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Chiral amine diastereomers are ubiquitous in pharmaceuticals and agrochemicals1, yet their preparation often relies on low-efficiency multi-step synthesis2. These valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Herein we characterize a multifunctional biocatalyst for amine synthesis, which operates using a mechanism that is, to our knowledge, previously unreported. This enzyme (EneIRED), identified within a metagenomic imine reductase (IRED) collection3and originating from an unclassified Pseudomonasspecies, possesses an unusual active site architecture that facilitates amine-activated conjugate alkene reduction followed by reductive amination. This enzyme can couple a broad selection of α,β-unsaturated carbonyls with amines for the efficient preparation of chiral amine diastereomers bearing up to three stereocentres. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by EneIRED, which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

Unspecific peroxygenases (UPOs) have emerged as attractive biocatalysts for selective oxygenations because, unlike cytochromes P450, they can be employed as easy-to-use lyophilized powders and depend only upon hydrogen peroxide as the external oxidant. The application of UPOs to a range of synthetic challenges relies on the characterization of activity and specificity of complementary enzymes. Here, we show that two UPOs, artUPO and rAaeUPO-PaDa-I-H, which are representative members of the “short” family I and “long” family II UPOs, display complementary activity in a series of scalable, preparative biotransformations of a diverse array of terpenes. The UPOs were also applied to the biotransformation of chrysanthemic-acid-derived fragments relevant in the agrichemical industry, culminating in the highly diastereoselective and enantioselective oxidation of a racemic synthetic pyrethroid derivative via kinetic resolution.

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans d-mannose (Man) and d-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.

The Pictet–Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet–Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila(OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.

Reductive amination is one of the most important methods for the synthesis of chiral amines. Here we report the discovery of an NADP(H)-dependent reductive aminase from Aspergillus oryzae(AspRedAm, Uniprot code Q2TW47) that can catalyse the reductive coupling of a broad set of carbonyl compounds with a variety of primary and secondary amines with up to >98% conversion and with up to >98% enantiomeric excess. In cases where both carbonyl and amine show high reactivity, it is possible to employ a 1:1 ratio of the substrates, forming amine products with up to 94% conversion. Steady-state kinetic studies establish that the enzyme is capable of catalysing imine formation as well as reduction. Crystal structures of AspRedAm in complex with NADP(H) and also with both NADP(H) and the pharmaceutical ingredient (R)-rasagiline are reported. We also demonstrate preparative scale reductive aminations with wild-type and Q240A variant biocatalysts displaying total turnover numbers of up to 32,000 and space time yields up to 3.73 g l−1d−1.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5–C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD. The three-dimensional structure of LinD from Castellaniella defragrans revealed a pentamer with active sites at the protomer interfaces. Furthermore, the structure of LinD in complex with the product geraniol provides initial mechanistic insights into this bifunctional enzyme. Site-directed mutagenesis confirmed active site amino acid residues essential for its dehydration and isomerization activity. These structural and mechanistic insights facilitate the development of hydrating catalysts, enriching the toolbox for novel bond-forming biocatalysis.

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature is yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.

This report reviews significant developments in applications of biological catalysis in synthetic organic chemistry for the year 2012. Hydrolytic enzymes have been coupled with ruthenium and iridium catalysts for the dynamic kinetic resolution (DKR) of exocyclic allylic alcohols, secondary alcohols and lactic acid esters. Alcohol dehydrogenases (ADHs) have been applied to the DKR of α-substituted β-esters and for the first time to the oxidative resolution of racemic aldehydes. Ene-reductases have been shown to catalyse double-bond isomerisation reactions, and have been combined with ADHs to furnish a closed-loop system for the conversion of cyclohexenol to cyclohexanone. ADHs have also been combined with transaminases for the conversion of primary alcohols to terminal amines. Methylaspartate ammonia lyase has been engineered to have a broad substrate specificity including derivatives of the native substrate mesaconate and large amine nucleophiles. An amino acid dehydrogenase has been engineered to accept ketones, rather than keto-acids, as substrates, allowing the enzymatic reductive amination of ketones using only ammonia and NADH. Cytochromes P450 have been engineered for the regio- and enantioselective hydroxylation of artemisinin, and one P450 has been shown to catalyse a nitration reaction. The application of prenyltransferase enzymes in biotransformations has been explored in detail, and dehalogenases have been applied to the tandem kinetic resolution of substrates, and also evolved in vitroto catalyse enantiocomplementary dehalogenations of 1,2,3-trichloropropane.

The enzyme 6-oxocamphor hydrolase (OCH) from Rhodococcus sp. NCIMB 9784 catalyses the cleavage of a carbon-carbon bond between two carbonyl groups in both mono- and bicyclic non-enolisable β-diketone substrates. In this mode OCH has been shown to effect the desymmetrisation of both bridged symmetrical bicyclic [2.2.1] and [2.2.2] systems and a series of 1-alkylbicyclo[3.3.0]octane-2,8-diones, yielding chiral substituted cyclopentanone and cyclohexanone products in high optical purity. In the present study, OCH has been challenged with a series of heteroannular substrates including 1-methylbicyclo[4.3.0]nonane-2,9-dione (7a-methylhexahydroindene-1,7-dione) in an effort to assess the competence of the enzyme for kinetic resolutions of asymmetric, racemic substrates. OCH was shown to catalyse the resolution of 1-methylbicyclo[4.3.0]nonane-2,9-dione with an E value of 2.9. The effect of increasing the length of the alkyl chain in the 1-position, or enlarging one of the rings, was to increase the enantioselectivity of the enzyme to 5.7 and 3.1 for the substrates 1-allylbicyclo[4.3.0]nonane-2,9-dione (7a-allylhexahydroindene-1,7-dione) and 1-methylbicyclo[5.3.0]decane-2,10-dione (8a-methyloctahydroazulene-1,8-dione), respectively. 1-Methylbicyclo[5.4.0]undecane-2,10-dione (9a-methyloctahydrobenzocycloheptene-1,9-dione) was not a substrate for OCH. These experiments constitute the first description of the resolution behaviour of such a retro-Claisenase enzyme, and suggest a maximum steric limit for substrate recognition by OCH.

A series of 1-alkylbicyclo[3.3.0]octane-2,8-diones was transformed by enzymatic retro-Claisen reaction using recombinant 6-oxocamphor hydrolase (OCH) overexpressed in Escherichia coli, to yield optically active 2,3-substituted cyclopentanones with enantiomeric excesses of up to >95 %. Whilst the parent substrate, bicyclo[3.3.0]octane-2,8-dione 12, was transformed only very slowly, derivatives 13, 14, 15, 16 and 30 with alkyl chains of varying length in the 1-position were converted rapidly to optically active products with typically 82 % de and up to >95 % enantiomeric excess. The results confirm the apparent requirement of OCH for non-enolisable diketone substrates, and offer a potential route to decorated cyclopentanone derivatives of multiple chiral centres. Computer modelling of 1-methylbicyclo[3.3.0]octane-2,8-dione into the active site of OCH suggested that the bicyclic [3.3.0] series substrates were accommodated in the active site in similar orientation with the natural enzyme substrate, 6-oxocamphor, and would thus yield the (2S,3S)-product series.

The enzymatic cleavage of C–C bonds in β-diketones is, comparatively, a little studied biochemical process, but one that has important relevance to human metabolism, bioremediation and preparative biocatalysis. In recent studies, four types of enzymes have come to light that cleave C–C bonds in the β-diketone functionality using different chemical mechanisms. OPH [oxidized poly(vinyl alcohol) hydrolase from Pseudomonas sp. strain VM15C], which cleaves nonane-4,6-dione to butyrate and pentan-2-one is a serine-triad hydrolase. Dke1 (diketone-cleaving enzyme from Acinetobacter johnsonii) is a dioxygenase, cleaving acetylacetone to methylglyoxal and acetate. Fumarylacetoacetate hydrolase cleaves fumarylacetoacetate to fumarate and acetoacetate using a water molecule, activated by a catalytic His/Asp dyad, aided by a calcium ion that both chelates the enol acid form of the substrate and indirectly positions the water for nucleophilic attack at a carbonyl group. 6-Oxocamphor hydrolase cleaves nonenolizable cyclic β-diketones and is a homologue of the crotonase superfamily, employing a catalytic His/Asp dyad to activate a water molecule for nucleophilic attack at a carbonyl group on one prochiral face of the diketone substrate, effecting desymmetrizations of symmetrical substrates.

Rhodococcus sp. NCIMB 9784 is a camphor-degrading Gram-positive organism originally isolated from activated sewage sludge. A 5.4 kbp portion of a proposed camphor degradation gene cluster from this organism was cloned and its nucleotide sequence determined. Four open reading frames (ORFs) were identified encoding proteins possibly involved in camphor metabolism; sequence alignment of the translation products suggested that the ORFs encode for a ferredoxin reductase, acyl-CoA ligase, epimerase and an acyl-CoA dehydrogenase. The last three activities are thought to be involved in the poorly understood late stage of camphor degradation. Our findings are entirely consistent with the proposed formation of a branched 9-carbon acid intermediate (3,4,4-trimethyl-5-oxo-trans-2-hexenoic acid) which has been isolated from the fermentation broth of camphor-grown cells.

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The biohydroxylation of a number of cyclic substrates (3–24) containing aromatic side chains was used to compare substrate specificity and selectivity of hydroxylation using microbial enzymes and whole-cell biocatalysts. In general, the regioselectivity of reaction was remarkably similar between the different catalysts in that little aromatic or benzylic, but significant aliphatic hydroxylation was observed. However, a more detailed investigation of isolated products showed complementary substrate specificity, functional group compatibility, and regioselectivity of hydroxylation. Substrate specificity and regioselectivity could be further modulated by small changes to the nature of the aromatic side chain, which appears to play an important role in substrate recognition.

Several sulfides and bicyclo[3.2.0]hept-2-en-6-one were enantioselectively oxidized to the corresponding sulfoxides and oxa lactones by a crude preparation of the two diketocamphane monooxygenases from Pseudomonas putida. The reactions were carried out in a membrane reactor with the use of poly(ethylene glycol)-N6-(2-aminoethyl)-NAD and coenzyme regeneration by the formate/formate dehydrogenase system.

Both NADH- and NADPH-dependent Baeyer-Villiger monooxygenase activities with potential uses as biocatalysts for biotransformations are present to different extents throughout the growth of Pseudomonas putidaNCIMB 10007 on (+)-camphor. The two activities give a different pattern of stereoselective oxygenations with various mono- and bicyclic ketone substrates.

The regio- and stereoselective biotransformation of bicyclo (3. 2. 0) hept-2-en-6-one by the NADH-dependent Baeyer-Villiger monooxygenase from camphorgrownPseudomonas putidaNCIMB 10007 has been shown to yield a chiral lactone not accessible by curently-used biocatalysts. The biotransformation can be conductedin vitrousing two alternative coupled enzyme systems (alcohol dehydrogenase and monooxygenase: formate dehydrogenase and monooxygenase) within siturecycling of NAD+/NADH.

Conditions have been devised to enable partially purified enzyme preparations from camphor-grownPseudomonas putidaNCIMB 10007 to undertake oxidative and reductive biotransformations of 5-hexylcyclopent-2-enone.

2,5-Diketocamphane 1,2-monooxygenase and 3,6-diketocamphane 1,6-monooxygenase isolated from (±)-camphor grown Pseudomonas putidaNCIMB 10007 catalysed two different types of Baeyer-Villiger oxidations. The former isozyme biotransformed various bicyclic [3.2.0] ketones and aryl alkyl sulfides with consistently greater regio- and/or enantioselectivity, yielding lactone and sulfoxide products with enantiomeric excesses > 55%.

The selective oxygenation of nonactivated carbon atoms is an ongoing synthetic challenge, and biocatalysts, particularly hemoprotein oxygenases, continue to be investigated for their potential, given both their sustainable chemistry credentials and also their superior selectivity. However, issues of stability, activity, and complex reaction requirements often render these biocatalytic oxygenations problematic with respect to scalable industrial processes. A continuing focus on Cytochromes P450 (P450s), which require a reduced nicotinamide cofactor and redox protein partners for electron transport, has now led to better catalysts and processes with a greater understanding of process requirements and limitations for both in vitro and whole-cell systems. However, the discovery and development of unspecific peroxygenases (UPOs) has also recently provided valuable complementary technology to P450-catalyzed reactions. UPOs need only hydrogen peroxide to effect oxygenations but are hampered by their sensitivity to peroxide and also by limited selectivity. In this Perspective, we survey recent developments in the engineering of proteins, cells, and processes for oxygenations by these two groups of hemoproteins and evaluate their potential and relative merits for scalable reactions.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulodadium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.