A Tandem Enzymatic sp2‐C‐Methylation Process: Coupling in Situ S‐Adenosyl‐l‐Methionine Formation with Methyl Transfer

A one‐pot, two‐step biocatalytic platform for the regiospecfic C‐methylation and C‐ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S‐adenosyl‐l ‐methionine (SAM), followed by alkylation of aromatic substrates by the C‐methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled ¹³CH_3, ¹³CD₃ and CD₃ groups from their corresponding SAM analogues formed in situ.     

Nucleic‐Acid‐Templated Enzyme Cascades

Cellular metabolism involves complex sequences of organized enzymatic reactions, known as metabolic pathways, that convert substrates into readily usable materials. In nature, these enzymatic complexes are organized in a well‐defined manner so that the cascade reactions are more rapid and efficient than they would be if the enzymes were randomly distributed in the cytosol. Development of artificial enzyme cascades that resemble nature's organization of sequentially assembled enzymes is of current interest due to its potential applications, from diagnostics to the production of high‐value chemicals. Nucleic acids and their nanostructures have been used to organize enzyme cascades and have been shown to enhance the efficiencies and rates of sequential reactions. Here we summarize the recent progress in the development of artificial enzyme cascades and sequential reactions by arranging enzymes on various DNA/RNA templates and discuss the future directions of this research endeavour.     

Stereospecific Reduction of Methyl o‐Chlorobenzoylformate at 300 g⋅L−1 without Additional Cofactor using a Carbonyl Reductase Mined from Candida glabrata

In order to search for oxidoreductases suitable for the preparation of methyl (R)‐o‐chloromandelate [(R)‐CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o‐chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g⋅L⁻¹ of CBFM was almost stoichiometrically converted to (R)‐CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram‐scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)‐CMM.     

Industrially Relevant Enzyme Cascades for Drug Synthesis and Their Ecological Assessment

Environmentally friendly and sustainable processes for the production of active pharmaceutical ingredients (APIs) gain increasing attention. Biocatalytic synthesis routes with enzyme cascades support many stated green production principles, for example, the reduced need for solvents or the biodegradability of enzymes. Multi-enzyme reactions have even more advantages such as the shift of the equilibrium towards the product side, no intermediate isolation, and the synthesis of complex molecules in one reaction pot. Despite the intriguing benefits, only a few enzyme cascades have been applied in the pharmaceutical industry so far. However, several new enzyme cascades are currently being developed in research that could be of great importance to the pharmaceutical industry. Here, we present multi-enzymatic reactions for API synthesis that are close to an industrial application. Their performances are comparable or exceed their chemical counterparts. A few enzyme cascades that are still in development are also introduced in this review. Economic and ecological considerations are made for some example cascades to assess their environmental friendliness and applicability.     

Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods.     

Efficient Biosynthesis of (R)‐ or (S)‐2‐Hydroxybutyrate from l‐Threonine through a Synthetic Biology Approach

An efficient multi‐enzyme cascade reaction for the synthesis of (R)‐ or (S)‐2‐hydroxybutyric acid [(R)‐ or (S)‐2‐HB] from l ‐threonine was developed by using recombinant Escherichia coli cells expressing separately or co‐expressing l ‐threonine deaminase from Escherichia coli K‐12 (ilvA), formate dehydrogenase (FDH) from Candida boidinii and l ‐lactate dehydrogenase (l ‐LDH) from Oryctolagus cuniculus or d ‐lactate dehydrogenase (d ‐LDH) from Staphylococcus epidermidis ATCC 12228. Up to 750 mM of l ‐threonine were completely transformed to (R)‐ or (S)‐2‐HB in optically pure form (>99% ee) with high isolated yields. This one‐pot multi‐enzyme transformation provides a new practical method for the synthesis of these important optically pure compounds.

     

Copper‐Catalyzed Three‐Component Synthesis of 2‐Iminodihydrocoumarins and 2‐Iminocoumarins

An efficient synthesis of N‐sulfonyl‐substituted 2‐imino‐3,4‐dihydrocoumarins and 2‐iminocoumarins via a copper‐catalyzed multicomponent reaction of sulfonyl azides with terminal alkynes and β‐(ortho‐hydroxyphenyl)‐α,β‐unsaturated ketones or ortho‐hydroxyphenylpropiolates has been developed. The cascade process involves trapping the keteimine by a nucleophilic addition and an intramolecular Michael addition. This methodology could well be extended to the concise synthesis of the polysubstituted piperidine scaffold.     

Gold‐ vs. Platinum‐Catalyzed Polycyclizations by O‐Acyl Migration. Solvent‐Free Reactions

Polycyclic derivatives incorporating a cyclopropyl group have been efficiently synthesized from propargyl acetates using platinum(II), gold(I) and gold(III) catalysis. These reactions which are also viable for the preparation of medium‐sized rings, proceed with a complete diastereocontrol and can also be run in neat conditions.     

Extended Catalytic Scope of a Well‐Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase

NADP(H)‐dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d ‐glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized ¹H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.     

Structures of the Apo and FAD‐Bound Forms of 2‐Hydroxybiphenyl 3‐monooxygenase (HbpA) Locate Activity Hotspots Identified by Using Directed Evolution

The FAD‐dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2‐hydroxybiphenyl (2HBP) to 2,3‐dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed‐evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD‐complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate‐binding site.     

Development of a thermostable glucose dehydrogenase by a structure-guided consensus concept

Instability under non-native processing conditions, especially at elevated temperatures, is a major factor preventing the widespread adoption of biocatalysts for industrial synthesis. A crucial distinction of many redox enzymes used to synthesize chiral compounds is the need for cofactors (e.g., NAD(P)(H)) for function. Because of the prohibitively high prices of nicotinamide cofactors, a robust cofactor-regenerating enzyme is required for the economical synthesis of fine chemicals by biocatalysis. Here we test the structure-guided consensus for the generation of a thermostable glucose dehydrogenase (GDH). The consensus sequence in combination with additional knowledge-based criteria was used to select amino acids for substitutions. Using this approach we generated 24 variants, 11 of which showed higher thermal stability than the wild-type GDH, a success rate of 46 %. Of the 24 variants, seven were located at the subunit interface-known to influence GDH stability-and six were more stable (86 % success). The best variants feature a half-life of approximately 3.5 days at 65 degrees C, in contrast to approximately 20 min at 25 degrees C for the wild type, thus enhancing stability 10(6)-fold. In addition, the three most stabilizing single mutations were transferred to two GDH homologues from Bacillus thuringiensis and Bacillus licheniformis. The thermal stability as measured by half-life and CD(222 nm) of the GDH variants was increased, as expected. The resulting stability changes provide further support for the view that these residues are critical for stability of GDHs and reinforce the success of the consensus approach for identifying stabilizing mutations.     

Functional Characterization of MpaG′, the O‐Methyltransferase Involved in the Biosynthesis of Mycophenolic Acid

Mycophenolic acid (MPA, 1 ) is a clinically important immunosuppressant. In this report, a gene cluster mpa′ responsible for the biosynthesis of 1 was identified from Penicillium brevicompactum NRRL 864. The S‐adenosyl‐L ‐methionine‐dependent (SAM‐dependent) O‐methyltransferase encoded by the mpaG′ gene was functionally and kinetically characterized in vitro. MpaG′ catalyzes the methylation of demethylmycophenolic acid (DMMPA, 6 ) to form 1 . It also showed significant substrate flexibility by methylating two structural derivatives of 6 prepared by organic synthesis.     

Functional Analysis of MycE and MycF,Two O‐Methyltransferases Involved in the Biosynthesis of Mycinamicin Macrolide Antibiotics

Mg motors : We characterized the in vitro function of MycE and MycF, two O‐methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S‐adenosyl‐L ‐methionine (SAM)‐dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg²⁺. With the reconstituted in vitro assays, the order of mycinamicin VI→III→IV in the post‐PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

     

New Palladium‐Catalyzed Cascades: 4‐exo‐dig Cyclocarbopalladation Reaction Followed by Suzuki–Miyaura or Sonogashira Cross‐Coupling

Herein we describe new accesses to dienynes and trienynes using new cascade reactions: 4‐exo‐dig cyclocarbopalladation followed by a Suzuki–Miyaura or Sonogashira cross‐coupling.     

One‐Pot Three‐Component Click Reaction of Cyclic Sulfates and Cyclic Sulfamidates

A one‐pot, three‐component methodology involving tandem azidation and click copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC) of cyclic sulfates or cyclic sulfamidates in the presence of sodium azides and alkynes is reported. The developed protocol takes also advantage of the concomitant use of microwave (MW) irradiation and heterogeneous catalysis. The protocol allows the fast and efficient preparation of (alkyl sulfate)‐ and (alkyl sulfamidate)‐1H‐1,2,3‐triazoles in a simple manner.     

Regeneration of Carbonyl Compounds by Oxidative Cleavage of Hydrazones with Zr(O‐t‐Bu)4/tert‐Butyl Hydroperoxide

A catalytic method with Zr(O‐t‐Bu)4 as the catalyst and tert‐butyl hydroperoxide as the oxidant is described to cleave phenylhydrazones 6—9 , 14—16 to the parent carbonyl compounds under mild and neutral oxidative conditions.