Diego Romano

Preparative Biotransformations: Oxidation of Alcohols

Oxidation of primary and secondary alcohols, to their corresponding aldehydes or carboxylic acids and ketones respectively, are among the most important reactions in organic synthesis. The oxidation occurs through the transfer of two-hydrogens, and can be achieved using metal oxides. Classical chemical oxidizing metal-based agents include: Ag-, Mn-, or Croxides (e.g. Jones, Sarett, Collins, and Cornforth reagents). Metal-free alternatives include the explosive hypervalent organoiodane (Dess–Martin reactions) or the S-based oxidants (Swern, Corey–Kim, and Pfitzner–Moffatt reactions). In addition, catalytic oxidations with metals such as Os, Ru, Rh, and Ir are also used, as are conventional Oppenauer oxidations. However, all of these reactions have some potential disadvantages in terms of their environmental impact (e.g. energy use, toxic byproducts etc.). Following the principles of green chemistry, a very useful and interesting alternative to convert alcohols into their corresponding carbonyl compounds is to use N-oxoammonium salts as oxidants. TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl) or derivatives thereof, when used in catalytic amounts, exploit cheap sodium hypochlorite as a stoichiometric oxidant and generate in situ oxoammonium salt. TEMPO is still very expensive, but cheaper derivatives have been introduced. Molecular oxygen is an ideal oxidant, but the application of chemocatalysis to mimic the activation of molecular oxygen at ambient temperature is far from practical to date. Green oxidations have been discussed in detail by Sheldon et al. Beside environmental and technical limitations, standard chemical oxidations of alcohols are still often poorly stereoselective. Biological oxidations of alcohols occur under mild conditions of temperature, pressure and pH, and may display their activity with high chemo-, regio-, and stereoselectivity. The preparative impact of enzyme-mediated oxidations in organic chemistry has been brilliantly reviewed recently, including the important biocatalytic systems for alcohol oxidation using dehydrogenases or oxidases. The present review gives an overview of the preparative methods for the oxidation of alcohols available for organic chemists, highlighting the advantages and the limitations of classical and innovative bioprocesses. When possible, suggestions for combining recent improvements of the techniques used in biocatalysis (high throughput screening, (meta)genomics, protein engineering, metabolic engineering, and bioprocess engineering) with green and efficient synthetic approaches are made. Alcohol Oxidation: The Biocatalysts, the Problems and the Solutions

Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini

An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15 beta,16 beta-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7 alpha- and 15 alpha-hydroxylation of 1 and 7 alpha-hydroxylation of 2, while B. malorum gave 7 beta-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glucose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7 alpha,15 alpha-dihydroxy-1 production were improved obtaining 5.8 gl(-1) (recovered product) from 7.0 gl(-1) of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton-proton and proton-carbon correlation techniques.

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates.

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase.

Esterases as stereoselective biocatalysts

Non-lypolitic esterases are carboxylester hydrolases with preference for the hydrolysis of water-soluble esters bearing short-chain acyl residues. The potential of esterases as enantioselective biocatalysts has enlarged in the last few years due to the progresses achieved in different areas, such as screening methodologies, overproduction of recombinant esterases, structural information useful for understanding the rational behind enantioselectivity, and efficient methods in protein engineering. Contributions of these complementary know-hows to the development of new robust enantioselective esterases are critically discussed in this review.

Newly isolated Streptomyces spp. as enantioselective biocatalysts: hydrolysis of 1,2‐O‐isopropylidene glycerol racemic esters

Aims:  To identify microbial strains with esterase activity able to enantioselectively hydrolyse esters of (R,S)‐1,2‐O‐isopropylidene glycerol.

An efficient continuous flow process for the synthesis of a non-conventional mixture of fructooligosaccharides.

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity. Moreover, the stability of this production system was ascertained by up to 7 days of continuous working. These results suggest the suitability of the proposed method for a large-scale production of the desired FOS mixture, in view of a foreseeable use as a novel prebiotic preparation.

New insights on the features of the vinyl phenol reductase from the wine-spoilage yeast Dekkera/Brettanomyces bruxellensis

Vinyl phenol reductase activity was assayed in extracts from 19 strains of Dekkera bruxellensis isolated from wine. In all strains, vinyl phenol reductase activity was insensitive to the presence/absence of 4-vinyl guaiacol, confirming that expression is not related to the presence of the substrate. D. bruxellensis CBS 4481 showed the highest vinyl phenol reductase activity toward 4-vinyl guaiacol. Vinyl phenol reductase from D. bruxellensis CBS 4481 was purified to mass spectrometric homogeneity, and sequenced by trypsinolysis and mass spectrometry. The sequence of the purified protein showed convincing homology with a Cu/Zn superoxide dismutase in the D. bruxellensis AWRI 1499 genome, and indeed it was found to possess both vinyl phenol reductase and superoxide dismutase activities. A bioinformatics analysis of the sequence of vinyl phenol reductase/superoxide dismutase from D. bruxellensis CBS 4481 reveals the presence in this protein of cofactor-binding structural features, that are absent in sequences of superoxide dismutases from related microorganisms, that do not display vinyl phenol reductase activity.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

One-pot chemoenzymatic synthesis of aldoximes from primary alcohols in water

A new synthetic method for the one-pot preparation of aldoximes in water was developed; the method is based on the combination of the enzymatic oxidation of primary alcohols to aldehydes using different acetic acid bacteria and in situ condensation of the aldehydes with hydroxylamine.

Enoate reductases from non conventional yeasts: bioconversion, cloning, and functional expression in Saccharomyces cerevisiae.

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of most of the strains reduced KIP via OYE and showed high stereoselectivity, producing R-levodione as major product. Competition by carbonyl reductase (CR) activity was observed in several strains. The best performing yeasts belong to Candida castellii, Kazachstania spencerorum and Kluyveromyces marxianus exhibited yields of levodione ≥77% up to 95% e.e., and. Candida freyschussii, the sole strain lacking the OYE gene, reduced KIP only to unsaturated alcohols via CR. Nine unedited OYE genes were cloned, sequenced, and heterologously expressed in Saccharomyces cerevisiae BY4741ΔOye2, a mutant that showed negligible OYE and CR activities. Compared with the corresponding wild-type yeasts, growing cells of the recombinant strains bioconverted KIP with improved yields of OYE products, minor competition by CR activity, and lower enantioselectivity. In particular, resting cells of recombinant S. cerevisae presented the best performance in KIP bioconversion. Based on the results herein reported, selected strains of non-conventional yeasts and novel OYE genes can be profitably used as innovative biocatalysts in asymmetric reductions.