R.B. Leveson-Gower

Antimicrobial resistance is an urgent global public health problem that has made the search for new antibiotics essential. Ribosomally synthesized and post-translationally modified peptides are a promising new class of antibiotics and in this work, we report site-selective modification of their dehydroamino acids by β-amination in order to increase water solubility: the singly modified thiopeptide Thiostrepton showed an increase up to 35-fold and minimum inhibitory concentration tests demonstrated that the antimicrobial activity was still good, albeit lower than the natural peptide.

Abstract

We report the efficient and site selective modification of non-canonical dehydroamino acids in ribosomally synthesized and post-transationally modified peptides (RiPPs) by β-amination. The singly modified thiopeptide Thiostrepton showed an up to 35-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity remained good, albeit lower than the unmodified peptide. Also the lanthipeptide nisin could be modified using this method.

Unnatural amino acids, and their synthesis via the late-stage functionalization (LSF) of peptides, play a crucial role in areas such as drug design and discovery. Historically, the LSF of biomolecules has predominantly utilized traditional synthetic methodologies that exploit nucleophilic residues, such as cysteine, lysine or tyrosine. In this study, we present a photocatalytic hydroarylation process targeting the electrophilic residue dehydroalanine (Dha). This residue possesses an α,β-unsaturated moiety and can be combined with various arylthianthrenium salts, both in batch and flow reactors. Notably, the flow setup proved instrumental for efficient scale-up, paving the way for the synthesis of unnatural amino acids and peptides in substantial quantities. Our photocatalytic approach, being inherently mild, permits the diversification of peptides even when they contain sensitive functional groups. The readily available arylthianthrenium salts facilitate the seamless integration of Dha-infused peptides with a wide range of arenes, drug blueprints, and natural products, culminating in the creation of unconventional phenylalanine derivatives. The synergistic effect of the high functional group tolerance and the modular characteristic of the aryl electrophile enables efficient peptide conjugation and ligation in both batch and flow conditions.

Nature Chemistry, Published online: 13 February 2024; doi:10.1038/s41557-023-01435-3

The use of biocatalysis to support early-stage drug discovery campaigns remains largely untapped. Here, engineered biocatalysts enable the synthesis of sp3-rich polycyclic compounds through an intramolecular cyclopropanation of benzothiophenes, affording a class of complex scaffolds potentially useful for fragment-based drug discovery campaigns.

Nature Communications, Published online: 09 February 2024; doi:10.1038/s41467-024-45523-5

Polyamides (PAs) or nylons are types of plastics with wide applications, but due to their accumulation in the environment, strategies for their deconstruction are of interest. Here, the authors screen 40 potential nylon-hydrolyzing enzymes (nylonases) using a mass spectrometry-based approach and identify a thermostabilized N-terminal nucleophile hydrolase as the most promising for further development, as well as crucial targets for progressing PA6 enzymatic depolymerization.

A novel promiscuous activity of the phenolic acid decarboxylase from Bacillus subtilis and other CO₂-binding enzymes is described. The enzymes catalyze the photocatalytic CO₂ reduction in the presence of a Ru(bpy)₃ photosensitizer, exhibiting high selectivity for CO₂ reduction over proton reduction (up to 93 %). Remarkably, BsPAD itself does not require an additional metal cofactor for this catalysis.

Abstract

Novel concepts to utilize carbon dioxide are required to reach a circular carbon economy and minimize environmental issues. To achieve these goals, photo-, electro-, thermal-, and biocatalysis are key tools to realize this, preferentially in aqueous solutions. Nevertheless, catalytic systems that operate efficiently in water are scarce. Here, we present a general strategy for the identification of enzymes suitable for CO₂ reduction based on structural analysis for potential carbon dioxide binding sites and subsequent mutations. We discovered that the phenolic acid decarboxylase from Bacillus subtilis (BsPAD) promotes the aqueous photocatalytic CO₂ reduction selectively to carbon monoxide in the presence of a ruthenium photosensitizer and sodium ascorbate. With engineered variants of BsPAD, TONs of up to 978 and selectivities of up to 93 % (favoring the desired CO over H₂ generation) were achieved. Mutating the active site region of BsPAD further improved turnover numbers for CO generation. This also revealed that electron transfer is rate-limiting and occurs via multistep tunneling. The generality of this approach was proven by using eight other enzymes, all showing the desired activity underlining that a range of proteins is capable of photocatalytic CO₂ reduction.

The stereocontrolled synthesis of stereogenic-at-phosphorus compounds is a long-standing challenge in organic chemistry that has received heightened research attention in recent years. None of the catalytic approaches taken to date have leveraged the rich manifold of transformations proceeding through nucleophilic dealkylation of phosphonium ion intermediates (e.g. Michaelis–Arbuzov, Pudovik, and Appel reactions). Here, we report enantioselective hydrogen-bond-donor-catalyzed Michaelis–Arbuzov reactions of dialkylphosphonites with hydrogen chloride to afford H-phosphinates, which are versatile P-chiral building blocks. Mechanistic and computational investigations reveal that the catalyst diminishes the reactivity of the chloride nucleophile, yet accelerates the rate-determining dealkylation step by preorganizing the phosphonium chloride resting state into a geometry that is primed to enter the SN2 transition state.

Genetically encoded cyclic peptide libraries are invaluable for peptide drug discovery. Here we report an enzymatic strategy for asparaginyl endopeptidase-mediated peptide ligation and cyclization, and its application in the construction of phage-displayed cyclic peptide libraries. Introduction of a low-reactive chloroacetyl group into the tripeptide recognition sequence of OaAEP1 allows intramolecular cyclization with Cys residues to generate macrocyclic peptides. By optimzing OaAEP1 activation conditions and OaAEP1-catalyzed peptide ligation, we establish an efficient OaAEP1-based enzymatic peptide ligation under acidic conditions. The OaAEP1-based enzymatic ligation is fully compatible with phage display and enables the construction of genetically encoded monocyclic and bicyclic peptide libraries. By using OaAEP1-based phage display, we identify macrocyclic peptide ligands targeting TEAD4 at the nanomolar level. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM,16-fold lower than its linear analogue, indicating the contribution of the bicyclic scaffold to its biological activity and demonstrating the utility of the technology platform in the discovery of high-affinity cyclic peptide ligands.

A re-examination of recent claims of room temperature colossal superparamagnetic behaviour of a material based on graphene oxide (GO) modified by well-known aminoferrocene has been undertaken. The synthetic claims of the well-known and commercially available aminoferrocene do not bear scrutiny, neither from new data obtained nor from the data presented in the original paper. The density functional theory-derived model developed to describe the apparent magnetic properties of the material is based on an artefact that occurs through poor choice of the starting model for the GO.

A simple protocol is outlined herein for rapid access to enantiopure unnatural amino acids from trivial glutamate and aspartate precursors. The method relies on Ag/Ni-electrocatalytic decarboxylative coupling and can be rapidly conducted in parallel (24 reactions at a time) to ascertain coupling viability followed by scale-up for the generation of useful quantities of UAAs for exploratory studies.

The transformation of pyridines into benzene derivatives is described, using a one-pot ANRORC process with soft nucleophiles such as malonate. Triflic anhydride activates the pyridine to ANRORC synthesis of a carbocyclic β-aminoester intermediate, which aromatizes on heating. The reaction has been exemplified with a room temperature protocol, along with direct syntheses of biologi-cally active, tertiary-alkylated and isotopically-labelled benzoates.

Bicyclic peptides are a powerful modality for the engagement of challenging drug targets such as protein-protein interactions. The most common crosslinkers used to generate bicyclic peptides are C3-symmetrical, with evenly positioned peptide loops facing radially outwards from a linker core to favour globular conformations. In contrast, linkers with alternative symmetries can potentially provide access to a more diverse conformational landscape of bicyclic peptides. Here, we use 1,2,3-tris(bromomethyl) benzene (1,2,3-TBMB) to access bicyclic peptides with multiple isomeric configurations, leading to conformations that differ substantially from both the parent linear peptides and the conventional bicyclization products formed with 1,3,5-TBMB, as observed in 2D NMR and CD experiments. Bicyclization at cysteine residues proceeds efficiently under standard aqueous buffer conditions, with broad substrate scope, compatibility with high-throughput screening, and clean conversion (>90%) of linear precursors to bicyclic products for 88 of the 106 diverse peptide sequences tested. We envisage that the 1,2,3-TBMB linker will be applicable to a variety of peptide screening techniques, thereby enabling the discovery of unconventional bicyclic peptides that can engage a broad range of novel drug targets.

Nature Chemical Biology, Published online: 02 February 2024; doi:10.1038/s41589-023-01532-x

Nonribosomal peptide synthetases produce valuable natural products but are challenging to engineer. Yeast surface display and fluorescence-activated cell sorting have now been combined to reprogram a condensation domain to recognize a noncanonical lipid substrate. This methodology may facilitate molecular tailoring of many biosynthetic assembly lines.

Nature Catalysis, Published online: 29 January 2024; doi:10.1038/s41929-023-01099-9

Chiral BINOL-phosphates have qualified as privileged Brønsted acid organocatalysts, providing solutions to many challenging enantioselective transformations for a wide range of substrates under mild reaction conditions. Here we revisit the story of their origins.

Modular type I polyketide synthases (PKSs) comprise a family of enzymes that synthesize a diverse class of natural products with medicinal applications. The biochemical features of these systems include the extension and processing of polyketide chains in a stepwise, stereospecific manner, organized by a series of modules divided into distinct catalytic domains. Previous work revealed that a primary hurdle for utilizing PKS modules to create diverse macrolactones hinges on the selectivity of the thioesterase (TE) domain. Herein, we generated a novel hybrid 12-membered macrolactone/lactam ring system employing an unnatural amide hexaketide intermediate in conjunction with an engineered TE S148C mutant from the pikromycin (Pik) biosynthetic pathway. This unnatural macrocycle was initially formed in severely attenuated yields compared to the native product generated from the natural hexaketide substrate. A step-wise directed evolution campaign generated Pik TE variants with enhanced selectivity for macrocycle formation over hydrolysis. Over three rounds of evolution, a series of mutant Pik TE proteins were identified, and further combinations of beneficial mutations carried from each round produced a composite variant with six-fold enhanced isolated yield of the hybrid macrocycle compared to the parent TE enzyme. This study offers new insights into the range of amino acid residues, both proximal and distal to the active site that impart improved selectivity and yield against the unnatural polyketide substrate and overcoming a key PKS pathway gatekeeper.

Carbocation-based rearrangements are vital for terpene versatility. AacSHC triterpene cyclase from Alicyclobacillus acidocaldarius acted as a promiscuous catalyst for multiple monoterpene rearrangements. Tailored AacSHC variants directed the specific isomerization of pinene with exceptional selectivities. Success involved restructuring the aromatic active pocket and reorganizing water clusters to stabilize demanding carbocationic intermediates.

Abstract

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-β-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

Alpha-ketoglutarate-dependent dioxygenases (αKGDs) are promising biocatalysts for selective oxidations, but their practical use is limited by substrate scope and stability. We engineered an αKGD to hydroxylate a non-native substrate with total turnovers exceeding those of reported αKGDs. The engineered αKGD enabled direct synthesis of a hydroxy-indanone to replace the previous five step synthesis en route to the oncology treatment belzutifan.

Abstract

Biocatalytic oxidations are an emerging technology for selective C−H bond activation. While promising for a range of selective oxidations, practical use of enzymes catalyzing aerobic hydroxylation is presently limited by their substrate scope and stability under industrially relevant conditions. Here, we report the engineering and practical application of a non-heme iron and α-ketoglutarate-dependent dioxygenase for the direct stereo- and regio-selective hydroxylation of a non-native fluoroindanone en route to the oncology treatment belzutifan, replacing a five-step chemical synthesis with a direct enantioselective hydroxylation. Mechanistic studies indicated that formation of the desired product was limited by enzyme stability and product overoxidation, with these properties subsequently improved by directed evolution, yielding a biocatalyst capable of >15,000 total turnovers. Highlighting the industrial utility of this biocatalyst, the high-yielding, green, and efficient oxidation was demonstrated at kilogram scale for the synthesis of belzutifan.

The single component flavin-dependent halogenase AetF halogenates a range of 1,1-disubstituted styrenes, often with high stereoselectivity, and AetF and homologues of this enzyme also halogenate terminal alkynes. These findings expand the scope of FDH catalysis, and mutagenesis studies and deuterium kinetic isotope effects provide insight into the unique utility of single component FDHs for biocatalysis.

Abstract

Single component flavin-dependent halogenases (FDHs) possess both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site-selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C−H halogenation. We find that AetF catalyzes halogenation of a range of 1,1-disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross-coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1′-disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis.

Science, Volume 383, Issue 6681, Page 421-426, January 2024.

Science, Volume 383, Issue 6681, Page 438-443, January 2024.