R.B. Leveson-Gower

Synergistic catalysis offers the unique possibility of simultaneous activation of both the nucleophile and the electrophile in a reaction. This review discusses developments in aminocatalysis and its synergistic combination with other synthetic platforms published since 2015. Through the four sections, a critical overview of the most common systems involving amino-organo, amino-metal, amino-photoredox and amino-electrocatalysis is provided, with particular emphasis on HOMO-raising and LUMO-lowering strategies and asymmetric transformations. For more details, see the Review by F. Pesciaioli and co-workers (DOI: 10.1002/chem.202200818)

The limonoids have attracted significant attention from the synthetic community owing to their striking structural complexity and medicinal potential. Recent efforts notwithstanding, synthetic access to many intact or ring-D seco limonoids still remains elusive. Here, we report the first de novo synthesis of gedunin, a ring-D seco limonoid with HSP90 inhibitory ac-tivity, that proceeds in thirteen steps. Two enabling features in our strategy are the application of modern catalytic transformations to set the key quaternary centers in the carbocyclic core and the application of site- and chemoselective enzymatic oxidation to establish the requisite oxidation pattern on the A ring. This work lays the foundation for efficient synthetic access to other limonoids and unnatural analogs to facilitate further pharmacological investigation of the family.

Biocatalysis offers many advantages for selective isotopic labelling of valuable small molecules, such as the deuterated amino acids utilised in protein NMR. Until recently, applications of biocatalytic deuteration systems have been restricted by their requirement for a supply of super-stoichiometric quantities of a specifically labelled 2H-pre-cursor, which can be both costly to purchase and complex to prepare. Overcoming this hurdle, we have demonstrated a novel and easy to use H2-driven biocatalytic platform for the incorporation of 2H-atoms across a number of molecular functional groups. By combining the biocatalytic deuteration catalyst with enzymes capable of reductive amination, we synthesised a suite of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. Notably, this strategy enables the introduction of a 15N-label, 2H-label, and chiral centre all in a single-step, and gives rise to amino acid isotopologues on a half gram scale for use directly in the preparation of isotopically labelled proteins. To demonstrate the applicability of the approach in the workflow of protein NMR chemists, we prepared L-[α-2H,15N, β-13C]-alanine and integrated it into a large (> 400 kDa) heat-shock protein, which was subsequently analysable by Methyl-TROSY techniques, revealing new structural information.

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. The high specificity of enzymes can enable one-pot reactions where the substrates, intermediates, and products react only with the intended enzyme. However, this same specificity can hinder the substrate scope of biocatalytic cascades because each constituent enzyme requires complementary activity. Here, we implement a substrate multiplexed screening (SUMS) approach to improve the substrate scope overlap of a two-enzyme cascade via directed evolution. This cascade leverages an L-threonine transaldolase, ObiH, to produce a range of β-OH amino acids that are subsequently decarboxylated to produce chiral 1,2-amino alcohols. Crucially, for the success of this cascade, we engineered a tryptophan decarboxylase to act efficiently on β-OH amino acids while avoiding activity on L-threonine, which is needed for ObiH activity. We leverage this exquisite selectivity with matched substrate scopes to produce a variety of chiral 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides. This route constitutes a new disconnection for the synthesis of β-adrenergic receptor agonists and shows how SUMS can be used to guide the development of promiscuous, C-C bond forming cascades.

Chemoenzymatic cascades offer a simple and efficient way to rapidly build structural complexity. A three-step one-pot process is reported in which a Wittig reaction, chiral-thiourea-mediated asymmetric conjugate addition, and a bioreduction step were combined to access chiral nitro alcohols from commercially available benzaldehyde derivatives in good overall yields and excellent diastereomeric and enantiomeric ratios.

Abstract

The combination of small-molecule catalysis and enzyme catalysis represents an underexploited area of research with huge potential in asymmetric synthetic chemistry due to both compatibility of reaction conditions and complementary reactivity. Herein, we describe the telescopic synthesis of chiral nitro alcohols starting from commercially available benzaldehyde derivatives through the one-pot three-step chemoenzymatic cascade combination of a Wittig reaction, chiral-thiourea-catalysed asymmetric conjugate addition, and ketoreductase-mediated reduction to access the corresponding target compounds in moderate to excellent overall isolated yields (36–80 %) and high diastereomeric and enantiomeric ratios (up to >97 : 3). This represents the first example of the combination of an organocatalysed asymmetric conjugate addition via iminium ion activation and a bioreduction step catalysed by ketoreductases.

Naturally occurring members of the thiopurine methyltransferase family have been found to accept synthetic methyl sulfates or methyl sulfonates as methyl donors for the stereoselective methylation of S-adenosylhomocysteine to form S-adenosylmethionine. This activity can be used for co-substrate regeneration in methyltransferase biocatalysis.

Abstract

Late-stage methylation is a key technology in the development of pharmaceutical compounds. Methyltransferase biocatalysis may provide powerful options to insert methyl groups into complex molecules with high regio- and chemoselectivity. The challenge of a large-scale application of methyltransferases is their dependence on S-adenosylmethionine (SAM) as a stoichiometric, and thus exceedingly expensive co-substrate. As a solution to this problem, we and others have explored the use of methyl halides as reagents for the in situ regeneration of SAM. However, the need to handle volatile electrophiles, such as methyl iodide (MeI), may also hamper applications at scale. As a more practical solution, we have now developed an enzyme-catalyzed process for the regeneration of SAM with methyl toluene sulfonate. Herein, we describe enzymes from the thiopurine methyltransferase family that accept sulfate- and sulfonate-based methyl donors to convert S-adenosylhomocysteine into SAM with efficiencies that rival MeI-based reactions.

A single multifunctional enzyme is reported that can promote biocatalytic cascades based on multiple stereoselective steps. Specifically, a 4-oxalocrotonate tautomerase (4-OT) enzyme can form enamine and iminium ion intermediates from aldehydes and enals to promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Abstract

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Sactipeptides are a class of microbial peptides possessing thioether crosslinks. We demonstrate sactipeptide engineering without compromising their post-translational modification introduced by sactisynthases. A variety of natural and hybrid sactipeptide constructs were generated and analyzed for the presence of thioether crosslinks, demonstrating the possibility to design variants with novel functionalities.

Abstract

Sactipeptides are ribosomally synthesized peptides containing a unique sulfur to α-carbon crosslink. Catalyzed by sactisynthases, this thioether pattern endows sactipeptides with enhanced structural, thermal, and proteolytic stability, which makes them attractive scaffolds for the development of novel biotherapeutics. Herein, we report the in-depth study on the substrate tolerance of the sactisynthase AlbA to catalyze the formation of thioether bridges in sactipeptides. We identified a possible modification site within the sactipeptide subtilosin A allowing for peptide engineering without compromising formation of thioether bridges. A panel of natural and hybrid sactipeptides was produced to study the AlbA-mediated formation of thioether bridges, which were identified mass-spectrometrically. In a proof-of-principle study, we re-engineered subtilosin A to a thioether-bridged, specific streptavidin targeting peptide, opening the door for the functional engineering of sactipeptides.

Communications Chemistry, Published online: 25 August 2022; doi:10.1038/s42004-022-00715-2

Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates, however the substrate selection mechanism is not yet known. Here, the authors investigate the substrate promiscuity of two histidine-incorporating CDPSs to generate an extensive library of products which complement the chemical realm of histidine-containing cyclic dipeptides.

A single multifunctional enzyme is reported that can promote biocatalytic cascades based on multiple stereoselective steps. Specifically, a 4-oxalocrotonate tautomerase (4-OT) enzyme can form enamine and iminium ion intermediates from aldehydes and enals to promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Abstract

Asymmetric catalytic cascade processes offer direct access to complex chiral molecules from simple substrates and in a single step. In biocatalysis, cascades are generally designed by combining multiple enzymes, each catalyzing individual steps of a sequence. Herein, we report a different strategy for biocascades based on a single multifunctional enzyme that can promote multiple stereoselective steps of a domino process by mastering distinct catalytic mechanisms of substrate activation in a sequential way. Specifically, we have used an engineered 4-oxalocrotonate tautomerase (4-OT) enzyme with the ability to form both enamines and iminium ions and combine their mechanisms of catalysis in a complex sequence. This approach allowed us to activate aldehydes and enals toward the synthesis of enantiopure cyclohexene carbaldehydes. The multifunctional 4-OT enzymes could promote both a two-component reaction and a triple cascade characterized by different mechanisms and activation sequences.

Nature Communications, Published online: 20 August 2022; doi:10.1038/s41467-022-32641-1

Epoxide ring opening reactions are important in both biological processes and synthetic applications. Here, the authors show that flavin cofactors can catalyze reductive and oxidative epoxide ring opening reactions and propose the underlying mechanisms.

Nature Communications, Published online: 23 August 2022; doi:10.1038/s41467-022-32710-5

De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build enzymes for efficient energy conversion. Here, the authors develop an artificial photosynthetic reaction center that functions without the need for sacrificial electron donors or acceptors.

In this work, we report a computationally driven approach to access enantiodivergent enzymatic carbene N–H bond insertions catalyzed by P411 enzyme variants. Computational modeling was employed to 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. By combining MD simulations and protein engineering, a reversed (R-selective) P411 enzyme variant, L5_FL-B3, was obtained in a single round of semi-rational directed evolution. L5_FL-B3 accepts a broad scope of amine substrates with excellent yields (up to >99%), high efficiency (up to 12,300 TTN) and good enantiocontrol (up to 7:93 er), which complements the previously engineered S-selective P411-L7_LF variant.

Nature Catalysis, Published online: 11 August 2022; doi:10.1038/s41929-022-00821-3

Enzymes for poly(ethylene terephthalate) (PET) deconstruction are of interest for plastics recycling, but reports on their directed evolution are missing. Now, an automated, high-throughput directed evolution platform is described, affording HotPETase that effectively achieves depolymerization above the glass transition temperature of PET.

Nature, Published online: 03 August 2022; doi:10.1038/s41586-022-05022-3

Biochemical and lipidomic analyses identify an anti-ferroptotic function of vitamin K and reveal ferroptosis suppressor protein 1 (FSP1) as the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle.

Nature, Published online: 11 August 2022; doi:10.1038/s41586-022-05167-1

An Asymmetric sp³–sp³ Cross-Electrophile Coupling Using ‘Ene’-Reductases

YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases. WT YfeX catalyzes N−H insertion (including aliphatic and secondary amines) in high yield, cyclopropanation, and most excitingly, Si−H insertion of dimethylphenylsilane. QM/MM calculations reveal details of the mechanism of the unusual Si−H insertion reaction.

Abstract

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N−H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si−H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si−H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

We present a de novo discovery of an efficient catalyst of the Morita–Baylis–Hillman (MBH) reaction by searching chemical space for molecules that lower the estimated barrier of the rate determining step using a genetic algorithm (GA) starting from randomly selected tertiary amines. We performed five independent GA searches that resulted in 448 unique molecules, for which we were able to locate 435 true transitions states at semiempirical level of theory. The predicted activation energies of all 435 molecules where all lower than that of DABCO, which is a popular catalyst of the MBH reaction. Virtually all the molecules contain an aziridine N as the catalytically active site, which is discovered by the GA since it is either not found in the initial population or discarded early only to be redisovered as the search progresses. Many of the GA searches also introduce a substituent with a hydrogen bond donor that helps to stabilize the transition state and thus lower the barrier. Two molecules are selected for further study based on their synthetic accessibility as predicted by the retrosynthesis package Manifold. For these two molecules we compute the entire free energy reaction profile at the DFT level and show that their rate determining barriers are 1.7 and 2.4 kcal/mol lower than that of DABCO. The molecule with the lowest barrier has higher barriers for the other steps compared to DABCO, but none of the barriers are competitive with the rate-determining barrier and is predicted to outperform DABCO. This demonstrates the power of free exploration of chemical a space compared to more constrained fragment-based approaches.

Nature Catalysis, Published online: 15 August 2022; doi:10.1038/s41929-022-00822-2

His-C2-directed modification is chemically challenging and rarely occurs in nature due to the low reactivity of this position. Now, the prenlytransferase LimF has been discovered and applied for geranylation of histidine-containing peptides and imidazole-containing small molecules, showcasing the versatility of this biocatalyst.