R.B. Leveson-Gower

Using a phylogenetic tree-building approach, five novel tyrosine ammonia lyases (TALs) and three novel phenylalanine/tyrosine ammonia lyases (PTALs) were identified. The enzymes were biochemically characterized and the optimal conditions for a whole cell E. coli biotransformation were determined. Under these conditions p-coumaric acid (p-CA) yields of 2.03 g/L after 8 hours by TAL _clu and 2.35 g/L after 24 hours by TAL _rpc could be achieved.

Abstract

p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TAL _clu ) has the highest affinity (K _m =0.019 mm) and conversion efficiency (k _cat/K _m= 1631 s⁻¹ ⋅ mm ⁻¹) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TAL _clu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TAL _rpc ) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.

Catalytic regioselective C−H silylation of a variety of aromatic heterocycles such as thiophene, furan and indole derivatives with secondary hydrosilanes was achieved by using a calcium catalyst. This protocol provides an efficient route to the synthesis of silylated heteroaromatic compounds without the need for an H₂ acceptor and free of transition metal. Calcium thiophenyl complex (2), proposed as the catalytic reaction intermediate, was isolated and structurally characterized.

Abstract

Treatment of mononuclear calcium hydride complex [(Tp^Ad,iPr)Ca(H)(THP)] (1) (Tp^Ad,iPr=hydrotris(3-adamantyl-5-isopropyl-pyrazolyl)borate, THP=tetrahydropyran) with 2-methylthiophene, 2-methylfuran, and 1-methyl-1H-indole in THF/hexane solution led to the formation of calcium thiophenyl (2), furanyl (3), and indolyl (4) complexes, via sp² C−H bond activation. The reaction of complex 1 with 2-methylpyridine and quinoline afforded calcium benzyl pyridinyl (5) and 1,2-dihydroquinolide (6) complexes, through sp³ C−H bond activation and hydride insertion reaction, respectively. Under mild conditions, the catalytic regioselective sp² C−H silylation of a series of aromatic heterocycles with secondary hydrosilane was achieved by the use of complex 1. This protocol offers a straightforward method for the synthesis of silylated heteroaromatic compounds without a hydrogen acceptor and free of transition metal.

The intrinsic resilience of biofilms to environmental conditions makes them an attractive plat-form for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiolo-gy and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using MC4100, a strain of Escherichia coli that forms bio-films poorly, we demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in this bacterium, with increasingly hydrophobic polymers outperforming less hydro-phobic polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expres-sion of the adhesin curli. In the presence of selected polymers MC4100 can reach levels of bio-mass production and curli expression similar or higher than PHL644, demonstrating that syn-thetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.

Automation has fuelled dramatic advances in fields such as proteomics and genomics (e.g., in preparation of proteins and nucleic acids),1,2 enabling non-experts to prepare, test and analyse complex biological molecules. However, the field of automated organic synthesis lags far behind, partly because of the complexity and variety of organic molecules. As a result, only a handful of relatively simple organic molecules, requiring a small number of synthetic steps, have been made in an automated fashion. Herein, we report an automated assembly-line synthesis that allows iterative, stereocontrolled formation of C(sp3)–C(sp3) bonds with high stereochemical fidelity and reproducibility, enabling access to complex organic molecules even by non-synthesis experts. This was achieved on a commercially available robotic platform capable of handling air sensitive reactants and performing low temperature reactions, which enabled six sequenced one-carbon homologations of organoboron substrates to be performed iteratively without human intervention. Together with other automated functional group manipulations, this methodology has been exploited to rapidly build the core fragment of the natural product (+)-kalkitoxin, thus leading the way towards automated organic synthesis.

Though chiral pool synthesis is widely accepted as a powerful strategy in complex molecule synthesis, the effectiveness of the approach is intimately linked to the range of available chiral building blocks and the functional groups they possess. To date, there is still a pressing need for new remote functionalization methods that would allow the installation of useful chem-ical handles on these building blocks to enable a broader spectrum of synthetic manipulations. Herein, we report the engi-neering of a P450BM3 variant for the regioselective C–H oxidation of sclareol at C6. The synthetic utility of the resulting product was demonstrated in a formal synthesis of ansellone B and the first total synthesis of the 2,3-seco-labdane excolide B.

Organocatalysts meet proteins: Enzymatic activities by natural organocatalytic cofactors, non-natural derivatives of them and artificial cofactors are reviewed.

Abstract

Catalytically active non-metal cofactors in enzymes carry out a variety of different reactions. The efforts to develop derivatives of naturally occurring cofactors such as flavins or pyridoxal phosphate and the advances to design new, non-natural cofactors are reviewed here. We report the status quo for enzymes harboring organocatalysts as derivatives of natural cofactors or as artificial ones and their application in the asymmetric synthesis of various compounds.

A deaminative reductive coupling of amino acid pyridinium salts with aryl bromides has been developed to enable efficient synthesis of noncanonical amino acids and diversification of peptides. This method transforms natural, commercially available lysine, ornithine, diaminobutanoic acid (DAB), and diaminopropanoic acid (DAP) to aryl alanines and homologated derivatives with varying chain lengths. Attractive features include scalability, tolerance of pharma-relevant (hetero)aryls and functional groups, applicability to both monomeric amino acid and short peptide substrates, and compatibility with biorthogonal handles useful for chemical biology. Furthermore, these cross-couplings can be conducted in microscale and nanoscale and are amenable to solid-phase peptide synthesis platforms. The success of this work relied on an academic/industry collaboration and high-throughput experimentation to identify complementary conditions that proved critical for achieving broad scope of aryl bromides and pyridinium substrates.

Deuterated amino acids have been recognized for their utility in drug development, for facilitating NMR analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogendeuterium (H/D) exchange. However, site-selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and UV-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of L-alloIle. We show an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cβ H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cβ-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.

Nature, Published online: 09 March 2022; doi:10.1038/s41586-022-04499-2

Tryptophan depletion, which occurs in tumours, results in in-frame translation across tryptophan-encoding codons by phenylalanine substitution.

Biochemical characterization of HheG-682, a new homolog of halohydrin dehalogenase HheG sharing 46% sequence identity, is presented.

Pericyclases, enzymes that catalyze pericyclic reactions, form an expanding family of enzymes that have biocatalytic utility. Despite the increasing number of pericyclases discovered, the Diels-Alder (DA) cyclization between a cyclopentadiene and an olefinic dienophile to form norbornene, which is among the best-studied cycloadditions in synthetic chemistry, has surprisingly no enzymatic counterpart to date. Here we report the discovery of a pathway featuring a norbornene synthase SdnG for the biosynthesis of sordaricin-the terpene precursor of antifungal natural product sordarin. Full reconstitution of sordaricin biosynthesis revealed a concise oxidative strategy used by Nature to transform an entirely hydrocarbon precursor into the highly functionalized substrate of SdnG for intramolecular Diels-Alder (IMDA) cycloaddition. SdnG generates the norbornene core of sordaricin and accelerates this reaction to suppress host-mediated redox modifications of the activated dienophile. Findings from this work expand the scopes of pericyclase-catalyzed reactions and P450-mediated terpene maturation.

TsrM is a B₁₂-dependent radical SAM enzyme catalyzing methylation reactions from carbon-atoms to nucleophilic atoms making it a unique and versatile alkylating agent. TsrM is able to directly transfer methyl groups on the less reactive carbon atom of the indole ring and to install several methyl groups on its substrate. TsrM has thus unique properties among radical SAM enzymes by notably catalyzing non-radical reactions. (SAM: S-adenosyl-L-methionine).

Abstract

B₁₂-dependent radical SAM enzymes are an emerging enzyme family with approximately 200,000 proteins. These enzymes have been shown to catalyze chemically challenging reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms. However, to date we have little information regarding their complex mechanisms and their biosynthetic potential. Here we show, using X-ray absorption spectroscopy, mutagenesis and synthetic probes that the vitamin B₁₂-dependent radical SAM enzyme TsrM catalyzes not only C- but also N-methyl transfer reactions further expanding its synthetic versatility. We also demonstrate that TsrM has the unique ability to directly transfer a methyl group to the benzyl core of tryptophan, including the least reactive position C4. Collectively, our study supports that TsrM catalyzes non-radical reactions and establishes the usefulness of radical SAM enzymes for novel biosynthetic schemes including serial alkylation reactions at particularly inert C−H bonds.

A one-pot, two-step enzymatic cascade reaction was developed for the synthesis of N-substituted chiral 1,2-amino alcohols from simple aldehydes and amines by coupling hydroxymethylation and reduction amination reactions. This methodology was then applied to rapidly access a key building block of various tetrahydroquinoline alkaloids of pharmaceutical importance.

Abstract

The chiral N-substituted 1,2-amino alcohol motif is found in many natural and synthetic bioactive compounds. In this study, enzymatic asymmetric reductive amination of α-hydroxymethyl ketones with enantiocomplementary imine reductases (IREDs) enabled the synthesis of chiral N-substituted 1,2-amino alcohols with excellent ee values (91–99 %) in moderate to high yields (41–84 %). Furthermore, a one-pot, two-step enzymatic process involving benzaldehyde lyase-catalyzed hydroxymethylation of aldehydes and subsequent asymmetric reductive amination was developed, offering an environmentally friendly and economical way to produce N-substituted 1,2-amino alcohols from readily available simple aldehydes and amines. This methodology was then applied to rapidly access a key synthetic intermediate of anti-malaria and cytotoxic tetrahydroquinoline alkaloids.