Detection of neutral and aminosugars from glycoproteins and polysaccharides as their alditol acetates

A new method for the preparation and separation of alditol acetates from neutral sugars has been applied to aminosugars. Reduced aminosugars were rapidly acetylated using 1-methylimidazole as the catalyst without removal of borate formed during reduction. The alditol acetates were separated by glass capillary gas chromatography on Silar 10C. The alditol acetates of aminosugars had retention times much longer than those of neutral sugars. However, the alditol acetates of the deamination products of aminosugars had shorter retention times and were resolved from those of neutral sugars. This method was used for the simultaneous detection of neutral and aminosugars in acid hydrolysates of chitin and the glycoproteins, ovalbumin and peroxidase.     

Analysis of biomass sugars using a novel HPLC method

The precise quantitative analysis of biomass sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time consuming but the alternative high-performance liquid chromatography (HPLC) method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This method was demonstrated for standard sugars, pretreated corn stover liquid and solid fractions. Our method can also be used to analyze dimeric sugars (cellobiose and sucrose).     

High resolution glass capillary columns for the gas chromatographic analysis of alditol acetates of neutral and amino sugars

The development of improved high resolulation glass capillary columns for separating the alditol acetates of neutral and amino sugars is described. Different capillary column treatments were evaluated for their activity toward amino sugar derivatives and for their effect on the efficiency of the completed column. The simple two-step procedure of leaching with aqueous hydrochloric acid and dynamic coating with SP-2330 was found to produce efficient and inert columns.     

Analysis of the amino acid and sugar composition of streptococcal cell walls by gas chromatography-mass spectrometry

A procedure for determining the amino acid and sugar composition of streptococcal peptidoglycan-polysaccharide complexes by capillary gas chromatography-mass spectrometry (GC-MS) was established. Amino acids are analysed as butyl heptafluorobutyl derivatives and sugars as alditol acetates. These two different groups of compounds are derivatized independently but chromatography in both cases utilizes the same OV-1701 fused-silica capillary column which simplifies GC-MS analysis. The butyl heptafluorobutyl procedure incorporates new pre- and post-derivatization clean-up steps. Additionally, selected-ion monitoring MS allows amino acids to be readily analysed without interference from background noise.     

Identification of partially methylated methyl glycosides by gas chromatography-mass spectrometry of trimethylsilyl derivatives. Application to mycobacterial glycolipid antigen analysis

Partially methylated glycosides play an important role in the stereospecificity of glycolipid antigen-antibody binding reactions. A method for the structural determination of partially methylated methyl glycosides is described. The proposed method, which is an alternative to that using alditol acetates, consists in the analysis of trimethylsilyl glycoside derivatives by gas chromatography-mass spectrometry in the electron impact (EI) mode. Seven partially methylated methyl rhamnopyranosides were synthesized, then purified by high-performance liquid chromatography. After trimethylsilylation, their EI mass spectra showed characteristic fragmentation patterns allowing the determination of the position and the number of methoxy groups in the sugar ring. This method was successfully applied to the structural elucidation of partially methylated sugars contained by a quantitatively minor phenolic glycolipid antigen isolated from Mycobacterium kansasii. Moreover, the carbons involved in the glycosidic linkages of the glycolipid tetrasaccharide moiety were also identified by this method after permethylation, methanolysis and trimethylsilylation.     

Tandem acetalation-acetylation of sugars and related derivatives with enolacetates under solvent-free conditions

Molecular iodine catalyzes acetalation and acetylation of reducing sugars and sugar glycosides with stoichiometric amounts of enol acetates under solvent-free conditions, thereby facilitating the synthesis of various types of orthogonally protected sugar derivatives in short time and good yields. The outcome of the reaction can be controlled by variation in temperature. Thus at lower temperature, it is possible to obtain the acetonide acetate as a single product whereas peracetate is the major product at higher temperature.     

Modified One‐Pot Protocol for the Preparation of Thioglycosides from Unprotected Aldoses via S‐Glycosyl Isothiouronium Salts*

An efficient one‐pot protocol for the direct preparation of thioglycosides starting from unprotected reducing sugars via S‐glycosyl isothiouronium salts is reported. In this one‐pot methodology, BF₃ · OEt₂ has been used as a general catalyst for both per‐O‐acetylation of sugars and conversion of sugar per‐O‐acetates into S‐glycosyl isothiouronium salts, which was allowed to react with alkylating agents in the presence of a base to furnish thioglycosides in excellent yield.     

气相色谱-质谱联用同时分析新型细菌多糖中的单糖和糖醛酸

对纯化的新型细菌多糖进行酸水解,用乙硫醇-三氟乙酸和醋酐-吡啶体系先后对酸水解物进行衍生,与之前报道不同的是糖醛酸得到有效衍生化。以木糖为内标,采用气相色谱-质谱联用(GC-MS)定量分析该多糖酸水解物中单糖和糖醛酸衍生物发现,该多糖的糖链由岩藻糖、葡萄糖、葡萄糖醛酸和半乳糖组成,其相对物质的量比为1.50:1.0:0.79:2.06;中性糖比例与糖醇乙酸酯化分析岩藻糖、葡萄糖和半乳糖的相对物质的量比(1.76:1.0:1.98)接近;糖醛酸咔唑法与该方法分析葡萄糖醛酸的含量分别为16.19%和14.85%。以上结果表明所建立的衍生化方法及GC-MS同时定量分析多糖酸水解物中单糖和糖醛酸的方法可行。此外还对葡萄糖醛酸的质谱裂解机理进行了阐述。     

A Simplified,One-Pot Preparation of Acetobromosugars from Reducing Sugars

ABSTRACT

Acetobromoglycoses continue to be important as glycosyl donors in the synthesis of simple glycosides as well as complex oligosaccharides. From reducing sugars they are usually prepared via their peracetates in two steps. In the first step, sugars are converted to their peracetates using pyridine and acetic anhydride^1,2 and the acetates are then converted in a second step to acetobromosugars using a solution of hydrogen bromide in glacial acetic acid(HBr/HOAc).² Although not in use very often Redemann and Niemann³ as well as Lemieux⁴ have described one-pot methods for the preparation of acetobromoglucose wherein the reducing sugar is first treated with acetic anhydride in the presence of sulfuric acid³ or perchloric acid⁴ respectively to afford the peracetate. Direct conversion of the peracetate to its 1-bromo-derivative, in yields ranging from 80-87%, was then achieved by either treating the solution of the peracetate with gaseous HBr³ or with bromine in the presence of red phosphorus.⁴ In another approach to a one-pot method Humoller⁵ prepared tri-O-acetyl-β-L-arabinopyranosyl bromide in 40% yield by passing anhydrous HBr gas through a suspension of L-arabinose in acetic anhydride. By an essentially similar method Dale⁶ obtained acetobromosugars in yields ranging from 26-77% depending upon the sugar. Because acetobromosugars are the compounds most frequently used for synthesizing numerous other sugar derivatives, we thought it would be worthwhile to report our recent observation that acetic anhydride and HBr/HOAc can be used with advantage for the preparation of acetobromosugars.     

稻子、小麦麸皮中可溶性非淀粉多糖的气相色谱法测定

讨论稻子、小麦麸皮中可溶性非淀粉多糖的气相色谱法测定。将稻子、小麦麸皮磨细后,用８０％乙醇萃取游离糖,剩余物在醋酸缓冲液（ｐＨ５．０）条件下分别用α－淀粉酶和α－葡萄糖苷酶酶解,除掉淀粉。非淀粉多糖用酸水解为各单糖,采用糖醇衍生化成乙酸酯的制备方法,用ＯＶ－１７０１石英毛细管色谱柱,可分离得到非淀粉多糖（ＮＳＰ）各组成单糖的色谱峰。并以自然界中不存在的阿洛糖作内标物,提高了定性定量分析的准确性,计算出了稻子、小麦麸皮中可溶性非淀粉多糖的含量。     

TMG-chitotriomycin, an enzyme inhibitor specific for insect and fungal beta-N-acetylglucosaminidases, produced by actinomycete Streptomyces anulatus NBRC 13369

A novel beta-N-acetylglucosaminidase (GlcNAcase) inhibitor named TMG-chitotriomycin (1) was isolated from the culture filtrate of Streptomyces anulatus NBRC13369. The strain produced 1 only when colloidal chitin was used as the sole carbon source in the production medium. The structure of 1 was determined by spectral and constitutive sugar analyses of the corresponding alditol derivatives to be an equilibrated mixture of alpha-d-N,N,N-triMeGlcNH2-(1,4)-beta-d-GlcNAc-(1,4)-beta-d-GlcNAc-(1,4)-d-GlcNAc and its C-2 epimer of the reducing end residue. TMG-chitotriomycin (1) showed potent and selective inhibition of insect and fungal GlcNAcases with no inhibition of mammalian and plant GlcNAcases. In contrast, the known GlcNAcase inhibitor nagstatin potently inhibited all GlcNAcases. It should be emphasized that synthesized d-N,N,N-triMeGlcNH2, which is the component sugar of 1, showed no inhibition of the insect Spodoptera litura GlcNAcase. These results suggest that the (GlcNAc)3 unit positioned at the reducing end of 1 is essential for its enzyme inhibitory activity. The unique inhibitory spectrum of 1 will be useful to study chitinolytic systems and to develop selective fungicides or pesticides.     

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry and their trace detection in complex matrices by gas chromatography-tandem mass spectrometry.

Bacterial cellular polysaccharides are composed of a variety of sugar monomers. These sugars serve as chemical markers to identify specific species or genera or to determine their physiological status. Some of these markers can also be used for trace detection of bacteria or their constituents in complex clinical or environmental matrices. Analyses are performed, in our hands, employing hydrolysis followed by the alditol acetate derivatization procedure. Substantial improvements have been made to sample preparation including simplification and computer-controlled automation. For characterization of whole cell bacterial hydrolysates, sugars are analyzed by gas chromatography-mass spectrometry (GC-MS). Simple chromatograms are generated using selected ion monitoring (SIM). Using total ion GC-MS, sugars can be readily identified. In more complex clinical and environmental samples, markers for bacteria are present at sufficiently low concentrations that more advanced instrumentation, gas chromatography-tandem mass spectrometry (GC-MS-MS), is preferred for optimal analysis. Using multiple reaction monitoring, MS-MS is used (replacing more conventional SIM) to ignore extraneous chromatographic peaks. Triple quadrupole and ion trap GC-MS-MS instruments have both been used successfully. Absolute chemical identification of sugar markers at trace levels is achieved, using MS-MS, by the product spectrum.     

Sequencing of oligosaccharides by collision-induced dissociation matrix-assisted laser desorption/ionization mass spectrometry

A study of the collision-induced dissociation post-source decay (PSD) spectra of free oligosaccharides is presented. These spectra, when obtained with helium as collision gas, show (1,5)X fragments containing the reducing end sugar. The presence of these fragments permits Y ions and, consequently, B and C peaks to be identified. This is a common behaviour from which it has been possible to delineate a general method for the easy assignment of the peaks in PSD spectra of underivatized neutral sugars, allowing the sequence of a real unknown to be obtained.     

Synthesis of the O-linked hexasaccharide containing β-D-Galf-(1→2)-β-D-Galf in Trypanosoma cruzi mucins

The hexasaccharide β-D-Galp-(1→2)-[β-D-Galp-(1→3)]-β-D-Galp-(1→6)-[β-D-Galf(1→2)-β-D-Galf(1→4)]-D-GlcNAc (1) is the largest carbohydrate structure released as alditol by reductive β-elimination from mucins of some strains of T. cruzi. The terminal β-D-Galp units are sites of sialylation by trans-sialidase which transfers sialic acid from the host to the parasite. Hexasaccharide 1 was synthesized by a [3 + 3]-convergent strategy based on a nitrile assisted glycosylation, using the trichloroacetimidate method. The β-D-Galf-(1→2)-β-D-Galf-D-GlcNAc synthon was sequentially constructed from the reducing end to the non-reducing end employing benzyl α-D-galactofuranoside as starting material for the internal Galf unit. The choice of this novel precursor, obtained in one-reaction step from galactose, allowed the introduction of an orthogonal and participating levulinoyl group at O-2. Thus, the diastereoselective construction of the Galf-β(1→4)-GlcNAc linkage by the trichloroacetimidate method of glycosylation was achieved. The (1)H NMR spectrum of alditol 2 was identical to the product released by β-elimination from the parasite mucin.     

Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring

Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.     

Production and Characterization of the Exopolysaccharides Produced by Agaricus brasiliensis in Submerged Fermentation

The aim of the work was to study the production of the exopolysaccharides by Agaricus brasiliensis and the isolation of exopolysaccharides (EPSs) with biological effects. A brasiliensis LPB03 was cultured in submerged fermentation in a medium containing glucose, yeast extract, hydrolyzed soybean protein, and salts (pH 6.1) at 29 degrees C and 120 rpm for 144 h. The maximum biomass and EPS yield was 7.80 +/- 0.01 and 1,430.70 +/- 26.75 mg/L, respectively. To isolate the produced EPSs, two methods were compared: (1) with alcohol precipitation and (2) treatment with tricloroacetic acid (TCA), followed by alcohol precipitation. The use of TCA facilitated the purification of the EPS, reducing the amount of the contaminant soy proteins. For monosaccharide identification, the EPSs were hydrolyzed, derivatized to alditol acetates, and analyzed by gas chromatography (GC) and GC-mass spectrometry, which showed the presence (in molar percentage) of mannose (58.7), galactose (21.4), and glucose (13.1) as major sugars, with lower amounts of rhamnose (3.9) and xylose (2.8). Scanning electron microscopy was used to observe the morphological structure of the EPS. The experiments in vivo including EPS in the mice diet during 8 weeks indicated the hipocholesteremic and hypoglycemic effects.     

Discoloration of cellulose solutions in <Emphasis Type="Italic">N</Emphasis>-methylmorpholine-<Emphasis Type="Italic">N</Emphasis>-oxide (Lyocell). Part 1: Studies on model compounds and pulps

N-Methylmorpholine-N-oxide monohydrate (NMMO) is used as solvent for cellulose in the Lyocell process as a modern industrial fiber-making technology. Undesired chemical side reactions and byproduct formation in the system cellulose/NMMO/water are known to cause detrimental effects, such as chromophore formation and discoloration of the resulting fibers. A detailed kinetic study on the influence of carbonyl structures on chromophore formation in NMMO melts was carried out employing UV spectroscopy. Different sugar model compounds, such as reducing or non-reducing sugars, and sugars with additional oxidized functions, were applied. The chromophore formation rate differed widely for various reducing sugar model compounds, with pentoses generally reacting faster than hexoses, and carbohydrates with protected reducing end being largely inert. The effect of carbonyl groups on chromophore generation has been studied further using oligomers and oxidized pulps with different contents of carbonyl groups. As in the case of model compounds, also for the pulps a linear correlation between carbonyl content and chromophore formation rate was established. A distinct effect of hemicelluloses was observed.     

Trace analysis of sugars by HPLC and post-column derivatization

Summary A HPLC system with post-column derivatization for the quantitative analysis of sugars in complex matrices is described. As reagent a 0.2% solution of thymol in concentrated sulfuric acid has been used. The reaction is sensitive with reducing as well as non reducing sugars whereas sugar alcohols are discriminated. With this reagent and separation of sugars at high pH values with an anion exchange column it is possible to detect sugars in the ng range. Hence, it is possible to characterize wines not only by their fructose and glucose content but also by differences in the distribution of the other not fermentable sugars like trehalose, arabinose, galacturonic and glucuronic acid.     

衍生化毛细管气相色谱法分离测定山梨醇和甘露醇

采用乙酸酐为衍生化试剂,吡啶为溶剂和催化剂,将山梨醇和甘露醇转化成易恽发的糖醇乙酰酯衍生物。用苯基氰丙基聚硅氧烷（BPX-70）毛细管色谱柱分离分析,使同分异构体的六元糖醇得到了很好的分离,并用内标法定量。山梨醇甘露醇的线性范围均为2.50g/L-12.5g/L,检测限分别为0.60ng和0.68ng。     

A Convenient,Highly Efficient One-Pot Preparation of Peracetylated Glycals From Reducing Sugars

ABSTRACT

A convenient, highly efficient, one-pot, three-step procedure has been developed for the synthesis of peracetylated glycal derivatives from various reducing sugars including D-glucose, D-galactose, L-rhamnose, L-arabinose, D-maltose, D-lactose, and maltotriose. This procedure involves peracetylation of the reducing sugars with acetic anhydride and HBr/acetic acid followed by the transformation of the anomeric acetates to the corresponding bromides with additional HBr/acetic acid and finally reductive elimination of the 1-bromo and 2-acetoxy groups with Zn/CuSO₄·5H₂O in acetic acid/water containing sodium acetate. The overall yields of purified peracetylated glycals from the corresponding sugars range from 50 - 98%.