R.B. Leveson-Gower

The chemical transformation of aromatic amino acids has emerged as an attractive alternative to non-selective lysine or cysteine labeling for the modification of biomolecules. However, this strategy has largely been limited by the scope of functional groups and biocompatible reaction conditions available. Herein, we report the implementation of near-infrared-activatable photocatalysts, TTMAPP and n-Pr-DMQA+, capable of generating fluoroalkyl radicals for selective tryptophan functionalization within simple and complex biological systems. At the peptide level, a diverse set of iodo-perfluoroalkyl reagents were used to install bioorthogonal handles for downstream applications or link inter- or intramolecular tryptophan residues for peptide stapling. We also found this photoredox transformation amenable to biotinylation of intracellular proteins in live cells for downstream confocal imaging and mass spectrometry-based analysis. Given the inherent tissue penetrant nature of near-infrared light we further demonstrated the utility of this technology to achieve photocatalytic protein fluoroalkylation in physiologically relevant tissue and tumor environments.

Science Advances, Volume 8, Issue 44, November 2022.

The chiral building block 5-amino-2-hydroxymethyltetrahydropyran 1a has been previously synthesized through a cumbersome 9-step synthesis from tri-O-acetyl-D-glucal, which renders access to nemtabrutinib (2), a BTK inhibitor currently being evaluated for the treatment of various hematologic malignancies, inefficient and wasteful. Herein, we describe the development of a protecting group-free, 2-step synthesis of 1a from Cyrene, a biorenewable feedstock. The improved synthesis involves a biocatalytic transamination reaction of Cyrene to install the desired amine-stereocenter in a single step with high diastereoselectivity. The enzymatic reaction is followed by a stereo-retentive reductive acetal opening reaction of the chiral cyrene amine intermediate 3a to furnish 1a. A mechanistic investigation of the acetal opening reaction is also described which uncovered unprecedented reaction conditions for the in-situ generation of diborane mediated by the sulfolane co-solvent. The streamlined synthesis of 1a from Cyrene resulted in a > 27% yield improvement and a significant reduction in the environmental impact of the synthesis.

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. Whilst there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation has been lacking. Here we describe the first in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modelling and site mutagenesis studies identified three key residues in the catalytic pocket. Moderate resolution map using single-particle cryogenic electron microscopy (CryoEM) reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and was applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide, Fludioxonil.

G-type halohydrin dehalogenases convert 5- and 6-membered cyclic epoxides with a range of small anionic C-, N-, O- and S-nucleophiles.

Abstract

Halohydrin dehalogenases are promiscuous biocatalysts, which enable asymmetric ring opening reactions of epoxides with various anionic nucleophiles. However, despite the increasing interest in such asymmetric transformations, the substrate scope of G-type halohydrin dehalogenases toward cyclic epoxides has remained largely unexplored, even though this subfamily is the only one known to display activity with these sterically demanding substrates. Herein, we report on the exploration of the substrate scope of the two G-type halohydrin dehalogenases HheG and HheG2 and a newly identified, more thermostable member of the family, HheG3, with a variety of sterically demanding cyclic epoxides and anionic nucleophiles. This work shows that, in addition to azide and cyanide, these enzymes facilitate ring-opening reactions with cyanate, thiocyanate, formate, and nitrite, significantly expanding the known repertoire of accessible transformations.

Facile reductions of carboxylic acids to aldehydes or alcohols can be effected under mild conditions upon initial conversion to their corresponding S-2-pyridyl thioesters. Upon treatment with a commercially available and air-stable nickel pre-catalyst and silane as a stoichiometric reductant, aldehydes are formed in moderate to good yields. Alternatively, the 1-pot conversion of acids to their thioester derivatives can be followed by reduction to the alcohol upon treatment with sodium borohydride. A variety of starting materials ranging from highly functionalized acids to educts from the Merck Informer Library can be transformed using these green reaction media.

Radical addition to the dehydroalanine (Dha) residue of a peptide could diversify the peptide sequence with noncanonical residues, but this strategy is currently limited by the lack of control over the stereochemistry. This work addresses this important challenge by applying chiral nickel catalysts to control the stereoselective radical addition to Dha on oligopeptides.

Abstract

Radical addition to dehydroalanine (Dha) represents an appealing, modular strategy to access non-canonical peptide analogues for drug discovery. Prior studies on radical addition to the Dha residue of peptides and proteins have demonstrated outstanding functional group compatibility, but the lack of stereoselectivity has limited the synthetic utility of this approach. Herein, we address this challenge by employing chiral nickel catalysts to control the stereoselectivity of radical addition to Dha on oligopeptides. The conditions accommodate a variety of primary and secondary electrophiles to introduce polyethylene glycol, biotin, halo-tag, and hydrophobic and hydrophilic side chains to the peptide. The reaction features catalyst control to largely override substrate-based control of stereochemical outcome for modification of short peptides. We anticipate that the discovery of chiral nickel complexes that confer catalyst control will allow rapid, late-stage modification of peptides featuring nonnatural sidechains.

Predicting the reaction kinetics, that is, how fast a reaction can happen in a solution, is essential information for many processes, such as industrial chemical manufacturing, refining, synthesis and separation of petroleum products, environmental processes in air and water, biological reactions in cells, biosensing, and drug delivery. Collision theory was originally developed to explain the reaction kinetics of gas reactions with no dilution. For a reaction in a diluted inert gas solution or a diluted liquid solution, diffusion often dominates the collision process. Thus, it is necessary to include diffusion in such a calculation. Traditionally the classical Smoluchowski rate is used as a starting point to predict the collision frequency of two molecules in a diluted solution. In this report, a different collision model is derived from the adsorption of molecules on a flat surface. A surprising result is obtained showing that the reaction order for biomolecular reaction should be 2 and 1/3 instead of 2, following a fractal reaction kinetics.

Late-stage halogenation of peptides has become feasible using a highly flexible halogenase that catalyses bromination of a wide range of amides and peptides. Upon optimization studies, even longer peptides carrying a terminal tryptophan residue were reasonably accepted leading to high conversions and remarkable selectivity. This novel bioorthogonal approach was exemplified by halogenating an RGD peptide derivative in the final step.

Abstract

The late-stage site-selective derivatisation of peptides has many potential applications in structure-activity relationship studies and postsynthetic modification or conjugation of bioactive compounds. The development of orthogonal methods for C−H functionalisation is crucial for such peptide derivatisation. Among them, biocatalytic methods are increasingly attracting attention. Tryptophan halogenases emerged as valuable catalysts to functionalise tryptophan (Trp), while direct enzyme-catalysed halogenation of synthetic peptides is yet unprecedented. Here, it is reported that the Trp 6-halogenase Thal accepts a wide range of amides and peptides containing a Trp moiety. Increasing the sequence length and reaction optimisation made bromination of pentapeptides feasible with good turnovers and a broad sequence scope, while regioselectivity turned out to be sequence dependent. Comparison of X-ray single crystal structures of Thal in complex with d-Trp and a dipeptide revealed a significantly altered binding mode for the peptide. The viability of this bioorthogonal approach was exemplified by halogenation of a cyclic RGD peptide.

Science Advances, Volume 8, Issue 41, October 2022.

Calculations suggest that a coordination switch of the sulfoxide intermediate is involved in the catalysis of ergothioneine synthase (EgtB). This coordination switch from S to O is driven by the S/π nonbonding electrostatic interactions, which efficiently promotes the observed stereoselective C−S bond formation while bypassing cysteine dioxygenation.

Abstract

The non-heme iron ergothioneine synthase (EgtB) is a sulfoxide synthase that catalyzes oxidative C−S bond formation in the synthesis of ergothioneine, which plays roles against oxidative stress in cells. Despite extensive experimental and computational studies of the catalytic mechanisms of EgtB, the root causes for the selective C−S bond formation remain elusive. Using quantum mechanics/molecular mechanics (QM/MM) calculations, we show herein that a coordination switch of the sulfoxide intermediate is involved in the catalysis of the non-heme iron EgtB. This coordination switch from the S to the O atom is driven by the S/π electrostatic interactions, which efficiently promotes the observed stereoselective C−S bond formation while bypassing cysteine dioxygenation. The present mechanism is in agreement with all available experimental data, including regioselectivity, stereoselectivity and KIE results. This match underscores the critical role of coordination switching in the catalysis of non-heme enzymes.

Cyclic amines are ubiquitous structural motifs found in pharmaceuticals and biologically active natural products, making methods for their elaboration via direct C–H functionalization of considerable synthetic value. Herein, we report the development of an iron-based biocatalytic strategy for enantioselective a-C–H functionalization of pyrrolidines via a carbene transfer reaction with diazoacetone. Currently unreported for organometallic catalysts, this transformation can be accomplished in high yields, high catalytic activity and high stereoselectivity (up to 99:1 e.r. and 20,350 TON) using engineered variants of cytochrome P450 CYP119 from Sulfolobus solfataricus. This methodology was further extended to enable enantioselective a-C–H functionalization in the presence of ethyl diazoacetate as carbene donor (up to 89:11 e.r. and 8,920 TON), and the two strategies were combined to achieve a one-pot as well as a tandem dual C–H functionalization of the cyclic amine substrate with enzyme-controlled diastereo- and enantiodivergent selectivity. This biocatalytic approach is amenable to gram-scale synthesis and can be applied to drug scaffolds for late-stage C–H functionalization. This work provides an efficient and tunable method for direct asymmetric a-C–H functionalization of saturated N-heterocycles which should offer new opportunities for the synthesis, discovery, and optimization of bioactive molecules.

The combination of photochemistry with enzyme catalysis offers exciting opportunities to induce new reactivities and to create novel enzymes for reactions other than their native ones. Recently, Hyster and co-workers demonstrated this for a photoenzymatic asymmetric Csp ³−Csp ³ cross-electrophile coupling, a reactivity previously unknown to enzymes.

Abstract

Enzymes have several advantages over conventional catalysts for organic synthesis. Over the last two decades, much effort has been made to further extend the scope of biocatalytic reactions available to synthetic chemists, particularly by expanding the repertoire of enzymes for abiological transformations. In this regard, exciting new developments in the area of photobiocatalysis enable now the introduction of non-natural reactivity in enzymes to solve long-standing synthetic challenges. A recently described example from the Hyster group demonstrates in an unprecedented way how the combination of photochemistry with enzyme catalysis empowers the catalytic asymmetric construction of Csp ³−Csp ³ bonds with high chemo- and enantioselectivity.

Of the different classes of halogenases characterized to date, flavin dependent halogenases (FDHs) are most commonly associated with site-selective halogenation of electron rich arenes and enol(ate) moieties in the biosynthesis of halogenated natural products. This capability has made them attractive biocatalysts, and extensive efforts have been devoted to both discovering and engineering these enzymes for different applications. We have established that engineered FDHs can catalyze different enantioselective halogenation processes, including halolactonization of simple alkenes with a tethered carboxylate nucleophile. In this study, we expand the scope of this reaction to include alcohol nucleophiles and a greater diversity of alkene substitution patterns to access a variety of chiral tetrahydrofurans. We also demonstrate that FDHs can be interfaced with ketoreductases to enable halocyclization using ketone substrates in one-pot cascade reactions and that the halocyclization products can undergo subsequent rearrangements to form novel hydroxylated and halogenated products. Together, these advances expand the utility of FDHs for enantio- and diastereoselective olefin functionalization.

The cepafungins are a class of highly potent and selective eukaryotic proteasome inhibitor natural products with potential to treat refractory multiple myeloma and other cancers. The structure-activity relationship of the cepafungins is not fully understood. This account chronicles the development of a chemoenzymatic approach to cepafungin I. A failed initial route involving derivatization of pipecolic acid prompted us to examine the biosynthetic pathway for the production of 4-hydroxylysine, which culminated in the development of a 9-step synthesis of cepafungin I. An alkyne-tagged analog enabled chemoproteomic studies of cepafungin and comparison of its effects on global protein expression in human multiple myeloma cells to the clinical drug bortezomib. A preliminary series of analogs elucidated critical determinants of potency in proteasome inhibition. Herein we report the chemoenzymatic syntheses of 13 additional analogs of cepafungin I guided by a proteasome-bound crystal structure, 5 of which are more potent than the natural product. Enzymatic strategies enabled the facile synthesis of oxidized amino acids in the macrocycle warhead as well as the tail fragment. Additional analogs were prepared by chemical methods to further explore the SAR at other regions of the scaffold. These studies reveal the criticality of the macrocyclic L-lysine oxidation regio-/stereochemistry introduced in the natural product biosynthesis relative to other possible lysine oxidation patterns found in nature. The lead analog was found to have seven-fold greater proteasome b5 subunit inhibitory activity and has been evaluated against several multiple myeloma and mantle cell lymphoma cell lines in comparison to the clinical drug bortezomib.

The ubiquity of C–H bonds presents an attractive opportunity to elaborate and build complexity in organic molecules. Methods for selective functionalization, however, often must differentiate among multiple chemically similar and, in some cases indistinguishable, C–H bonds within the same molecule. An advantage of enzymes is that they can be finely tuned using directed evolution to achieve control over divergent C–H functionalization pathways. Here, we present engineered enzymes that effect a new-to-nature C–H alkylation (C–H carbene insertion) with unparalleled selectivity: two complementary carbene C–H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an α-cyanocarbene into the α-amino C(sp3)–H bonds or the ortho-arene C(sp2)–H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold (nine mutations, less than 2% of the sequence) were needed to adjust the enzyme’s control over the site-selectivity of cyanomethylation. The X-ray crystal structure of the selective C(sp3)–H alkylase, P411-PFA, reveals an unprecedented helical disruption which alters the shape and electrostatics in the enzyme active site. Overall, this work demonstrates the advantages of using enzymes as C–H functionalization catalysts for divergent molecular derivatization.

Catalytic olefin metathesis can be performed in live microalgae, converting the fatty acids stored in lipid organelles to polymer building blocks and chemicals in vivo.

Abstract

Sustainable sources are key to future chemicals production. Microalgae are promising resources as they fixate carbon dioxide to organic molecules by photosynthesis. Thereby they produce unsaturated fatty acids as established raw materials for the industrial production of chemical building blocks. Although these renewable feedstocks are generated inside cells, their catalytic upgrading to useful products requires in vitro transformations. A synthetic catalysis inside photoautotrophic cells has remained elusive. Here we show that a catalytic conversion of renewable substrates can be realized directly inside living microalgae. Organometallic catalysts remain active inside the cells, enabling in vivo catalytic olefin metathesis as new-to-nature transformation. Stored lipids are converted to long-chain dicarboxylates as valuable building blocks for polymers. This is a key step towards the long-term goal of producing desired renewable chemicals in microalgae as living “cellular factories”.

N-Methylated and -alkylated unsaturated heterocycles are privileged scaffolds in pharmaceuticals that are often tedious to synthesize. Now, promiscuous and engineered enzymes can be used to access such molecules through alkylation with high regioselectivity, high yield and on a preparative scale using simple starting materials.

Abstract

Methods for regioselective N-methylation and -alkylation of unsaturated heterocycles with “off the shelf” reagents are highly sought-after. This reaction could drastically simplify synthesis of privileged bioactive molecules. Here we report engineered and natural methyltransferases for challenging N-(m)ethylation of heterocycles, including benzimidazoles, benzotriazoles, imidazoles and indazoles. The reactions are performed through a cyclic enzyme cascade that consists of two methyltransferases using only iodoalkanes or methyl tosylate as simple reagents. This method enables the selective synthesis of important molecules that are otherwise difficult to access, proceeds with high regioselectivity (r.r. up to >99 %), yield (up to 99 %), on a preparative scale, and with nearly equimolar concentrations of simple starting materials.

“My favorite way to spend a holiday is to do nothing … If I were not a scientist, I would be a school teacher …” Find out more about Apparao Draksharapu in his Introducing … Profile.

Despite the very low acidity of the inert α C−H bonds (pK _a≈42.6), direct asymmetric α-C(sp ³ )−H addition of N-unprotected propargylic amines to trifluoromethyl ketones has been achieved by using a chiral pyridoxal as the carbonyl catalyst, producing a broad variety of chiral alkynyl β-aminoalcohols in high yields with excellent stereoselectivities (up to 84 % yield, >20 : 1 dr, 99 % ee).

Abstract

Direct asymmetric functionalization of the inert α C−H bonds of N-unprotected propargylic amines is a big challenge in organic chemistry, due to the low acidity (pK _a≈42.6) of the α C−H bonds and interruption of the nucleophilic NH₂ group. By using a chiral pyridoxal as carbonyl catalyst, we have successfully realized direct asymmetric α-C−H addition of N-unprotected propargylic amines to trifluoromethyl ketones, producing a broad range of chiral alkynyl β-aminoalcohols in 54–84 % yields with excellent stereoselectivities (up to 20 : 1 dr and 99 % ee). The α C−H bonds of propargylic amines are greatly activated by the pyridoxal catalyst via the formation of an imine intermediate, resulting in the increase of acidity by up to 10²² times (from pK _a 42.6 to pK _a 20.1), which become acidic enough to be deprotonated under mild conditions for the asymmetric addition. This work presented an impressive example for asymmetric functionalization of inert C−H bonds enabled by an organocatalyst.

Polyethylene terephthalate (PET) is the most common polyester plastic in the packaging industry, and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases (PETases), have been found to hydrolyze this polymer at different temperatures, with the enzyme from I. sakaiensis (IsPETase) having optimal catalytic activity at 40ºC. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between 3 conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218 (S/I). When these residues are inserted into other PETases in place of the otherwise strictly conserved His/Phe (H/F) residues found at their respective positions, they enhance activity and decrease Topt. Herein, we combine conventional molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S/I and H/F variants of IsPETase, as well as three other mesophilic and thermophilic PETases, at their respective temperature and pH optima. Our simulations show that the S/I insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved Trp, as well as expanding the conformational plasticity of this Trp side chain, allowing the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PETases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.

Described are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red-light (660 nm) photocatalysis is used to initiate a cascade of DHTz-oxidation, intramolecular Diels-Alder reaction, and elimination to release phenolic compounds. Silarhodamine (SiR) dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially-available conjugates of Hoechst dye (SiR-H) and Taxol (SiR-T) are used to localize SiR to the nucleus and tubulin, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM of SiR and 40 µM of the photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate determining elimination step. In cellular studies, this uncaging process is successful at low concentration of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as photocatalyst and the fluorescent reporter for tubulin depolymerization, and with confocal microscopy it was possible to visualize tubulin depolymerization in real time as the result of photocatalytic uncaging in live cells.