To address this issue, we report cobalt porphyrin substituted myoglobin (CoMb) as a homogeneous catalyst for photo-driven CO₂ to CO conversion in water. The activity and product selectivity were optimized by varying pH and concentrations of the enzyme and the photosensitizer. Up to 2000 TON(CO) was attained at low enzyme concentrations with low product selectivity (15 %), while a product selectivity of 74 % was reached by increasing the enzyme loading but with a compromised TON(CO). The efficiency of CO generation and overall TON(CO) were further improved by introducing positively charged residues (Lys or Arg) near the active stie of CoMb, which demonstrates the value of tuning the enzyme secondary coordination sphere to enhance the CO₂-reducing performance of a protein-based photocatalytic system.

Nature, Published online: 13 March 2023; doi:10.1038/d41586-023-00761-3

Zoologist Zuofu Xiang’s research in Asia helps governments to protect the populations and teaches him the value of cooperation.

Science Advances, Volume 9, Issue 10, March 2023.

This study significantly expands the scope of multiple noncanonical amino acid (ncAA) mutagenesis in mammalian cells by developing a tryptophanyl-tRNA synthetase/tRNA pair that efficiently decodes the TGA stop codon. It can be combined with three previously established pairs to create new ways to incorporate up to three distinct ncAAs into a protein in mammalian cells. Using this technology, two distinct cytotoxic drugs were site-specifically conjugated to a full-length humanized antibody.

Abstract

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Here, we report the design and application of multi-step enzymatic cascades to synthesize enantioenriched epoxides and vicinal aromatic triols from simple biomass-derived starting materials in one pot. These artificial metabolic pathways involve a tailor-made aldolase, a highly evolved cofactor-independent peroxyzyme, and when needed a specifically chosen epoxide hydrolase. These attractive biocatalytic cascades can be performed under environmentally benign conditions, such as the use of aqueous media and mild temperatures, and do not require the isolation of reaction intermediates. Good to excellent conversions, high enantioselectivity, and moderate to good product yields are achieved.

Abstract

Multi-enzymatic cascades exploiting engineered enzymes are a powerful tool for the tailor-made synthesis of complex molecules from simple inexpensive building blocks. In this work, we engineered the promiscuous enzyme 4-oxalocrotonate tautomerase (4-OT) into an effective aldolase with 160-fold increased activity compared to 4-OT wild type. Subsequently, we applied the evolved 4-OT variant to perform an aldol condensation, followed by an epoxidation reaction catalyzed by a previously engineered 4-OT mutant, in a one-pot two-step cascade for the synthesis of enantioenriched epoxides (up to 98 % ee) from biomass-derived starting materials. For three chosen substrates, the reaction was performed at milligram scale with product yields up to 68 % and remarkably high enantioselectivity. Furthermore, we developed a three-step enzymatic cascade involving an epoxide hydrolase for the production of chiral aromatic 1,2,3-prim,sec,sec-triols with high enantiopurity and good isolated yields. The reported one-pot, three-step cascade, with no intermediate isolation and being completely cofactor-less, provides an attractive route for the synthesis of chiral aromatic triols from biomass-based synthons.

Nature, Published online: 08 March 2023; doi:10.1038/s41586-023-05781-7

Structural and biochemical studies of the Mycobacterium smegmatis hydrogenase Huc provides insights into how [NiFe] hydrogenases oxidize trace amounts of atmospheric hydrogen and transfer the electrons liberated via quinone transport.

Proceedings of the National Academy of Sciences, Volume 120, Issue 9, February 2023.

Here we report functionalized peptides that can participate in disulfide and acyl-hydrazone chemistry. DCLs of such molecules yield self-assembling fibres. We incorporated cell adhesion-promoting sequences into the scaffold that can undergo hydrogelation to obtain materials that present biologically relevant ligands. The importance of this work lies in the methodology for fabricating tailor-made materials through a modular approach.

Abstract

Dynamic covalent chemistry (DCC) has proven to be a valuable tool in creating fascinating molecules, structures, and emergent properties in fully synthetic systems. Here we report a system that uses two dynamic covalent bonds in tandem, namely disulfides and hydrazones, for the formation of hydrogels containing biologically relevant ligands. The reversibility of disulfide bonds allows fiber formation upon oxidation of dithiol-peptide building block, while the reaction between NH−NH₂ functionalized C-terminus and aldehyde cross-linkers results in a gel. The same bond-forming reaction was exploited for the “decoration” of the supramolecular assemblies by cell-adhesion-promoting sequences (RGD and LDV). Fast triggered gelation, cytocompatibility and ability to “on-demand” chemically customize fibrillar scaffold offer potential for applying these systems as a bioactive platform for cell culture and tissue engineering.

Biased esterification of nucleosides with borate preferentially removes those reactants from biocatalytic equilibrium systems, with the resulting cyclic borate esters acting as reversible enzyme inhibitors. This effect can be used to manipulate glycosylation equilibria and facilitate biocatalytic access to nucleoside analogues.

Abstract

Biocatalytic nucleoside (trans-)glycosylations catalyzed by nucleoside phosphorylases have evolved into a practical and convenient approach to the preparation of modified nucleosides, which are important pharmaceuticals for the treatment of various cancers and viral infections. However, the obtained yields in these reactions are generally determined exclusively by the innate thermodynamic properties of the nucleosides involved, hampering the biocatalytic access to many sought-after target nucleosides. We herein report an additional means for reaction engineering of these systems. We show how apparent equilibrium shifts in phosphorolysis and glycosylation reactions can be effected through entropically driven, biased esterification of nucleosides and ribosyl phosphates with inorganic borate. Our multifaceted analysis further describes the kinetic implications of this in situ reactant esterification for a model phosphorylase.

A general one-pot in vivo biocatalytic cascade for the production of α,ω-diamines as important nylon monomers was developed, starting from cheap and readily available cycloalkanes. The desired α,ω-diamines were successfully produced with the highest biosynthesis productivity to date, thus providing an ecologically viable alternative to the current industrial process for manufacturing α,ω-diamines.

Abstract

Aliphatic α,ω-diamines (DAs) are important monomer precursors that are industrially produced by energy-intensive, multistage chemical reactions that are harmful to the environment. Therefore, the development of sustainable green DA synthetic routes is highly desired. Herein, we report an efficient one-pot in vivo biocatalytic cascade for the transformation of cycloalkanes into DAs with the aid of advanced techniques, including the RetroBioCat tool for biocatalytic route design, enzyme mining for finding appropriate enzymes and microbial consortia construction for efficient pathway assembly. As a result, DAs were successfully produced by the designed microbial consortia-based biocatalytic system. In particular, the highest biosynthesis productivity record of 1,6-hexanediamine was achieved when using either cyclohexanol or cyclohexane as a substrate. Thus, the developed biocatalytic process provides a promising alternative to the dominant industrial process for manufacturing DAs.

An efficient organocatalytic method to synthetically versatile chiral triflones with two non-adjacent stereogenic centers was developed. The work uses, for the first time, α-aryl vinyl triflones as Michael acceptors in stereoselective catalysis. Control over the absolute configuration is achieved by a catalyst-controlled stereoselective C−C bond formation-protonation sequence.

Abstract

Trifluoromethylsulfones (triflones) are useful compounds for synthesis and beyond. Yet, methods to access chiral triflones are scarce. Here, we present a mild and efficient organocatalytic method for the stereoselective synthesis of chiral triflones using α-aryl vinyl triflones, building blocks previously unexplored in asymmetric synthesis. The peptide-catalyzed reaction gives rise to a broad range of γ-triflylaldehydes with two non-adjacent stereogenic centers in high yields and stereoselectivities. A catalyst-controlled stereoselective protonation following a C−C bond formation is key to control over the absolute and relative configuration. Straightforward derivatization of the products into, e.g., disubstituted δ-sultones, γ-lactones, and pyrrolidine heterocycles highlights the synthetic versatility of the products.

Nature Communications, Published online: 24 February 2023; doi:10.1038/s41467-023-36756-x

Detoxification enzymes are crucial for the survival of animals in new environments. Here, the authors study the molecular mechanism behind the catalytic diversification of a major family of tetrapod detoxification enzymes—the FMOs—during evolution.