TEMPO-mediated oxidation of cellulose III

Various cellulose samples converted into cellulose III by two different ammonia treatments, either liquid or gaseous, were reacted with catalytic amounts of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO), sodium hypochlorite, and sodium bromide in water. A substantial increase in the reactivity of cellulose III samples was observed in comparison to those in cellulose I, and a relationship between oxidation conditions and cellulose primary hydroxyl groups accessibility was directly established. For the characterization, we have used several methods, mainly (13)C NMR, methylene blue adsorption, FTIR, and conductometric titration. In all samples, the primary alcohol groups were selectively oxidized into carboxyl groups, provided the sodium hypochlorite is added dropwise and the reaction is performed at constant pH 10.     

Chlorination and oxidation of heparin and hyaluronan by hypochlorous acid and hypochlorite anions: effect of sulfate groups on reaction pathways and kinetics

Hypochlorous acid (HOCl), produced in inflammatory conditions by the enzyme myeloperoxidase, and its anion hypochlorite (OCl⁻) exist in vivo at almost equal concentrations. Their reactions with hyaluronan and heparin (as a model for sulfated glycosaminoglycans in the extracellular matrix) have been studied as a function of pH. The major product in these reactions is the chloramide derivative of the glycosaminoglycans. Spectral, chloramide yield, and kinetic measurements show sharply contrasting behavior of heparin and hyaluronan and the data allow the calculation of second-order rate constants for the reactions of both HOCl and OCl⁻ for all reaction pathways leading to the formation of chloramides and also oxidation products. By comparison with hyaluronan, it can be demonstrated that both N-sulfate and O-sulfate groups in heparin influence the proportions of these pathways in this glycosaminoglycan. Evidence is also given for further oxidation pathways involving a reaction of HOCl with the chloramide product of hyaluronan but not with heparin. The significance of these results for the mechanisms of inflammation, particularly for fragmentation of extracellular matrix glycosaminoglycans, is discussed.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.     

Chitin nanocrystals prepared by TEMPO-mediated oxidation of alpha-chitin

Chitin nanocrystals dispersed in water were successfully prepared by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) mediated oxidation of alpha-chitin in water at pH 10 under specific conditions, followed by ultrasonic treatment. When the amount of NaClO added as co-oxidant in the oxidation was 5.0 mmol/g of chitin, the weight percentage of the water-insoluble fraction in the TEMPO-oxidized chitin was 90%, and its carboxylate content reached 0.48 mmol/g. Since the TEMPO-oxidized chitin thus prepared had a crystallinity as high as that of the original alpha-chitin, the C6 carboxylate groups formed by TEMPO-mediated oxidation can be regarded as being present only on the chitin crystallite surfaces. No N-deacetylation occurred on the TEMPO-oxidized chitins. When the TEMPO-oxidized chitin was subjected to ultrasonic treatment in water, mostly individualized chitin nanocrystals were obtained, and the average nanocrystal length and width were 340 and 8 nm, respectively.     

Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose

Never-dried and once-dried hardwood celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the celluloses reached approximately 1.5 mmol/g, the oxidized cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized cellulose nanofibers 3-4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with those in the dispersed states of the cellulose nanofibers in water.     

Effect of temperature modulations on TEMPO-mediated regioselective oxidation of unprotected carbohydrates and nucleosides

Regioselective oxidation of unprotected and partially protected oligosaccharides is a much sought-after goal. Herein, we report a notable improvement in the efficiency of TEMPO-catalyzed oxidation by modulating the temperature of the reaction. Mono-, di-, and tri-saccharides are oxidized regioselectively in yields of 75 to 92%. The present method is simple to implement and is also applicable for selective oxidations of other mono- and poly-hydroxy compounds including unprotected and partially protected nucleosides.     

TEMPO-mediated oxidation of native cellulose. The effect of oxidation conditions on chemical and crystal structures of the water-insoluble fractions

Cellulose cotton linter was oxidized with sodium hypochlorite with catalytic amounts of sodium bromide and 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) under various conditions. After this TEMPO-mediated oxidation, water-insoluble fractions were collected and characterized in terms of carboxylate and aldehyde contents, crystallinities and crystal sizes, degrees of polymerization, morphology, and water retention values. Carboxylate and aldehyde groups were introduced into the water-insoluble fractions up to about 0.7 and 0.3 mmol/g, respectively, by the oxidation, where recovery of the water-insoluble fractions were generally higher than 80%. Crystallinities and crystal sizes of cellulose I were nearly unchanged during the oxidation, and thus, carboxylate and aldehyde groups were introduced selectively on crystal surfaces and in disordered regions of the water-insoluble fractions. Water retention values of cotton linter can be increased from 60% to about 280% through the introduction of hydrophilic carboxylate groups and morphological changes from fibrous forms to short fragments by the TEMPO-mediated oxidation.     

Nonisothermal denaturation kinetics of human hair and the effects of oxidation

Human hair as alpha-keratin fiber exhibits a complex morphology, which for the context of this investigation is considered as a filament/matrix-composite, comprising the intermediate filaments (IF) and a variety of amorphous protein components as matrix. Differential scanning calorimetry (DSC) under aqueous conditions was used to analyze the denaturation of the alpha-helical material in the IFs and to assess the changes imparted by repeated, oxidative bleaching processes. The DSC curves were submitted to kinetic analysis by applying the Friedman method and assuming first order kinetics. It was found that the course of the denaturation process remains largely unchanged through oxidation, despite the fact that pronounced decreases of denaturation temperature as well as of enthalpy occur. In parallel, the reaction rate constant at the denaturation temperature, k(TD), increases with repeated treatments, that is with cumulative chemical modification. However, this effect is in fact small compared to the overall change of k(T) through the denaturation process. This leads to conclude that once the temperature rise in combination with the chemical change has induced a suitable drop of the viscosity of the matrix around the IFs, denaturation of the remaining helical material occurs along a pathway that is largely independent of temperature and of the pretreatment history. This emphasizes the kinetic control of the matrix over the denaturation process of the helical segments in the filament/matrix composite.     

Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous slurries of cotton linters. The water-insoluble fibrous fractions thus obtained in the yields of more than 78% were characterized by solid-state ¹³C-NMR, X-ray diffraction and scanning electron microscopic analyses for evaluation of distribution of carboxylate groups formed in the TEMPO-oxidized celluloses. The patterns of solid-state ¹³C-NMR spectra revealed that the oxidation occurred at the C6 primary hydroxyl groups of cellulose. X-ray diffraction and scanning electron microscopic analyses showed that such C6 oxidation took place at the surfaces of cellulose I crystallites without any oxidation at the C6 of inside cellulose I crystallites. Thus, carboxylate and aldehyde groups introduced into the TEMPO-oxidized celluloses are densely present on the surfaces of cellulose I crystallites. In addition, the obtained results revealed that the shoulder signal due to non-crystalline C6 carbons at about 63 ppm in solid-state ¹³C-NMR spectra of native celluloses is ascribed to those of surfaces of cellulose I crystallites or those of cellulose microfibrils.     

The solvent effects on the kinetics of bacterial formate dehydrogenase reaction

The increase in concentration of organic cosolvents results in a 2-2.5-fold increase of the maximal reaction rate and a decrease of Michaelis constant for formate of NAD(+)-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. These parameters, however, are not affected with the increase of ionic strength. For the logarithm of both Vmax and Km a linear function of the reciprocal of solvent dielectric permittivity was found. The decrease of Km is possibly due to the dielectric screening effect on the substrate binding energy. The increase in Vmax is explained by a model based on a solvent-dependent electrostatic image force, acting on the charges moved in the course of the catalytic step of the enzyme reaction.     

Bromide-free TEMPO-mediated oxidation of primary alcohol groups in starch and methyl alpha-D-glucopyranoside

TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl)-mediated oxidation of potato starch and methyl alpha-D-glucopyranoside (MGP) was performed in the absence of sodium bromide (NaBr) as co-catalyst, solely using sodium hypochlorite (NaOCl) as the primary oxidant. The low reaction rate associated with a bromide-free process was increased by performing the oxidation at increased temperatures. The reaction proceeded stoichiometrically and with high selectivity and with only minor depolymerisation, provided that temperature and pH were kept < or = 20 degrees C and < 9.0, respectively. At 20 degrees C and pH 8.5, the reaction rate was comparable to that of a corresponding oxidation catalysed by NaBr at 2 degrees C. Consequently, this is a simple approach to raise the TEMPO/NaOCl reaction rate under bromide-free conditions while still maintaining good product properties. At higher oxidation temperatures (> or = 25 degrees C) and under more alkaline conditions (pH > or = 9.0) degradation of the starch skeleton occurred. Simultaneously, side-reactions of the nitrosonium ion lowered the yield of the oxidation. Despite the absence of the NaBr catalyst, the reaction rate-controlling step was found to be the oxidation of the primary hydroxyl groups with the nitrosonium ion. The reaction was first-order in MGP and in TEMPO.     

TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

Assignment of charge movements to electrogenic reaction steps of Na,K-ATPase by analysis of salt effects on the kinetics of charge movements

Na,K-ATPase-enriched membrane fragments adsorbed to lipid bilayers were used to study electrogenic Na+ movements induced by enzyme phosphorylation when ATP was photo-released from inactive caged ATP, and simultaneously by externally applied alternating voltages which allowed the measurement of small ATP-induced membrane admittance changes. A detailed analysis of frequency dependence of the capacitance and conductance increments showed that the observed process consists of more than one electrogenic step. The frequency dependence could be described by the sum of two Lorentzian functions and a constant term. The faster process (approximately 2000 s(-1)) was assigned to the release of the first extracellular Na+ ion. The corner frequency of the slower Lorentzian (about 30 s(-1)) coincided with the reciprocal exponential time constant of the falling phase of the transient current, which can be assigned to the conformational transition. Preferentially, the slower process showed a dependence on the ion concentration of choline salts with different anions. The effectiveness of the used chaotropic anions to decelerate the kinetics decreased in agreement with the Hofmeister series, I- > Br- > Cl-. This observation matches their effect on the partition between two phosphoenzyme states of the Na,K-ATPase, as established previously.     

TEMPO-mediated oxidation of maltodextrins and D-glucose: effect of pH on the selectivity and sequestering ability of the resulting polycarboxylates

Maltodextrins were oxidized to polyglucuronic acids with the ternary oxidation system: NaOCl-NaBr-2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). The chemoselective oxidation at the primary alcohol groups was shown to be strongly pH dependent. Oxidation of polysaccharides was best achieved at pH 9.5 in order to minimize depolymerization, whereas oxidation of oligosaccharides required stronger alkaline conditions (pH 11-11.5). The resulting sodium polyglucuronates present interesting sequestering properties, the best of which being obtained from maltodextrins with the highest degrees of polymerization. The same oxidation process allowed the convenient conversion of D-glucose to D-glucaric acid in high yield (> 90%), under strongly basic conditions (pH > 11.5).     

Viscoelastic evaluation of average length of cellulose nanofibers prepared by TEMPO-mediated oxidation

Dynamic viscoelasticity measurements were performed for aqueous dispersions of cellulose nanofibers prepared by TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation and subsequent mechanical disintegration in water. The frequency dependence of the storage and loss moduli of 0.02% (w/v) dispersions of TEMPO-oxidized cellulose nanofibers in water showed terminal relaxation behavior at relatively lower angular frequencies. This strongly suggests that each cellulose nanofiber in the dispersion behaves as a semiflexible rod-like macromolecular chain or colloidal particle. Furthermore, a clear boundary was observed between the terminal relaxation and rubbery plateau regions. The longest viscoelastic relaxation time, τ, was estimated from the angular frequency, corresponding to the boundary point, and the average length of the cellulose nanofibers, L, was estimated using the equation τ = πη(s)L(3)/[18k(B)T ln(L/d)]. The equation gave a value of L = 2.2 μm, which was in good agreement with TEM observations.     

Quantitative analysis of the effects of hyaluronan and aggrecan concentration and hyaluronan size on the elasticity of hyaluronan-aggrecan solutions

We determined elasticity (G') and viscosity (G') of various aggrecan-hyaluronan solutions using a controlled-stress rheometer with high (10 Hz) to low (0.1 Hz) frequencies. Aggrecan solution (50 mg/ml) alone showed little elasticity at any frequency, but the addition of 3300 kDa hyaluronan at 0.001-0.1 mg/ml markedly increased the elasticity, but not the viscosity, at all frequencies. Increasing hyaluronan concentration at >0.1 mg/ml did not further increase the elasticity of the aggrecan solution, and the elasticity of the aggrecan-hyaluronan complex solution reached a plateau at a 500:1 (w/w) ratio. In studies with increasing concentrations of aggrecan and a constant concentration (0.5 mg/ml) of 3300 kDa hyaluronan, aggrecan induced elasticity only at >20 mg/ml, indicating the presence of a critical concentration for elasticity. In the presence of 50 mg/ml aggrecan, 1000 kDa hyaluronan had far less effect on the elasticity of the aggrecan solution than did 3300 kDa hyaluronan. These findings suggest that only approximately 50% reduction in aggrecan concentration (<20 mg/ml), or reduced hyaluronan size (<1000 kDa)--compared with their physiological levels in young cartilage--can abolish the elastic network of the aggrecan-hyaluronan complex.     

透明质素的标准化研究

透明质素(Hyaluronan)是一种被广泛应用于临床医学领域的粘多糖类物质。有关它的药用标准相继出台。本文引用了欧洲药典2002版以及删拟定的草案对透明质酸钠制定的药用标准。同时提出了建立我国的相应标准所需的检测项目和期望采用的测试手段,为我国开展相关研究提供可供参考的工作平台。     

Enzymatic oxidation of gallocatechin and epigallocatechin: effects of C-ring configuration on the reaction products

Tea leaf is rich in pyrogallol-type catechins, and their oxidation is important in the generation of black tea polyphenols. In the present study, the enzymatic oxidation of three pyrogallol-type catechins, (+)- and (−)-gallocatechins and (−)-epigallocatechin, was compared. The reactions yielded unstable quinone products, which were trapped as condensation products with o-phenylenediamine. The oxidation of (+)-gallocatechin proceeded very slowly compared to the reaction of (−)-epigallocatechin, and yielded a proepitheaflagallin-type dimer as the major product, though oxidation of (−)-epigallocatechin gave predominantly dehydrotheasinensin C. The cis-configuration of the C-3 hydroxyl group and the B-ring of (−)-epigallocatechin was apparently crucial for rapid and selective production of dehydrotheasinensin C. Oxidation of (−)-gallocatechin proceeded in a manner similar to that of (+)-gallocatechin, and produced an enantiomer of the (+)-gallocatechin product. The results suggest that enzymes catalyze oxidation of the pyrogallol B-ring to the o-quinone, with subsequent non-enzymatic coupling reactions proceed under highly steric control.     

Modulation of vesicle adhesion and spreading kinetics by hyaluronan cushions

The adhesion of giant unilamellar phospholipid vesicles to planar substrates coated with extracellular matrix mimetic cushions of hyaluronan is studied using quantitative reflection interference contrast microscopy. The absolute height of the vesicle membrane at the vicinity of the substrate is measured by considering, for the first time, the refractive indices of the reflecting media. The thickness of the cushion is varied in the range of approximately 50-100 nm, by designing various coupling strategies. On bare protein-coated substrates, the vesicles spread fast (0.5 s) and form a uniform adhesion disk, with the average membrane height approximately 4 nm. On thick hyaluronan cushions (>80 nm), the membrane height is approximately the same as the thickness of the cushion, implying that the vesicle lies on top of the cushion. On a thin and inhomogeneous hyaluronan cushion, the adhesion is modified but not prevented. The spreading is slow ( approximately 20 s) compared to the no-cushion case. The average membrane height is approximately 10 nm and the adhesion disk is studded with blisterlike structures. Observations with fluorescent hyaluronan indicate that the polymer is compressed under, rather than expelled from, the adhesion disk. The adhesion energy density is approximately threefold higher in the no-cushion case (1.2 microJ/m(2)) as compared to the thin-cushion case (0.54 microJ/m(2)). In the thin-cushion case, the presence of short ( approximately 4 nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state--the membrane height ranges from zero to approximately 250 nm. The minimal model system presented here mimics in vitro the hyaluronan-modulated early stages of cell adhesion, and demonstrates that the presence of a polymer cushion influences both the final equilibrium adhesion-state and the spreading kinetics.