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1.
Chem Commun (Camb) ; 56(73): 10746-10749, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32789356

RESUMO

Here we propose a general strategy to label carbohydrates with N-methyl-anthranilic acid at the anomeric position. Through two examples, we demonstrate that the generated glycoprobes are suitable for fluorescence-based binding/competition assays. Our approach is expected to readily generate series of glycoprobes dedicated to screening assays for the discovery of drugs targeting carbohydrate-protein interactions.

2.
Org Biomol Chem ; 18(25): 4831-4842, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32608461

RESUMO

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. Biosynthesis of PGs is complex and involves a large number of glycosyltranferases. We report herein for the first time the synthesis of a collection of various sulfoforms of the disaccharide GlcA-1,3-ß-d-Gal and trisaccharides GlcNAc-1,4-α-d-GlcA-1,3-ß-d-Gal and GalNAc-1,4-ß-d-GlcA-1,3-ß-d-Gal using a regioselective glycosylation. Preliminary results on the impact of sulfation of these disaccharides upon recombinant chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate chain initiation is also reported.

3.
Anal Bioanal Chem ; 412(17): 4195-4207, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32377866

RESUMO

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL-1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL-1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors. Graphical abstract.

4.
Carbohydr Res ; 475: 56-64, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30836261

RESUMO

The activity of eukaryote hydrolase-type of hyaluronidases was studied using a miniaturized capillary electrophoresis (CE) assay developed in our laboratory. Few nanoliters of reagents are sufficient and no labeling is required for this assay. The effect of natural and original synthetic effectors of hyaluronidase was evaluated. These di- and trisaccharides from linkage region of proteoglycans were synthesized in 30-40 steps from monomeric units using classical protection, deprotection, glycosylation and deoxygenation reactions. The influence of the chain length (di/trisaccharide), the modification type (methoxy/deoxy) and its position (2/4/6) was studied. The inhibition and/or activation percentages were determined at two concentrations of effectors; 0.2 mM and 2 mM. The half maximal effective concentration (EC50) values were evaluated (n = 2) for the most effective inhibitors (∼1 mM) and activators (∼0.2 mM). Results showed that hyaluronidase was mostly inhibited in a concentration-dependent fashion by a deoxy modification and activated by a methoxy modification. Trisaccharides were found to be more effective on hyaluronidase activity than disaccharides. Position 4 was found to be more favorable for hyaluronidase activity than position 6 and the activity in position 2 was negligible. For a better understanding of the enzyme function mode, the inhibition constant (Ki) was also evaluated by CE (Ki ∼ 2 mM). These results are of great interest especially as few activators of hyaluronidase are presented in the literature.


Assuntos
Eletroforese Capilar , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Hialuronoglucosaminidase/antagonistas & inibidores , Oligossacarídeos/farmacologia , Animais , Configuração de Carboidratos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Hialuronoglucosaminidase/metabolismo , Oligossacarídeos/química , Relação Estrutura-Atividade
5.
Org Biomol Chem ; 15(45): 9653-9669, 2017 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29116283

RESUMO

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. The biosynthesis of PGs is complex and involves a large number of glycosyltranferases. Here we present a structure-activity study of human ß4GalT7, which transfers the first Gal residue onto a xyloside moiety of the linkage region. An efficient and regiocontrolled synthesis of a library of modified analogs of 4-methylumbelliferyl xyloside (XylMU) is reported herein. Hydroxyl groups at the position C-2, C-3 or C-4 have been epimerized and/or replaced by a hydrogen or a fluorine, while the anomeric oxygen was replaced by either a sulfur or a sulfone. The effect of these compounds on human ß4GalT7 activity in vitro and on GAG biosynthesis in cellulo was then evaluated.


Assuntos
Galactosiltransferases/metabolismo , Glicosídeos/biossíntese , Bibliotecas de Moléculas Pequenas/metabolismo , Configuração de Carboidratos , Glicosídeos/química , Humanos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
6.
Rapid Commun Mass Spectrom ; 31(23): 2003-2010, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28901031

RESUMO

RATIONALE: Chondroitin sulfate (CS) glycosaminoglycans are bioactive sulfated polysaccharides comprising repeating units of uronic acid and N-acetyl galactose sulfated at various positions. The optimal length and sulfation pattern of the CS bioactive sequences remain elusive so that structure-activity relationships cannot be easily established. Development of efficient analytical methods allowing the differentiation of the various sulfation patterns of CS sequences is therefore of particular importance to correlate their biological functions to the sulfation pattern. METHODS: Discrimination of different oligomers (dp2 to dp6) of synthetic chondroitin sulfate isomers was evaluated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative-ion mode from deprotonated and alkali adduct species. In addition, ion mobility mass spectrometry (IMS-MS) was used to study the influence of both the degree of polymerization and sulfate group location on the gas-phase conformation of CS oligomers. RESULTS: ESI-MS/MS spectra of chondroitin sulfate isomers show characteristic product ions exclusively from alkali adduct species (Li, Na, K and Cs). Whatever the alkali adducts studied, MS/MS of chondroitin oligosaccharides sulfated at position 6 yields a specific product ion at m/z 139 while CS oligosaccharides sulfated at position 4 show a specific product ion at m/z 154. Being observed for the different CS oligomers di-, tetra- and hexasaccharides, these fragment ions are considered as diagnostic ions for chondroitin 6-O-sulfate and chondroitin 4-O-sulfate, respectively. IMS-MS experiments reveal that collision cross-sections (CCS) of CS oligomers with low charge states evolved linearly with degrees of polymerization indicating a similar gas-phase conformation. CONCLUSIONS: This study allows the fast and unambiguous differentiation of CS isomers sulfated at position 6 or 4 for both saturated and unsaturated analogues from MS/MS experiments. In addition, the CCS linear evolution of CS oligomers in function of the degree of polymerization indicates that no folding occurs even for hexasaccharides.

7.
Anal Chim Acta ; 951: 140-150, 2017 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-27998482

RESUMO

The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km, Vmax and IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 µL min-1 and an ESI voltage at -4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL-1 and 150.4 ± 0.1 nM s-1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL-1 and Vmax of 155.7 ± 0.2 nM s-1. IC50 of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.


Assuntos
Eletroforese Capilar , Hialuronoglucosaminidase/química , Espectrometria de Massas , Cinética
8.
Org Biomol Chem ; 14(33): 7962-71, 2016 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-27492660

RESUMO

Efficient and stereocontrolled preparation of a library of variously sulfated biotinylated tetra- and pentasaccharides possessing the backbone of the partial linkage region plus the first chondroitin sulfate mono- or disaccharide unit (d-GlcA)n-ß-d-(1,3)-GalNAc-ß-d-(1,4)-GlcA-ß-d-(1,3)-Gal-ß-d-(1,3)-Gal (n = 0 or 1) is reported herein for the first time. The synthesis of these compounds was achieved using common key intermediates and a disaccharide building block obtained by semisynthesis. Stereoselective glycosylation, selective protection/deprotection steps, efficient reduction of the N-trichloroacetyl group into the corresponding N-acetyl group, efficient sulfation strategy, deprotection and biotinylation afforded target oligomers in good yield with high purity.


Assuntos
Condroitina/química , Monossacarídeos/síntese química , Oligossacarídeos/síntese química , Proteoglicanas/química , Biotinilação , Configuração de Carboidratos , Monossacarídeos/química , Oligossacarídeos/química , Proteoglicanas/síntese química , Estereoisomerismo
9.
J Biol Chem ; 290(12): 7658-70, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25568325

RESUMO

Among glycosaminoglycan (GAG) biosynthetic enzymes, the human ß1,4-galactosyltransferase 7 (hß4GalT7) is characterized by its unique capacity to take over xyloside derivatives linked to a hydrophobic aglycone as substrates and/or inhibitors. This glycosyltransferase is thus a prime target for the development of regulators of GAG synthesis in therapeutics. Here, we report the structure-guided design of hß4GalT7 inhibitors. By combining molecular modeling, in vitro mutagenesis, and kinetic measurements, and in cellulo analysis of GAG anabolism and decorin glycosylation, we mapped the organization of the acceptor binding pocket, in complex with 4-methylumbelliferone-xylopyranoside as prototype substrate. We show that its organization is governed, on one side, by three tyrosine residues, Tyr(194), Tyr(196), and Tyr(199), which create a hydrophobic environment and provide stacking interactions with both xylopyranoside and aglycone rings. On the opposite side, a hydrogen-bond network is established between the charged amino acids Asp(228), Asp(229), and Arg(226), and the hydroxyl groups of xylose. We identified two key structural features, i.e. the strategic position of Tyr(194) forming stacking interactions with the aglycone, and the hydrogen bond between the His(195) nitrogen backbone and the carbonyl group of the coumarinyl molecule to develop a tight binder of hß4GalT7. This led to the synthesis of 4-deoxy-4-fluoroxylose linked to 4-methylumbelliferone that inhibited hß4GalT7 activity in vitro with a Ki 10 times lower than the Km value and efficiently impaired GAG synthesis in a cell assay. This study provides a valuable probe for the investigation of GAG biology and opens avenues toward the development of bioactive compounds to correct GAG synthesis disorders implicated in different types of malignancies.


Assuntos
Inibidores Enzimáticos/química , Galactosiltransferases/metabolismo , Xilosidases/antagonistas & inibidores , Domínio Catalítico , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Galactosiltransferases/química , Humanos , Cinética , Modelos Moleculares , Sondas Moleculares
10.
Carbohydr Res ; 402: 35-43, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25486221

RESUMO

The synthesis of biotinylated conjugates of oligomers of the basic repeating unit of chondroitin sulfate E (CS-E) with the sequence [GlcA-4,6-disulfated GalNAc]n is reported herein for the first time. An efficient and stereocontrolled preparation of di-, tetra-, and hexasaccharide derivatives was achieved using a common key disaccharide intermediate in an iterative way. An unexpected and never reported side reaction on the carbonyl group of the levulinate ester was observed during a coupling reaction. These complex molecules should be useful to study their interactions with various proteins.


Assuntos
Biotinilação , Sulfatos de Condroitina/química , Dissacarídeos/química , Hidroxilação , Estereoisomerismo , Especificidade por Substrato
11.
Carbohydr Res ; 353: 33-48, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22525101

RESUMO

The synthesis of a collection, as biotinylated conjugates, of various sulfoforms of the trisaccharide ß-D-GlcpA-(1→3)-ß-D-Galp-(1→3)-ß-D-Galp, structures encountered in the linkage region of proteoglycans, is reported herein for the first time. An efficient and stereocontrolled preparation was achieved using common key intermediates in a divergent manner. These molecules should be useful probes to study the substrate specificity of the glycosyltransferases involved at the bifurcation point in the biosynthesis of proteoglycans.


Assuntos
Oligossacarídeos/química , Oligossacarídeos/síntese química , Proteoglicanas/química , Sequência de Carboidratos , Dados de Sequência Molecular
12.
Chemistry ; 15(37): 9561-78, 2009 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-19575349

RESUMO

Controlled acid hydrolysis of polymeric chondroitin sulfate of bovine origin afforded in good yield a basic disaccharide fragment that was used for the first time as a starting material for the expeditious preparation of a set of building blocks that in turn act as versatile synthons for the efficient and stereocontrolled construction of a collection of size-defined chondroitin oligomers (from di- to octasaccharides). This step economy process allows their preparation as reducing species, fitted with a fluorophore, or as biotinylated conjugates; all useful tools for the preparation of microarrays, or as probes for the study of the biosynthesis of chondroitin sulfate.


Assuntos
Sulfatos de Condroitina/química , Oligossacarídeos/química , Polímeros/síntese química , Animais , Biotinilação , Bovinos , Glicosilação , Oligossacarídeos/síntese química , Polímeros/química , Estereoisomerismo
13.
Chemistry ; 15(37): 9579-95, 2009 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-19621396

RESUMO

An efficient, stereocontrolled, and highly divergent approach for the preparation of oligomers of chondroitin sulfate (CS) A, C, D, E, K, L, and M variants, starting from a single precursor easily obtained by semisynthesis from abundant natural polymer is reported for the first time. Common intermediates were designed that allowed the straightforward construction of O-sulfonated species either on the D-galactosamine unit (CS-A, -C, and -E) or on both D-glucuronic acid and D-galactosamine units (CS-D and CS-K, -L, and -M). This strategy represents a successful improvement and brings a definitive answer toward the synthesis of such complex molecules with numerous relevant biological functions.


Assuntos
Sulfatos de Condroitina/síntese química , Polímeros/síntese química , Sulfatos de Condroitina/química , Galactosamina/química , Ácido Glucurônico/química , Glicosilação , Oligossacarídeos/química , Polímeros/química , Estereoisomerismo
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