Derivatization Of Sugars Research Articles

Monosaccharide composition of lignocellulosic matrix—Optimization of microwave-assisted acid hydrolysis condition

Compositional analysis of polysaccharides is crucial in many applications of analytical chemistry, and typically involves hydrolysis as initial step, to break glycosidic bonds and release individual monosaccharide units. We developed a new method for the microwave-assisted acid hydrolysis of polysaccharides, here applied for determining the composition of the polysaccharide fraction within the lignocellulosic matrix of Pinus sylvestris. The optimisation of reaction conditions was carried out using a GC–MS method after a double-step sugar derivatization process.Under the optimized conditions, specifically, employing 2 M TFA, 40 min, and 120 °C, a high depolymerisation yield was achieved with minimal degradation. Furthermore, the amount of polysaccharides detected in pine wood aligns with the results in existing literature, substantiating the determined chemical composition.The synergy of microwave-assisted acid hydrolysis and full factorial design not only facilitated the determination of the optimal hydrolysis conditions but also enabled the assessment of the parameters influencing the polysaccharide hydrolysis process and their relationships. This approach made it feasible to achieve maximum hydrolysis efficiency while minimizing analyte degradation, employing a method that reduces environmental impact through the use of microwave energy. This strategy effectively reduced the required reaction temperature, time, and reagent quantities.

Polyacetylenes with xanthine oxidase inhibitory activity from the medicinal and edible fruits of Cyclocodon lancifolius (Roxburgh) Kurz

Five new polyacetylene derivatives (1–5), cyclocodonlandiynosides A–E, and eight known analogues (6–13) were isolated and identified from the fruits of Cyclocodon lancifolius. Their structures were established via spectroscopic and chemical methods, including NMR, HRESIMS, enzymatic hydrolysis, Mo2(OAc)4-induced circular dichroism and sugar derivatization. Compound 1 contains a nitrogenous fragment, which was rarely found in C14 polyacetylenes. Compounds 3 and 4 are polyacetylene glucosides possessing novel aglycones. All the isolated polyacetylenes (except 12) were screened for their xanthine oxidase (XO) inhibitory activity. All the tested compounds, at the concentration of 62.5 μg/mL, showed XO inhibiting effects. Among them, 13 and 3 showed the most potent XO inhibitory activity with IC50 values of 87.65 and 96.32 μM, compared to the positive control allopurinol with an IC50 value of 19.25 μM.

Derivatization of sugars with N,O-dimethylhydroxylamine. Efficient RP-HPLC separation of sugar mixtures

Sugars were derivatized with N,O-dimethylhydroxylamine (DMHA) using a simple procedure. The disaccharides lactose and chitobiose and the human milk tetrasaccharides lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) were used as examples. The β-glycosylamines were formed exclusively in good yields (80–84%). The derivatives were very well suited for RP-HPLC, giving rise to single peaks for each sugar, without the usual complications caused by mutarotation. The LNT- and LNnT-derivatives separated very well on an ordinary RP-HPLC column, despite their close structural similarity. Also, three human milk pentasaccharides (LNF I, II and III) were derivatized with DMHA. Again, good separation of these isomers was obtained. The DMHA derivatization was easily reversed. The free oligosaccharides were recovered quantitatively by mild acidic hydrolysis. To demonstrate usefulness on a preparative scale, an LNDI-rich human milk oligosaccharide fraction was derivatized, and three HPLC fractions (one major and two minor) were collected. Hydrolysis and desalting gave saccharides LNDI, LNnDII, and LNDII, the latter mixed with minor amounts of LNnDI.

Development and Validation of HPLC-DAD Method with Pre-Column PMP Derivatization for Monomeric Profile Analysis of Polysaccharides from Agro-Industrial Wastes.

The instrumental analysis of complex mixtures of sugars often requires derivatization to enhance the method’s selectivity and sensitivity. 1-Phenyl-3-methyl-5-pyrazolone (PMP) is a common sugar derivatization agent used in high-performance liquid chromatography (HPLC). Although many C18 column applications for PMP–sugar derivative analysis have been developed, their transferability is not straightforward due to variations in column chemistry and preparation technology. The aim of this study was to develop and validate an application for Zorbax Extend C18 columns for the analysis of 8 neutral and 2 acidic sugars commonly found in plant polysaccharides. The method was further compared to well-established alditol acetates and m-hydroxydiphenyl methods and employed for sugar profiling of selected agro-industrial wastes. The most influential separation factors were the mobile-phase pH and acetonitrile content, optimized at 8.0 and a 12–17% gradient, respectively. The method showed excellent linearity, repeatability and intermediate precision. High sensitivity was achieved, especially for neutral sugars, with an accuracy error range of 5–10% relative standard deviation. The sugar profiling results were highly correlated to the reference method for neutral sugars. The HPLC method was highly applicable for the evaluation of polysaccharides in selected wastes and showed advantages in terms of simplicity, accuracy in acidic sugar determination and suitability for their simultaneous analysis with neutral sugars.

Reversible derivatization of sugars with carbobenzyloxy groups and use of the derivatives in solution-phase enzymatic oligosaccharide synthesis

Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.

Regioselective Synthesis of Palm‐Based Sorbitol Esters as Biobased Surfactant by Lipase from Thermomyces lanuginosus in Nonaqueous Media

AbstractThis paper describes the regioselective production of palm‐based sorbitol monoesters via esterification catalyzed by Lipozyme® TL IM (Thermomyces lanuginosus lipase adsorbed onto silica gel, Novozymes, Inc., Franklington, NC, USA). Effects of various reaction parameters including types of solvent, substrate molar ratio, molecular sieve and lipase concentration, temperature, reaction time, and fatty acid chain length were investigated. Approximately 76% conversion of sorbitol to sorbitol esters was achieved within 24 h under optimal conditions: sorbitol (0.4 M), fatty acid (0.8 M), 20 wt% Lipozyme® TL IM in 100 mL tert‐butanol at 55 °C for 24 h in the presence of 25 wt% 3 Å molecular sieve as water absorbent. The reactions were conducted in an orbital incubator shaker at a shaking rate of 200 rpm. Lipozyme® TL IM was highly regioselective, esterifying exclusively at sorbitol's primary hydroxyl groups, producing 1‐O‐ and 6‐O‐sorbitol monoesters. The biocatalyst also exhibited substrate selectivity toward shorter chain acyl donors, with caprylic acid exhibiting the highest conversion of sorbitol. In addition, Lipozyme® TL IM was reused up to four successive reaction cycles without significant loss of activity. The biocatalytic process reported in this paper is a one‐step process to produce biobased surfactants that does not involve the use of toxic or expensive solvents that are commonly employed for derivatization of sugars, or pre‐derivatization of the substrates molecules.

Neuroprotective Glycosylated Cyclic Lipodepsipeptides, Colletotrichamides A-E, from a Halophyte-Associated Fungus, Colletotrichum gloeosporioides JS419.

Glutamate neurotoxicity has been implicated in neuronal death in both acute CNS injury and in chronic neurodegenerative diseases. Five unique cyclic depsipeptides with neuroprotective activity, colletotrichamides A-E (1-5), were isolated from cultures of a halophyte Suaeda japonica-associated fungus, Colletotrichum gloeosporioides JS419. Spectroscopic analysis revealed that they were glycosylated cyclic lipodepsipeptides. Their relative configurations were determined by ROESY and J-based configuration analysis, and absolute configurations were established by chemical reactions including modified Mosher's method, advanced Marfey's method, and sugar derivatization. This is the first report of a glycosylated dimethyl-trioxygenated dodecanoyl moiety, and the relative as well as absolute stereochemistry was elucidated herein for the first time. Colletotrichamide C exhibited strong neuroprotective activity against glutamate in hippocampal HT22 cells.

Investigation of a side reaction occurring during N-linked glycan labeling by cationic tags

Capillary electrophoresis–mass spectrometry was applied for the analysis of oligosaccharides and N-linked glycans with an attached charge label facilitating electrophoretic migration and electrospray ionization efficiency. Several different labeling strategies have been tested with different tags and tagging reactions including reductive amination and hydrazone formation. However, a formation of multiple labeled N-linked glycans was observed by CE-MS in a positive ion mode when positively charged labels such as aliphatic amines containing a quaternary ammonium group were attached to N-linked glycans by reductive amination. A reaction mechanism explaining a side reaction occurring during the labeling and the multiple product formation was proposed and confirmed by using isotopically labeled N-acetylglucosamine. Finally, it was confirmed that derivatization of sugars via a hydrazone formation can be a simpler method with a high reaction yield suitable for high sensitive CE-ESI/MS analyses of N-linked glycans.

Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity

This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the β-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H2O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (KM; Vmax) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars.

Elaboration of Glycopolymer-Functionalized Micelles from anN-Vinylpyrrolidone/Lactide-Based Reactive Copolymer Platform

Glycopolymer-corona-based micelles are obtained in a one-pot procedure, through reaction of D-mannosamine or D-glucosamine with the N-succinimidyl (NS) esters of a poly(D,L-lactide)-block-poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (PLA-b-P(NAS-co-NVP)) amphiphilic copolymer (presenting quasi-alternating NAS/NVP units) in dimethyl sulfoxide (DMSO), followed by nanoprecipitation and dialysis against water. The glycopolymer micelles exhibit a higher CMC and size than those obtained from unmodified copolymer, due to increased hydrophilicity of the external block as a result of sugar derivatization, and the availability of the sugars at the micelle surface is evidenced through interactions with Concanavalin A (Con A) lectin, as attested by turbidimetric measurements and enzyme-linked lectin assay (ELLA). Interestingly, the glycopolymer micelles can be further used for hydrophobic molecule encapsulation and release, as shown with imiquimod, while keeping their interactions with con A intact. It is concluded that the PLA-based amphiphilic/reactive copolymer represents a versatile platform for glycopolymer-based micelle constructs for drug/vaccine delivery.

Monitoring changes in the chemical composition of dissolvable tobacco products

Dissolvable tobacco products are smokeless, spit-free, made from finely milled tobacco, and dissolve in the mouth of a user. In 2009, R.J. Reynolds Tobacco Co. released a line of dissolvable tobacco products in several test markets. In late 2010, the dissolvables were reformulated and several changes in their composition were determined and are reported here. Our analytical approach featured gas chromatography-mass spectrometry preceded by four separate methods for sample preparation: solvent extraction using acetone, trimethylsilyl derivatization of sugars, solid-phase microextraction of semi-volatiles and liquid–liquid extraction for the quantitative analysis of nicotine. Taken together, these methods identified several changes in the formulation of the initial products and their reformulated versions. One major change in the new dissolvables is that “mellow” and “fresh” flavors have been replaced by a “mint” flavor. Additionally, the strips are no longer dominated by glycerol, but rather threitol. Total nicotine and (free nicotine) concentrations for the orbs, sticks, and strips were found to be 3.35 mg g−1 (1.242 mg g−1), 2.6 mg g−1 (0.850 mg g−1), and 2.74 mg g−1 (0.572 mg g−1), respectively. Total nicotine and free nicotine concentrations have significantly increased in some cases.

Three New Iridoid Glycosides from the Fruit of <i>Gardenia jasminoides</i> var. <i>radicans</i>

Three new iridoid glycosides, 6″-O-trans-feruloylgenipin gentiobioside (1), 2'-O-trans-p-coumaroylgardoside (2), 2'-O-trans-feruloylgardoside (3), were isolated from the fruit of Gardenia jasminoides var. radicans MAKINO (Rubiaceae). The structures of these compounds were elucidated on the basis of MS, NMR spectra analysis, glycoside hydrolysis, and sugar derivatization coupled with HPLC analysis.

A spectrophotometric method for quantitative determination of xylose in fermentation medium

Abstract Monitoring the consumption of sugars during fermentation is a key to optimizing product formation and maintaining a healthy environment for microorganisms. Difficulty arises in the availability of a rapid, inexpensive and sensitive method for the detection of sugars because fermentation media are a complex mix of nutrients, cell debris, waste and target products. A method involving reaction-based UV–Vis spectrophotometry for the quantitative determination of xylose as the target sugar was developed. Factors affecting xylose concentration measurements such as hydrochloric acid concentration, heating time and the amount of Fe 3+ catalyst were investigated. A continuous scan revealed the working wavelength to be 671 nm. The effect of other components in the fermentation broth was found to be negligible. Absorbance shows a linear relationship with xylose concentration within a range of 0.1–0.5 g/L. Xylose concentrations from fermentation samples obtained at specific time intervals (0–168 h) were determined with the method and compared with YSI 2700, an enzyme electrode, HPLC-ELSD method, currently a common technique for measuring xylose and GC aldononitrile sugar derivatization method. Dilution is necessary for comparable xylose concentrations with YSI 2700 and HPLC-ELSD. Xylose concentration measurements obtained with the UV–Vis spectrophotometric method although quantitatively comparable to HPLC-ELSD xylose measurements were easily and conveniently obtained compared to YSI 2700, HPLC-ELSD and GC derivatization methods.

Separation and determination of sugars by reversed-phase high-performance liquid chromatography after pre-column microwave-assisted derivatization

In this study the possibility of derivatizing sugars using microwave irradiation was investigated. The amount of reagent, irradiation intensity, and derivatization time were optimized. In the derivatization of sugars with p-nitroaniline the reaction is complete within 5 min at 600 W when the p-nitroaniline-to-sugar and NaBH3CN-to-sugar mole ratios were above 1.4 and 3.1, respectively. A Doehlert design was used to optimize the mobile phase for separation of p-nitroaniline-labeled sugars; and the best separation was obtained by use of 0.01 mol L-1 acetate buffer at pH 4.40 containing 11.0% acetonitrile. Analysis using this method was highly sensitive and analysis time was short. Finally, a food sample was analyzed using the proposed method.

Engineered Urdamycin Glycosyltransferases Are Broadened and Altered in Substrate Specificity

Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.

Amino acid and amino sugar determination by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate followed by high-performance liquid chromatography and fluorescence detection

A method for the derivatization of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for use with HPLC was tested. The reproducibility, linearity and limits of detection are similar to those obtained with other methods, such as the o-phthalaldehyde and 9-fluorenylmethoxycarbonyl methods. The ease of sample preparation and the possibility of amino sugar derivatization and resolution are the reasons why this method was chosen for simultaneous amino acid and amino sugar analysis. The need for a previous step of amino sugar reduction was studied and it was concluded that it is not necessary.

Effects of nonenzymatic glycation of subfragment-1 of myosin on interactions with actin.

Nonenzymatic bonding of reducing sugars to subfragment-1 of myosin (S-1) resulted in a reduction in actin-activated S-1 ATPase activity. Fructose caused a greater reduction than glucose. The Km for binding of actin to S-1 was significantly increased with sugar derivatization. In addition, sugar derivatization lowered the ability of S-1 to promote polymerization of G-actin. Western blot analysis demonstrated that glucose was nonenzymatically incorporated into the 50 and 20 kilodalton (kDa) fragments of S-1 with preponderance in the 20-kDa fragment. The reduced affinity of derivatized myosin for actin is indicated by the increased Km, the reduced ability to stimulate actin polymerization, and the positive Western blot reaction in the 20-kDa fragment.