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1.
PLoS One ; 13(9): e0204472, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30260999

RESUMO

The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes.


Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Estabilidade de Medicamentos , Dinorfinas/administração & dosagem , Dinorfinas/sangue , Dinorfinas/farmacocinética , Feminino , Glicolipídeos/administração & dosagem , Glicolipídeos/química , Técnicas In Vitro , Lipossomos/administração & dosagem , Lipossomos/química , Nanopartículas/química , Antagonistas de Entorpecentes/administração & dosagem , Antagonistas de Entorpecentes/sangue , Antagonistas de Entorpecentes/farmacocinética , Peptídeos/sangue , Estabilidade Proteica , Proteólise , Ratos , Ratos Sprague-Dawley , Receptores Opioides kappa/antagonistas & inibidores , Espectrometria de Massas por Ionização por Electrospray
2.
Molecules ; 23(8)2018 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-30060568

RESUMO

An efficient study of carbohydrate-protein interactions was achieved using multivalent glycodendrimer library. Different dendrimers with varied peripheral sugar densities and linkers provided an arsenal of potential novel therapeutic agents that could be useful for better specific action and greater binding affinities against their cognate protein receptors. Highly effective click chemistry represents the basic method used for the synthesis of mannosylated dendrimers. To this end, we used propargylated scaffolds of varying sugar densities ranging from 2 to 18 for the attachment of azido mannopyranoside derivatives using copper catalyzed click cycloaddition. Mannopyranosides with short and pegylated aglycones were used to evaluate their effects on the kinetics of binding. The mannosylated dendrons were built using varied scaffolds toward the accelerated and combined "onion peel" strategy These carbohydrates have been designed to fight E. coli urinary infections, by inhibiting the formation of bacterial biofilms, thus neutralizing the adhesion of FimH type 1 lectin present at the tip of their fimbriae against the natural multiantennary oligomannosides of uroplakin 1a receptors expressed on uroepithelial tissues. Preliminary DLS studies of the mannosylated dendrimers to cross- link the leguminous lectin Con A used as a model showed their high potency as candidates to fight the E. coli adhesion and biofilm formation.


Assuntos
Antibacterianos/síntese química , Biofilmes/efeitos dos fármacos , Dendrímeros/síntese química , Lectinas/química , Manose/química , Oligossacarídeos/química , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Azidas/química , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Química Click , Concanavalina A/química , Concanavalina A/metabolismo , Reação de Cicloadição , Dendrímeros/metabolismo , Dendrímeros/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/metabolismo , Expressão Gênica , Glicosilação , Humanos , Lectinas/metabolismo , Modelos Biológicos , Polietilenoglicóis/química , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Uroplaquina Ia/genética , Uroplaquina Ia/metabolismo , Urotélio/efeitos dos fármacos , Urotélio/metabolismo , Urotélio/microbiologia
3.
Rapid Commun Mass Spectrom ; 32(17): 1573-1582, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29920820

RESUMO

RATIONALE: Acetaminophen (APAP) is a well-known analgesic, deemed a very safe over-the-counter medication. However, it is also the main cause of acute liver failure (ALF) in the Western world, via the formation of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), and its covalent attachment to liver proteins. The aim of this study was to develop a sensitive and robust quantitative assay to monitor APAP-protein binding to human serum albumin (HSA) in patient samples. METHODS: A combination of isotope dilution, peptic digestion and solid-phase extraction coupled to liquid chromatography/multiple reaction monitoring (LC/MRM) was employed. An external calibration curve with surrogate modified protein spiked into blank serum was used for absolute quantitation. Samples were analyzed by LC/MRM to measure the modified active site peptide of HSA. The LC/MRM assay was validated and successfully applied to serum samples from patients suffering from APAP-induced ALF. RESULTS: Accuracy ranged from 83.8-113.3%, within-run coefficient of variation (CV) ranged from 0.3-6.9%, and total CVs from 1.6-10.6%. Patient samples ranged from 0.12-3.91 nmol/mL NAPQI-HSA; in-between the assay dynamic range of 0.11-50.13 nmol/mL serum. In vivo median concentrations were found to be 0.62 nmol/mL and 0.91 nmol/mL for non-spontaneous survivors (n = 25) and individuals with irreversible liver damage (n = 10), respectively (p-value = 0.028), demonstrating significant potential as a biomarker for ALF outcome. CONCLUSIONS: A fast and sensitive assay was developed to accurately quantify NAPQI-HSA as a biomarker for APAP-related covalent binding in human serum.


Assuntos
Acetaminofen/efeitos adversos , Cromatografia Líquida/métodos , Falência Hepática Aguda/sangue , Albumina Sérica Humana/análise , Espectrometria de Massas em Tandem/métodos , Acetaminofen/administração & dosagem , Adulto , Estudos de Coortes , Feminino , Humanos , Falência Hepática Aguda/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Albumina Sérica Humana/metabolismo
4.
Biochimie ; 146: 127-138, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29248541

RESUMO

Despite its natural abundance in lenses of vertebrates the physiological function(s) of the galectin-related inter-fiber protein (GRIFIN) is (are) still unclear. The same holds true for the significance of the unique interspecies (fish/birds vs mammals) variability in the capacity to bind lactose. In solution, ultracentrifugation and small angle X-ray scattering (at concentrations up to 9 mg/mL) characterize the protein as compact and stable homodimer without evidence for aggregation. The crystal structure of chicken (C-)GRIFIN at seven pH values from 4.2 to 8.5 is reported, revealing compelling stability. Binding of lactose despite the Arg71Val deviation from the sequence signature of galectins matched the otherwise canonical contact pattern with thermodynamics of an enthalpically driven process. Upon lactose accommodation, the side chain of Arg50 is shifted for hydrogen bonding to the 3-hydroxyl of glucose. No evidence for a further ligand-dependent structural alteration was obtained in solution by measuring hydrogen/deuterium exchange mass spectrometrically in peptic fingerprints. The introduction of the Asn48Lys mutation, characteristic for mammalian GRIFINs that have lost lectin activity, lets labeled C-GRIFIN maintain capacity to stain tissue sections. Binding is no longer inhibitable by lactose, as seen for the wild-type protein. These results establish the basis for detailed structure-activity considerations and are a step to complete the structural description of all seven members of the galectin network in chicken.


Assuntos
Galectinas/química , Animais , Sítios de Ligação , Metabolismo dos Carboidratos , Galinhas , Cristalografia por Raios X , Galectinas/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Soluções
5.
Phys Chem Chem Phys ; 19(43): 29333-29343, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29075707

RESUMO

Copper-loaded organo-montmorillonite showed improved affinity towards hydrogen under ambient conditions. Clay ion exchange with a propargyl-ended cation followed by thiol-yne coupling with thioglycerol resulted in a porous structure with a 6 fold higher specific surface area, which dramatically decreased after copper incorporation. X-ray diffraction and photoelectron spectrometry, nuclear magnetic resonance (1H and 13C) and CO2-thermal programmed desorption revealed strong sulfur:Cu0 and oxygen:Cu0 interactions. This was explained in terms of structure compaction that 'traps' Cu0 nanoparticles (CuNPs) and reduces their mobility. Transmission electron microscopy showed predominant 1.0-1.5 nm CuNPs. Hydrogen capture appears to involve predominantly physical interaction, since differential scanning calorimetry measurements gave low desorption heat and almost complete gas release between 20 °C and 75 °C. Possible hydrogen condensation within the compacted structure should hinder gas diffusion inside CuNPs and prevent chemisorption. These results allow safe hydrogen storage with easy gas release to be envisaged even at room temperature under vacuum. The reversible capture of hydrogen can be even more attractive when using natural inorganic supports and commercial plant-derived dendrimers judiciously functionalized, even at the expense of porosity.

6.
Chem Rev ; 117(15): 9839-9873, 2017 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-28682060

RESUMO

Among other classes of biomolecules, carbohydrates and glycoconjugates are widely involved in numerous biological functions. In addition to addressing the related synthetic challenges, glycochemists have invested intense efforts in providing access to structures that can be used to study, activate, or inhibit these biological processes. Over the past few decades, aminooxylated carbohydrates have been found to be key building blocks for achieving these goals. This review provides the first in-depth overview covering several aspects related to the syntheses and applications of aminooxylated carbohydrates. After a brief introduction to oxime bonds and their relative stabilities compared to related C═N functions, synthetic aspects of oxime ligation and methodologies for introducing the aminooxy functionality onto both glycofuranosyls and glycopyranosyls are described. The subsequent section focuses on biological applications involving aminooxylated carbohydrates as components for the construcion of diverse architectures. Mimetics of natural structures represent useful tools for better understanding the features that drive carbohydrate-receptor interaction, their biological output and they also represent interesting structures with improved stability and tunable properties. In the next section, multivalent structures such as glycoclusters and glycodendrimers obtained through oxime ligation are described in terms of synthetic design and their biological applications such as immunomodulators. The second-to-last section discusses miscellaneous applications of oxime-based glycoconjugates, such as enantioselective catalysis and glycosylated oligonucleotides, and conclusions and perspectives are provided in the last section.


Assuntos
Carboidratos/química , Carboidratos/síntese química , Glicoproteínas/química , Oligonucleotídeos/química , Estereoisomerismo
7.
Histochem Cell Biol ; 147(2): 285-301, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28013366

RESUMO

A hallmark of endogenous lectins is their ability to select a few distinct glycoconjugates as counterreceptors for functional pairing from the natural abundance of cellular glycoproteins and glycolipids. As a consequence, assays to assess inhibition of lectin binding should necessarily come as close as possible to the physiological situation, to characterize an impact of a synthetic compound on biorelevant binding with pharmaceutical perspective. We here introduce in a proof-of-principle manner work with sections of paraffin-embedded tissue (jejunum, epididymis) and labeled adhesion/growth-regulatory galectins, harboring one (galectin-1 and galectin-3) or two (galectin-8) types of lectin domain. Six pairs of synthetic lactosides from tailoring of the headgroup (3'-O-sulfation) and the aglycone (ß-methyl to aromatic S- and O-linked extensions) as well as three bi- to tetravalent glycoclusters were used as test compounds. Varying extents of reduction in staining intensity by synthetic compounds relative to unsubstituted/free lactose proved the applicability and sensitivity of the method. Flanking cytofluorimetric assays on lectin binding to native cells gave similar grading, excluding a major impact of tissue fixation. The experiments revealed cell/tissue binding of galectin-8 preferentially via one domain, depending on the cell type so that the effect of an inhibitor in a certain context cannot be extrapolated to other cells/tissues. Moreover, the work with the other galectins attests that this assay enables comprehensive analysis of the galectin network in serial tissue sections to determine overlaps and regional differences in inhibitory profiles.


Assuntos
Galectinas/química , Galectinas/metabolismo , Citometria de Fluxo , Galectinas/classificação , Glicosídeos/síntese química , Glicosídeos/química , Glicosídeos/metabolismo , Humanos , Lectinas/química , Lectinas/metabolismo , Ligação Proteica
8.
Chemosphere ; 168: 1648-1657, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27939664

RESUMO

Hematite-SBA-16 mixture (HS) exhibited high catalytic activity in Orange-G (OG) ozonation in water. Total OG discoloration was achieved in half the time required with hematite or SBA-16 alone, all UV-Vis bands disappeared in less than 2 min. Liquid chromatography- Mass spectrometry (LC-MS) revealed that OG ozonation triggers via both hydroxylation and desulfonation of the aromatic rings into specific intermediates. Prolonged ozonation in the presence of hematite and SBA-16 alone resulted in different distributions of common derivatives. The latter were not detected after 25 min ozonation with HS. Stochastic modeling of the evolution in time of the UV-Vis bands of OG revealed strong binary interaction between the initial pH and catalyst concentration. This was explained in terms of reciprocal contributions of: i. the catalytic properties of hematite in spite of its low porosity; ii. the high specific surface area of SBA-16 for adsorption and surface reaction notwithstanding its low intrinsic catalytic activity. The weak basicity of SBA-16 surface seems to play a key-role in adsorption. These findings are of great interest for envisaging flexible oxidative treatments, where Fe3+ containing soils or mixtures of sand and rust may also act as catalyst for total mineralization of various azo-dyes, regardless to their structures.


Assuntos
Compostos Azo/química , Corantes/química , Compostos Férricos/química , Ozônio/química , Dióxido de Silício/química , Adsorção , Catálise , Oxirredução , Poluentes Químicos da Água/química
9.
Infect Immun ; 84(7): 2059-2075, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27113360

RESUMO

Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 µg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections.


Assuntos
Cápsulas Bacterianas/imunologia , Glicoconjugados/imunologia , Polissacarídeos Bacterianos/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus suis/imunologia , Vacinas Conjugadas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Feminino , Imunização , Switching de Imunoglobulina , Imunomodulação , Camundongos , Oligodesoxirribonucleotídeos , Sorogrupo , Infecções Estreptocócicas/mortalidade , Infecções Estreptocócicas/prevenção & controle , Streptococcus suis/classificação , Suínos
10.
Molecules ; 21(4): 448, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-27049377

RESUMO

Most classical dendrimers are frequently built-up from identical repeating units of low valency (usually AB2 monomers). This strategy necessitates several generations to achieve a large number of surface functionalities. In addition, these typical monomers are achiral. We propose herein the use of sugar derivatives consisting of several and varied functionalities with their own individual intrinsic chirality as both scaffolds/core as well as repeating units. This approach allows the construction of chiral, dense dendrimers with a large number of surface groups at low dendrimer generations. Perpropargylated ß-D-glucopyranoside, serving as an A5 core, together with various derivatives, such as 2-azidoethyl tetra-O-allyl-ß-D-glucopyranoside, serving as an AB4 repeating moiety, were utilized to construct chiral dendrimers using "click chemistry" (CuAAC reaction). These were further modified by thiol-ene and thiol-yne click reactions with alcohols to provide dendritic polyols. Molecular dynamic simulation supported the assumption that the resulting polyols have a dense structure.


Assuntos
Química Click , Dendrímeros/química , Compostos de Sulfidrila/química , Álcoois/química , Dendrímeros/síntese química , Estrutura Molecular , Polímeros/química , Propriedades de Superfície
11.
Nanoscale ; 8(9): 5106-19, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26868181

RESUMO

Low G1 generation polyamine dendrimers built around programmable, flexible, and short tetraethyleneglycol branches were readily prepared in a divergent manner using a combination of orthogonal AB3 or AB5 units and highly efficient chemical transformations based on Cu(i) catalyzed alkyne-azide cycloaddition (CUAAC) and thiol-ene click reactions. The constructs showed that the G1 polyamines with only twelve and eighteen amine surface groups can successfully deliver siRNA in human cells, with transfection efficiency comparable to that of Lipofectamine 2000®. Measurements of cell viability following transfection of plasmid DNA and siRNA showed that the dendritic polyamines are less cytotoxic than Lipofectamine 2000® and are thus preferable for biological applications.


Assuntos
Dendrímeros , Portadores de Fármacos , Nanopartículas/química , Plasmídeos , Poliaminas , Polietilenoglicóis , RNA Interferente Pequeno , Transfecção/métodos , Dendrímeros/síntese química , Dendrímeros/química , Dendrímeros/farmacocinética , Dendrímeros/farmacologia , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Células MCF-7 , Plasmídeos/química , Plasmídeos/farmacocinética , Plasmídeos/farmacologia , Poliaminas/síntese química , Poliaminas/química , Poliaminas/farmacocinética , Poliaminas/farmacologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/farmacologia
12.
Chembiochem ; 17(9): 843-51, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26792008

RESUMO

The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1.


Assuntos
Cisteína/metabolismo , Endorribonucleases/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Pirimidinonas/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Endorribonucleases/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinonas/química , Pirimidinonas/farmacologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Resposta a Proteínas não Dobradas/efeitos dos fármacos
13.
Rapid Commun Mass Spectrom ; 29(18): 1632-40, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26467115

RESUMO

RATIONALE: An isotopic labeling strategy based on derivatizing amine-containing metabolites has been developed using light ((12) C6 ) and heavy ((13) C6 ) N-benzoyloxysuccinimide reagents for semi-targeted metabolomic applications. METHODS: Differentially labeled samples were combined and analyzed simultaneously by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS) to compare relative amounts of amine-containing metabolites. The selectivity of the reaction was determined with model metabolites and was shown to also be applicable to thiol and phenol moieties. The potential for relative quantitation was evaluated in cell extracts and the method was then applied to quantify metabolic perturbations occurring in human cultured cells under normal vs. oxidative stress conditions. RESULTS: A total of 279 derivatized features were detected in HL60 cell extracts, 77 of which yielded significant concentration changes upon oxidative stress treatment. Based on accurate mass measurements and MS/MS spectral matching with reference standard solutions, 10 metabolites were clearly identified. Derivatized compounds were found to have diagnostic fragment ions from the reagent itself, as well as structurally informative ions useful for metabolite identification. CONCLUSIONS: This simple derivatization reaction can be applied to the relative quantitation of amine-, thiol- and phenol-containing compounds, with improved sensitivity and chromatographic peak shapes due to the increased hydrophobicity of polar metabolites not readily amenable to reversed-phase LC/MS analysis.


Assuntos
Cromatografia Líquida/métodos , Metabolômica/métodos , Succinimidas/química , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Células HL-60 , Humanos , Marcação por Isótopo
14.
Chem Soc Rev ; 44(12): 3924-41, 2015 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-25556902

RESUMO

This review represents the first of its kind in that it is mainly devoted to the use of carbohydrates as both scaffolds and building blocks for the construction of a wide range of novel, dense, and chiral dendrimers. It deviates from several previous reviews describing solely carbohydrates as functional surface groups, mostly devoted to biological applications. A brief overview of the most recent synthetic strategies in dendrimer design will be presented for the purpose of comparing their differences, similitudes, and advantages. A particular emphasis will be devoted to the general family of core molecules or scaffolds possessing a large number of functional groups from which, carbohydrates clearly emanate for their wide structural diversities, abundant chiral centers, and the relative ease with which their functional groups can be selectively manipulated. This beneficial characteristic relies on the fact that carbohydrates exist in enantiomeric states, several conformations, anomeric configurations, range of functional groups, and as three to seven carbon units.

15.
Chembiochem ; 16(1): 126-39, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25407851

RESUMO

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective ß-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.


Assuntos
Amino Açúcares/química , Galectina 1/química , Galectina 3/química , Galectinas/química , Glicosídeos/química , Acetilglucosamina/química , Alquinos/química , Amino Açúcares/síntese química , Azidas/química , Sequência de Carboidratos , Catálise , Reação de Cicloadição , Galectina 1/antagonistas & inibidores , Galectina 3/antagonistas & inibidores , Galectinas/antagonistas & inibidores , Glicosídeos/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Nitrofenilgalactosídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Triazóis/química
16.
J Mass Spectrom ; 49(12): 1223-33, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25476939

RESUMO

We present in this manuscript the characterization of the exact glycation sites of the Thomsen-Friedenreich antigen-BSA vaccine (TF antigen:BSA) prepared using a Michael addition reaction between the saccharide antigen as an electrophilic acceptor and the nucleophilic thiol and L-Lysine ε-amino groups of BSA using different ligation conditions. Matrix laser desorption ionization time-of-flight mass spectrometry of the neoglycoconjugates prepared with TF antigen:protein ratios of 2:1 and 8:1, allowed to observe, respectively, the protonated molecules for each neoglycoconjugates: [M + H](+) at m/z 67,599 and 70,905. The measurements of these molecular weights allowed us to confirm exactly the carbohydrate:protein ratios of these two synthetic vaccines. These were found to be closely formed by a TF antigen:BSA ratios of 2:1 and 8:1, respectively. Trypsin digestion and liquid chromatography coupled with electrospray ionization mass spectrometry allowed us to identify the series of released glycopeptide and peptide fragments. De novo sequencing affected by low-energy collision dissociation tandem mass spectrometry was then employed to unravel the precise glycation sites of these neoglycoconjugate vaccines. Finally, we identified, respectively, three diagnostic and characteristic glycated peptides for the synthetic glycoconjugate possessing a TF antigen:BSA ratio 2:1, whereas we have identified for the synthetic glycoconjugate having a TF:BSA ratio 8:1 a series of 14 glycated peptides. The net increase in the occupancy sites of these neoglycoconjugates was caused by the large number of glycoforms produced during the chemical ligation of the synthetic carbohydrate antigen onto the protein carrier.


Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Vacinas Anticâncer/química , Cisteína/química , Glicoconjugados/química , Soroalbumina Bovina/química , Sequência de Aminoácidos , Animais , Bovinos , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
17.
Chem Commun (Camb) ; 50(87): 13300-3, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25227948

RESUMO

Both convergent and divergent strategies for the synthesis of "onion peel" glycodendrimers are reported which resulted in one of the best multivalent ligands known against the virulent factor from a bacterial lectin isolated from Pseudomonas aeruginosa.


Assuntos
Dendrímeros/química , Dendrímeros/síntese química , Glicerídeos/química , Glicerídeos/síntese química , Dendrímeros/farmacologia , Glicerídeos/farmacologia , Lectinas/química , Ligantes , Estrutura Molecular , Pseudomonas aeruginosa/química , Fatores de Virulência/antagonistas & inibidores
18.
Chem Res Toxicol ; 27(9): 1632-9, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25168029

RESUMO

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.


Assuntos
Acetaminofen/química , Benzoquinonas/química , Iminas/química , Albumina Sérica/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Pepsina A/metabolismo , Peptídeos/análise , Peptídeos/química , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Albumina Sérica/metabolismo , Espectrometria de Massas em Tandem
19.
Chem Commun (Camb) ; 50(16): 1983-5, 2014 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-24416763

RESUMO

An orthogonal coupling strategy was developed by combining thiol-ene and SN2 reactions, which was subsequently applied to the accelerated synthesis of multifunctional dendrimers using carbohydrate building blocks. In surface plasmon resonance (SPR) studies, the ß-d-galactopyranoside-coated dendrimer exhibited nM binding affinity with the bacterial LecA lectin extracted from Pseudomonas aeruginosa.


Assuntos
Dendrímeros/síntese química , Compostos de Sulfidrila/química , Adesinas Bacterianas/química , Dendrímeros/química , Estrutura Molecular , Pseudomonas aeruginosa/química , Ressonância de Plasmônio de Superfície
20.
Org Biomol Chem ; 11(40): 6906-18, 2013 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-24057051

RESUMO

Three small families of hydrolytically stable thioaryl glycosides were prepared as inhibitors of the LecA (PA-IL) virulence factor corresponding to the carbohydrate binding lectin from the bacterial pathogen Pseudomonas aeruginosa. The monosaccharidic arylthio ß-d-galactopyranosides served as a common template for the major series that was also substituted at the O-3 position. Arylthio disaccharides from lactose and from melibiose constituted the other two series members. In spite of the fact that the natural ligand for LecA is a glycolipid of the globotriaosylceramide having an α-d-galactopyranoside epitope, this study illustrated that the ß-d-galactopyranoside configuration having a hydrophobic aglycon could override the requirement toward the anomeric configuration of the natural sugar. The enzyme linked lectin assay together with isothermal titration microcalorimetry established that naphthyl 1-thio-ß-d-galactopyranoside () gave the best inhibition with an IC50 twenty-three times better than that of the reference methyl α-d-galactopyranoside. In addition it showed a KD of 6.3 µM which was ten times better than that of the reference compound. The X-ray crystal structure of LecA with was also obtained.


Assuntos
Adesinas Bacterianas/metabolismo , Pseudomonas aeruginosa/química , Tioglicosídeos/farmacologia , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Tioglicosídeos/síntese química , Tioglicosídeos/química
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