Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.     

Modular synthesis of heparin oligosaccharides

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di- and trisaccharide modules in a fully selective fashion. Installation of the alpha-glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans-uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII-binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.     

Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern.     

A modular strategy toward the synthesis of heparin-like oligosaccharides using monomeric building blocks in a sequential glycosylation strategy

A novel flexible assembly strategy is described for the modular synthesis of heparin and heparan sulfates. The reported strategy uses monomeric building blocks to construct the oligosaccharide chain to attain a maximum degree of flexibility. In the assembly, 1-hydroxyl glucosazido- and 1-thio uronic acid donors are combined in a sequential glycosylation protocol using sulfonium triflate activator systems. The key 1-thio uronic acids were obtained in an efficient manner from diacetone glucose employing a chemo- and regioselective oxidation of partially protected glucose and idose thioglycosides.     

Toward the assembly of heparin and heparan sulfate oligosaccharide libraries: efficient synthesis of uronic acid and disaccharide building blocks

The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine-glucuronic acid (GlcN-GlcA) or iduronic acid (GlcN-IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation.     

Characterization of neutral sugars and uronic acids after methanolysis and trimethylsilylation for recognition of plant gums

The main standard neutral sugars and uronic acids that occur as components of plant gums were methanolysed and silylated for study by gas chromatography—electron impact and chemical ionization mass spectrometry (GC—EI-MS and GC—CI-MS). The 25 TMSi methylglycosides, components of the chromatograms obtained were studied and identified by their mass spectra and/or comparison with the corresponding standards. In addition, an unusual uronic acid (4-O-methylglucuronic acid) and the lactone forms of glucuronic acid are reported. A classification of the ions in both EI-MS and CI-MS which allows differentiation between sugar classes and their tautomeric forms is given. The sample preparation method and the results of the above identification were applied to the analysis of some plant gums and a seventeenth century ink sample.     

Simultaneous Determination of Aldoses and Uronic Acids of Citrus Pectin by LC with Precolumn Derivatization and UV Detection

The simultaneous determination of aldoses and uronic acids is now available by liquid chromatography. The procedure involves acid hydrolysis followed by derivatization with 3-amino-9-ethylcarbazole, which was first employed for aldoses and uronic acids derivatization. The usefulness of this method is seen in the ability to analyze commercially available citrus pectin. The results show that citrus pectin consists of xylose, glucose, arabinose, rhamnose, galactose and galacturonic acid in molar ratios of 0.3:1.0:1.8:2.8:7.4:50.9, which was consistent with the result obtained by GC. The described method is suitable for routine analysis of pectin or other polysaccharides containing uronic acids.     

Validated spectrophotometric and fluorimetric methods for analysis of clozapine in tablets and urine

Five spectrophotometric methods and one fluorimetric method have been developed and validated for the analysis of clozapine. The spectrophotometric methods were based on the charge-transfer complexation reaction between clozapine as electron donor and each of iodine as sigma-acceptor or 7,7,8,8-tetracyanoquinondimethane (TCNQ), 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), tetracyanoethane (TCNE), and p-chloranilic acid (pCA) as pi-acceptors. The obtained complexes were measured spectrophotometrically at 365, 843, 460, 414, and 520 nm for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The fluorimetric method was based on the oxidation of clozapine in the presence of perchloric acid by cerium (IV), and subsequent measuring the fluorescence of the produced cerium (III) fluorimetrically at lambda(excitation) 260 and lambda(emission) 355 nm. Under the optimum assay conditions, Beer's law was obeyed at concentrations ranged from 4-200 microg mL(-1) for the spectrophotometric methods and from 24-250 ng mL(-1) for the fluorimetric method. The limits of detection for the spectrophotometric methods were 1.12, 1.76, 2.22, 0.95, and 13.26 microg mL(-1) for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The limit of detection for the fluorimetric method was 6.69 ng mL(-1). The proposed methods were successfully applied to the analysis of clozapine in tablets with good recoveries. The fluorimetric method could also be applied to the analysis of clozapine in spiked urine samples. The molar ratios and the reaction mechanisms were investigated.     

Benzylidene Acetal Protecting Group as Carboxylic Acid Surrogate: Synthesis of Functionalized Uronic Acids and Sugar Amino Acids

Direct oxidation of the 4,6‐O‐benzylidene acetal protecting group to C‐6 carboxylic acid has been developed that provides an easy access to a wide range of biologically important and synthetically challenging uronic acid and sugar amino acid derivatives in good yields. The RuCl₃–NaIO₄‐mediated oxidative cleavage method eliminates protection and deprotection steps and the reaction takes place under mild conditions. The dual role of the benzylidene acetal, as a protecting group and source of carboxylic acid, was exploited in the efficient synthesis of six‐carbon sialic acid analogues and disaccharides bearing uronic acids, including glycosaminoglycan analogues.     

Tandem mass spectrometric strategies for determination of sulfation positions and uronic acid epimerization in chondroitin sulfate oligosaccharides

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating (HexA-GalNAc sulfate) disaccharides, the functions of which depend on patterns of sulfation and uronic acid epimerization. The correlation of biological activities with structure requires a strategy to determine the sequences of CS oligosaccharides without the need for total isolation. Tandem mass spectrometry has enabled the development of proteomics, based on CID fragmentation of ions produced from complex mixtures of proteolytic peptides, and has the potential for rapid sequencing of CS and other glycosaminoglycan classes. The most challenging aspects of CS sequencing are to distinguish GalNAc residues sulfated at the 4- versus the 6-position and uronic acid epimers. This work describes the utility of (1) reducing terminal derivatives and (2) control of precursor ion charge state for tandem mass spectrometric strategies for determining GalNAc sulfation positional isomers of CS. The capability of tandem MS to differentiate uronic acid epimers is also shown, providing evidence that complete or nearly complete information on CS covalent structure may be obtained using tandem MS.     

柱前衍生高效液相色谱法分析海带岩藻聚糖的单糖及糖醛酸组成

建立了一种柱前衍生高效液相色谱法同时测定海带中岩藻聚糖的单糖及糖醛酸组成的方法.岩藻聚糖经4 mol/L三氟乙酸降解后,用1-苯基-3-甲基-5-吡唑啉酮(PMP)进行衍生化,并采用配有紫外检测器的反相高效液相色谱仪在250 nm波长下进行测定,实现了7种单糖和糖醛酸的良好分离.结果表明:该岩藻聚糖样品由5种单糖和2种...     

Exploitation of synthetic surfactant vesicles for enhanced fluorescence of metal chelates

The use of synthetic surfactant vesicles as a means for enhancing the photoluminescence of metal chelates is illustrated and a sensitive fluorimetric determination of aluminium with quinolin-8-ol-5-sulphonic acid in vesicles of didodecyldimethylammonium bromide is given. The influence of different variables on the organized aluminium-quinolin-8-ol-5-sulphonic acid reaction is investigated and possible mechanisms of the observed enhancing effects are discussed. The limit of detection is 1 μg l^?1 of aluminium. The optimized fluorimetric method has been successfully applied to the determination of aluminium in tap waters and dialysis fluids.     

Total Synthesis of the Gastroprotective Substance AI-77-B and of Analogues

The Diels-Alder adducts 8 of furan to 1-cyanovinyl acetate were converted into (Methyl 3-chloro)-5-O-(3-chlorobenzoyl)-2, 3-dideoxy-α-dl-arabino-hexofuranosid)uronic acid ((α)- 18a ) and into (methyl 3-azido-5-O-(3-chlorobenzoyl_-2,3-dideoxy-α-dl-ribo-hexofuranosid)uronic acid ((α)- 41a ). These compounds were condensed to (3S)-3-[(1′S)-1′-amino-3′-methylbutyl]-3,4-dihydro-8-hydroxyisocoumarin hydrochloride ((?)- 2 ); the resulting mixtures of diastereoisomeric amides were transformed and separated to give the gastroprotective substance AI-77- B ((?)- 1 ) and analogues.     

The Glycosylation Mechanisms of 6,3‐Uronic Acid Lactones

Uronic acids are important constituents of polysaccharides found on the cell membranes of different organisms. To prepare uronic‐acid‐containing oligosaccharides, uronic acid 6,3‐lactones can be employed as they display a fixed conformation and a unique reactivity and stereoselectivity. Herein, we report a highly β‐selective and efficient mannosyl donor based on C‐4 acetyl mannuronic acid 6,3‐lactone donors. The mechanism of glycosylation is established using a combination of techniques, including infrared ion spectroscopy combined with quantum‐chemical calculations and variable‐temperature nuclear magnetic resonance (VT NMR) spectroscopy. The role of these intermediates in glycosylation is assayed by varying the activation protocol and acceptor nucleophilicity. The observed trends are analogous to the well‐studied 4,6‐benzylidene glycosides and may be used to guide the development of next‐generation stereoselective glycosyl donors.     

Fluorimetric determination of phytic acid based on the activation of the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II)

Phytic acid exerts an activation effect on the oxidation of 2,2'-dipyridyl ketone hydrazone catalysed by Cu(II) ion and the oxidation product is highly fluorescent. A fixed time method for the fluorimetric determination of phytic acid based on this effect is described. The calibration graph is linear over the range 0.05-0.6 mg l-1 phytic acid, resulting in a limit of detection of 0.03 mg l-1 phytic acid. The relative standard deviation is in the range 1.4-1.8%, depending on the sample analysed. The method was successfully applied to the determination of phytic acid in human urine (20 samples) and food samples (nine different products). The results obtained for urine samples ranged from 0.31 to 3.6 mg l-1 phytic acid and for food samples from 3.8 to 22 mg g-1 phytic acid. This is the first procedure to be reported for the determination of phytic acid based on fluorimetric measurements.     

Structural studies of the Klebsiella type 57 capsular polysaccharide.

The structure of the capsular polysaccharide from Klebsiella type 57 has been investigated. Methylation analysis, uronic acid degradation, modified Smith degradation and graded acid hydrolysis were the principle methods used. Pure oligomeric fragments were isolated using the three methods of degradation and characterized by chemical and physical methods. These studies show the structure to consist of a tetrasaccharide repeating unit (all sugar residues have the D-configuration and are pyranosidic).     

氨基苯甲酸衍生化高效液相色谱法 分析多糖中的单糖及糖醛酸组成

衍生反相离子对色谱法同时分离检测多糖中单糖及糖醛酸组成的方法,筛选出适合于p-AMBA糖衍生物分离的色谱柱,考察了流动相组成对9种单糖和两种糖醛酸的p-AMBA衍生化产物的保留值及分离的影响,优化了反应温度和反应时间等衍生化条件,并应用优化的分析方法测定了螺旋藻中的单糖和糖醛酸的组成。采用紫外检测时,方法的检出限为 (2.55～13.4)×107mol/L;采用荧光检测时,方法的检出限为(3.38～176)×108 mol/L。     

流动注射时间扫描荧光分析法测定青霉素V钾

研究了H2SO4颜色反应用于青霉素V钾(PMPP)荧光测定的新方法。PMPP为弱荧光物质,与浓H2SO4反应后荧光显著增强。结合流动注射进样技术,借助时间扫描荧光方式,提出了流动注射时间扫描荧光分析法测定PMPP的新方法。在最大激发236.0 nm、最大发射波长306.0 nm处,PMPP在3.6×10-5g/L~4.0×10-2g/L范围内与荧光强度呈良好的线性关系,相关系数为0.9999。方法检出限为1.0×10-5g/L,相对标准偏差为0.6%(n=11,ρ=1.0×10-3g/L),进样量为0.18 mL。方法已用于药物及尿样中PMPP的测定。     

Mannopyranosyl uronic acid donor reactivity

The reactivity of a variety of mannopyranosyl uronic acid donors was assessed in a set of competition experiments, in which two (S)-tolyl mannosyl donors were made to compete for a limited amount of promoter (NIS/TfOH). These experiments revealed that the reactivity of mannuronic acid donors is significantly higher than expected based on the electron-withdrawing capacity of the C-5 carboxylic acid ester function. A 4-O-acetyl-β-(S)-tolyl mannuronic acid donor was found to have similar reactivity as per-O-benzyl-α-(S)-tolyl mannose.     

Molecular properties of hemicelluloses located in the surface and inner layers of hardwood and softwood pulps

The molecular properties of hemicelluloses located in the surface and inner layers of fibers present in hardwood and softwood pulps, together with the effects of different bleaching processes on these properties, have been investigated in this study. In order to separate the hemicelluloses located in these two layers, fibers were subjected to mechanical peeling and then separated by filtration into surface (filtrate) and inner layer materials. The materials thus obtained were characterized with respect to their polysaccharide compositions and uronic acid contents. The molar mass parameters of the hemicelluloses (extracted by alkali) were determined by employing size-exclusion chromatography in combination with off-line MALDI mass spectrometry. For all of the pulps examined, the relative content of xylan was found to be greater in the surface layer of the fiber than in the corresponding inner layer. The xylan polymers of the surface layer exhibited higher molar masses and lower frequencies of uronic acid side groups than did the xylans in the inner fiber layer. In connection with ozone treatment, hexenuronic acid residues in the surface layer xylan were removed to a greater extent than in the case of the inner layers, indicating a gradient for the reaction with ozone across the fiber wall. The xylan polymer remaining on the surface of the softwood pulps after completion of the chlorine dioxide bleaching process was predominantly uncharged.