Euglenatides, potent antiproliferative cyclic peptides isolated from the freshwater photosynthetic microalga Euglena gracilis

By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC 50 values of 4.3 μM in Aspergillus fumigatus and 0.29 μM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products

Nouvelles transaminases et procédés multienzymatiques innovants pour la synthèse hautement sélective d’amines et d’aminoalcools chiraux

This thesis had two main objectives. On the one hand, the search for new ATA via the screening of enzyme libraries from biodiversity and on the other hand the development of new biocatalytic processes associating AL and TA. We have developed two original screening assays allowing the high-throughput screening of TA collections against a variety of amine donor and acceptor substrates. These assays were implemented to screen TA collections constructed by genome mining in collaboration with the Génoscope institute of Evry. From the pool of hits, a sub-collection of 41 ATAs was constituted for further studies. Within this sub-collection, 7 ATA with remarkable properties were purified and extensively characterised. The screening of these different groups of TAs was carried out within the framework of collaborations. This made possible to demonstrate the interest of these new biocatalysts for the synthesis of chiral amines. For example, a very efficient and highly enantioselective synthesis of an amine was achieved on a gram scale. Preliminary molecular modelling work confirmed the sequence-structure-activity relationships of these new ATA. In addition, an important part of this research work was devoted to the development of linear or cyclic AL-TA cascades for the synthesis of a variety of γ-amino alcohols. This work allowed us to prepare a new analogue of 4-hydroxyproline, two new stereoisomers of D-4-hydroxyglutamic acid, the (2S,3R,4S) isomer of 4-hydroxyisoleucine and two analogues of this molecule. The development of these processes allowed us to elucidate the different factors responsible for controlling the stereoselectivity of the AL-TA cyclic cascades.Ce travail de thèse avait deux objectifs principaux. D’une part, la recherche de nouvelles ATA via le criblage de banques d’enzymes issues de la biodiversité et d’autre part le développement de nouveaux procédés biocatalytique associant AL et TA. Nous avons développé deux tests de criblages originaux permettant le criblage haut débit des collections de TA envers une variété de substrats donneurs d’amines et d’accepteurs. Ces tests ont été mis en œuvre pour cribler des collections de TA construites par exploration génomique dans le cadre d’une collaboration avec le Génoscope d’Evry. Parmi l’ensemble des hits, une sous collection de 41 ATA a été constituée pour des études plus approfondies. Au sein de cette sous collection, 7 ATA aux propriétés remarquables ont été purifiées et largement caractérisées. Le criblage de ces différents groupes de TA a été mis en œuvre dans le cadre de collaborations et a notamment permis de démontré l’intérêt de ces nouveaux biocatalyseurs pour la synthèse d’amines chirales. Par exemple, un procédé de synthèse très efficace et hautement énantiosélectif d’une amine a été réalisée à l’échelle du gramme. Des travaux préliminaires de modélisation moléculaire ont confirmé les relations séquence-structure-activité de ces nouvelles ATA. Un part importante de ce travail de recherche a été consacré à la mise au point de cascades linéaires ou cycliques AL-TA pour la synthèse d’une variété de γ-aminoalcools. Ces travaux nous ont permis de préparer un nouvel analogue de la 4-hydroxyproline, deux nouveaux stéréisomères de l’acide D-4-hydroxyglutamique, l’isomère (2S,3R,4S) de la 4-hydroxyisoleucine ainsi que deux analogues de cette molécule. La mise au point de ces procédés nous a permis d’élucider les différents facteurs responsables du contrôle de la stéréosélectivité des cascades cycliques AL-TA

Nouvelles transaminases et procédés multienzymatiques innovants pour la synthèse hautement sélective d’amines et d’aminoalcools chiraux

This thesis had two main objectives. On the one hand, the search for new ATA via the screening of enzyme libraries from biodiversity and on the other hand the development of new biocatalytic processes associating AL and TA. We have developed two original screening assays allowing the high-throughput screening of TA collections against a variety of amine donor and acceptor substrates. These assays were implemented to screen TA collections constructed by genome mining in collaboration with the Génoscope institute of Evry. From the pool of hits, a sub-collection of 41 ATAs was constituted for further studies. Within this sub-collection, 7 ATA with remarkable properties were purified and extensively characterised. The screening of these different groups of TAs was carried out within the framework of collaborations. This made possible to demonstrate the interest of these new biocatalysts for the synthesis of chiral amines. For example, a very efficient and highly enantioselective synthesis of an amine was achieved on a gram scale. Preliminary molecular modelling work confirmed the sequence-structure-activity relationships of these new ATA. In addition, an important part of this research work was devoted to the development of linear or cyclic AL-TA cascades for the synthesis of a variety of γ-amino alcohols. This work allowed us to prepare a new analogue of 4-hydroxyproline, two new stereoisomers of D-4-hydroxyglutamic acid, the (2S,3R,4S) isomer of 4-hydroxyisoleucine and two analogues of this molecule. The development of these processes allowed us to elucidate the different factors responsible for controlling the stereoselectivity of the AL-TA cyclic cascades.Ce travail de thèse avait deux objectifs principaux. D’une part, la recherche de nouvelles ATA via le criblage de banques d’enzymes issues de la biodiversité et d’autre part le développement de nouveaux procédés biocatalytique associant AL et TA. Nous avons développé deux tests de criblages originaux permettant le criblage haut débit des collections de TA envers une variété de substrats donneurs d’amines et d’accepteurs. Ces tests ont été mis en œuvre pour cribler des collections de TA construites par exploration génomique dans le cadre d’une collaboration avec le Génoscope d’Evry. Parmi l’ensemble des hits, une sous collection de 41 ATA a été constituée pour des études plus approfondies. Au sein de cette sous collection, 7 ATA aux propriétés remarquables ont été purifiées et largement caractérisées. Le criblage de ces différents groupes de TA a été mis en œuvre dans le cadre de collaborations et a notamment permis de démontré l’intérêt de ces nouveaux biocatalyseurs pour la synthèse d’amines chirales. Par exemple, un procédé de synthèse très efficace et hautement énantiosélectif d’une amine a été réalisée à l’échelle du gramme. Des travaux préliminaires de modélisation moléculaire ont confirmé les relations séquence-structure-activité de ces nouvelles ATA. Un part importante de ce travail de recherche a été consacré à la mise au point de cascades linéaires ou cycliques AL-TA pour la synthèse d’une variété de γ-aminoalcools. Ces travaux nous ont permis de préparer un nouvel analogue de la 4-hydroxyproline, deux nouveaux stéréisomères de l’acide D-4-hydroxyglutamique, l’isomère (2S,3R,4S) de la 4-hydroxyisoleucine ainsi que deux analogues de cette molécule. La mise au point de ces procédés nous a permis d’élucider les différents facteurs responsables du contrôle de la stéréosélectivité des cascades cycliques AL-TA

A continuous colorimetric screening assay for amine-transaminases

International audienceTransaminases (TA) which offer a highly stereoselective access to chiral amino pharmaceuticals and bioactive compounds have gained considerable attention in the past few years.1–3 The availability of efficient screening assays is of high importance to detect, in wild type or mutant enzymes libraries, the wanted TA for a specific application. Therefore, in the course of our research aimed at the design of new TA-catalysed processes,4–6 we have developed a new screening method for amine-TA. This method is based on the use of hypotaurine (HPT) as amino donor substrate, which is converted upon transamination into 2-oxoethylsulfinic acid, itself instantaneously decomposed into sulfite and acetaldehyde. Sulfite ions can then be easily detected by spectrophotometry at 412 nm using Ellman’s reagent, thus allowing direct kinetic measurements. As shown below, two complementary assays were developed based on this titration method.The direct assay allows to detect a HPT-TA, ie an enzyme active with any chosen acceptor and HPT as donor. These HPT-TA catalyse an irreversible transamination, provided that they don’t accept acetaldehyde as substrate. In the coupled assay, L- or D-Ala is used as generic donor substrate of amine-TA and is regenerated using an auxiliary HPT-TA. This coupled reaction thus allows the activity measurement and can afford an equilibrium shift. We will present the development and application of this method for the screening of a collection of 375 Amine-TA from biodiversity. The direct assay implementation allowed to identify several highly active HPT-TA, whereas the enzyme with the most restricted substrate spectrum proved suitable for use as the auxiliary enzyme in the coupled assay. This latter general screening assay was then successfully employed for the activity assessment of diverse amine-TA with Ala and a variety of acceptor substrates.References:1 D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H.; D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H. (2018), Catalysts, 8: 254.2 Gomm, A.; O’Reilly, E. (2018), Curr. Opin. Chem. Biol., 43: 106–112.3 Ferrandi, E. E.; Monti, D. (2017), World J Microbiol Biotechnol., 34:13.4 Guérard-Hélaine, C.; Heuson, E.; Ndiaye, M.; Gourbeyre, L.; Lemaire, M.; Hélaine, V.; Charmantray, F.; Petit, J.-L.; Salanoubat, M.; Berardinis, V. de; et al. (2017), Chem. Commun., 53: 5465–5468.5 Heuson, E.; Petit, J.-L.; Debard, A.; Job, A.; Charmantray, F.; Berardinis, V. de; Gefflaut, T. (2015), Appl Microbiol Biotechnol, 100: 397–408.6 Heuson, E.; Charmantray, F.; Petit, J.-L.; de Berardinis, V.; Gefflaut, T. (2019), Advanced Synthesis & Catalysis, 361: 778–785

A continuous colorimetric screening assay for amine-transaminases

Transaminases (TA) which offer a highly stereoselective access to chiral amino pharmaceuticals and bioactive compounds have gained considerable attention in the past few years.1–3 The availability of efficient screening assays is of high importance to detect, in wild type or mutant enzymes libraries, the wanted TA for a specific application. Therefore, in the course of our research aimed at the design of new TA-catalysed processes,4–6 we have developed a new screening method for amine-TA. This method is based on the use of hypotaurine (HPT) as amino donor substrate, which is converted upon transamination into 2-oxoethylsulfinic acid, itself instantaneously decomposed into sulfite and acetaldehyde. Sulfite ions can then be easily detected by spectrophotometry at 412 nm using Ellman’s reagent, thus allowing direct kinetic measurements. As shown below, two complementary assays were developed based on this titration method.The direct assay allows to detect a HPT-TA, ie an enzyme active with any chosen acceptor and HPT as donor. These HPT-TA catalyse an irreversible transamination, provided that they don’t accept acetaldehyde as substrate. In the coupled assay, L- or D-Ala is used as generic donor substrate of amine-TA and is regenerated using an auxiliary HPT-TA. This coupled reaction thus allows the activity measurement and can afford an equilibrium shift. We will present the development and application of this method for the screening of a collection of 375 Amine-TA from biodiversity. The direct assay implementation allowed to identify several highly active HPT-TA, whereas the enzyme with the most restricted substrate spectrum proved suitable for use as the auxiliary enzyme in the coupled assay. This latter general screening assay was then successfully employed for the activity assessment of diverse amine-TA with Ala and a variety of acceptor substrates.References:1 D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H.; D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H. (2018), Catalysts, 8: 254.2 Gomm, A.; O’Reilly, E. (2018), Curr. Opin. Chem. Biol., 43: 106–112.3 Ferrandi, E. E.; Monti, D. (2017), World J Microbiol Biotechnol., 34:13.4 Guérard-Hélaine, C.; Heuson, E.; Ndiaye, M.; Gourbeyre, L.; Lemaire, M.; Hélaine, V.; Charmantray, F.; Petit, J.-L.; Salanoubat, M.; Berardinis, V. de; et al. (2017), Chem. Commun., 53: 5465–5468.5 Heuson, E.; Petit, J.-L.; Debard, A.; Job, A.; Charmantray, F.; Berardinis, V. de; Gefflaut, T. (2015), Appl Microbiol Biotechnol, 100: 397–408.6 Heuson, E.; Charmantray, F.; Petit, J.-L.; de Berardinis, V.; Gefflaut, T. (2019), Advanced Synthesis & Catalysis, 361: 778–785

A continuous colorimetric screening assay for amine-transaminases

Transaminases (TA) which offer a highly stereoselective access to chiral amino pharmaceuticals and bioactive compounds have gained considerable attention in the past few years.1–3 The availability of efficient screening assays is of high importance to detect, in wild type or mutant enzymes libraries, the wanted TA for a specific application. Therefore, in the course of our research aimed at the design of new TA-catalysed processes,4–6 we have developed a new screening method for amine-TA. This method is based on the use of hypotaurine (HPT) as amino donor substrate, which is converted upon transamination into 2-oxoethylsulfinic acid, itself instantaneously decomposed into sulfite and acetaldehyde. Sulfite ions can then be easily detected by spectrophotometry at 412 nm using Ellman’s reagent, thus allowing direct kinetic measurements. As shown below, two complementary assays were developed based on this titration method.The direct assay allows to detect a HPT-TA, ie an enzyme active with any chosen acceptor and HPT as donor. These HPT-TA catalyse an irreversible transamination, provided that they don’t accept acetaldehyde as substrate. In the coupled assay, L- or D-Ala is used as generic donor substrate of amine-TA and is regenerated using an auxiliary HPT-TA. This coupled reaction thus allows the activity measurement and can afford an equilibrium shift. We will present the development and application of this method for the screening of a collection of 375 Amine-TA from biodiversity. The direct assay implementation allowed to identify several highly active HPT-TA, whereas the enzyme with the most restricted substrate spectrum proved suitable for use as the auxiliary enzyme in the coupled assay. This latter general screening assay was then successfully employed for the activity assessment of diverse amine-TA with Ala and a variety of acceptor substrates.References:1 D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H.; D. Patil, M.; Grogan, G.; Bommarius, A.; Yun, H. (2018), Catalysts, 8: 254.2 Gomm, A.; O’Reilly, E. (2018), Curr. Opin. Chem. Biol., 43: 106–112.3 Ferrandi, E. E.; Monti, D. (2017), World J Microbiol Biotechnol., 34:13.4 Guérard-Hélaine, C.; Heuson, E.; Ndiaye, M.; Gourbeyre, L.; Lemaire, M.; Hélaine, V.; Charmantray, F.; Petit, J.-L.; Salanoubat, M.; Berardinis, V. de; et al. (2017), Chem. Commun., 53: 5465–5468.5 Heuson, E.; Petit, J.-L.; Debard, A.; Job, A.; Charmantray, F.; Berardinis, V. de; Gefflaut, T. (2015), Appl Microbiol Biotechnol, 100: 397–408.6 Heuson, E.; Charmantray, F.; Petit, J.-L.; de Berardinis, V.; Gefflaut, T. (2019), Advanced Synthesis & Catalysis, 361: 778–785

De la biocatalyse à la catalyse hybride, les enzymes au cœur de procédés sélectifs et durables

National audienc

Biocatalysed Synthesis of Chiral Amines: Continuous Colorimetric Assays for Mining Amine-Transaminases

International audienceIn the course of our research aimed at the design of new biocatalytic processes for the enantioselective synthesis of chiral amines, we have developed new continuous assays for the screening of amine-transaminases collections. These assays are based on the use of hypotaurine as irreversible amino donor. This β-aminosulfinic acid is converted upon transamination into 2-oxoethylsulfinic acid, which instantaneously decomposes into acetaldehyde and sulfite ions that can be easily detected by spectrophotometry using Ellman's reagent. Two complementary assays were developed based on this titration method. Firstly, a direct assay allowed detecting various transaminases able to use hypotaurine as amino donor. In a second coupled assay, L-alanine is used as generic donor substrate of aminetransaminases and is regenerated using an auxiliary hypotaurine-transaminase. The powerful and complementary nature of both assays was demonstrated through the screening of a collection of 549 amine-transaminases from biodiversity, thus allowing the discovery of a variety of valuable new biocatalysts for use in synthetic processes

Pyruvate Aldolases Catalyze Cross-Aldol Reactions between Ketones: Highly Selective Access to Multi-Functionalized Tertiary Alcohols

International audienceTertiary alcohols are widely represented in nature and among bioactive molecules. Their importance is attested by the continuous efforts to meet the challenge of their stereoselective synthesis. In this context, we propose an enzymatic approach, involving class II pyruvate aldolases. These enzymes are shown to catalyze selective cross-aldol reactions between pyruvic acid or derivatives as nucleophiles and a series of ketones as electrophiles. This catalytic activity is exemplified by the highly stereoselective preparation of seven branched ketols with good yields. One of them was readily converted into a constrained 4-hydroxyproline analogue in a multienzymatic one-pot one-step process

Stereoselective synthesis of γ-hydroxy-α-amino acids through aldolase–transaminase recycling cascades

International audienceEfficient bi-enzymatic cascades combining aldolases and α-transaminases were designed for the synthesis of γ-hydroxy-α-amino acids. These recycling cascades provide high stereoselectivity, atom economy, and an equilibrium shift of the transamination. L-syn or anti-4-hydroxyglutamic acid and D-anti-4,5-dihydroxynorvaline were thus prepared in 83–95% yield in one step from simple substrates