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1. Galf-Specific Neolectins: Towards Promising Diagnostic Tools

Author

Mateja Seničar, Benoît Roubinet, Pierre Lafite, Laurent Legentil, Vincent Ferrières, Ludovic Landemarre, and Richard Daniellou

Subjects
galactofuranosidase, neolectin bioengineering, site-directed mutagenesis, neoglycoprotein, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Published
2024
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2. The melanoma tumor glyco-code impacts human dendritic cells’ functionality and dictates clinical outcomes

Author

Eleonora Sosa Cuevas, Benoît Roubinet, Stephane Mouret, Michel Thépaut, Florence de Fraipont, Julie Charles, Franck Fieschi, Ludovic Landemarre, Laurence Chaperot, and Caroline Aspord

Subjects
glycan, lectin, human DC subsets, melanoma, immune subversion, Immunologic diseases. Allergy, RC581-607
Abstract

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients’ clinical outcome and DC subsets’ functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs’ functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor’ hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.

Published
2023
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3. Unique CLR expression patterns on circulating and tumor-infiltrating DC subsets correlated with clinical outcome in melanoma patients

Author

Eleonora Sosa Cuevas, Jenny Valladeau-Guilemond, Stephane Mouret, Benoît Roubinet, Florence de Fraipont, Ludovic Landemarre, Julie Charles, Nathalie Bendriss-Vermare, Laurence Chaperot, and Caroline Aspord

Subjects
CLR, human DC subsets, melanoma, immune subversion, glycan, cDC1s, Immunologic diseases. Allergy, RC581-607
Abstract

Subversion of immunity by tumors is a crucial step for their development. Dendritic cells (DCs) are strategic immune cells that orchestrate anti-tumor immune responses but display altered functions in cancer. The bases for such DCs’ hijacking are not fully understood. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids. DCs express glycan-binding receptors, named C-type lectin receptors (CLR), allowing them to sense changes in glycan signature of their environment, and subsequently trigger a response. Recognition of tumor glycans by CLRs is crucial for DCs to shape antitumor immunity, and decisive in the orientation of the response. Yet the status of the CLR machinery on DCs in cancer, especially melanoma, remained largely unknown. We explored CLR expression patterns on circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs of melanoma patients, assessed their clinical relevance, and further depicted the correlations between CLR expression profiles and DCs’ features. For the first time, we highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Furthermore, melanoma tumor cells directly altered CLR expression profiles of healthy DC subsets, and this was associated with specific glycan patterns (Man, Fuc, GlcNAc) that may interact with DCs through CLR molecules. Notably, specific CLR expression profiles on DC subsets correlated with unique DCs’ activation status and functionality and were associated with clinical outcome of melanoma patients. Higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better survival, whereas elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Thus, melanoma tumor may shape DCs’ features by exploiting the plasticity of the CLR machinery. Our study revealed that melanoma manipulates CLR pathways to hijack DC subsets and escape from immune control. It further paved the way to exploit glycan-lectin interactions for the design of innovative therapeutic strategies, which exploit DCs’ potentialities while avoiding hijacking by tumor, to properly reshape anti-tumor immunity by manipulating the CLR machinery.

Published
2022
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4. Insightful Improvement in the Design of Potent Uropathogenic E. coli FimH Antagonists

Author

Leila Mousavifar, Meysam Sarshar, Clarisse Bridot, Daniela Scribano, Cecilia Ambrosi, Anna Teresa Palamara, Gérard Vergoten, Benoît Roubinet, Ludovic Landemarre, Julie Bouckaert, and René Roy

Subjects
uropathogenic Escherichia coli (UPEC), FimH, antagonists, mannosides, glycocomimetics, crystallography, Pharmacy and materia medica, RS1-441
Abstract

Selective antiadhesion antagonists of Uropathogenic Escherichia coli (UPEC) type-1 Fimbrial adhesin (FimH) are attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent urinary tract infections (UTIs) caused by UPECs. A rational small library of FimH antagonists based on previously described C-linked allyl α-D-mannopyranoside was synthesized using Heck cross-coupling reaction using a series of iodoaryl derivatives. This work reports two new members of FimH antagonist amongst the above family with sub nanomolar affinity. The resulting hydrophobic aglycones, including constrained alkene and aryl groups, were designed to provide additional favorable binding interactions with the so-called FimH “tyrosine gate”. The newly synthesized C-linked glycomimetic antagonists, having a hydrolytically stable anomeric linkage, exhibited improved binding when compared to previously published analogs, as demonstrated by affinity measurement through interactions by FimH lectin. The crystal structure of FimH co-crystallized with one of the nanomolar antagonists revealed the binding mode of this inhibitor into the active site of the tyrosine gate. In addition, selected mannopyranoside constructs neither affected bacterial growth or cell viability nor interfered with antibiotic activity. C-linked mannoside antagonists were effective in decreasing bacterial adhesion to human bladder epithelial cells (HTB-9). Therefore, these molecules constituted additional therapeutic candidates’ worth further development in the search for potent anti-adhesive drugs against infections caused by UPEC.

Published
2023
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5. Lectin Analysis of SARS-CoV-2-Positive Nasopharyngeal Samples Using GLYcoPROFILE® Technology Platform

Author

Mateja Seničar, Benoît Roubinet, Richard Daniellou, Thierry Prazuck, and Ludovic Landemarre

Subjects
SARS-CoV-2, COVID-19, nasopharyngeal samples, lectins, GLYcoPROFILE® technology, Medicine (General), R5-920
Abstract

Nasopharyngeal samples are currently accepted as the standard diagnostic samples for nucleic acid amplification testing and antigenic testing for the SARS-CoV-2 virus. In addition to the diagnostic capacity of SARS-CoV-2-positive crude nasopharyngeal samples, their qualitative potential for direct glycan-specific analysis, in order to uncover unique glycol profiles, was assessed. In this study we provide glycan characterization of SARS-CoV-2-positive and -negative nasopharyngeal samples directly from lectin interactions. Although with limited throughput, this study evaluated the clinical sensitivity and specificity of the GLYcoPROFILE® technology platformon45crude nasopharyngeal samples collected between November 2020 and April 2022. Each GLYcoPROFILE® of 39 SARS-CoV-2-positive samples was compared toglycoprofiling on a panel of 10 selected lectins and the results were paralleled with SARS-CoV-2-negative samples’ results. The GLYcoPROFILE® showed a clear distinction between positive and negative samples with WFA, GSL-II, PHA-L (GlcNAc-specific) and BPA (GalNAc-specific) highlighted as relevant lectins in SARS-CoV-2-positive samples. In addition, a significant, positive statistical correlation was found for these lectins (p < 0.01).

Published
2022
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6. Human galectin-1 and galectin-3 promote Tropheryma whipplei infection

Author

Diyoly Ayona, Sandra Madariaga Zarza, Ludovic Landemarre, Benoît Roubinet, Philippe Decloquement, Didier Raoult, Pierre-Edouard Fournier, and Benoit Desnues

Subjects
galectin-1, galectin-3, glycans, glycosylation, infection, tropheryma whipplei, macrophage, Diseases of the digestive system. Gastroenterology, RC799-869
Abstract

Tropheryma whipplei, is an actinobacterium that causes different infections in humans, including Whipple’s disease. The bacterium infects and replicates in macrophages, leading to a Th2-biased immune response. Previous studies have shown that T. whipplei harbors complex surface glycoproteins with evidence of sialylation. However, the exact contribution of these glycoproteins for infection and survival remains obscure. To address this, we characterized the bacterial glycoprofile and evaluated the involvement of human β-galactoside-binding lectins, Galectin-1 (Gal-1) and Galectin-3 (Gal-3) which are highly expressed by macrophages as receptors for bacterial glycans. Tropheryma whipplei glycoproteins harbor different sugars including glucose, mannose, fucose, β-galactose and sialic acid. Mass spectrometry identification revealed that these glycoproteins were membrane- and virulence-associated glycoproteins. Most of these glycoproteins are highly sialylated and N-glycosylated while some of them are rich in poly-N-acetyllactosamine (Poly-LAcNAc) and bind Gal-1 and Gal-3. In vitro, T. whipplei modulates the expression and cellular distribution of Gal-1 and Gal-3. Although both galectins promote T. whipplei infection by enhancing bacterial cell entry, only Gal-3 is required for optimal bacterial uptake. Finally, we found that serum levels of Gal-1 and Gal-3 were altered in patients with T. whipplei infections as compared to healthy individuals, suggesting that galectins are also involved in vivo. Among T. whipplei membrane-associated proteins, poly-LacNAc rich-glycoproteins promote infection through interaction with galectins. T. whipplei modulates the expression of Gal-1 and Gal-3 both in vitro and in vivo. Drugs interfering with galectin–glycan interactions may provide new avenues for the treatment and diagnosis of T. whipplei infections.

Published
2021
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7. Laminarin Effects, a β-(1,3)-Glucan, on Skin Cell Inflammation and Oxidation

Author

Hélène Ozanne, Hechmi Toumi, Benoît Roubinet, Ludovic Landemarre, Eric Lespessailles, Richard Daniellou, and Annabelle Cesaro

Subjects
laminarin, skin aging, skin cells, oxidative stress, inflammation, lectin, Chemistry, QD1-999
Abstract

Laminarin, a β-(1,3)-glucan from the seaweed Laminaria digitata, is a polysaccharide which provides anti-inflammatory and anti-oxidative properties. Its influence on both human dermal fibroblasts adult (HDFa) and normal human epidermal keratinocytes (NHEK) has not been established yet. Herein, laminarin effects were examined on skin cells’ mitochondrial and antioxidant activities. Cytokines, hyaluronic acid, and procollagen type I secretions and interaction mechanisms were explored after a maximum of 72 h treatment with laminarin. Our results demonstrated a decrease in mitochondrial activities with 72 h treatment with laminarin from 500 µg.mL−1 for NHEK cells and from 100 µg.mL−1 for HDFa cells without cytotoxicity. No variation of hyaluronic acid or type I procollagen was observed for all laminarin concentrations, while an antioxidant effect was found against reactive oxygen species (ROS) from 1 µg.mL−1 for HDFa cells in both H2O2 and UVA radiation conditions, and from 10 µg.mL−1 and 1 µg.mL−1 for NHEK cells in both H2O2 and UVA radiation conditions, respectively. Laminarin treatment modulated both cells surface glycosylation and cytokine secretions of skin cells. Overall, our data suggest a positive effect of β-(1,3)-glucan on skin cells on oxidative stress and inflammation induced by environmental factors. Of note, these effects are through the modulation of glycan and receptors interactions at the skin cells surface.

Published
2020
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8. Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors

Author

Eva-Maria Krammer, Clarisse Bridot, Sonia Serna, Begoña Echeverria, Shubham Semwal, Benoît Roubinet, Kim van Noort, Ruud H.P. Wilbers, Gleb Bourenkov, Jérôme de Ruyck, Ludovic Landemarre, Niels Reichardt, and Julie Bouckaert

Subjects
core fucose, crystal structure, cooperativity, paucimannose, Cell Biology, multivalency, bacterial adhesion, Biochemistry, oligomannose-3, oligomannose-6, FimH, kinetics, N-glycan, ddc:540, EPS, Laboratory of Nematology, Molecular Biology, Laboratorium voor Nematologie
Abstract

The journal of biological chemistry 299(5), 104627 (2023). doi:10.1016/j.jbc.2023.104627, The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1—Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4′-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity., Published by Soc., Bethesda, Md.

Published
2023
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9. Homo- and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins

Author

David Goyard, Benoît Roubinet, Federica Vena, Ludovic Landemarre, and Olivier Renaudet

Subjects
010405 organic chemistry, General Chemistry, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences
Abstract

Click chemistry gives access to unlimited set of multivalent glycoconjugates to explore carbohydrate-protein interactions and discover high affinity ligands. In this study, we have created supramolecular systems based on a carrier protein that was grafted by Cu(I)-catalyzed azide-alkyne cycloaddition with tetravalent glycodendrons presenting αGal, βGal and/or αFuc. Binding studies of the homo- (4 a-c) and heterovalent (5) neoglycoproteins (neoGPs) with the LecA and LecB lectins from P. aeruginosa has first confirmed the interest of the multivalent presentation of glycodendrons by the carrier protein (IC

Published
2021

10. Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases

Author

Amélie Saumonneau, Coralie Assailly, Benoît Roubinet, Franck Daligault, Ludovic Landemarre, Francesca François, Cyrille Grandjean, Laurent Fontaine, Sagrario Pascual, paul lottin, Eva-Maria Krammer, frederica vena, Sébastien G. Gouin, David Deniaud, Véronique Montembault, Charles Tellier, Clarisse Bridot, Dimitri Alvarez-Dorta, Julie Bouckaert, Université de Lille, CNRS, Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation [CEISAM], Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Unité de fonctionnalité et ingénierie de protéines [UFIP], Institut des Molécules et Matériaux du Mans [IMMM], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Unité de fonctionnalité et ingénierie de protéines (UFIP), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Institut des Molécules et Matériaux du Mans (IMMM), Le Mans Université (UM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), GLYcoDiag, ANR-17-CE07-0028,HICARE,Inhibiteurs hétérovalents de sialidases et trans-sialidases(2017), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
Stereochemistry, enzymes, Neuraminidase, Sialidase inhibitor, Human pathogen, 010402 general chemistry, medicine.disease_cause, Sialidase, 01 natural sciences, Catalysis, Catalytic Domain, inhibitors, multivalency, sialidases, Streptococcus pneumoniae, medicine, [CHIM]Chemical Sciences, chemistry.chemical_classification, Transition (genetics), 010405 organic chemistry, Chemistry, Organic Chemistry, General Chemistry, Clostridium perfringens, Orders of magnitude (mass), 0104 chemical sciences, Inhibitory potency, Enzyme, Biochemistry, Vibrio cholerae
Abstract

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi‐ and polyvalent compounds are developed, based on the transition‐state analogue 2‐deoxy‐2,3‐didehydro‐N‐acetylneuraminic (DANA). Poly‐DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate‐binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA. 27;9

Published
2021
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11. Laminarin Effects, a β-(1,3)-Glucan, on Skin Cell Inflammation and Oxidation

Author

Benoît Roubinet, Annabelle Cesaro, Ludovic Landemarre, Eric Lespessailles, Richard Daniellou, Hélène Ozanne, Hechmi Toumi, Imagerie Multimodale Multiéchelle et Modélisation du Tissu Osseux et articulaire (I3MTO), Université d'Orléans (UO), and Centre Hospitalier Régional d'Orléans (CHRO)

Subjects
Aging, medicine.medical_treatment, Pharmaceutical Science, Inflammation, Dermatology, Polysaccharide, medicine.disease_cause, lcsh:Chemistry, 03 medical and health sciences, chemistry.chemical_compound, Laminarin, 0302 clinical medicine, Hyaluronic acid, medicine, oxidative stress, Chemical Engineering (miscellaneous), Cytotoxicity, skin aging, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Reactive oxygen species, skin cells, Molecular biology, 3. Good health, Cytokine, lcsh:QD1-999, chemistry, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, inflammation, 030220 oncology & carcinogenesis, lectin, Surgery, medicine.symptom, laminarin, Oxidative stress
Abstract

Laminarin, a &beta, (1,3)-glucan from the seaweed Laminaria digitata, is a polysaccharide which provides anti-inflammatory and anti-oxidative properties. Its influence on both human dermal fibroblasts adult (HDFa) and normal human epidermal keratinocytes (NHEK) has not been established yet. Herein, laminarin effects were examined on skin cells&rsquo, mitochondrial and antioxidant activities. Cytokines, hyaluronic acid, and procollagen type I secretions and interaction mechanisms were explored after a maximum of 72 h treatment with laminarin. Our results demonstrated a decrease in mitochondrial activities with 72 h treatment with laminarin from 500 &micro, g.mL&minus, 1 for NHEK cells and from 100 &micro, 1 for HDFa cells without cytotoxicity. No variation of hyaluronic acid or type I procollagen was observed for all laminarin concentrations, while an antioxidant effect was found against reactive oxygen species (ROS) from 1 &micro, 1 for HDFa cells in both H2O2 and UVA radiation conditions, and from 10 &micro, 1 and 1 &micro, 1 for NHEK cells in both H2O2 and UVA radiation conditions, respectively. Laminarin treatment modulated both cells surface glycosylation and cytokine secretions of skin cells. Overall, our data suggest a positive effect of &beta, (1,3)-glucan on skin cells on oxidative stress and inflammation induced by environmental factors. Of note, these effects are through the modulation of glycan and receptors interactions at the skin cells surface.

Published
2020

12. Fluorescent Photoswitchable Diarylethenes for Biolabeling and Single-Molecule Localization Microscopies with Optical Superresolution

Author

Masahiro Irie, Heydar Shojaei, Michael Weber, Mariano L. Bossi, Mark Bates, Vladimir N. Belov, Benoît Roubinet, and Stefan W. Hell

Subjects
Single molecule localization, chemistry.chemical_classification, Aqueous solution, Chemistry, Carboxylic acid, Nanotechnology, 02 engineering and technology, General Chemistry, 010402 general chemistry, 021001 nanoscience & nanotechnology, Ring (chemistry), Photochemistry, 01 natural sciences, Biochemistry, Fluorescence, Superresolution, Catalysis, 0104 chemical sciences, Colloid and Surface Chemistry, Microscopy, 0210 nano-technology, Conjugate
Abstract

A modular assembly of water-soluble diarylethenes (DAEs), applicable as biomarkers for optical nanoscopy, is reported. Reversibly photoswitchable 1,2-bis(2-alkyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes possessing a fluorescent "closed" form were decorated with one or two methoxy group(s) attached to the para-position(s) of phenyl ring(s) and two, four, or eight carboxylic acid groups. Antibody conjugates of these DAEs feature low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and photophysical properties (high emission efficiencies and low cycloreversion quantum yields) enabling their application in superresolution microscopy. Images of tubulin, vimentin, and nuclear pore complexes are presented. The superresolution images can also be acquired by using solely 488 nm light without additional photoactivation with UV light. These DAEs exhibit reversible photoswitching without requiring any additives to the imaging media and open new paths toward the modular design of fluorescent dyes for bioimaging with optical superresolution.

Published
2017

13. Reinvestigation of the Synthesis of 'Covalent-Assembly' Type Probes for Fluoride Ion Detection. Identification of Novel 7-(Diethylamino)coumarins with Aggregation-Induced Emission Properties

Author

Benoît Roubinet, Pierre-Yves Renard, Anthony Romieu, Valentin Quesneau, Institut de Chimie Moléculaire de l'Université de Bourgogne [Dijon] (ICMUB), Centre National de la Recherche Scientifique (CNRS)-Université de Bourgogne (UB)-Institut de Chimie du CNRS (INC), Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Chemistry, Organic Chemistry, Substituent, 02 engineering and technology, Tetraphenylethylene, Alkylation, 021001 nanoscience & nanotechnology, 010402 general chemistry, 01 natural sciences, Biochemistry, Combinatorial chemistry, Fluorescence, 0104 chemical sciences, Adduct, chemistry.chemical_compound, Salicylaldehyde, Covalent bond, Drug Discovery, Moiety, 0210 nano-technology, Fluoride
Abstract

International audience; An unprecedented C-3 functionalization of 4-(diethylamino)salicylaldehyde through a Friedel-Crafts type alkylation reaction has been discovered during the synthesis of “covalent-assembly”-based fluorescent probes for detection of fluoride ions. The resulting Friedel-Crafts adduct was successfully used for the preparation of two novel 8-substituted 7-(diethylamino)coumarin dyes. The photophysical study of these fluorophores has enabled us to highlight their remarkable aggregation-induced emission (AIE) properties characterized by a yellow-orange emission of aggregates in water. Therefore, 4-(tert-butyldimethylsilyloxy)benzyl substituent was identified as a novel AIE-active moiety which could be seen as a possible alternative to popular tetraphenylethylene (TPE).

Published
2019

14. Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli

Author

David Deniaud, Medy C. Nongbe, Ludovic Landemarre, Benoît Roubinet, Madeleine Cauwel, Sébastien G. Gouin, Julie Bouckaert, Adeline Sivignon, François-Xavier Felpin, Nicolas Barnich, Clarisse Bridot, Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte (M2iSH), Institut National de la Recherche Agronomique (INRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), GLYcoDiag, Institut des Sciences Moléculaires (ISM), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1 (UB)-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des Adaptations Métaboliques à l'Exercice en Conditions Physiologiques et Pathologiques (AME2P), Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-UFR Sciences et Techniques des Activités Physiques et Sportives - Clermont-Auvergne (UFR STAPS - UCA), Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Université Clermont Auvergne [2017-2020] (UCA [2017-2020]), Laboratoire de Synthèse Organique (Hétérochimie organique, organoéléments et matériaux) (LSOHOOM), Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Université de Lille, CNRS, Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 [UGSF], Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], Bouckaert, Julie, Université de Nantes - Faculté des Sciences et des Techniques, Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte - Clermont Auvergne (M2iSH), Institut National de la Recherche Agronomique (INRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne (UCA)-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne), Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), CNRS, UMR 8576, GLYcoDIAG, Université d'Orléans (UO), Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Laboratoire des Adaptations Métaboliques à l’Exercice en conditions Physiologiques et Pathologiques (AME2P), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre de Recherche en Nutrition Humaine d'Auvergne (CRNH d'Auvergne)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), and Université Montesquieu - Bordeaux 4-Université Sciences et Technologies - Bordeaux 1-École Nationale Supérieure de Chimie et de Physique de Bordeaux (ENSCPB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Paper, Specific detection, [SDV]Life Sciences [q-bio], Nanofibers, 010402 general chemistry, 01 natural sciences, Catalysis, Bacterial Adhesion, Mannans, chemistry.chemical_compound, Feces, Cell Line, Tumor, Materials Chemistry, Humans, Cellulose, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, ComputingMilieux_MISCELLANEOUS, Adhesins, Escherichia coli, biology, Escherichia coli K12, 010405 organic chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Metals and Alloys, Lectin, Inflammatory Bowel Diseases, General Chemistry, [CHIM.ORGA] Chemical Sciences/Organic chemistry, 3. Good health, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Bacterial Typing Techniques, Gastrointestinal Microbiome, Bacterial adhesin, [SDV] Life Sciences [q-bio], [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Biochemistry, Covalent bond, Nanofiber, Ceramics and Composites, biology.protein, Fimbriae Proteins, Protein Binding
Abstract

International audience; We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E.coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens. The worldwide spread of antibiotic resistances raises serious health problems, and has driven the identification of new virulence factors and development of alternative antibacterial therapeutics. Mannose-binding FimH adhesin, expressed by Escherichia coli strains has been extensively studied as a target for disrupting bacterial attachment to the host cells. 1 Impressive results were obtained in the context of urinary tract infections (UTI), a prevalent infection type generally mediated by the attachment of uropathogenic E. coli strains (UPEC) to the highly mannosylated uroplakin transmembrane protein of urothelial lining cells. FimH antagonists orally administered to in vivo UTI mouse models, were shown to decrease the E.coli load in the bladder by several orders of magnitude, 2-4 making them competitive with conventional antibiotic treatment. 5,6 This concept was more recently extended to Crohn's disease (CD), an inflammatory disorder of the intestine where an altered gut microbiota, particularly the presence of adherent-invasive E. coli strains (AIEC), is suspected to play a key role in the pathogenesis. 7 Synthetic derivatives of heptylmannoside (HMan), a nanomolar FimH antagonist, 8 were shown to lower the AIEC bacterial level, signs of colitis and gut inflammation when administered per os (10 mg/kg) in CD mouse models. 9,10 Sensitive and rapid diagnostic systems are essential to evaluate the presence of E. coli expressing FimH adhesin in gut microenvironments in order to properly stratify patients before treatment. While the high prevalence of UPEC in the normally sterile urinary tract environment facilitates diagnosis, the AIEC niche is more complex, located at the ileal mucosa in 21 to 63% of CD patients, 11,12 within an ecological community of hundreds of symbiotic microorganisms. Furthermore, no specific biomarkers are currently effective at distinguishing AIEC from other commensal E. coli of the gut microbiota. Previously, it has been shown that AIEC pathobiont possesses specific allelic variants in the fimH gene, conferring them a high ability to adhere in vitro and to colonize the gut of CEABAC10 mice. 13 Establishing an approach to discriminate the strong mannose-binders from other bacteria would therefore be of tremendous importance for efficient diagnosis. In this work, we developed a heterogeneous support to specifically trap and accumulate pathogenic E.coli from biological fluids. Heptyl-α-D-mannoside (HMan) was grafted by click chemistry techniques onto cellulose nanofibers (CN) and cellulose paper (CP). HMan was previously identified as a potent binder of the isolated FimH lectin domain. 8 It should be noted that the lectin domain represents the high-affinity state of FimH under mechanical force and that full-length FimH display a lower affinity for mannosides. 14,15 Covalently functionalized CN or CP were characterized by Fourier transform spectroscopy (FTIR), elemental analysis, X-ray photo-electron spectrometry (XPS), and scanning electron microscopy (SEM). HMan-CN was first compared in vitro against CN grafted with lower FimH affinity ligands i.e. Man-CN lacking the hydrophobic heptyl chain, and HGlc-CN an analog with a glucose sugar that is not recognized by FimH lectin (Scheme 1). In addition, we switched the anomeric oxygen atom to a sulfur and synthesized HSMan-CN to prevent potential sugar hydrolysis from the surface by mannosidases. The modified CN were first assessed for their faculty to bind FimH and to prevent AIEC adhesion to intestinal cells. HMan-CN was then orally administered to the CEABAC10 mouse model of CD to assess its capacity to decrease AIEC in the feces of AIEC-infected mice and to act as a potential CD therapeutic. HM was next coated on CP and the capacity of the HMan-CP to selectively catch AIEC in solution was analyzed. Scheme 1. Chemical synthesis of the sugar-coated cellulose fibers.

Published
2019

15. Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases

Author

Benoît Roubinet, Charles Tellier, Julien Bernard, Clarisse Bridot, Coralie Assailly, Franck Daligault, Clémence Petitot, Cyrille Grandjean, Julie Bouckaert, Blanka Didak, Amélie Saumonneau, Xibo Yan, Mike Maillasson, Sébastien G. Gouin, David Deniaud, Ralf Blossey, Yoan Brissonnet, Ludovic Landemarre, Chimie Et Interdisciplinarité : Synthèse, Analyse, Modélisation (CEISAM), Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Unité de fonctionnalité et ingénierie de protéines (UFIP), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), IMPACT - Interactions Moléculaires Puces ACTivités, Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), GLYcoDiag, Ingénierie des Matériaux Polymères (IMP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet - Saint-Étienne (UJM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Unité de Biotechnologie, Biocatalyse et Biorégulation (U3B), Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS), University of Bern, ANR-17-CE07-0028,HICARE,Inhibiteurs hétérovalents de sialidases et trans-sialidases(2017), Université de Nantes (UN)-Université de Nantes (UN)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), GLYcoDIAG, Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet [Saint-Étienne] (UJM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Nantes - Faculté des Sciences et des Techniques, Transport des assimilats (TA), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Centre de recherche de Cancérologie et d'Immunologie / Nantes - Angers (CRCINA), Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Immunobiology of Human αβ and γδ T cells and Immunotherapeutic Applications (CRCINA - Département INCIT - Equipe 1), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), Université de Lille, CNRS, Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF], and Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576

Subjects
chemistry.chemical_classification, 010405 organic chemistry, Glycoconjugate, Organic Chemistry, enzymes, glycoclusters, glycosidases, inhibition, sialidases, Virulence, Pathogenic bacteria, General Chemistry, 010402 general chemistry, medicine.disease_cause, Sialidase, 01 natural sciences, Catalysis, 0104 chemical sciences, Enzyme, Biochemistry, chemistry, medicine, [CHIM]Chemical Sciences, Catalytic efficiency, Eukaryotic cell
Abstract

International audience; Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.

Published
2019

16. Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super‐Resolution RESOLFT Microscopy

Author

Stefan W. Hell, Masahiro Irie, Shamil Nizamov, Philipp Alt, Vladimir N. Belov, Mariano L. Bossi, Benoît Roubinet, Heydar Shojaei, Marcel Leutenegger, and Sebastian Schnorrenberg

Subjects
RESOLFT, photochromic dyes, 010402 general chemistry, Photochemistry, 01 natural sciences, Catalysis, law.invention, Optical microscope, law, Microscopy, Specific staining, optical microscopy, water solubility, Aqueous solution, 010405 organic chemistry, Chemistry, Communication, Resolution (electron density), Optical Microscopy | Very Important Paper, General Chemistry, Fluorescence, Superresolution, Communications, 0104 chemical sciences, diarylethenes, fluorescence
Abstract

Reversibly photoswitchable 1,2‐bis(2‐ethyl‐6‐phenyl‐1‐benzothiophene‐1,1‐dioxide‐3‐yl)perfluorocyclopentenes (EBT) having fluorescent “closed” forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology‐related super‐resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.

Published
2016

17. Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with 'Universal Hydrophilizer' or Hydroxyl Groups

Author

Mariano L. Bossi, Matthias Bischoff, Vladimir N. Belov, Benoît Roubinet, Stefan Stoldt, Stefan W. Hell, Shamil Nizamov, Gyuzel Yu. Mitronova, Claudia Geisler, and Sergey Yan

Subjects
0301 basic medicine, Allylic rearrangement, Aqueous solution, Dipeptide, 010405 organic chemistry, Organic Chemistry, Adhesion, 01 natural sciences, Combinatorial chemistry, 0104 chemical sciences, Hydroxylation, Rhodamine, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, chemistry, Reactivity (chemistry), Solubility
Abstract

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

Published
2018

18. New insights into the water-solubilization of thiol-sensitive fluorogenic probes based on long-wavelength 7-hydroxycoumarin scaffolds

Author

Pierre-Yves Renard, Benoît Roubinet, Anthony Romieu, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
chemistry.chemical_classification, Quenching (fluorescence), [CHIM.ORGA]Chemical Sciences/Organic chemistry, Process Chemistry and Technology, General Chemical Engineering, Chemical modification, Ether, [CHIM.CATA]Chemical Sciences/Catalysis, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, Photochemistry, Coumarin, Fluorescence, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, chemistry.chemical_compound, chemistry, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Thiol, Phenol, Moiety, ComputingMilieux_MISCELLANEOUS, [CHIM.CHEM]Chemical Sciences/Cheminformatics
Abstract

The synthesis and photophysical properties of novel water-soluble phenol-based fluorophores derived from 3-benzothiazolyl-7-hydroxycoumarin and emitting in the range 485–631 nm are described. Further conversion into thiol-sensitive fluorogenic probes through the chemical modification of their hydroxyl group was next investigated. Depending on the type of thiol-reactive quenching moiety used (2,4-dinitrobenzenesulfonyl ester, 2,4-dinitrophenyl ether or benzoquinone-type Michael acceptors) and the water-solubilizing group(s) pre-introduced into the coumarin core, dramatic differences in the thiol-induced fluorescence activation of these pro-fluorophores under physiological conditions were observed. Results for this comparative study provide valuable informations for the selection of the most suitable structural features for designing 7-hydroxycoumarin-based long-wavelength fluorescent probes for thiol bioimaging.

Published
2014

19. Carboxylierte photoschaltbare Diarylethene als Biomarkierungen für hochauflösende RESOLFT-Mikroskopie

Author

Vladimir N. Belov, Masahiro Irie, Marcel Leutenegger, Philipp Alt, Heydar Shojaei, Sebastian Schnorrenberg, Benoît Roubinet, Shamil Nizamov, Mariano L. Bossi, and Stefan W. Hell

Subjects
010405 organic chemistry, General Medicine, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences
Abstract

Reversibel photoschaltbares 1,2-Bis(2-ethyl-6-phenyl-1-benzothiophen-1,1-dioxid-3-yl)perfluorcyclopenten (EBT) mit fluoreszierender “geschlossener” Form wurde mit vier oder acht Carboxygruppen versehen und an Antikorper gebunden. Die carboxylierten Derivate wiesen geringe Aggregation, effizientes Photoschalten in wassrigen Puffern, gezieltes Farben von zellularen Strukturen und gute photophysikalische Eigenschaften auf. Abwechselnde Bestrahlung mit UV und blauem Licht relativ geringer Intensitat fuhrte zu reversibler photochemischer Isomerisierung zwischen zwei stabilen Strukturen uber mehrere dutzend Schaltzyklen. Dies ermoglichte die Verwendung der Farbstoffe fur hochauflosende RESOLFT-Mikroskopie (“reversible switchable optical linear fluorescence transitions”). Hierbei konnte eine optische Auflosung von 75 nm an zellularen Tubulin-Filamenten erzielt werden.

Published
2016

20. A FRET-​based probe for fluorescence sensing of sulfide​/sulfite analytes, using a novel long-​wavelength water-​soluble 7-​hydroxycoumarin as reporter fluorophore

Author

Benoît Roubinet, Anthony Romieu, Emilie Petit, Pierre-Yves Renard, Laetitia Bailly, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Fluorophore, Sulfide, Carboxylic acid, Inorganic chemistry, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, 010402 general chemistry, 01 natural sciences, Biochemistry, chemistry.chemical_compound, Sulfite, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Drug Discovery, Moiety, chemistry.chemical_classification, 010405 organic chemistry, Chemistry, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Organic Chemistry, [CHIM.CATA]Chemical Sciences/Catalysis, Fluorescence, Combinatorial chemistry, 0104 chemical sciences, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, Förster resonance energy transfer, Linker, [CHIM.CHEM]Chemical Sciences/Cheminformatics
Abstract

International audience; A FRET-based fluorescent probe for the detection of sulfide and/or sulfite in aqueous buffer, was constructed by connecting the quencher moiety nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) to a water-soluble 3-(2-benzimidazolyl)-7-hydroxycoumarin carboxylic acid through a piperazine linker. This probe exhibits good selectivity and high sensitivity for sulfite and sulfide over cysteine and other potential analytes. Furthermore, this is one of the few examples of fluorogenic coumarins whose solubility in water is maintained upon its conversion into reaction-based fluorescent probes.

Published
2015

21. New 3-(Heteroaryl)-2-iminocoumarin-based Borate Complexes: Synthesis, Photophysical Properties, and Rational Functionalization for Biosensing/Biolabeling Applications

Author

Pierre-Yves Renard, Anthony Romieu, Benoît Roubinet, Frédéric Boschetti, Mathieu Moreau, Gilles Ulrich, Raymond Ziessel, Cédrik Massif, Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille Institut (CLIL), CheMatech - Macrocycle Design Technologies, Institut de chimie et procédés pour l'énergie, l'environnement et la santé (ICPEES), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie Organique Fine (IRCOF), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université de Rouen Normandie (UNIROUEN), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), and Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Aminocoumarins, Boron Compounds, Macrocyclic Compounds, Carboxylic acid, Substituent, Ligands, 010402 general chemistry, 01 natural sciences, Catalysis, Fluorescence, chemistry.chemical_compound, Macrocyclic ligands, Coumarins, Borates, Organic chemistry, Molecule, Moiety, [CHIM]Chemical Sciences, Dyes, Fluorescent Dyes, Boron, chemistry.chemical_classification, Molecular Structure, 010405 organic chemistry, Coumarin dyes, Organic Chemistry, Chemical modification, Serum Albumin, Bovine, General Chemistry, Combinatorial chemistry, 0104 chemical sciences, chemistry, Amine gas treating, Biosensor
Abstract

International audience; Members of a series of boron difluoride complexes with 3‐(heteroaryl)‐2‐iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N‐aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two‐step method. These novel fluorogenic dyes belong to the family of “Boricos” (D. Frath et al., Chem. Commun.­ 2013, 49, 4908–4910) and are the first examples of phenol‐based fluorophores of which the photophysical properties in the green‐yellow spectral range are dramatically improved by N,N‐chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4‐dinitrophenyl (DNP) ethers, which led to a dramatic “OFF‐ON” fluorescence response upon reaction with thiols. Additionally, to expand the scope of these “7‐hydroxy‐Borico” derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and “Borico”‐DOTA‐like scaffolds in an effort to design novel monomolecular multimodal fluorescence‐ radioisotope imaging agents.

Published
2015

22. Back Cover: Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy (Angew. Chem. Int. Ed. 49/2016)

Author

Marcel Leutenegger, Heydar Shojaei, Philipp Alt, Benoît Roubinet, Sebastian Schnorrenberg, Shamil Nizamov, Vladimir N. Belov, Masahiro Irie, Stefan W. Hell, and Mariano L. Bossi

Subjects
Materials science, Optical microscope, law, RESOLFT, Microscopy, Cover (algebra), General Chemistry, Photochemistry, Superresolution, Fluorescence, Catalysis, law.invention
Published
2016

24. A Synthetic Route to 3-(Heteroaryl)-7-hydroxycoumarins Designed for Biosensing Applications

Author

Arnaud Chevalier, Benoît Roubinet, Pierre-Yves Renard, and Anthony Romieu

Subjects
Protease, 010405 organic chemistry, Chemistry, medicine.medical_treatment, Organic Chemistry, 010402 general chemistry, Condensation reaction, 01 natural sciences, Fluorescence, 0104 chemical sciences, Diselenide, medicine, Organic chemistry, Physical and Theoretical Chemistry, Biosensor
Abstract

A straightforward method to synthesize 3-(2-benzimidazolyl)-7-hydroxycoumarins, based on the condensation reaction of 7-acetoxy-3-formylcoumarin with various C- and/or N-substituted ortho-phenylenediamine derivatives is presented. This unusual approach proved particularly effective for introducing different hydrophilic groups (carboxylic or sulfonic acids or trimethylalkylammonium moieties) onto the heteroaryl scaffold, leading to cyan-green emitting coumarins that were both water-soluble and strongly fluorescent under physiological conditions. The further extension of this condensation reaction to bis(2-aminophenyl)diselenide enabled the first synthesis of 3-(2-benzoselenazolyl)-7-hydroxycoumarin. The potential utility of these new 7-hydroxycoumarins was demonstrated through the synthesis and spectroscopic and analyte-responsive behavior of fluorogenic probes suitable for sensing biologically relevant thiols and urokinase, a protease that plays a key role in cancer invasion and metastasis.

Published
2014

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