Applications of biocatalytic CC bond reductions in the synthesis of flavours and fragrances.

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of CC bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving CC bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems.

Chemo-enzymatic synthesis and biological activity evaluation of propenylbenzene derivatives.

Propenylbenzenes, including isosafrole, anethole, isoeugenol, and their derivatives, are natural compounds found in essential oils from various plants. Compounds of this group are important and valuable, and are used in the flavour and fragrance industries as well as the pharmaceutical and cosmetic industries. The aim of this study was to develop an efficient process for synthesising oxygenated derivatives of these compounds and evaluate their potential biological activities. In this paper, we propose a two-step chemo-enzymatic method. The first step involves the synthesis of corresponding diols 1b-5b from propenylbenzenes 1a-5avia lipase catalysed epoxidation followed by epoxide hydrolysis. The second step involves the microbial oxidation of a diasteroisomeric mixture of diols 1b-5b to yield the corresponding hydroxy ketones 1c-4c, which in this study was performed on a preparative scale using Dietzia sp. DSM44016, Rhodococcus erythropolis DSM44534, R. erythropolis PCM2150, and Rhodococcus ruber PCM2166. Application of scaled-up processes allowed to obtain hydroxy ketones 1-4c with the following yield range 36-62.5%. The propenylbenzene derivatives thus obtained and the starting compounds were tested for various biological activities, including antimicrobial, antioxidant, haemolytic, and anticancer activities, and their impact on membrane fluidity. Fungistatic activity assay against selected strains of Candida albicans results in MIC50 value varied from 37 to 124 µg/mL for compounds 1a, 3a-c, 4a,b, and 5a,b. The highest antiradical activity was shown by propenylbenzenes 1-5a with a double bond in their structure with EC50 value ranged from 19 to 31 µg/mL. Haemolytic activity assay showed no cytotoxicity of the tested compounds on human RBCs whereas, compounds 2b-4b and 2c-4c affected the fluidity of the RBCs membrane. The tested compounds depending on their concentration showed different antiproliferative activity against HepG2, Caco-2, and MG63. The results indicate the potential utility of these compounds as fungistatics, antioxidants, and proliferation inhibitors of selected cell lines.

Trametes hirsuta as an Attractive Biocatalyst for the Preparative Scale Biotransformation of Isosafrole into Piperonal.

Piperonal is a compound of key industrial importance due to its attractive olfactory and biological properties. It has been shown that among the fifty-six various fungal strains tested, the ability to cleave the toxic isosafrole into piperonal through alkene cleavage is mainly found in strains of the genus Trametes. Further studies involving strains isolated directly from different environments (decaying wood, fungal fruiting bodies, and healthy plant tissues) allowed the selection of two Trametes strains, T. hirsuta Th2_2 and T. hirsuta d28, as the most effective biocatalysts for the oxidation of isosafrole. The preparative scale of biotransformation with these strains provided 124 mg (conv. 82%, isolated yield 62%) and 101 mg (conv. 69%, isolated yield 50.5%) of piperonal, respectively. Due to the toxic impact of isosafrole on cells, preparative scale processes with Trametes strains have not yet been successfully performed and described in the literature.

Stereoselective synthesis of whisky lactone isomers catalyzed by bacteria in the genus Rhodococcus.

Whisky lactone is a naturally occurring fragrance compound in oak wood and is widely used as a sensory additive in food products. However, safe and efficient methods for the production of its individual enantiomers for applications in the food industry are lacking. The aim of this study was to develop an efficient and highly stereoselective process for the synthesis of individual enantiomeric forms of whisky lactones. The proposed three-step method involves (1) column chromatography separation of a diastereoisomeric mixture of whisky lactone, (2) chemical reduction of cis-and trans-whisky lactones to corresponding syn-and anti-diols, and (3) microbial oxidation of racemic diols to individual enantiomers of whisky lactone. Among various bacteria in the genera Dietzia, Gordonia, Micrococcus, Rhodococcus, and Streptomyces, R. erythropolis DSM44534 and R. erythropolis PCM2150 effectively oxidized anti-and syn-3-methyl-octane-1,4-diols (1a-b) to corresponding enantiomerically pure cis-and trans-whisky lactones, indicating high alcohol dehydrogenase activity. Bio-oxidation catalyzed by whole cells of these strains yielded enantiomerically pure isomers of trans-(+)-(4S,5R) (2a), trans-(-)-(4R,5S) (2b), and cis-(+)-(4R,5R) (2d) whisky lactones. The optical density of bacterial cultures and the impact of the use of acetone powders as catalysts on the course of the reaction were also evaluated. Finally, the application of R. erythropolis DSM44534 in the form of an acetone powder generated the enantiomerically enriched cis-(-)-(4S,5S)-isomer (2c) from the corresponding syn-diol (1b). The newly developed method provides an improved approach for the synthesis of chiral whisky lactones.

Chemoenzymatic Synthesis of the Most Pleasant Stereoisomer of Jessemal.

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.

"A Study in Yellow": Investigations in the Stereoselectivity of Ene-Reductases.

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.

Ene-reductase transformation of massoia lactone to δ-decalactone in a continuous-flow reactor.

The demand for natural food flavorings increases every year. Biotransformation has become an attractive approach to obtain natural products. In this work, enantiomerically pure (R)-(+)-δ-decalactone was obtained by reduction of the C=C double bond of natural massoia lactone in a continuous-flow reactor. Of 13 different ene-reductases isolated, purified and tested, OYE3 was found to be the most efficient biocatalyst. The selected biocatalyst, either in the form of purified enzyme, cell lysate, whole cells or immobilized cells, was tested in the batch system as well as in the packed-bed flow bioreactor. The biotransformation performed in batch mode, using Ca2+-alginate immobilized cells of Escherichia coli BL21(DE3)/pET30a-OYE3, furnished the desired product with complete conversion in 30 min. The process was intensified using a continuous-flow reactor-membrane filtration system (flow 0.1 mL/min, substrate concentration 10 mM, pH 7, 24 °C) with cell lysate as biocatalyst combined with a cofactor regeneration system, which allowed obtaining > 99% bioconversion of massoia lactone.

Bacterial Biotransformation of Oleic Acid: New Findings on the Formation of γ-Dodecalactone and 10-Ketostearic Acid in the Culture of Micrococcus luteus.

Microbial conversion of oleic acid (1) to form value-added industrial products has gained increasing scientific and economic interest. So far, the production of natural lactones with flavor and fragrance properties from fatty acids by non-genetically modified organisms (non-GMO) involves whole cells of bacteria catalyzing the hydration of unsaturated fatty acids as well as yeast strains responsible for further ß-oxidation processes. Development of a non-GMO process, involving a sole strain possessing both enzymatic activities, significantly lowers the costs of the process and constitutes a better method from the customers' point of view regarding biosafety issues. Twenty bacteria from the genus of Bacillus, Comamonas, Dietzia, Gordonia, Micrococcus, Pseudomonas, Rhodococcus and Streptomyces were screened for oxidative functionalization of oleic acid (1). Micrococcus luteus PCM525 was selected as the sole strain catalyzing the one-pot transformation of oleic acid (1) into natural valuable peach and strawberry-flavored γ-dodecalactone (6) used in the food, beverage, cosmetics and pharmaceutical industries. Based on the identified products formed during the process of biotransformation, we clearly established a pathway showing that oleic acid (1) is hydrated to 10-hydroxystearic acid (2), then oxidized to 10-ketostearic acid (3), giving 4-ketolauric acid (4) after three cycles of ß-oxidation, which is subsequently reduced and cyclized to γ-dodecalactone (6) (Scheme 1). Moreover, three other strains (Rhodococcus erythropolis DSM44534, Rhodococcus ruber PCM2166, Dietzia sp. DSM44016), with high concomitant activities of oleate hydratase and alcohol dehydrogenase, were identified as efficient producers of 10-ketostearic acid (3), which can be used in lubricant and detergent formulations. Considering the prevalence of γ-dodecalactone (6) and 10-ketostearic acid (3) applications and the economic benefits of sustainable management, microbial bioconversion of oleic acid (1) is an undeniably attractive approach.

Conversion of Oleic Acid into Azelaic and Pelargonic Acid by a Chemo-Enzymatic Route.

A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in 44% isolation yield after three steps, avoiding column chromatography purifications. In the first step, the lipase-mediated generation of peroleic acid in the presence of 35% H2O2 is employed for the self-epoxidation of the unsaturated acid to the corresponding oxirane derivative. This intermediate is submitted to in situ acid-catalyzed opening, to afford 9,10-dihydroxystearic acid, which readily crystallizes from the reaction medium. The chemical oxidation of the diol derivative, using atmospheric oxygen as a stoichiometric oxidant with catalytic quantities of Fe(NO3)3∙9∙H2O, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and NaCl, affords 9,10-dioxostearic acid which is cleaved by the action of 35% H2O2 in mild conditions, without requiring any catalyst, to give pelargonic and azelaic acid.

Stereoselectivity Switch in the Reduction of α-Alkyl-ß-Arylenones by Structure-Guided Designed Variants of the Ene Reductase OYE1.

Ene reductases from the Old Yellow Enzyme (OYE) family are industrially interesting enzymes for the biocatalytic asymmetric reduction of alkenes. To access both enantiomers of the target reduced products, stereocomplementary pairs of OYE enzymes are necessary, but their natural occurrence is quite limited. A library of wild type ene reductases from different sources was screened in the stereoselective reduction of a set of representative α-alkyl-ß-arylenones to investigate the naturally available biodiversity. As far as the bioreduction of the ethyl ketone derivatives concerns, the results confirmed the distinctiveness of the OYE3 enzyme in affording the reduced product in the (S) configuration, while all the other tested ene reductases from the Old Yellow Enzymes family showed the same stereoselectivity toward the formation of corresponding (R) enantiomer. A possible determinant role of the "hot spot" residue in position 296 for the stereoselectivity control of these reactions was confirmed by the replacement of Phe296 of OYE1 with Ser as found in OYE3. Further investigations showed that the same stereoselectivity switch in OYE1 could be achieved also by the replacement of Trp116 with Ala and Val, these experimental results being rationalized by structural and docking studies. Moreover, an additive effect on the stereoselectivity of OYE1 was observed when coupling the selected mutations in position 296 and 116, thus providing two extremely enantioselective variants of OYE1 (W116A-F296S, W116V-F296S) showing the opposite stereoselectivity of the wild type enzyme. Lastly, the effects of the mutations on the bioreduction of carvone enantiomers were investigated as well.

Exploiting the vicinal disubstituent effect on the diastereoselective synthesis of γ and δ lactones.

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.

Old Yellow Enzyme homologues in Mucor circinelloides: expression profile and biotransformation.

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.

One-Pot Multi-Enzymatic Synthesis of the Four Stereoisomers of 4-Methylheptan-3-ol.

The use of pheromones in the integrated pest management of insects is currently considered a sustainable and environmentally benign alternative to hazardous insecticides. 4-Methylheptan-3-ol is an interesting example of an insect pheromone, because its stereoisomers are active towards different species. All four possible stereoisomers of this compound were prepared from 4-methylhept-4-en-3-one by a one-pot procedure in which the two stereogenic centres were created during two sequential reductions catalysed by an ene-reductase (ER) and an alcohol dehydrogenase (ADH), respectively.

Exploitation of a Multienzymatic Stereoselective Cascade Process in the Synthesis of 2-Methyl-3-Substituted Tetrahydrofuran Precursors.

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 α-bromo-α,ß-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 ß-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.

A Rapid and High-Throughput Assay for the Estimation of Conversions of Ene-Reductase-Catalysed Reactions.

A fast and sensitive colorimetric assay (FRED, fast and reliable ene-reductases detection) that allows the estimation of levels of conversion of ene-reductase (ER)-catalysed reactions has been developed. The activated olefin is reduced by ER at the expense of NAD(P)H cofactor, whose regeneration is carried out in situ by the glucose/glucose dehydrogenase system. Subsequently, the consumption of the co-substrate glucose is determined colorimetrically by a multienzymatic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic analysis, to primary screening of enzyme libraries and optimisation of ERs' performances under different reaction conditions.

Identification of fungal ene-reductase activity by means of a functional screening.

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.

Investigation of the stereochemical course of ene reductase-catalysed reactions by deuterium labelling.

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.

Synthesis of robalzotan, ebalzotan, and rotigotine precursors via the stereoselective multienzymatic cascade reduction of α,ß-unsaturated aldehydes.

A stereoselective synthesis of bicyclic primary or secondary amines, based on tetralin or chroman structural moieties, is reported. These amines are precursors of important active pharmaceutical ingredients such as rotigotine (Neupro), robalzotan, and ebalzotan. The key step is based on a multienzymatic reduction of an α,ß-unsaturated aldehyde or ketone to give the saturated primary or secondary alcohol, in a high yield and with a high ee. The catalytic system consists of the combination of an ene-reductase (ER; i.e., OYE2 or OYE3 belonging to the Old Yellow Enzyme family) with an alcohol dehydrogenase (ADH), applying the in situ substrate feeding product removal technology. By this system the formation of the allylic alcohol side product and the racemization of the chirally unstable α-substituted aldehyde intermediate are minimized. The primary alcohols were elaborated via a Curtius rearrangement. The combination of OYE2 with a Prelog or an anti-Prelog ADH allowed the preparation of the secondary alcohols with ee > 99% and de > 87%. The absolute configuration of the primary amines was unambiguously assigned by comparison with authentic samples. The stereochemistry of secondary alcohols was assigned by X-ray crystal structure and NMR analysis of Mosher esters.

Substrate scope and synthetic applications of the enantioselective reduction of α-alkyl-ß-arylenones mediated by Old Yellow Enzymes.

The ene-reductases mediated bioreduction of a selection of open-chain α-alkyl-ß-aryl enones afforded the corresponding saturated α-chiral ketones in high yield and optical purity in several cases. The stereo-electronic requirements of the reaction have been investigated, considering the nature and location of substituents on the aromatic ring as well as the steric hindrance at the α-position and adjacent to the carbonyl functionality. The general considerations drawn allow us to guide the design of α,ß-unsaturated ketones to be employed as substrates of ene-reductases in future preparative applications. An interesting case of orthogonality between enzyme-based and substrate-based stereocontrol within the highly homologous ene-reductases from Saccharomyces species (OYE1-3) has been reported and rationalized with the help of computational docking studies. Furthermore, to demonstrate the synthetic versatility of the reaction, the key chiral precursors of biologically active compounds such as (2'R)-stenusines and (S)-iopanoic acid were obtained. The very robust protocol allowed us to run the reactions on preparative scale in quantitative yields, with a simple work-up and no chromatographic purification steps.