R.B. Leveson-Gower

Boronic acids are essential building blocks used for the synthesis of bioactive molecules, the generation of chemical libraries and the exploration of structure-activity relationships. As a result, more than ten thousand boronic acids are commercially available. Medicinal chemists are therefore facing a challenge; which of them should they select to maximize information obtained by the synthesis of new target molecules. The present article aims to help them to make the right choices. The boronic acids used frequently in the synthesis of bioactive molecules were identified by mining several large molecular and reaction databases and their properties were analyzed. Based on the results a diverse set of boronic acids covering well the bioactive chemical space was selected and is suggested as a basis for library design for the efficient exploration of structure-activity relationships. A Boronic Acid Navigator web tool which helps chemists to make their own selection is also made available at https://bit.ly/boronics.

We present the application of the genetic code expansion technique to achieve the site-specific modification of the sensing region of a nanopore. The rationally designed conformation of unnatural amino acid (UAA) residues provides a favorable geometric orientation for the interactions of peptides and pore. The chemical environment of the sensing region facilitates the direct discrimination of the mixtures of peptides containing hydrophobic amino acids.

Abstract

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

A concerted strategy involving enzyme-instructed self-assembly to target overexpressed alkaline phosphatases in cancer cells and bioorthogonal reactions for controllable extracellular and intracellular activation of Ru^II-based imaging probes and photosensitizers is presented. The emission enhancement, lifetime extension, and (photo)cytotoxicity of the resulting Ru^II supramolecular assemblies were explored extracellularly and intracellularly.

Abstract

In this article, we report a novel targeting strategy involving the combination of an enzyme-instructed self-assembly (EISA) moiety and a strained cycloalkyne to generate large accumulation of bioorthogonal sites in cancer cells. These bioorthogonal sites can serve as activation triggers in different regions for transition metal-based probes, which are new ruthenium(II) complexes carrying a tetrazine unit for controllable phosphorescence and singlet oxygen generation. Importantly, the environment-sensitive emission of the complexes can be further enhanced in the hydrophobic regions offered by the large supramolecular assemblies, which is highly advantageous to biological imaging. Additionally, the (photo)cytotoxicity of the large supramolecular assemblies containing the complexes was investigated, and the results illustrate that cellular localization (extracellular and intracellular) imposes a profound impact on the efficiencies of photosensitizers.

Nature, Published online: 15 May 2023; doi:10.1038/d41586-023-01625-6

Work felt unmanageable, and I was always angry and constantly tired. Here’s how I stepped back, says Kelly Korreck.

Self-replicating molecules provide a simple approach for investigating fundamental processes in scenarios of the emergence of life. Although homochirality is an important aspect of life and of how it emerged, the effects of chirality on self-replicators have received only little attention so far. Here we report several self-assembled self-replicators with chiral selectivity, that emerge spontaneously and grow only from enantiopure material. These require a relatively small number of chiral units in the replicators (down to 8) and in the precursors (down to a single chiral unit), compared to the only other chiral selective replicator reported previously. One replicator was found to incorporate material of its own handedness with high fidelity when provided with a racemic mixture of precursors, thus sorting (L)- and (D)-precursors into (L)- and (D)-replicators. Systematic studies reveal that the presence or absence of chiral selectivity depends on structural features (ring size of the replicator) that appear to impose constraints on its supramolecular organization. This work reveals new aspects of the little researched interplay between chirality and self-replication and represents another step towards the de novo synthesis of life.

Communications Chemistry, Published online: 11 May 2023; doi:10.1038/s42004-023-00885-7

Prebiotic environments rich in boron have been postulated to be ideal for abiotic RNA synthesis, but the effects of boron on amino acid polymerization are unclear. Here, boric acid is shown to enable the polymerization of amino acids at acidic and near-neutral pH levels.

A methyltransferase from the green alga Chlamydomonas reinhardtii was identified and engineered for late-stage modifications of the carbon skeleton of terpenes. A variant with three changes in the amino acid sequence was able to produce methylated derivatives of linear terpenoids with high selectivity by C-methylation of an unactivated alkene. This opens new avenues for the modification of carbon scaffolds in various applications, as demonstrated by Bernhard Hauer et al. in their Communication (e202301601). Artwork: Verena Resch, Graz.

Nature, Published online: 10 May 2023; doi:10.1038/s41586-023-06052-1

After 600 rounds of selection, anaerobic snowflake yeast evolved to be macroscopic, becoming around 20,000 times larger (approximately mm scale) and about 10,000-fold more biophysically tough, while retaining a clonal multicellular life cycle.

The stereocontrolled cationic cyclization cascade is a vital step in the modular biogenesis of terpenes, as it defines the carbon skeleton's three-dimensional structure in one atom-economical step. While nature has adopted this strategy for eons, state-of-the-art synthetic routes to asymmetrically access cyclic terpenes still rely predominantly on sequential multi-step scaffold remodelling. Herein, we bridge this long-standing methodological gap by unlocking the target-oriented synthesis ability of the squalene-hopene cyclase. Our mechanistic insights show that the biocatalytic head-to-tail cyclization is highly customizable by mechanism-guided enzyme engineering and substrate-focused setup engineering. As a result, we demonstrate two- or three-step hybrid synthetic routes of pheromones, fragrances, and drug candidates by merging a stereocontrolled cyclization with interdisciplinary synthetic and catalytic methods. This biomimetic strategy significantly reduces the synthesis effort to terpenes and provides rapid access to thousands of head-to-tail-fused scaffolds.

Nature Communications, Published online: 06 May 2023; doi:10.1038/s41467-023-38328-5

Recently, a pipeline for the design of protein-binding proteins using only the structure of the target protein was reported. Here, the authors report that the incorporation of deep learning methods into the original pipeline increases experimental success rate by ten-fold.

Discovery of the biosynthetic gene cluster for the α-diazoester natural product azaserine is reported. Isotope feeding and biochemical experiments implicate generation of a hydrazonoacetic acid intermediate that is oxidized and transferred to l-serine. This pathway represents a distinct biosynthetic strategy for diazo formation.

Abstract

Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.

Nature, Published online: 03 May 2023; doi:10.1038/s41586-023-06027-2

The α-diazoester azaserine can be produced by Streptomyces albus engineered with a biosynthetic gene cluster and act as the carbene precursor for coupling with intracellularly produced styrene to generate unnatural amino acids containing a cyclopropyl group.

Separation not required: Mass spectrometry without prior chromatographic separation, carried out on a single-quadrupole HPLC-MS, can be used for the qualitative and quantitative analysis of diverse biotransformations. This flow-injection analysis mass spectrometry (FIA-MS) approach represents an attractive alternative to more traditional photometric, fluorometric, and chromatographic methods for screening enzymatic reactions.

Abstract

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1–50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Sequence-based functional annotation of enzymes is an essential step in the discovery and development of new biocatalysts. The vinylglycine ketimine (VGK) subfamily of pyridoxal-phosphate dependent enzymes plays critical roles in sulfur metabolism and is home to a growing range of secondary metabolic enzymes that synthesize noncanonical amino acids. However, discovery of useful new enzymes has been slowed because functional assignments within the VGK sub-family are convoluted by pervasive mis-annotation. Here, we used a whole-cell substrate multiplexed screening approach to rapidly measure catalytic activities of 40 homologs in the VGK subfamily. This strategy gives direct information on enzyme specificity without having to purify each enzyme or measure individual kinetic constants. We identified a thermostable cystathionine γ-lyase from Thermobifida fusca and performed mechanistic and structural studies. For biocatalytic applications, we identified a well-behaved, thermostable, and promiscuous amino acid γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). We showed CahyGS can catalyze a stereoselective γ-addition into L-allylglycine, providing preparative-scale access to a unique set of γ-branched amino acids. High resolution crystal structures of CahyGS show an open-closed transition associated with ligand binding and provide a basis for subsequent engineering. Together, these data show how multiplexed screening approaches aid in the rapid deconvolution of enzyme function and identify enzymes with useful properties for enzymology and biocatalysis.

A bioinspired approach using enzyme electrocatalysis for the efficient direct reduction of CO₂ at low concentrations to formate using Carbonic Anhydrase co-immobilized with Formate Dehydrogenase in a mesoporous indium tin oxide electrode is described.

Abstract

The electrolysis of dilute CO₂ streams suffers from low concentrations of dissolved substrate and its rapid depletion at the electrolyte-electrocatalyst interface. These limitations require first energy-intensive CO₂ capture and concentration, before electrolyzers can achieve acceptable performances. For direct electrocatalytic CO₂ reduction from low-concentration sources, we introduce a strategy that mimics the carboxysome in cyanobacteria by utilizing microcompartments with nanoconfined enzymes in a porous electrode. A carbonic anhydrase accelerates CO₂ hydration kinetics and minimizes substrate depletion by making all dissolved carbon available for utilization, while a highly efficient formate dehydrogenase reduces CO₂ cleanly to formate; down to even atmospheric concentrations of CO₂. This bio-inspired concept demonstrates that the carboxysome provides a viable blueprint for the reduction of low-concentration CO₂ streams to chemicals by using all forms of dissolved carbon.