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1.
Open Biol ; 6(8)2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27558933

RESUMEN

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Epítopos/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteínas/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/química , Sitios de Unión , Cristalografía por Rayos X , Variación Genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/farmacología , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Modelos Moleculares , Biblioteca de Péptidos , Unión Proteica , Proteínas/genética , Proteínas/metabolismo , Relación Estructura-Actividad , Vía de Señalización Wnt
2.
AAPS J ; 17(1): 17-23, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25338740

RESUMEN

The A2 harmonization team, a part of the Global Bioanalysis Consortium (GBC), focused on defining possible tiers of chromatographic-based bioanalytical method performance. The need for developing bioanalytical methods suitable for the intended use is not a new proposal and is already referenced in regulatory guidance language. However, the practical implementation of approaches that differ from the well-established full validation requirements has proven challenging. Advances in technologies, the need to progress drug development more efficiently, and emerging new drug compound classes support the use of categorized tiers of bioanalytical methods. This paper incorporated the input from an international team of experienced bioanalysts to surmise the advantages and the challenges of tiered approaches and to provide recommendations on paths forward.


Asunto(s)
Cromatografía/métodos , Diseño de Fármacos , Preparaciones Farmacéuticas/análisis , Humanos , Cooperación Internacional , Tecnología Farmacéutica/métodos , Estudios de Validación como Asunto
3.
Artículo en Inglés | MEDLINE | ID: mdl-24695212

RESUMEN

Simeprevir (also known as TMC435 or TMC435350) is a novel hepatitis C protease inhibitor. A validated high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the sensitive and selective quantification of simeprevir in human EDTA plasma is described. During assay development, special attention was given to light instability of the drug in plasma and blood. The method consisted of precipitation of plasma proteins with acetonitrile after which the supernatant was analyzed using electrospray LC-MS/MS. The linearity was confirmed in the concentration range from 2.00 to 2000ng/mL, with 50-fold dilution extending to 100,000ng/mL. The precision of this assay, expressed as CV, ranged between 4.4% and 8.5% over the entire concentration range with assay accuracy between -0.3% and 8.5%. The method was applied successfully in many clinical studies to document the pharmacokinetics of simeprevir in plasma from healthy volunteers and patients.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Hepatitis C/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/sangre , Inhibidores de Proteasas/sangre , Sulfonamidas/sangre , Espectrometría de Masas en Tándem/métodos , Monitoreo de Drogas/métodos , Humanos , Sensibilidad y Especificidad , Simeprevir
4.
Protein Eng Des Sel ; 25(5): 251-9, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22454505

RESUMEN

Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides mimicking defined epitopes of the bone modulator protein sclerostin, which has been identified as a negative regulator of the Wnt pathway. For a fast exploration of activity defining epitopes, we produced a set of synthetic peptide constructs mimicking native sclerostin, in which intervening loops from the cystine-knot protein sclerostin were truncated and whose sequences were optimized for fast and productive refolding. We found that the second loop within the cystine knot could be replaced by unnatural sequences, both speeding up folding, and increasing yield. Subsequently, we used these constructs to pan the HuCAL phage display library for antibodies capable of binding the native protein, thereby restricting recognition to the desired epitope regions. It is shown that the antibodies that were obtained recognize a complex epitope in the protein that cannot be mimicked with linear peptides. Antibodies selected against peptides show similar recognition specificity and potency as compared with antibodies obtained from full-length recombinant protein.


Asunto(s)
Epítopos/inmunología , Proteínas/inmunología , Vía de Señalización Wnt/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Cistina/química , Mapeo Epitopo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Resonancia por Plasmón de Superficie
5.
Anal Chem ; 76(24): 7304-9, 2004 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-15595873

RESUMEN

Several methods estimating the partitioning over biological membranes and thus the biological activity of potential oral drug molecules have been developed and are described in the literature. A previous study suggested that fast micellar liquid chromatography on a monolithic column could be one of them. For a set of diverse pharmaceuticals, retention by this fast chromatographic method was determined, besides other parameters also thought or established to describe oral permeability or absorption, e.g., from the Caco-2 permeability method. In view of a high-throughput determination of membrane permeability, a study was made of which information fast micellar liquid chromatography is providing and to what degree this system can replace other methods, i.e., deliver similar information. The retention with this fast method, which is mainly based on hydrophobic interactions, proved useful to sort substances into classes of Caco-2 and percent intestinal absorption.


Asunto(s)
Cromatografía Liquida/métodos , Membranas Artificiales , Permeabilidad , Preparaciones Farmacéuticas/metabolismo , Células CACO-2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Absorción Intestinal/fisiología , Micelas
6.
Curr Protein Pept Sci ; 4(4): 253-60, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-14529532

RESUMEN

Due to the advantageous properties of synthetic molecules compared to biological ones biological molecules in diagnostic tests are replaced increasingly by synthetic ones, usually synthetic peptides or related molecules. The replacement of biological antigens by synthetic peptides is most advanced at present, as well as the use of site-specific antibodies induced with synthetic peptides. Moreover recent results indicate that synthetic molecules may also replace antibodies. Ultimately this will lead to diagnostic assays built of synthetic molecules only.


Asunto(s)
Diseño de Fármacos , Péptidos , Animales , Anticuerpos Monoclonales/inmunología , Técnicas Químicas Combinatorias/métodos , Mapeo Epitopo/métodos , Epítopos de Linfocito B/química , Humanos , Sueros Inmunes/inmunología , Inmunohistoquímica , Pruebas Inmunológicas/métodos , Imitación Molecular , Biblioteca de Péptidos , Péptidos/síntesis química , Péptidos/química , Análisis por Matrices de Proteínas/métodos , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína
7.
J Org Chem ; 66(25): 8297-301, 2001 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-11735506

RESUMEN

This paper describes the spontaneous and reversible assembly of approximately 20 kDa synthetic hydrogen-bonded assemblies via the formation of 144 cooperative hydrogen bonds. These nanostructures ( approximately 3.0 x 5.5 nm), consisting of 27 different components, have been carefully characterized using a combination of (1)H NMR spectroscopy, MALDI-TOF MS using Ag(+)-labeling, gel permeation chromatography, and CD spectroscopy.

8.
J Am Chem Soc ; 123(42): 10153-63, 2001 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-11603964

RESUMEN

The amplification of supramolecular chirality has been studied in dynamic chiral hydrogen-bonded assemblies 1(3).(CA)(6) using "Sergeants and Soldiers" experiments. Previously, we have shown that chiral centers present in either the dimelamine component 1 or the cyanurate component CA quantitatively induce one handedness (M or P) in the assembly. This offers the possibility to study the amplification of chirality under two different kinetic regimes. When chiral dimelamines 1 are used, the exchange of chiral components and (M/P)-interconversion, i.e., interconversion between the (M)- and (P)-isomers of assembly 1(3).(CA)(6), take place via identical pathways (condition A). When chiral cyanurates CA are used, the exchange of chiral components occurs much faster than (M/P)-interconversion (condition B). Experimentally, a much stronger chiral amplification is observed under condition B. For example, the observed chiral amplification for a mixture of chiral and achiral components (40:60) is 46% under condition B and 32% under condition A. Kinetic models were developed to fit the experimental data and to simulate chiral amplification in dynamic systems in general. These simulations show that it is theoretically possible that the diastereomeric excess in a dynamic system is more than 99% with less than 1% chiral component present!


Asunto(s)
Calixarenos , Enlace de Hidrógeno , Modelos Químicos , Barbitúricos/química , Cinética , Fenoles/química , Estereoisomerismo , Termodinámica , Triazinas/química
9.
J Am Chem Soc ; 123(31): 7518-33, 2001 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-11480972

RESUMEN

In this paper we describe model calculations for the self-assembly of N,N-disubstituted melamines 1 and N-substituted cyanuric acid or 5,5-disubstituted barbituric acid derivatives 2 into linear or crinkled tapes and cyclic rosettes via cooperative hydrogen bond formation. The model description considers all possible stereoisomeric tape structures consisting of two to eight different components (270 different species in total) and one cyclic hexameric rosette structure. Furthermore, eight steric parameters (R(12)-R(28)) are included that represent the different types of steric interactions within the assemblies. Most importantly, the model calculations clearly show that the tape/rosette ratio is very sensitive to changes in parameters that directly affect the internal energy of the rosette structure. In this respect, three parameters have been characterized, i.e., the basic equilibrium constant K(0) for the bimolecular association of a melamine and cyanurate, the equilibrium constant K(r)/K(0) for the cyclization of a linear hexamer, and the parameter R(12)-a(Z)b, representing attractive or repulsive interactions between adjacent melamine and cyanurate moieties. For example, an increase in K(0) from 100 to 10,000 M(-1) ([A](0) = [B](0) = 10 mM, K(r) = 0.01 M) or in K(r) from 0.001 to 0.1 M ([A](0) = [B](0) = 10 mM, K(0) = 1000 M(-1)) raises the concentration of the rosette from <5 to approximately 90% or from approximately 10 to approximately 85%, respectively. Similarly, a change in R(12)-a(Z)b from 1.0 (no repulsive or attractive interactions) to 1.5 (slight attractive interaction) raises the rosette fraction of the mixture from 25% to 45%. In sharp contrast to this, the model calculations show that parameters that only affect the internal energy of the tapes (R(13)--R(28)) hardly change the tape/rosette ratio. For example, by changing R(13)-a(EE)a from 1.0 (no repulsive or attractive interactions) to 0.001 (maximum repulsion), the rosette fraction in the mixture changes by no more than 8%. Including all possible sterics that occur only in tapes (i.e., R(13)--R(28)), the maximum change in rosette fraction is no more than 16%. These predictions can be rationalized by considering that any change in the stability of the tapes only affects the rosette concentration by means of shifting the equilibrium between free 1 and 2 and the rosette. Since there are 270 different tapelike structures in equilibrium, this mixture represents the best buffer solution in the world. These model calculations seem to conflict with the concept of peripheral crowding as put forward by Whitesides et al., which states that bulky substituents on the periphery of the melamine (and cyanurate) components can be used to shift the tape/rosette equilibrium completely toward the rosette structure. Computer simulations (CHARMm 24.0) show that linear tapes with bulky substituents are severely distorted from planarity, while the corresponding rosette remains planar. Therefore, tapelike structures with bulky substituents are expected to have a much higher solubility than the corresponding rosettes, which can explain the observed crystal data.

10.
Glycobiology ; 11(8): 663-76, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11479277

RESUMEN

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.


Asunto(s)
Fluorocarburos/química , Ácidos Siálicos/química , Acilación , Aminas/química , Animales , Cromatografía de Gases y Espectrometría de Masas/métodos , Lactonas/química , Metilación , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Ácidos Neuramínicos/química , Propionatos/metabolismo , Azúcares Ácidos/química , Sulfatos/química
11.
Proc Natl Acad Sci U S A ; 98(18): 10042-5, 2001 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-11526228

RESUMEN

Multichromophoric hydrogen-bonded assemblies 1(3) small middle dot(BAR)(6) are studied that bear a remarkably close resemblance to commelinin, a naturally occurring assembly responsible for an intense blue color of flowers. The incorporated chromophores exhibit a hypsochromic shift in the UV/visible (Vis) absorption maximum (Delta lambda(max) = 14 nm) compared with the free chromophores. In addition, the chiroptical properties of incorporated chromophores can be rationally controlled by changing the supramolecular chirality of the assembly. These properties have been used to study the stability of this type of assembly with UV and CD spectroscopy at concentrations far below the NMR sensitivity threshold (10(-4) M). The determined C(50%) values of 2-3 microM in benzene show the extremely high stability of these hydrogen-bonded assemblies.


Asunto(s)
Pirimidinonas/química , Dicroismo Circular , Cristalografía por Rayos X , Enlace de Hidrógeno , Sustancias Macromoleculares , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Pigmentos Biológicos/química , Plantas/química , Espectrofotometría Ultravioleta
12.
Curr Top Med Chem ; 1(5): 443-62, 2001 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11899107

RESUMEN

Over the last years, there has been an exponentially growing need and interest to bring pharmacokinetic expertise into discovery. In order to allow a multidisciplinary selection and a higher attrition rate, both the in vivo and in vitro pharmacokinetic parameters of an ever increasing number of tentative new chemical entities are evaluated in an earlier phase of Drug Discovery. A higher attrition rate at the beginning of the pipeline should result in a lower attrition rate at a later stage in development. In this process, the bioanalytical laboratory has become increasingly important. Analytical strategies needed to be adapted to cope with novel experimental designs such as cassette dosing, cassette analysis or 96-well techniques. At the same time, HT-synthesis programs surfaced a broader variety of chemical classes to be investigated, disfavoring further generalization of analytical approaches. Progress in lab automation, improved chromatographic techniques and the proliferation of LC-MS/MS enabled the analyst to deal with these challenges much faster and with a higher level of confidence. Quality standards regarding method development and method validation, setting the boundaries for more than a decade, needed to be titrated to reach an optimal balance between speed and quality. This review will give an illustrative overview of the bioanalytical techniques and strategies used to support Drug Discovery, together with some pitfalls related to the overzealous use of new techniques.


Asunto(s)
Técnicas de Química Analítica/métodos , Evaluación Preclínica de Medicamentos/métodos , Farmacocinética , Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/normas , Cromatografía Liquida/normas , Evaluación Preclínica de Medicamentos/normas , Cromatografía de Gases y Espectrometría de Masas/normas , Control de Calidad
13.
Nature ; 408(6809): 181-4, 2000 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-11089967

RESUMEN

Chiral molecules have asymmetric arrangements of atoms, forming structures that are non-superposable mirror images of each other. Specific mirror images ('enantiomers') may be obtained either from enantiomerically pure precursor compounds, through enantioselective synthesis, or by resolution of so-called racemic mixtures of opposite enantiomers, provided that racemization (the spontaneous interconversion of enantiomers) is sufficiently slow. Non-covalent assemblies can similarly adopt chiral supramolecular structures, and if they are held together by relatively strong interactions, such as metal coordination, methods analogous to those used to obtain chiral molecules yield enantiomerically pure non-covalent products. But the resolution of assemblies formed through weak interactions, such as hydrogen-bonding, remains challenging, reflecting their lower stability and significantly higher susceptibility to racemization. Here we report the design of supramolecular structures from achiral calix[4]arene dimelamines and cyanurates, which form multiple cooperative hydrogen bonds that together provide sufficient stability to allow the isolation of enantiomerically pure assemblies. Our design strategy is based on a non-covalent 'chiral memory' concept, whereby we first use chiral barbiturates to induce the supramolecular chirality in a hydrogen-bonded assembly, and then substitute them by achiral cyanurates. The stability of the resultant chiral assemblies in benzene, a non-polar solvent not competing for hydrogen bonds, is manifested by a half-life to racemization of more than four days at room temperature.

14.
Anal Biochem ; 284(2): 201-16, 2000 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-10964402

RESUMEN

In a previous work (Zanetta et al. Glycobiology 9, 255-266 (1999)), it was reported that all constituents of gangliosides could be obtained as heptafluorobutyrate derivatives after methanolysis in a single gas chromatography analysis. This report demonstrates that gas chromatography coupled with mass spectrometry in the electron impact mode allows identification and quantification of long-chain bases and fatty acids without interference from monosaccharides. On the basis of ions specific for families and for individual compounds, sphingosines, sphinganines, and phytosphingosines (including ramified, unsaturated, hydroxylated, and etherified compounds) can be identified. Fatty acid methyl esters, including linear, ramified, unsaturated, and hydroxylated species, are identified and quantified in the same way. Possible extensions of this method to the fatty moiety of other lipids (alkylacylglycerol and dimethyl acetal) are discussed.


Asunto(s)
Fluorocarburos/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Glucolípidos/análisis , Animales , Bacterias/química , Ésteres/análisis , Ácidos Grasos/química , Glucolípidos/química , Hidroxilación , Ratas , Levaduras/química
16.
Chemistry ; 6(22): 4104-15, 2000 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-11128274

RESUMEN

Herein we describe our results on the characterization of a wide variety of different hydrogen-bonded assemblies by means of a novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique with Ag+ labeling. The labeling technique with Ag+ ions is extremely mild and provides a nondestructive way to generate charged assemblies that can be detected by mass spectrometry. Up to now more than 25 different single (1(3).2(3)), double (3(3).2(6)), and tetrarosettes (4(3).2(12)) have been successfully characterized by the use of this method. The success of the method entirely depends on the presence of a suitable binding site for the Ag+ ion. A variety of functionalities has been identified that provide strong binding sites for Ag+, either acting in a cooperative way (pi-arene and pi-alkene donor functionalities) or individually (cyano and crown ether functionalities). The method works well for assemblies with molecular weights between 2,000 and 8,000 Da, and most likely far beyond this limit.

17.
Org Lett ; 2(26): 4121-4, 2000 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-11150179

RESUMEN

Small peptide fragments functionalized with dimelamine units spontaneously form well-defined assemblies. The hydrogen-bond donating and accepting sites in the peptide units are perfectly compatible with the hydrogen-bond assembly motif and slightly stabilize the assembly via additional hydrogen-bond formation.

18.
Glycobiology ; 9(3): 255-66, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10024663

RESUMEN

We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved.


Asunto(s)
Cromatografía de Gases/métodos , Fluorocarburos/química , Glucolípidos/química , Glicoproteínas/química , Metilglicósidos/química , Monosacáridos/aislamiento & purificación , Acilación , Secuencia de Carbohidratos , Cromatografía de Gases y Espectrometría de Masas/métodos , Datos de Secuencia Molecular
19.
Anal Biochem ; 267(2): 300-8, 1999 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-10036134

RESUMEN

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.


Asunto(s)
Glicoproteínas/análisis , Monosacáridos/análisis , Cromatografía de Gases , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Polisacáridos/análisis
20.
Glycoconj J ; 16(10): 617-27, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10972140

RESUMEN

A major impediment in the analysis of glycosaminoglycans is the difficulty to cleave quantitatively the glycosidic bonds because of the stabilisation of glycosidic bonds and of the relative instability of the liberated constituents. This manuscript describes a modified procedure of methanolysis in the presence of barium acetate, reducing the destruction of uronic acids and increasing the cleavage yield. The reaction products could be identified and analysed quantitatively by GC and GC/MS of the heptafluorobutyrate derivatives of O-methyl glycosides of monosaccharides (for keratan sulphate and chondroitin sulphate B), or as a mixture of O-methyl glycosides of monosaccharides and of disaccharides (for the other sulphated glycosaminoglycans). Quantitative molar ratio between the different monosaccharide constituents (including the linkage region constituents) could be obtained, even when proteoglycans also contain classical N-glycans or O-glycans.


Asunto(s)
Fluorocarburos/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Metanol/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Acilación , Artefactos , Bario/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Cromatografía en Capa Delgada , Disacáridos/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos , Heparina/química , Heparina/metabolismo , Sulfato de Queratano/química , Sulfato de Queratano/metabolismo , Metilglicósidos/química , Metilglicósidos/metabolismo , Monosacáridos/análisis , Polisacáridos/análisis , Polisacáridos/química , Ácidos Urónicos/metabolismo
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