R.B. Leveson-Gower

Benzolactone enamides, a number of which incorporate a methylated oxime moiety, are produced by a range of organisms, and constitute a family of cytotoxic natural products. Here, we determine how this capped oxime group is installed during assembly of the model polyketide lobatamide by a modular trans-AT polyketide synthase and provide molecular insight into the responsible mono-oxygenase domain by X-ray crystallography.

Abstract

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

Fusicoccane diterpenoids display intriguing biological activities, including the ability to act as molecular glue modulators of 14-3-3 protein–protein interaction. However, their innate structural complexity and diverse oxygenation patterns present enormous synthetic challenges. Here, a modular chemoenzymatic approach to this natural product family that combines de novo skeletal construction and late-stage hybrid C–H oxidations is presented. A convergent fragment coupling strategy allowed rapid access to a key tricyclic intermediate, which was subjected to chemical and enzymatic C–H oxidations to modularly prepare five oxidized family members. Complementarily, a biomimetic skeletal remodeling was conceived to render five rearranged fusicoccanes with unusual bridgehead double bonds synthetically accessible for the first time.

We demonstrate how it is possible to design a modular programmable inert-atmosphere Schlenkputer (Schlenk-line-computer) for the synthesis and manipulation of the most highly reactive compounds including those which are air- and moisture sensitive or pyrophoric. To do this we have designed and built a programmable Schlenk Line using the Chemputer architecture for the inertization of glassware which can achieve a vacuum line pressure of 1.5 10^-3 mbar and integrated a range of automated Schlenk glassware for the handling, storage, and isolation of reactive compounds at sub ppm levels of O2 and H2O. Utilising this hardware in conjunction with our platform has allowed the automation of a range of common organometallic reaction types for the synthesis of four highly reactive compounds from across the periodic table: [Cp2TiIII(MeCN)2]+ , CeIII{N(SiMe3)2}3, B(C6F5)3 and {DippNacNacMgI}2 which are variously sensitive to temperature, pressure, water and oxygen. Automated purification by crystallisation, filtration and sublimation are each demonstrated along with analysis using inline NMR or reaction sampling for UV/Vis. Finally, we demonstrate automated ultra-low temperature reactivity, down to −90 °C as well as safe handling and quenching of alkali metal reagents, using dynamic feedback from an in-situ temperature probe.

Introducing a new bioconjugation method by employing aqueous 1,3-dipolar cycloaddition reaction between nitrile oxide and dehydroalanine is shown. This approach facilitates a mild and fast protein modification without disruption of protein function. In addition, this can be applicable to the installation of a fluorescent molecule on the protein.

Abstract

Here we describe a novel catalyst-free 1,3-dipolar cycloaddition bioconjugation approach for chemical modification of proteins. The dehydroalanine (Dha)-containing protein reacts with nitrile oxides generated in situ through 1,3-dipolar cycloaddition in fully aqueous-buffered systems. This leads to the formation of a new isoxazoline ring at a pre-defined site (Dha) of the protein. Furthermore, the 1-pyrene isoxazoline-installed annexin V acts as a fluorescent probe, which successfully labels the outer cellular membranes of human cholangiocarcinoma (HuCCA-1) cells for detection of apoptosis.

A new series of easily synthesized, air and moisture stable compounds called bispyrrolidine diboronates (BPDB) is reported. Under Lewis acid activation, they can catalyze highly exo-selective and enantioselective Diels–Alder reactions. For diactivated dienophiles, BPDB can also selectively activate one of the two carbonyl groups based on steric effect, resulting in highly regioselective asymmetric Diels–Alder reactions.

Abstract

A highly enantioselective catalytic system for exo-Diels–Alder reactions was developed based on the newly discovered bispyrrolidine diboronates (BPDB). Activated by various Lewis or Brønsted acids, BPDB can catalyze highly stereoselective asymmetric exo-Diels–Alder reactions of monocarbonyl-based dienophiles. When 1,2-dicarbonyl-based dienophiles are used, the catalyst can sterically distinguish between the two binding sites, which leads to highly regioselective asymmetric Diels–Alder reactions. BPDB can be prepared as crystalline solids on a large scale and are stable under ambient condition. Single-crystal X-ray analysis of the structure for acid-activated BPDB indicated that its activation involves cleavage of a labile B←N bond.

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.

Decades of antibiotic misuse have led to alarming levels of antimicrobial resistance, and the development of alternative diagnostic and therapeutic strategies to delineate and treat infections is a global priority. In particular, the nosocomial, multi-drug resistant “ESKAPE” pathogens such as Gram-positive methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus spp (VRE) urgently require alternative treatments. Here, we developed light-activated molecules, based on conjugation of the FDA-approved photosensitizer riboflavin to the Gram-positive specific ligand vancomycin, to enable targeted antimicrobial photodynamic therapy. The riboflavin-vancomycin conjugate proved to be a potent and versatile antibacterial agent, enabling the rapid, light-mediated, killing of MRSA and VRE with no significant off-target effects. The attachment of riboflavin on vancomycin also led to an increased in antibiotic activity against S. aureus and VRE. Simultaneously, we evidenced for the first time that the flavin sub-unit undergoes an efficient photo-induced bond cleavage reaction to release vancomycin, thereby acting as a photo-removable protecting group for drug-delivery.

Science, Volume 380, Issue 6646, Page 706-712, May 2023.

Pt(II)−NHC complexes are used in alkene hydrosilylation reactions. Some of the examined compounds display excellent catalytic activity, outperforming Pt(0)−NHC pre-catalysts. Our study explores the catalyst structure-activity relationship and provides new mechanistic insights into this industrially important transformation. A sustainable protocol, featuring efficient platinum removal, allows us to access a series of organosilanes in very good to excellent yields.

Abstract

Herein, we report the catalytic activity of a series of platinum(II) pre-catalysts, bearing N-heterocyclic carbene (NHC) ligands, in the alkene hydrosilylation reaction. Their structural and electronic properties are fully investigated using X-ray diffraction analysis and nuclear magnetic resonance spectroscopy (NMR). Next, our study presents a structure-activity relationship within this group of pre-catalysts and gives mechanistic insights into the catalyst activation step. An exceptional catalytic performance of one of the complexes is observed, reaching a turnover number (TON) of 970 000 and a turnover frequency (TOF) of 40 417 h⁻¹ at 1 ppm catalyst loading. Finally, an attractive solvent-free and open-to-air alkene hydrosilylation protocol, featuring efficient platinum removal (reduction of residual Pt from 582 ppm to 5.8 ppm), is disclosed.

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.

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.