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Continuous flow synthesis of 3-hydroxy-2-pyrone from galactaric acid.

The review will discuss the methods that have been optimized so far for the enzymatic hydrolysis of soapstock into enriched mixtures of free fatty acids, in order to offer a sustainable alternative to the procedure which is currently employed at the industrial level for converting soapstock into the by-product known as acid oil (or olein, i.e., free fatty acids removed from raw vegetable oil, dissolved in residual triglycerides). The further biocatalyzed manipulation of soapstock or of the corresponding acid oil for the production of biodiesel and fine chemicals (surfactants, plasticizers, and additives) will be described, with specific attention given to processes performed in continuous flow mode. The valorization of soapstock as carbon source in industrial lipase production will be also considered.

The asymmetric hydrogenation of conjugated tetrasubstituted alkenes with transition-metal catalysts is a challenging reaction, especially for substrates bearing a halide substituent. We describe a two-step multienzymatic reduction of a series of α-halo β-alkyl tetrasubstituted cyclic enones, affording halohydrins with three contiguous stereogenic centers, in good yield and with a high stereoselectivity. The reduction is catalyzed by a stereospecific ene-reductase (OYE2-3 or NemA) and a highly enantioselective alcohol dehydrogenase (ADH). The use of two enantiodivergent ADHs allows the control of the diastereoselectivity. The absolute stereochemical configurations of the products have been determined from the analysis of single-crystal structures (Flack's parameter). The enantiomeric excess (ee) has been determined by derivatization of the products with (R) Mosher's acid. Lastly, we extended our methodology also to a nonhalogenated substrate: the α-methyl ketoisophorone was reduced by two distinct enantiodivergent ene-reductases (flavin mononucleotide- and F 420-dependent), affording each enantiomer of the saturated ketone with ee > 98%.

We describe the asymmetric synthesis of the most pleasant enantiomer of Jessemal fragrance. The key steps are (i) the one-pot reduction of an α-chloro-tetrasubstituted cyclohexenone to give the chlorohydrin, catalyzed by two stereoselective redox enzymes (an ene-reductase and an alcohol dehydrogenase); (ii) the regioselective epoxide ring-opening with organocuprate or organolithium nucleophiles. Density functional theory calculations together with the Curtin-Hammett principle allowed the rationalization of the regioselectivity.

Ene-reductases from the Old Yellow Enzyme (OYE) superfamily are a well-known and efficient biocatalytic alternative for the asymmetric reduction of C=C bonds. Considering the broad variety of substituents that can be tolerated, and the excellent stereoselectivities achieved, it is apparent why these enzymes are so appealing for preparative and industrial applications. Different classes of C=C bonds activated by at least one electron-withdrawing group have been shown to be accepted by these versatile biocatalysts in the last decades, affording a vast range of chiral intermediates employed in the synthesis of pharmaceuticals, agrochemicals, flavours, fragrances and fine chemicals. In order to access both enantiomers of reduced products, stereodivergent pairs of OYEs are desirable, but their natural occurrence is limited. The detailed knowledge of the stereochemical course of the reaction can uncover alternative strategies to orient the selectivity via mutagenesis, evolution, and substrate engineering. An overview of the ongoing studies on OYE-mediated bioreductions will be provided, with particular focus on stereochemical investigations by deuterium labelling.

The combination of a biocatalytic asymmetric C=C reduction with a simple sequence of chemical transformations was implemented in a new chemoenzymatic synthesis of various substituted aryloxyalkanoic acids, used as weed-killing agrochemicals or chiral precursors. By careful selection of the biocatalyst, either enantiomer of the product could be obtained in good yield and moderate to good ee. The method relies on the use of simple and commercially available starting materials, and requires neither purified enzymes nor chromatographic separations.

Production of azelaic and pelargonic acids from renewable high-oleic soapstock, a by-product of vegetable oil refining industry.

Trifluoroacetic acid catalysed lactonization of vicinal disubstituted γ-hydroxyesters was investigated in different solvents. The reaction kinetics, monitored by NMR spectroscopy, showed that: (i) the vic-disubstituent effect is stereoselective since the anti diastereoisomer ring closes substantially more rapidly than the syn isomer ring; (ii) the anti-vic effect is much stronger than the classical Thorpe-Ingold effect (known also as the gem-disubstituent effect), instead the syn diastereoisomers have rate constants comparable to that of the gem-disubstituted ester; (iii) the vic-effect can be enhanced by increasing the steric hindrance of one of the two substituents or carrying out the reaction in non-polar solvents. DFT computations of energy barriers (ΔG‡) were in good agreement with the experimental data. The distortion/interaction-activation strain model together with the Winstein-Holness kinetic scheme gave more insights into the origin of the vic-effect. An application of this effect consists of the diastereomeric resolution of disubstituted γ and δ lactones, among which are the naturally occurring Nicotiana t. lactone, the whisky and cognac oak lactones, and the Aerangis lactone. Both cis and trans diastereoisomers of these lactones were isolated in good yield and with high diastereomeric excess (de >92%). The selectivities of the diastereomeric resolution process, determined by NMR spectroscopy, are reported as well.

The text describes the main techniques used in the pharmaceutical field to mask the bitter taste of active pharmaceutical ingredients and make the product more pleasant for oral intake by the patient.

Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions.

Leather cyclohexanol (4-(isopropyl)cyclohexanol) and woody acetate (4-(tert-butyl)cyclohexyl acetate) are commercialized for functional perfumery applications as mixtures of cis- and trans-isomers. The cis-isomers are more potent odorants than the corresponding trans counterparts, but they are the less favoured products in most of the classical synthetic routes. Known stereoselective routes to cis-4-alkylcyclohexanols are characterized by a high environmental burden and/or troublesome reaction work-up. In this work, we examine the use of commercial alcohol dehydrogenases (ADHs) to produce cis-4-alkylcyclohexanols, including the two derivatives with isopropyl and tert-butyl substituents, by the stereoselective reduction of the corresponding ketones. High conversions and diastereoisomeric excess values were achieved with five of the eighteen tested ADHs. To complete the synthetic approach to woody acetate, Candida antarctica A (CALA) was employed as a catalyst for the enzymatic acetylation of cis-4-(tert-butyl)cyclohexanol. In order to provide a technological upgrade to the production of the most odorous isomers of the two commercial fragrances, we designed a continuous-flow process based on the combination of in-line enzymatic steps with in-line work-up, effectively providing samples of cis-leather cyclohexanol and cis-woody acetate with high diastereoisomeric purity.

The chemoselective hydrogenation of alkenes in the presence of alkynes is a very challenging transformation to achieve with traditional chemical methods. The development of an effective procedure to perform this transformation would enrich the tool-kit available to organic chemists for the development of useful synthetic routes, and the creation of novel structural motifs. The reduction of activated alkene bonds by ene-reductases (ERs) is completely chemoselective, because of the mechanism of the reaction. Thus, we investigated the use of ERs belonging to the Old Yellow Enzyme family for the reduction of ?,?-unsaturated aldehydes with a conjugated C?C triple bond at the ? position. This reaction was exploited as the key step for the development of an effective homologation route to convert aryl and alkyl substituted propynals and butynones into 4-alkynals and 4-alkynols, avoiding some troublesome or hazardous steps of known synthetic routes. (Figure presented.).

The integration of biocatalytic steps in retrosynthetic analysis of a target molecule offers multiple advantages, such as reduction of the environmental footprint of the process, viability of milder and safer reaction conditions, and accessibility of transformations that are challenging with traditional chemical synthesis. Herein, six chemo-enzymatic routes are described for the synthesis of a fluorinated D-phenylalanine derivative, precursor of the blockbuster antidiabetic drug sitagliptin. All routes start from the same aldehyde precursor and involve at least one biocatalytic step, including reductive amination, transamination, deracemisation, hydroamination, and alkene reduction. The target molecule was obtained in 2–5 steps from the aldehyde, with ee up to >99% and in 36–62% isolated yield. Furthermore, as part of one of the routes, the first example of a fully biocatalytic conversion of a cinnamic acid derivative to the corresponding D-phenylalanine (formal D-selective hydroamination) is reported.

Chiral &beta, nitroalcohols are important building blocks in organic chemistry. The synthetic approach that is based on the enzyme-mediated reduction of &alpha, nitroketones has been scarcely considered. In this work, the use of commercial alcohol dehydrogenases (ADHs) for the reduction of aromatic and aliphatic nitroketones is investigated. High conversions and enantioselectivities can be achieved with two specific ADHs, affording either the (S) or (R)-enantiomer of the corresponding nitroalcohols. The reaction conditions are carefully tuned to preserve the stability of the reduced product, and to avoid the hydrolytic degradation of the starting substrate. The further manipulation of the enantioenriched nitroalcohols into Boc-protected amminoalcohols is also described.

The zygomycete fungus Syncephalastrum racemosum shows the remarkable capability of reducing carboxylic acids to primary alcohols in water medium, at ambient temperature and pressure. The reaction does not require molecular hydrogen, and in most cases affords quantitative transformations. The results herein reported highlight the basic molecular scaffold that can be accepted by the fungus, the effects due to substituents, and also the possibility that carboxylic acids can be generated in the reaction medium by enzymatic hydrolysis of the corresponding methyl esters. This biocatalysed reduction implements the scarcely supplied enzymatic toolbox for the conversion of carboxylic groups into primary alcohols, which can be exploited for the optimisation of sustainable synthetic procedures. (C) 2015 Elsevier B.V. All rights reserved.

The Trp 116 mutants of Old Yellow Enzyme 1 that catalyse the reduction of (Z)-?-aryl-?-cyanoacrylates give the opposite enantioselectivity according to the nature of the amino acid in position 116. Small amino acids (e.g., alanine) make the substrate bind to the enzyme?s active site in a "classical" orientation, affording the (S)-enantiomer of the reduced product. When the size of the amino acid increases (e.g., leucine), a "flipped" binding mode is adopted by the substrate, which is converted into the corresponding (R)-derivative. With bulky amino acids (e.g., tryptophan in the wild-type) the reduction does not occur. The enantiomerically enriched cyanopropanoates thus prepared can be converted into the corresponding (S)- and (R)-?-aryl-?-lactams, precursors of inhibitory neurotransmitters belonging to the class of ?-aminobutyric acids, by a simple functional group interconversion in a sequential one-pot procedure.

The most odorous stereoisomers of the chiral commercial fragrance Muguesia® are prepared by a very effective linear biocatalysed cascade reaction, in which a suitable unsaturated ketone is submitted to the sequential action of two enzymes, an ene-reductase and an alcohol dehydrogenase, which are added together to the same reaction vessel with the cofactor regeneration system. Two stereogenic centres in 1,2 relative position are thus created under high stereochemical control by a two-step one-pot enzymatic procedure.

A chemo-enzymatic catalytic system, comprised of Bobbitt's salt and laccase from Trametes versicolor, allowed the [1,3]-oxidative rearrangement of endocyclic allylic tertiary alcohols into the corresponding enones under an Oxygen atmosphere in aqueous media. The yields were in most cases quantitative, especially for the cyclopent-2-en-1-ol or the cyclohex-2-en-1-ol substrates without an electron withdrawing group (EWG) on the side chain. Transpositions of macrocyclic alkenols or tertiary alcohols bearing an EWG on the side chain were instead carried out in acetonitrile by using an immobilized laccase preparation. Dehydro-Jasmone (R), dehydro-Hedione (R), dehydro-Muscone and other fragrance precursors were directly prepared with this procedure, while a synthetic route was developed to easily transform a cyclopentenone derivative into trans-Magnolione (R) and dehydro-Magnolione (R). The rearrangement of exocyclic allylic alcohols was tested as well, and a dynamic kinetic resolution was observed: alpha,beta-unsaturated ketones with (E)-configuration and a high diastereomeric excess were synthesized. Finally, the 2,2,6,6-tetramethyl-1-piperidinium tetrafluoroborate (TEMPO+BF4-)/laccase catalysed oxidative rearrangement was combined with the ene-reductase/ alcohol dehydrogenase cascade process in a one-pot three-step synthesis of cis or trans 3-methylcyclohexan-1-ol, in both cases with a high optical purity.

A three-step biocatalytic procedure is described for the conversion of methyl and ethyl cyclopentene- and cyclohexenecarboxylates into both the enantiomers of the corresponding chiral 3-oxoesters, which are useful building blocks for the synthesis of active pharmaceutical ingredients. The allylic hydroxylation of the starting cycloalkenecarboxylates is carried out by using R. oryzae resting cells entrapped in alginate beads, in acetate buffer solution at 25 °C. The oxidation of the intermediate allylic alcohols to unsaturated ketones, performed by the laccase/TEMPO system, and the ene-reductase mediated hydrogenation of the alkene bond were carried out in the same reaction vessel in a sequential mode at 30 °C. Being entirely biocatalytic, our multistep procedure provides considerable advantages in terms of selectivity and environmental impact over reported chemical methods.

Enantiopure 2-methyl-3-substituted tetrahydrofurans are key precursors of several biologically active products (drugs, flavors, and agrochemicals). Thus, a stereocontrolled and efficient methodology for the obtainment of these synthons is highly desirable. We exploited a two-step multienzymatic stereoselective cascade reduction of alpha-bromo-alpha,beta-unsaturated ketones to give the corresponding bromohydrins in good yields, with high ee and de values. The cascade process is catalyzed by an ene-reductase and an alcohol dehydrogenase. Further manipulations of these bromohydrins, by two diastereodivergent routes, allowed the preparation of the tetrahydrofuran synthons. One route is based on a lipase catalyzed cleavage of the protecting group. The second route is characterized by a camphor sulfonic acid mediated isomerization of a beta-hydroxyepoxide to give the tetrahydrofuran-2-ol. Finally, the synthesis of the most odorous and pleasant stereoisomer of the roasted meat aroma, i.e., (2S,3R)-2-methyl-3-thioacetate tetrahydrofuran, is reported as well.

The reduction of C=C double bond, a key reaction in organic synthesis, is mostly achieved by traditional chemical methods. Therefore, the search for enzymes capable of performing this reaction is rapidly increasing. Old Yellow Enzymes (OYEs) are flavin-dependent oxidoreductases, initially isolated from Saccharomyces pastorianus. In this study, the presence and activation of putative OYE enzymes was investigated in the filamentous fungus Mucor circinelloides, which was previously found to mediate C=C reduction. Following an in silico approach, using S. pastorianus OYE1 amminoacidic sequence as template, ten putative genes were identified in the genome of M. circinelloides. A phylogenetic analysis revealed a high homology of McOYE1-9 with OYE1-like proteins while McOYE10 showed similarity with thermophilic-like OYEs. The activation of mcoyes was evaluated during the transformation of three different model substrates. Cyclohexenone, α-methylcinnamaldehyde and methyl cinnamate were completely reduced in few hours and the induction of gene expression, assessed by qRT-PCR, was generally fast, suggesting a substrate-dependent activation. Eight genes were activated in the tested conditions suggesting that they may encode for active OYEs. Their expression over time correlated with C=C double bond reduction.

This work contains a complete account of the preparation of all the ten enantiopure isomers of irone, the odoriferous principle of orris root oil, by means of enzyme-mediated approaches, starting from commercial racemic Irone alpha ® . To cite this article: E. Brenna et al., C. R. Chimie 6 (2003).

The stereoselective reduction of suitably substituted C═C bonds mediated by enzymes, called ene reductases, has received great attention in the last decade. Some successful applications of this biocatalysed procedure to the synthesis of chiral active pharmaceutical ingredients have been reported in the literature. The generation of suitable models to be used for predicting the stereochemical outcome of this kind of reductions is a challenging task. In the last years we have exploited deuterium labelling to investigate the stereochemical course of the enzyme-mediated reductions of a wide collection of substrates belonging to well-defined chemical classes. The results of this research have allowed us to draw conclusions on the relationship between the structural characteristics of the substrate and the binding mode it adopts in the enzyme active site. The collected data can be exploited to create an empirical model to rationalise and predict the stereoselectivity of old yellow enzyme (OYE)-catalysed reductions.

A multistep stereoselective synthesis of each stereoisomer of Nicotiana tabacum lactone is reported. A two steps reduction of an α,β-unsaturated ketoester gives the corresponding key intermediate ethyl 4-hydroxy-3-methylpentanoate. This one pot synthesis was catalyzed by a multienzymatic system comprising an ene-reductase (ER) and an alcohol dehydrogenase (ADH). This cascade process was highly chemoselective and stereoselective. In the last step, treatment of the hydroxyester with trifluoroacetic acid gives the γ-lactone in a very high overall yield (up to 78%) and with an excellent stereoselectivity (de > 94%, ee > 98%). The access to each stereoisomer was achieved by a substrate engineering approach and by selecting a Prelog or an anti-Prelog ADH. Furthermore, computational studies of the binding modes of the substrates into the catalytic site of ene-reductases have been carried out, giving an insight of the observed enantiodivergence.

Bioeconomy stresses the need of green processes promoting the development of new methods for biocatalyzed alkene reductions. A functional screening of 28 fungi belonging to Ascomycota, Basidiomycota, and Zygomycota isolated from different habitats was performed to analyze their capability to reduce C C double bonds towards three substrates (cyclohexenone, α-methylnitrostyrene, and α-methylcinnamaldehyde) with different electron-withdrawing groups, i.e., ketone, nitro, and aldehyde, respectively. Almost all the fungi showed this reducing activity. Noteworthy Gliomastix masseei , Mucor circinelloides , and Mucor plumbeus resulted versatile and effective, being able to reduce all the model substrates quickly and with high yields.

The four stereoisomers of the rose oxide analogue Doremox®were prepared in enantiomerically enriched form by enantiospecific bakers' yeast reduction of suitable derivatives and by lipase-mediated kinetic resolution of diol precursors.Key words: yeast, lipase, odorant, reduction, kinetic resolution.

The enantioselective reduction of α-methylene nitrile derivatives catalysed by ene-reductases affords the corresponding (R)-2-arylpropanenitriles with high conversion values. The reaction is investigated either in aqueous medium (with an organic cosolvent or by loading the substrate onto hydrophobic resins) or in a biphasic ionic liquid–water system. The use of ionic liquids, herein with isolated ene-reductases, is found to improve the work-up and the substrate recovery method. The synthetic manipulation of the final chiral nitrile derivatives indicates how this biocatalysed method can be exploited for the preparation of a wide range of chiral compounds.

The OYE1-3-mediated reductions of some ?,?-difunctionalised alkenes, showing on the double bond a nitrile and ester group, are submitted to a careful stereochemical analysis, in order to identify which of the two electron-withdrawing groups (EWGs) is responsible for the activation of the CC double bond towards reduction and for establishing hydrogen bond interactions within the binding pocket of the enzymes. The results show that for most of these substrates the activating EWG is the CN moiety linked to the prostereogenic olefinic carbon atom. The final stereochemical outcome can be explained through the empirical model which has been recently developed for difunctionalised alkenes activated by carbonyl/carboxyl containing EWGs. In a single case the activation is due to the COOR group linked to the less substituted olefinic carbon atom: an alternative empirical model is established for this kind of substrates, taking into consideration the OYE-catalysed reductions of ?,??-disubstituted-?-monofunctionalised alkenes. © 2014 Elsevier B.V.

Coffee contains a huge amount of volatile components which contribute to the characteristic flavour. These materials are generated during the heating process. No one of these materials can be considered the ‘impact flavour’. Accordingly, the whole flavour depends upon the presence of an elevated number of components. However, at least four of the materials of are essential for a ‘good’ coffee.

Modern perfumery was born nearly a century ago, when synthetic organic chemistry made available a great variety of products, besides those traditionally obtained by extraction from nature: pure or enriched compounds isolated from natural sources, semi-synthetic products prepared from natural starting materials, aromatic compounds obtained by total synthesis. The most relevant results achieved in the last decade are herein presented for several types of odorous substances.

Enantiomerically pure 1,3-diols 1-3 were obtained by a chemoenzymatic approach ( lipase PS from Burkholderia cepacia). These diols were converted into useful chiral synthons, which could be considered homologues of glyceraldehyde and glyceric acid acetonides. Applications of these synthons to the de novo synthesis of sugars and preparation of conagenin carboxylic moiety were shown. Hydroxy ketone 4 was chosen as a model system for another synthetic evolution: it was obtained in enantiomerically pure form by enzymatic resolution and converted into chiral tetrahydropyranes, such as the stereoisomers of the commercial fragrance Gyrane.

During the past years biocatalytic production of fine chemicals has been expanding rapidly. Flavours and fragrances belong to many different structural classes and therefore represent a challenging target for academic and industrial research. Here, we present a condensed overview of the potential offered by biocatalysis for the synthesis of natural and natural-identical odorants, highlighting relevant biotransformations using microorganisms and isolated enzymes. The industrial processes based on biocatalytic methods are discussed in terms of their advantages over classical chemical synthesis and extraction from natural sources. Recent applications of the biocatalytic approach to the preparation of the most important fine odorants are comprehensively covered.

[structure: see text] The work describes the enzyme-mediated preparation and the odor evaluation of the single stereoisomers of the commercial odorants Muguesia and Pamplefleur. The synthetic approach to Muguesia stereoisomers helped to clear the assignment of the relative configuration of intermediate diols 5. The odor response of Pamplefleur isomers was found to be rather unusual. No stereoisomer prevailed, but each one played a definite role in establishing the odor sensation of the final blend.

The synthesis and application of three examples of a new class of chiral (C(2)) atropisomeric diphosphines characterized by two interconnected five-membered heteroaromatic rings, with hindered rotation around the interanular bond, are described. Optically pure (+)- and (-)-2,2'-bis(diphenylphosphino)-4,4',6,6'-tetramethyl-3,3'-bibenzo[b]thiophene (tetraMe-bitianp) (1a) and the parent unsubstituted system (+)- and (-)-bitianp (1b) were synthesized. They were found to be optically stable at 100 degrees C and were successfully employed as ligands in the Ru(II)-catalyzed hydrogenation of alpha- and beta-oxo esters to the corresponding alpha- and beta-hydroxy esters and in the hydrogenation of olefinic substrates. The optical and chemical yields were comparable with those reported for the same Ru(II)-binap-catalyzed reactions carried out under the same experimental conditions. The 2,2'-bis(diphenylphosphino)-3,3'-bibenzo[b]furan (1c), the oxygenated analogue of bitianp, was found to be configurationally unstable at room temperature. Complete structural X-ray elucidation of the Pd complexes of 1a-c is reported. The advantages of these biheteroaromatic ligands over the classical biaryl systems are discussed.