R.B. Leveson-Gower

Rieske oxygenases (ROs) are exciting enzymes exhibiting an outstanding broad reaction scope. The driving force behind these reactivities is the activation of molecular oxygen via a multicomponent electron transfer system. Because of enzyme instability and complexity, ROs are commonly restricted to whole-cell applications. This study aims to evaluate the catalytic performance of ROs in a novel cell-free reaction setup.

Abstract

Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio- and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.

Achieving substrate-selectivity is a central element of nature’s approach to synthesis; relying on the ability of a catalyst to discriminate, based on small structural changes, which molecules will move forward in a synthesis. This approach can be challenging to duplicate in the laboratory, but can be powerful when realized. In this work, substrate-selective catalysis is leveraged to discriminate between two intermediates that exist in equilibrium, subsequently directing the final cyclization to arrive at either the linear or angular tricyclic core common to subsets of azaphilone natural products. By using a flavin-dependent monooxygenase (FDMO) in sequence with an acyl transferase (AT), the conversion of several orcinaldehyde substrates directly to the corresponding linear tricyclic azaphilones in a single reaction vessel was achieved. Furthermore, mechanistic studies support that a substrate equilibrium together with enzyme substrate-selectivity play an import role in the selectivity of the final cyclization step. A panel of azaphilone natural products and derivatives thereof were synthesized using this strategy.

Nature, Published online: 31 May 2023; doi:10.1038/s41586-023-05945-5

A study biochemically and structurally characterizes a lanmodulin from Hansschlegelia quercus with an oligomeric state sensitive to rare-earth ionic radius.

Science Advances, Volume 9, Issue 22, May 2023.

Discovery of new to nature ‘de novo’ macrocyclic peptides has been greatly facilitated by the integration of genetic recoding approaches with peptide display technologies. Perhaps most important among the changes that can made to a peptide to allow its use in a biological setting is macrocyclisation, which has beneficial impacts on target affinity, selectivity, stability, and cell permeability. However, introducing macrocyclisation into a linear sequence is unlikely to be successful unless the sequence is already primed to adpot an appropriate conformation. As a result it is important to include cyclisation already at the discovery stage, meaning there is a need for more diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transfering from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, also allowing easy access to spontaneously forming bicyclic peptides, and so is a valuable addition to the mRNA display toolbox.

A concise and chemoenzymatic synthesis of cyctetryptomycin A and B (1-2) using a Zr-catalyst and CttpC is reported. The recent discovery of cyctetryptomycin A and B (1-2) and their intriguing neuroprotective activities necessitated scalable synthesis to facilitate structure-activ-ity relationship studies. However, the synthesis of cyctetryptomycin A and B (1-2) solely by chemical reactions has proven to be impractical. Therefore, a novel approach using a short and chemoenzymatic strategy was developed to prepare cyctetryptomycin A and B (1-2) via Zr-catalyzed construction of two consecutive quaternary chiral carbons and CttpC-catalyzed direct oxidative coupling of the tryptophan moiety.

Computational modelling was employed to rationally guide protein engineering toward controlling the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the fleeting carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate.

Abstract

We report a computationally driven approach to access enantiodivergent enzymatic carbene N−H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. This H-bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselective N-nucleophilic attack by the amine substrate. By combining MD simulations and site-saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineered S-selective P411 enzymes. The resulting variant, L5_FL-B3, accepts a broad scope of amine substrates for N−H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93 er).

Nature Chemistry, Published online: 01 June 2023; doi:10.1038/s41557-023-01224-y

Ribosomal incorporation of non-α-amino acid monomers into proteins is largely restricted to in vitro translation. Now, pyrrolysyl-transfer RNA synthetase variants have been shown to acylate tRNAs with α-thio acids, malonic acids, and N-formyl amino acids. This work represents a key step towards the programmed ribosomal synthesis of sequence-defined non-protein polymers in cellulo.

Joining ribosomal subunits with a small RNA linker has recently given rise to tethered ribosomes. Their ability to process orthogonal mRNAs (o-mRNA) independent of endogenous ribosomes allowed the identification of otherwise lethal, gain-of-function mutations. Thus, engineering tethered ribosomes promises to significantly expand the scope of building blocks amenable to translation and, ultimately, enable the sequence-selective synthesis of tailor-made (bio)polymers.

Abstract

The ribosome is the core element of the translational apparatus and displays unrivaled fidelity and efficiency in the synthesis of long polymers with defined sequences and diverse compositions. Repurposing ribosomes for the assembly of nonproteinogenic (bio)polymers is an enticing prospect with implications for fundamental science, bioengineering and synthetic biology alike. Here, we review tethered ribosomes, which feature inseparable large and small subunits that can be evolved for novel function without interfering with native translation. Following a tutorial summary of ribosome structure, function, and biogenesis, we introduce design and optimization strategies for the creation of orthogonal and tethered ribosomes. We also highlight studies, in which (rational) engineering efforts of these designer ribosomes enabled the evolution of new functions. Lastly, we discuss future prospects and challenges that remain for the ribosomal synthesis of tailor-made (bio)polymers.

Cooperative chemoenzymatic catalysis has emerged as a uniquely powerful strategy for the synthesis of value-added small molecules and materials. This review will present a selection of recent notable accomplishments in this research area followed by a brief perspective on its promising future.

Abstract

The application of enzymes in synthetic organic chemistry has emerged as a powerful means to generate molecular complexity in a highly selective, efficient, and sustainable manner. While enzymes have increasingly been incorporated into synthetic sequences for numerous academic and industrial applications on their own and in sequential processes, their utility in cooperative catalysis with small molecule catalytic platforms has recently drawn increased attention across the field of organic synthesis. In this review, we present a selection of notable accomplishments in cooperative chemoenzymatic catalysis and provide a perspective on its future directions.

A novel and sustainable tandem catalysis system for asymmetric synthesis is disclosed, which is fabricated by bio-inspired self-assembly of artificial arthropod exoskeletons (AAEs) or fungi cell walls (AFCWs) containing two different types of catalysts (enzyme and metal nanoparticles). The heterogeneous AAE/AFCW systems, which contain chitosan as the main structural component, co-catalyze dynamic kinetic resolution of primary amines via a tandem racemization/enantioselective amidation reaction process to give the corresponding amides in high yields and excellent ee. The heterogeneous AAE/AFCW systems can catalyze multiple reaction cycles without metal leaching. The use of natural-based and biocompatible structural components makes the AAE/AFCW systems fully biodegradable and renewable fulfilling important green-chemistry requirements.

Abstract

A novel and sustainable tandem-catalysis system for asymmetric synthesis is disclosed, which is fabricated by bio-inspired self-assembly of artificial arthropod exoskeletons (AAEs) or artificial fungi cell walls (AFCWs) containing two different types of catalysts (enzyme and metal nanoparticles). The heterogeneous integrated enzyme/metal nanoparticle AAE/AFCW systems, which contain chitosan as the main structural component, co-catalyze dynamic kinetic resolution of primary amines via a tandem racemization/enantioselective amidation reaction process to give the corresponding amides in high yields and excellent ee. The heterogeneous AAE/AFCW systems display successful heterogeneous synergistic catalysis at the surfaces since they can catalyze multiple reaction cycles without metal leaching. The use of natural-based and biocompatible structural components makes the AAE/AFCW systems fully biodegradable and renewable, thus fulfilling important green chemistry requirements.

Nature Reviews Chemistry, Published online: 24 May 2023; doi:10.1038/s41570-023-00495-w

Enzyme conformational plasticity plays an important part in expanding the functional diversity of a limited repertoire of sequences. This Review discusses the role of flexible loops in enzyme evolution, focusing on both examples from natural evolution and engineering success stories.

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.

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.