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.

Nitrogenase catalyzes the multi-electron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxi-dation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, βSer188, was mutated to a non-ligating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av βSer188Ala MoFeP) could still grow and fix nitrogen as quickly as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that βSer188 plays a role in protecting the P-cluster under non-ideal conditions. Here, we investigated the protective role of βSer188 both in vivo and in vitro by characterizing the ability of Av βSer188Ala cells to grow under subop-timal conditions (high oxidative stress or Fe limitation) and by characterizing the ability of Av βSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that βSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) protects the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the ex-panding functional roles of non-Cys ligands to FeS clusters.

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.

Bioremediation. The discovery of MG8, an efficient polyethylene terephthalate (PET) hydrolase enzyme from the human saliva metagenome, is reported by Worawan Bhanthumnavin, Chayasith Uttamapinant et al. in their Research Article (e202203061). Aside from its plastic degradation capability, MG8 was further engineered via genetic code expansion into a covalent binder of PET plastic and can be used to attach protein payloads to PET and other polyesters.

Two analogues of geranylgeranyl diphosphate with shifted double bond were enzymatically converted with twelve diterpene synthases. The double bond shift causes a change of reactivity that results in the formation of many diterpenes with novel skeletons. A total number of 28 new diterpenes is reported and their mechanism of formation is discussed.

Abstract

Two analogues of the diterpene precursor geranylgeranyl diphosphate with shifted double bonds, named iso-GGPP I and iso-GGPP II, were enzymatically converted with twelve diterpene synthases from bacteria, fungi and protists. The changed reactivity in the substrate analogues resulted in the formation of 28 new diterpenes, many of which exhibit novel skeletons.

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.

Nature Chemistry, Published online: 05 September 2022; doi:10.1038/s41557-022-01021-z

Oligonucleotide catalysts such as ribozymes and DNAzymes can cleave RNA efficiently and specifically but are typically dependent on high concentrations of divalent cations, limiting their biological applications. A modular XNAzyme catalyst composed of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) has now been developed that can cleave long (>5 kb), highly structured mRNAs under physiological conditions and enables allele-specific catalytic RNA knockdown inside cells.

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.