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Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

International audience; In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes.

International audience; The structural diversity of terpenes is particularly notable and many studies are carried out to increase it further. In the terpene biosynthetic pathway this diversity is accessible from only two common precursors, i. e. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Methods recently developed (e. g. the Terpene Mini Path) have allowed DMAPP and IPP to be obtained from a two-step enzymatic conversion of industrially available isopentenol (IOH) and dimethylallyl alcohol (DMAOH) into their corresponding diphosphates. Easily available IOH and DMAOH analogues then offer quick access to modified terpenoids thus avoiding the tedious chemical synthesis of unnatural diphosphates. The aim of this minireview is to cover the literature devoted to the use of these analogues for widening the accessible terpene chemical space.

International audience; Terpenoids constitute the largest class of natural compounds and are extremely valuable from an economic point of view due to their extended physicochemical properties and biological activities. Due to recent environmental concerns, terpene extraction from natural sources is no longer considered as a viable option, and neither is the chemical synthesis to access such chemicals due to their sophisticated structural characteristics. An alternative to produce terpenoids is the use of biotechnological tools involving, for example, the construction of enzymatic cascades (cell- free synthesis) or a microbial bio-production thanks to metabolic engineering techniques. Despite outstanding successes, these approaches have been hampered by the length of the two natural biosynthetic routes (the mevalonate and the methyl erythritol phosphate pathways), leading to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), the two common universal precursors of all terpenoids. Recently, we, and others, developed what we called the terpene mini- path, a robust two enzyme access to DMAPP and IPP starting from their corresponding two alcohols, dimethylallyl alcohol and isopentenol. The aim here is to present the potential of this artificial bio-access to terpenoids, either in vitro or in vivo, through a review of the publications appearing since 2016 on this very new and fascinating field of investigation.

All naturally produced terpenes are derived from two universal C5 diphosphate precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Various prenyl transferases use DMAPP ...

Background Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established—mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes. Results The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule. Conclusion IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance. Electronic supplementary material The online version of this article (10.1186/s12934-019-1074-4) contains supplementary material, which is available to authorized users.

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

International audience; Out of 107 fungal strains belonging to three phyla (Ascomycota, Basidiomycota and Zygomycota) and 46 genera, 86 exhibited Baeyer–Villiger Monooxygenase (BVMO) activity against racemic bicyclo[3.2.0]hep-tenone. The strains were classified into three ‘‘profiles’’ based on regio- and enantioselectivity. Statistical analyses of our results, extended by literature data, showed that these profiles could be related to the taxonomic classification of the strains, and suggest that the BVMOs from the Zygomycota phylum may be different in their primary structures from established ones.

a b s t r a c t We performed a directed evolution study with a metagenome-derived epoxide hydrolase (EH), termed Kau2. Homology models of Kau2 were built; we selected one of them and used it as a guide for satu- ration mutagenesis experiments targeted at specific residues within the large substrate binding pocket. During the molecular evolution process, we found several enzyme variants with higher enantioselec- tivity or enhanced enantioconvergence toward para-Chlorostyrene oxide. Improved enantioselectivities by up to a factor of 5, reaching an E-value of up to 130 with the R-enantiomer as the residual epoxide, were achieved by replacing amino acid pairs at the positions 110 and 113, or 290 and 291, which are positions located in the vicinity of two presumed binding sites for the epoxide enantiomers. The (R)-para- Chlorophenylethane-1,2-diol product exhibited a high enantiomeric excess (ee) of 97% at 50% conversion of the racemic epoxide for the most enantioselective variant. Further, five amino acid substitutions were sufficient to substantially increase the degree of enantioconvergence and to lower the E-value to 17 for the final evolved EH variant, enabling the production of the R-diol with an ee-value of 93% at 28 ◦ C in a complete conversion of the racemic epoxide. Higher eep-values of up to 97% were determined in enan- tioconvergent reactions using lower temperatures. The EH activities of whole cells were found to be within the range of 74-125% of the wild-type activity for all investigated variants. We show in this report that the metagenome-derived Kau2 EH is amenable to the redesign of its enantioselectivity and regio- selectivity properties by directed evolution using a homology model as a guide. The generated enzyme variants should be useful for the production of the chiral building blocks (R)-para-Chlorostyrene oxide and (R)-para-Chlorophenylethane-1,2-diol. © 2013 Elsevier B.V. All rights reserved.

During a program dedicated to the biocatalytic access of structurally useful α-hydroxy ketones, we made the unexpected observation that epoxy enol acetates derived from (+)- and (–)-menthone stereoselectively rearranged on exposure to silica through a diastereoselective process, providing easy access to the complete set of α-hydroxymenthone stereoisomers. The absolute configurations of the four stereoisomers were unambiguously established by IR-VCD and X-ray diffraction, thus clarifying some literature discrepancy. Two of the four stereoisomers could also be obtained through astereoconvergent process, thus avoiding the inherent 50 % yield limitations of such diastereoselective reactions. A solvent-free version allowing an extremely rapid reaction (< 2 h) is also described. Finally, the observed diastereoselection was investigated by DFT calculations.

We used the recombinant CotA-laccase from the bacterium Bacillus subtilis to investigate the biotransformation of the commercial anthraqui- nonic dye Acid Blue 62. Kinetics of dye biotransfor- mation at pH 6 follow a Michaelis-Menten model. NMR and several MS techniques allowed the iden- tification of intermediates and final products of the enzymatic biotransformation. The main final product obtained, 1-((4-amino-9,10-dioxo-3-sulfo-9,10-dihy- droanthracen-1-yl)diazenyl)-4-cyclohexylamino-9,10- dioxo-9,10-dihydroanthracene-2-sulfonic acid, is formed through the creation of an azo link and has been previously identified as an intermediate com- pound in the biodegradation of Acid Blue 62 by crude fungal preparations. The identification of 1,4- diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthracene- 2-sulfonic acid and of cyclohexanone, in reaction mixtures with CotA-laccase and also its presence in reactions performed with the LAC3 laccase from the fungus Trametes sp. C30, suggest the occurrence of coupling reactions between the intermediate prod- ucts of dye oxidation. Based on these results, we pro- pose a mechanistic pathway for the biotransforma- tion of Acid Blue 62 by laccases. A bioassay based on the inhibitory effects of the dye and its enzymatic products on the growth of Saccharomyces cerevisiae shows the importance of laccases in reducing dye toxicity.

Quercetinase (quercetin 2,3-dioxygenase, EC 1.13.11.24) is produced by various filamentous fungi when grown on rutin as the sole carbon and energy source. From a rutin based liquid culture of Penicillium olsonii, we purified a quercetinase with a specific activity of 175U mg(-1). The enzyme is a monomeric glycoprotein of approximately 55 kDa, containing 0.9+/-0.1 copper atoms per protein. Its substrate specificity is restricted to the flavonol family of flavonoids. It is completely inhibited by diethyldithiocarbamate at a concentration of 100 nM and 1H-2-benzyl-3-hydroxy-4-oxoquinolin is a competitive inhibitor with a K(I) of 4 microM. The cDNA poquer1 was cloned and sequenced. It encodes a 365 amino acids long enzyme with a strong sequence identity with the Aspergillus japonicus quercetinase (Q7SIC2). Like the enzyme from A. japonicus, only one of the two cupin domains of the Penicillium olsonii quercetinase is able to bind a metal atom.

Epoxide hydrolase Kau2 overexpressed in Escherichia coli RE3 has been tested with ten different racemic and meso,-disubstituted aromatic epoxides. Some of the tested substrates were bi-functional, and most of them are very useful building blocks in synthetic chemistry applications. As a general trend Kau2 proved to be an extremely enantioselective biocatalyst, the diol products and remaining epoxides of the bioconversions being obtained – with two exceptions – in nearly enantiomerically pure form. Furthermore, the reaction times were usually very short (around 1 h, except when stilbene oxides were used), and the use of organic co-solvent was well tolerated, enabling very high substrate concentrations (up to 75 g/L) to be reached. Even extremely sterically demanding epoxides such as cis-and trans-stilbene oxides were transformed on a reasonable time scale. All reactions were successfully conducted on a 1-g preparative scale, generating diol-and epoxide-based chiral synthons with very high enantiomeric excesses and isolated yields close to the theoretical maximum. Thus we have here demonstrated the usefulness and versatility of lyophilizedEscherichia coli cells expressing Kau2 epoxide hydrolase as a highly enantioselective biocatalyst for accessing very valuable optically pure aromatic epoxides and diols through kinetic resolution of racemates or desymmetrization of meso epoxides.

International audience; Epoxide hydrolase Kau2 overexpressed in Escherichia coli RE3 has been tested with ten different racemic and meso,-disubstituted aromatic epoxides. Some of the tested substrates were bi-functional, and most of them are very useful building blocks in synthetic chemistry applications. As a general trend Kau2 proved to be an extremely enantioselective biocatalyst, the diol products and remaining epoxides of the bioconversions being obtained – with two exceptions – in nearly enantiomerically pure form. Furthermore, the reaction times were usually very short (around 1 h, except when stilbene oxides were used), and the use of organic co-solvent was well tolerated, enabling very high substrate concentrations (up to 75 g/L) to be reached. Even extremely sterically demanding epoxides such as cis-and trans-stilbene oxides were transformed on a reasonable time scale. All reactions were successfully conducted on a 1-g preparative scale, generating diol-and epoxide-based chiral synthons with very high enantiomeric excesses and isolated yields close to the theoretical maximum. Thus we have here demonstrated the usefulness and versatility of lyophilizedEscherichia coli cells expressing Kau2 epoxide hydrolase as a highly enantioselective biocatalyst for accessing very valuable optically pure aromatic epoxides and diols through kinetic resolution of racemates or desymmetrization of meso epoxides.

We report here on the use of a biphasic reaction solvent during the laccase catalysed transformation of sinapinic and ferulic acids. As compared to buffer alone, higher product selectivity, higher yields and higher product stability were obtained simply using ethyl acetate as added non-miscible co-solvent. Reactions run in biphasic medium are marginally longer but offer an easy separation protocol allowing to obtain the highest yield reported so far for the synthesis of the two bis-lactone lignans originating from sinapinic and ferulic acids.

The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.

Quercetinase is produced by various filamentous fungi when grown on rutin as sole carbon and energy source. We investigated on the effect of 10 phenolics and two sugars, structurally related to substrates and products of the rutin catabolic pathway, on the induction of a quercetinase activity in Penicillium olsonii. Neither the sugars (glucose and rhamnose, two constituents of rutin), nor phenolics such as protocatechuic acid, salicylic acid, 4-hydroxy-benzoic acid and phloroglucinol were inducers. Rutin (maximum activity 150 nmol/min/mL after 5 days), quercetin (70 nmol/min/mL, 3 days), phloroglucinol carboxylic acid (60 nmol/min/mL, 3 days), 2-protocatechuoylphloroglucinolcarboxylic acid (50 nmol/min/mL, 5 days), 2,6-dihydroxy-carboxylic acid (90 nmol/min/mL, 7 days) and 2,4-dihydroxy-carboxylic acid (30 nmol/min/mL, 7 days) were demonstrated to be quercetinase inducers. We propose that rutin, quercetin and 2-protocatechuoyl-phloroglucinol carboxylic acid, the product of the reaction catalysed by quercetinase, act as inducers after their catabolic transformation in phloroglucinol carboxylic acid.

The four diastereoisomers of 1-fluoro-2-amino-indane have been synthesized in high enantiomeric excess with lipase resolution of cis-2-azido-1-indanol as a key step.

Medium improvement for quercetinase production by Penicillium olsonii was investigated using a fractional factorial design in two sets of eight experiments with seven variable components each. The tested components were: rutin concentration, nitrogen source and its concentration, KH 2 PO 4 , NaCl, MgSO 4 and yeast extract concentrations, inoculum size, copper and metal salts concentrations and pH value. A final optimisation step, varying only yeast extract concentration, allowed six-fold improvement of the quercetinase activity with respect to initial medium, reaching a maximum activity of 0.708 μmol/min/ml after 9 days of cultivation.

Enantiomerically pure cyclic ( S )-diethyl 3-hydroxy-1-alkenyl phosphonates 1 – 3 were obtained by enzymatic resolutions. The corresponding ( R )-acetates were also obtained with high enantiomeric excess, except in the case of the five-membered ring compound. The most efficient enzyme (lipase Amano AK or Amano PS) in each case depends on the structure of the substrate (five to seven-membered ring compounds).

Enzymatic resolution of racemic 3-bromo-cyclohept-2-enol 2 with lipozyme affords enantiomerically pure (S)-(−)-2 whose absolute configuration was determined by chemical correlation, and further allowed an enantioselective synthesis of (S)-(+)-diethyl (3-hydroxy-cyclohept-1-enyl)phosphonate 1.

The borane adduct of (2-hydroxypropyl)diphenylphosphane 1 was resolved by enantioselective esterification with CAL-B. (S)-1 was thus obtained with 91% enantiomeric excess. Its absolute configuration was determined by chemical correlation from (S)-propylene oxide.

During a screening procedure for the discovery of a strong gamma-decalactone producer from ricinoleic acid, we observed that the yeast Pichia guilliermondii accumulated transiently 8-hydroxy-3Z,5Z-tetradecadienoic acid 1 during gamma-decalactone biosynthesis in the stationary phase of growth. The structural elucidation of 1 was based on nuclear magnetic resonance, infrared, ultraviolet and gas chromatography-mass spectrometry experiments. The occurrence of 1 is discussed in relation with previously proposed gamma-decalactone biosynthetic pathways.

A high intensity direct current was applied for more than 10 min onto a bacterial suspension of Pseudomonas oleovorans ATCC 29347 suspended in silicone oil. The application of a gradually increased electric field from 0 to 2500 V x cm(-1) resulted in a decrease of the optical density of the bacterial suspension and the occurrence of a peak current of several hundred microA for living cells instead of a linear increase (few microA) for killed or lyophilised cells. This procedure is not only a rapid way of investigating the living state of cell cultures but also an efficient experimental tool to study the cellular effects of a controlled electrical stress.

Cyclic dialkyl (3-oxo-1-cycloalkenyl) phosphonates were subjected to baker's yeast-mediated enantioselective reductions to afford the corresponding dialkyl (3-hydroxy-1-alkenyl) phosphonates. The six- and seven-membered ring enones were reduced with moderate to good enantiomeric excesses, whereas the five-membered ring substrate always yielded the double bond reduced compound. The use of different reduction conditions did not improve the ee's markedly, but it was found, for the six-membered analogues, that the alkyl groups held by phosphorus influence dramatically the enantioselectivity of the reduction, leading to up to 95% enantiomeric excess.

Laccases are blue-copper enzymatic proteins involved in the transformation of polyphenolic compounds, such as lignin, into forest litter. A method to measure their activity was developed using an evergreen oak litter as a model system. We considered factors that affect the extraction of these enzymes and the measurement of their activity. The factorsstudied included powdering of the leaves, adding polyvinylpolypyrrolidone or Tween 80, adsorption of enzymes on centrifugation pellets, concentration of the extracts, nature and molarity of the extraction solvent, amount of powdered leaves used for enzymatic extraction, extraction time, pH, and temperature during the measurement of the activity.

Indole is cleanly bioconverted into a trimer upon laccase action. The formed compound has been identified as 2,2-bis(3′-indolyl)indoxyl (1) by X-ray diffraction study. Optimisation of the reaction through the use of dioxygen overpressure and TEMPO as mediator allowed a yield of more than 50% of isolated product for 1. 2,2-Bis(3′-indolyl)indoxyl is a natural compound that has been isolated from bacterial sources. However, this is the first report on a clearly identified enzyme that could be involved in its (bio)synthesis. A mechanism, based on the initial formation of a transient indole hydroperoxide, is proposed to account for the laccase catalysed synthesis of 1.

The straightforward chemo-enzymatic synthesis of methyl 9( S )-HODE (dimorphecolic acid methyl ester) and methyl 9( S )-HOTE, from linoleic and α-linolenic acids, using barley seeds lipoxygenase under oxygen pressure and at high substrate concentration, is described.

(±)-trans and cis Cyclohexane-1,2-diols have been shown to undergo a double stereoinversion process to give trans (S,S)-cyclohexane-1,2-diols on incubation with the fungus C. cassiicola.

The optimization of 13-S-hydroperoxy-9Z, 11E-octadecadienoic acid synthesis is described using lipoxygenase-1 from soybeans at high substrate concentration. The optimal values of the tested parameters are as follows: oxygen pressure 2.5 bar, temperature 5°C, pH 11, enzyme concentration 4 mg/ml and substrate concentration 0.1 M. All these values were used in a single reaction, allowing chemoenzymatic synthesis of gram amounts of (+)-coriolic acid (99%, e.e. 97%) with a 54% yield starting from linoleic acid.

The enzymatic esterification of diethyl (3-hydroxy-1-butenyl) phosphonate 1 with different enzymes has been carried out, and allows the preparation of (S)-1 and (R)-diethyl (3-acetoxy-1-butenyl) phosphonate 2 with very high enantiomeric excess. The absolute conguration of 1 was determined by independent synthesis from (S)-ethyl lactate.

The optimization of 13-S-hydroperoxy-9Z, 11E-octadecadienoic acid synthesis is described using lipoxygenase-1 from soybeans at high substrate concentration. The optimal values of the tested parameters are as follows: oxygen pressure 2.5 bar, temperature 5°C, pH 11, enzyme concentration 4 mg/ml and substrate concentration 0.1 M. All these values were used in a single reaction, allowing chemoenzymatic synthesis of gram amounts of (+)-coriolic acid (99%, e.e. 97%) with a 54% yield starting from linoleic acid.

Enzymatic desymmetrization of the title compound 1 is reported using various commercially available lipases in hydrolysis and alcoholysis reactions or ester synthesis. In this area, lipase Amano AK ( Pseudomonas sp.) proved to be the best lipase whatever the experimental conditions used. The monoacetate product 2 is indifferently obtained with more than 95% enantiomeric excess (ee) as the levorotatory enantiomer 2a or the dextrorotatory one 2b .

An efficient preparation of pure ethyl Z- and E-alpha,alpha-difluoro-4-phenyl-3-butenoate 1a and 1b together with the corresponding acids 2a and 2b is described. The procedures involve stereocontrolled additions of *CF2CO2Et to phenylacetylene or beta-bromostyrene. Compound 1a is easily obtained by addition of *CF2CO2Et to phenylacetylene via a mechanism where the stereochemistry is controlled by an electron-transfer process to produce predominantly the Z vinyl anion. The product 1b is obtained by *CF2CO2Et addition-elimination to Z- or E-beta-bromostyrenes via a mechanism where the stereochemistry is controlled by steric factors in the conformational equilibration of the intermediates.

We report here the first chemical synthesis of three depsides related to quercetinase‐catalyzed degradation of kaempferol, quercetin, and myricetin. The three depsides were constructed through the coupling of suitably protected phloroglucinol carboxylic acid and hydroxy‐perbenzylated, derivatives of gallic, protocatechuic, and 4‐hydroxy benzoic acids. The three synthesized target compounds proved to be identical to their natural counterparts, arising from quercetinase action on corresponding flavonols.

Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Michaël-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation (MS2 mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation (MS3 mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC-MS/MS, using initially the neutral loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.

Various optically active hydroxy derivatives of polyunsaturated fatty acids were easily oxidised to their corresponding keto derivatives using Dess-Martin periodinane. The reaction was run on the millimolar scale with good yields and without appreciable isomerisation of the surrounding double bonds. Reduction of these keto compounds to yield back the starting alcohols, but now as racemic mixtures, was also conducted using CeCl3–NaBH4, once again without noticeable modification of the stereochemistry of the double bonds. These reactions proved the usefulness of the chemoenzymatic access to oxylipins through the use of lipoxygenases with various regiospecificity, combined with chemical transformations of the formed hydro(pero)xides.

A new tannase substrate, protocatechuic acid p-nitrophenyl ester, 5, was synthesized using modern synthetic methods. The synthesis was designed to be performed by non-specialized chemists. It only involves four steps, three of which are protection-deprotection, and uses standard methods of separation and purification, such as recrystallization and column chromatography over silica. Under tannase action, protocatechuic acid p-nitrophenyl ester, 5, releases p-nitrophenol, which is easily measured spectrophotometrically either at 350 nm for pH values

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak ( Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the K m was 7.1 μM. The structural gene, lac1 , was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

Using Pseudomonas fluorescens lipase in vinyl acetate, the methyl 4,6-O-benzylidene-α-D-pyranosides 1 and 6–8 have been esterified with very high selectivity to afford the C-2 acetates 3 and 10–12, respectively. In contrast the methyl 4,6-O-benzylidene-β-D-pyranosides 2 and 9 are acetylated under the same conditions to give the C-3 esters 5 and 13 with high/exquisite regioselectivity.

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Biocatalysis is nowadays an important tool available to organist chemist to get access to fine chemicals and especially enantiomerically enriched synthons and molecules. Within this framework, the PhD work described in this dissertation was conducted in order to get insights about a newly discovered epoxide hydrolase (EH) from a metagenomic analysis of a biofilter. In order to conduct directed mutagenesis on the so-called Kau2-EH, a model of the enzyme was constructed based on the 3D structure of murine EH. The choice of this template was dictated by comparative inhibition studies aimed at differentiating three otherwise closely Kau2-sequence-related EHs with known crystal structure (potato-, murine- and human-EHs) and showing inhibition behavior the closest to the one found for Kau2. The enzyme was previously shown to display interesting biocatalytic properties such a nearly perfect kinetic resolution of trans-methyl-styrene-oxide and a nearly perfect enantioconvergent transformation of cis-methyl-styrene-oxide. Thus, bioconversion studies dedicated to the evaluation Kau2-EH substrate chemical space were undertaken. The enzyme proved to be particularly useful when using 1,2-disubstituted cis- or trans-aromatic epoxides bearing an aromatic ring (phenyl, p-methoxy-phenyl) on one of the epoxide-bearing carbon atom and various chemical groups (methyl- or ethyl-esters, cyano, chloro- or bromo-methyl or phenyl) on the second carbon atom. For nine of the ten tested substrates very high enantioselectivities were observed allowing nearly perfect kinetic resolutions of eight of them and a nearly perfect desymmetrization of the ninth. Only cis-methyl-glycidate proved to be not a substrate of Kau2-EH. In the all other nine cases a preparative scale reaction could be conducted on the 1g scale, at high to very high substrate concentration (25 to 75 g/L) and in short periods of time (1 to 4h) except for cis-stilbene-oxide (24 h). Finally and in order to get access to fundamental kinetic constants, a preliminary stopped-flow analysis of Kau2-EH behavior was undertaken using trans-stilbene-oxide as substrate., Les chimistes organiciens disposent à l'heure actuelle des outils de la biocatalyse afin d'accéder aux produits de la chimie fine et en particulier à des synthons et des molécules optiquement enrichies. Dans ce cadre, le travail de thèse présenté dans ce mémoire a été conduit afin d'enrichir notre connaissance sur une époxyde hydrolase (EH) découverte après analyse métagénomique d'un bio-filtre. Afin de pouvoir mener une étude de mutagénèse dirigée de sorte à améliorer certaines propriétés de cette enzyme appelée Kau2-EH, un modèle de l'enzyme a été élaboré sur la base de la structure tridimensionnelle de l'EH de souris. Le choix de cette matrice fait suite à des études d'inhibition comparées visant à déterminer laquelle des trois EHs, dont la structure tridimensionnelle était connue (pomme de terre, souris, homme) et dont la séquence était proche de celle de Kau2-EH, présentait l’inhibition la plus proche de celle observée pour Kau2-EH. Il avait été montré précédemment que Kau2-EH présentait un intérêt en biocatalyse permettant une résolution cinétique quasi-parfaite de l'oxyde de trans-méthyl-styrène et une transformation énantioconvergente, elle aussi quasi-parfaite, de l'oxyde de cis-méthyl-styrène. Ainsi des études de bioconversion dédiées à l'évaluation de la diversité des substrats de Kau2-EH ont été réalisées. Cette enzyme se révéla être particulièrement performante lors de l'utilisation d'époxydes cis- et trans-1,2-disubstitués portant sur un des atomes de carbone de la fonction époxyde un groupement phényle ou p-méthoxy-phényle et sur l'autre un groupement variable (méthyl- ou éthyl-ester, cyano, bromo- ou chloro-méthyle, phényle). Pour neuf des dix substrats testés des énantiosélectivités très élevées ont été trouvées permettant des résolutions cinétiques quasi-parfaites de huit d'entre eux et la désymétrisation quasi-parfaite du neuvième. Seul le cis-méthyl-glycidate ne fut pas un substrat de Kau2-EH. Dans les neufs cas précédents une réaction préparative à l'échelle du gramme a pu être conduite à très haute concentration en substrat (de 25 à 75 g/L) et sur une courte période de temps (de 1 à 4h) sauf pour l'oxyde de cis-stilbène (24 h). Finalement et afin d'accéder aux constantes cinétiques fondamentale, une étude préliminaire de « stopped-flow » du comportement de Kau2-EH a été réalisée en utilisant l'oxyde de trans-stilbène comme substrat.