High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2- aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.      

Open Pore Block of Connexin26 and Connexin32 Hemichannels by Neutral,Acidic and Basic Glycoconjugates

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26–Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.     

Open Pore Block of Connexin26 and Connexin32 Hemichannels by Neutral, Acidic and Basic Glycoconjugates

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.     

Open pore block of connexin26 and connexin32 hemichannels by neutral, acidic and basic glycoconjugates

The mechanisms of molecular discrimination by connexin channels are of acute biological and medical importance. The availability of affinity or open-pore blocking reagents for reliable and specific study of the connexin permeability pathway, would make possible the rigorous cellular and physiological studies required to inform, in molecular terms, the underlying role of intercellular communication pathways in development and disease. Previous work utilized a series of glucosaccharides labeled with an uncharged fluorescent aminopyridine (PA-) group to establish steric constraints to permeability through connexin hemichannels. In that work, the smallest probe permeable through homomeric Cx26 and heteromeric Cx26-Cx32 channels was the PA-disaccharide, and the smallest probe permeable through homomeric Cx32 channels was the PA-trisaccharide. The larger impermeable probes did not block permeation of the smaller probes. Building on this work, a new set of glucosaccharide probes was developed in which the label was one of a homologous series of novel anthranilic acid derivatives (ABG) that carry negative or positive formal charge or remain neutral at physiological pH. When the PA-label of the smallest impermeant PA-derivatized oligosaccharides was replaced by ABG label, the resulting probes acted as reversible, high-affinity inhibitors of large molecule permeation through connexin pores in a size and connexin-specific manner.     

Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method

Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-Aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.     

Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling

Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.     

Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates

The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.     

Unique anthranilic acid chemistry facilitates profiling and characterization of Ser/Thr-linked sugar chains following hydrazinolysis

A novel method for the analysis of Ser/Thr-linked sugar chains was made possible by the virtue of unique anthranilic acid (AA, 2-aminobenzoic acid [2AA]) chemistry for labeling carbohydrates in aqueous salt solutions (K. R. Anumula, Anal. Biochem. 350 (2006) 1-23). The protocol for profiling of Ser/Thr carbohydrates by hydrazinolysis was made simple by eliminating intermediary isolation steps involved in a sample preparation such as desalting and various chromatographic purification schemes. A 6-h hydrazinolysis was carried out at 60 degrees C for O-linked oligosaccharides and at 95 degrees C for total oligosaccharides (N-linked with some O-linked). Following evaporation of hydrazine (<10 min), the oligosaccharides were N-acetylated and derivatized with AA in the same reaction mixture containing salts. Presumably, the glycosyl-hydrazines/hydrazones present in the mixture did not interfere with AA labeling. Because AA is the most fluorescent and highly reactive tag for labeling carbohydrates, the procedures described are suitable for the analysis of a limited amount of samples ( approximately 5 microg) by the current high-resolution high-performance liquid chromatography (HPLC) methods. HPLC conditions developed for the separation of O-linked sugar chains based on size on an amide column were satisfactory for quantitative profiling and characterization. Common O-linked sugar chains found in fetuin, equine chorionic gonadotropin, and glycophorin can be analyzed in less than 50 min. In addition, these fast profiling methods were comparable to profiling by PNGase F (peptide N-glycosidase from Flavobacterium meningosepticum) digestion in terms of time, effort, and simplicity and also were highly reproducible for routine testing. The procedures for the release of sugar chains by hydrazinolysis at the microgram level, labeling with fluorescent tag AA, and profiling by HPLC should be useful in characterization of carbohydrates found in glycoproteins.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Structures, function, and transformational changes of the sugar chains of glycohormones

Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid-stimulating hormone, and human follicle-stimulating hormone are closely related family of proteins which share a common alpha-subunit. However, their sugar moieties are quite different. hCG contains five acidic asparagine-linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N-1 and N-2) and one monoantennary (N-3) complex-type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N-1, N-2, and N-3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the alpha- and beta-subunits of hCG revealed that alpha contains 1 mol each of N-2 and N-3, while beta contains 1 mol each of N-1 and N-2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones. hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocarcinoma patients contain five additional neutral oligosaccharides. In contrast, hCGs from invasive-mole patients contain three of the five oligosaccharides, specifically found in choriocarcinoma hCGs. The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fuc alpha 1----6GlcNAc group and by ectopic expression and subsequent modification of N-acetylglucosaminyltransferase IV. The appearance of tumor-specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.     

Synthesis of new N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid derivatives and their evaluation as anticancer agents

The N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid 6 and a series of its ester and amide derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human cancer cells. Ester derivatives 13 and 18 exhibited potent growth inhibitory activity with GI(50) values at nanomolar concentrations. Among amide derivatives, N-anthraniloylglycinate 19 shown moderate inhibitory activity in the full panel cancer cell line screening.     

Sugar Chips immobilized with synthetic sulfated disaccharides of heparin/heparan sulfate partial structure

Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.     

Synthesis of fluorescent nucleoside analogs as probes for 2′-deoxyribonucleoside kinases

We are reporting on the synthesis of fluorescent nucleoside analogs with modified sugar moieties (e.g., sugars other than ribose and 2′-deoxyribose). Four novel derivatives of the fluorescent thymidine analog 6-methyl-3-(β-D-2′-deoxyribofuranosyl) furano-[2,3-d]pyrimidin-2-one were synthesized via Sonogashira reaction and subsequent copper-catalyzed cycloaddition. These compounds represent promising tools for studying nucleoside metabolism inside living cells, as well as for screening directed evolution libraries of 2′-deoxyribonucleoside kinases with new and improved activity for the corresponding nucleoside analogs.     

Fluorescent Labels for the Analysis of Mono- and Oligosaccharides

Various fluorophores used for improving chromatographic and electrophoretic separation and detection sensitivity in the analysis of reducing mono- and oligosaccharides are described. Complex bimodal labels that bear, in addition to fluorescent moieties, some additional functional groups are also discussed.     

Fluorescent labels for analysis of mono- and oligosaccharides

Various fluorophores used for improving the chromatographic and electrophoretic separation and the detection sensitivity in the analysis of reducing mono- and oligosaccharides are described. Complex bimodal labels that, in addition to fluorescent moieties, bear some additional functional groups are also discussed.     

The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device.

Various monosaccharides, oligosaccharides and small polysaccharides were labelled covalently at their reducing end groups with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS), and the resulting fluorescent derivatives were separated by high-resolution PAGE. The electrophoretic mobilities of the labelled saccharides are related largely to the compounds' Mr values, but they are also influenced by the individual chemical structures of the saccharides. Various positional isomers and some epimers, for instance galactose and glucose, were resolved. Oligosaccharide and small polysaccharide derivatives, prepared from an enzymic digest of starch, each differing in size by a single hexose residue and with a range of degrees of polymerization from 2 to 26, were all resolved in a single gel. The method was relatively rapid and simple to perform. It enabled multiple samples to be analysed in parallel with high sensitivity. The fluorescent-labelling procedure was virtually quantitative. As little as 1 pmol of ANTS-labelled saccharide was detected photographically when the gels were illuminated by u.v. light. When the gels were viewed using an imaging system based on a cooled charge-coupled device, as little as 0.2 pmol was detected. The method may be useful for the structural analysis of the carbohydrate moieties of glycoconjugates and other naturally occurring oligosaccharides.     

Synthesis and evaluation of fluorescent heterocyclic aminoadamantanes as multifunctional neuroprotective agents

A series of fluorescent heterocyclic adamantane amines were synthesised with the goal to develop novel fluorescent ligands for neurological assay development. These derivatives demonstrated multifunctional neuroprotective activity through inhibition of the N-methyl-d-aspartate receptor/ion channel, calcium channels and the enzyme nitric oxide synthase. It also exhibited a high degree of free radical scavenging potential. N-(1-adamantyl)-2-oxo-chromene-3-carboxamide (8), N-adamantan-1-yl-5-dimethyl-amino-1-naphthalenesulfonic acid (11) and N-(1-cyano-2H-isoindol-2-yl) adamantan-1-amine (12) were found to possess a high degree of multifunctionality with favourable physical-chemical properties for bioavailability and blood-brain barrier permeability. The ability of these heterocyclic adamantane amine derivatives as nitric oxide synthase inhibitors, calcium channel modulators, NMDAR inhibitors and effective antioxidants, indicate that they may find application as multifunctional drugs in neuroprotection.     

Diagnostic methods for the determination of iduronic acid in oligosaccharides

A high-performance liquid chromatography (HPLC) method with pulsed amperometric detection (PAD) was used for the determination of the acid hydrolysis products of L-iduronic acid containing oligosaccharides isolated from biological sources. This HPLC-PAD method was compared with gas chromatographic (GLC) methods. Since acid hydrolysis of oligosaccharides can produce a number of products, several uronic acid derivatives were prepared by chemical synthesis. These well characterized standards in conjunction with mass spectrometry allowed for the identification of most of the products of methanolysis or hydrolysis of glycosamino-glycans, which included chondroitin sulfates A and B (dermatan sulfate), heparin, and hyaluronic acid. (4 M) HCl in methanol 100 degrees C for 24 h was found to be optimum for GLC and 1 M aqueous HCl for 4 h at 100 degrees C for HPLC-PAD. All of the monosaccharides, hexosamines, and uronic acids could be separately identified in a single chromatographic step using either technique. Good resolution, high sensitivity (low microgram samples) and rapid analysis makes these methods particularly useful for the determination of small amounts of glycosaminoglycans and other glycoconjugates found in samples isolated from biological sources. These two techniques are specifically designed to allow the qualitative determination of the carbohydrate content and composition of samples whose carbohydrate composition and content is completely unknown.     

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)- alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non- reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta- glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl- hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.      

Hydroxyl-specific fluorescence labeling of ABP-deoxyguanosine, PhIP-deoxyguanosine, and AFB1-formamidopyrimidine with BODIPY-FL

Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.