R.B. Leveson-Gower

We report on a readily available indole thiolate organocatalyst that, upon excitation with light, can activate via single electron reduction a wide variety of typically inert electron-rich substrates, including strong C−F, C−Cl, and C−O bonds in both aromatic and aliphatic substrates. The protocol is also useful for the borylation and phosphorylation of inert substrates, exhibiting high functional group tolerance.

Abstract

Due to their strong covalent bonds and low reduction potentials, activating inert substrates is challenging. Recent advances in photoredox catalysis offered a number of solutions, each of which useful for activating specific inert bonds. Developing a general catalytic platform that can consistently target a broad range of inert substrates would be synthetically useful. Herein, we report a readily available indole thiolate organocatalyst that, upon excitation with 405 nm light, acquires a strongly reducing power. This excited-state reactivity served to activate, by single-electron reduction, strong C−F, C−Cl, and C−O bonds in both aromatic and aliphatic substrates. This catalytic platform was versatile enough to promote the reduction of generally recalcitrant electron-rich substrates (E_red<−3.0 V vs SCE), including arenes that afforded 1,4-cyclohexadienes. The protocol was also useful for the borylation and phosphorylation of inert substrates with a high functional group tolerance. Mechanistic studies identified an excited-state thiolate anion as responsible of the highly reducing reactivity.

Protein tyrosine phosphatases are crucial regulators of cellular signaling. Their activity is regulated by the motion of a conserved loop, the WPD-loop, from a catalytically inactive open to a catalytically active closed conformation. WPD-loop motion optimally positions a catalytically critical residue into the active site, and is directly linked to the turnover number of these enzymes. Crystal structures of chimeric PTPs constructed by grafting parts of the WPD-loop sequence of PTP1B onto the scaffold of YopH showed WPD-loops in a wide-open conformation never previously observed in either parent enzyme. This wide-open conformation has, however, been observed upon binding of small molecule inhibitors to other PTPs, suggesting the potential of targeting it for drug discovery efforts. Here, we have performed simulations of both enzymes and show that there are negligible energetic differences in the chemical step of catalysis, but significant differences in the dynamical properties of the WPD-loop. Detailed interaction network analysis provides insight into the molecular basis for this population shift to a wide-open conformation. Taken together, our study provides insight into the links between loop dynamics and chemistry in these YopH variants specifically, and how WPD-loop dynamic can be engineered through modification of the internal protein interaction network.

Science, Volume 380, Issue 6650, Page 1150-1154, June 2023.

Science, Volume 380, Issue 6650, Page 1188-1192, June 2023.

Diels-Alderases perform an essential step in the biosynthesis of bioactive spirotetronates. To expand the understanding of such enzymes a cyclase library was created, which made it possible to identify a novel spirotetronate cyclase from a metagenome mining approach. Structural elucidation of both the enzyme by X-ray crystallography and the product by NMR helped us to gain further insights into the essential features of how these enzymes perform complex cyclisations.

Abstract

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.

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.

Enzymes can offer an enticing tool for building complex chemical scaffolds through succinct routes and under mild conditions. Yet, the common application of biocatalysts in organic synthesis is often hampered by unpredictable substrate scope and scalability challenges, deterring the planning of biocatalytic steps at the retrosynthetic planning stage. Herein, we detail a method using a sequence similarity network to curate a library of non-heme iron (NHI)-dependent enzymes capable of performing complexity generating biocatalytic transformations. In the course of this study, we probed the substrate scope of TropC-like enzymes to furnish a range of beta-hydroxytropolone products. The potential to access diverse scaffolds was investigated and a variety of tropolone-containing molecules were prepared on milligram-scale. Furthermore, chemoenzymatically generated tropolones were transformed through a variety of chemistries to achieve the total synthesis of stipitaldehyde, an abbreviated formal synthesis of deoxyepolone B, and additional tropolone building blocks with a high density of functional handles. This work lays the foundation for using NHI enzymes in retrosynthetic planning of complex molecules and natural product analogues.

Nature Chemistry, Published online: 12 June 2023; doi:10.1038/s41557-023-01235-9

The metal-dependent, bifunctional isoprenyl diphosphate synthase PcIDS1 from the leaf beetle Phaedon cochleariae integrates substrate, product and metal-ion concentrations to tune its dynamic reactivity. Now structural and functional analyses reveal that this enzyme uses both catalytic centres to form geranyl pyrophosphate, while one domain is inactivated during farnesyl pyrophosphate production.

Nature Catalysis, Published online: 08 June 2023; doi:10.1038/s41929-023-00963-y

Pyridoxal 5′-phosphate (PLP)-dependent enzymes that catalyse Mannich reactions were unknown. Now, it is reported that the PLP-dependent enzyme LolT catalyses a 5-endo-trig Mannich cyclization reaction during the pyrrolizidine core scaffold formation in loline biosynthesis, and its crystal structure is solved.

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.