Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.     

TBAF-catalyzed deacylation of cellulose esters: Reaction scope and influence of reaction parameters

In order to expand its utility and understand how to carry it out most efficiently, the scope of the highly regioselective, tetrabutylammonium fluoride (TBAF) catalyzed deacylation of cellulose acetates has been investigated, including the influence of key process parameters: solvent, temperature, and water content. Reactions in DMSO, THF, MEK and acetone afforded similar extents of deacylation and regioselectivity. Reaction with TBAF in DMSO at 50 °C for 18 h was the most efficient process providing regioselective deacylation at O-2/3. All results were consistent with our previous mechanistic proposals. Furthermore, we demonstrate that TBAF-catalyzed deacylation is also effective and regioselective with cellulose acetate, butyrate, and hexanoate triesters, and even with a cellulose ester devoid of alpha protons, cellulose tribenzoate. These reactions displayed regioselectivity for deacylation at O-2/3 similar to that observed earlier with cellulose acetate (DS 2.4).     

Homogeneous esterification of cellulose in the lithium chloride-N,N-dimethylacetamide solvent system: effect of temperature and catalyst

Commercial rayon grade cellulose was dissolved in the lithium chloride-N,N-dimethylacetamide (LiCl-DMAc) solvent system and esterified with acetic anhydride using p-toluenesulfonyl chloride (p-TsCl) and pyridine as catalysts. The reaction temperature was varied from 28 to 70 degrees C and the time of reaction from 2 to 24 h. Full substitution took place at 60 and 70 degrees C at respective reaction times of 10 and 8 h for p-TsCl, and 10 and 6 h for pyridine. Esterification of cellulose followed a second-order reaction path. The rate constants at different reaction temperatures and the activation energy for the reaction are reported. Mechanisms for these reactions using the two catalysts are also suggested. The degrees of substitution (DS) of the esters prepared using both catalysts show that pyridine is a better catalyst than p-TsCl. Molecular weights of the esters, determined viscosimetrically, show that some degradation in the cellulose chain occurred at a reaction temperature of 70 degrees C. Hence, the optimum temperature for esterification appears to be 50-60 degrees C at 10 h reaction time to obtain full degree of acetyl substitution.     

几种硫酸化试剂和溶剂对菊糖硫酸化改性的影响

为制取硫酸化菊糖,以硫酸钡比浊法测定硫酸基取代度(DS)、红外光谱测定含硫基团的特征吸收峰、核磁共振碳谱(13C NMR)判断硫酸根取代位置等方法,比较了以N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)和吡啶(Py)三种溶剂,氯磺酸(CA)和三氧化硫(SO3)两种硫酸化试剂对菊糖硫酸酯化的影响.结果表明:以吡啶为溶剂、氯磺酸为硫酸化试剂的方法(CA-Py)与SO3-Py、CA-DMF三种硫酸化方法均获得了硫酸化菊糖,产品均显示不对称S=O键伸缩振动(约1255 cm-1)和对称的C-O-S键伸缩振动(约810 cm-1)特征吸收峰;三种方法的DS分别为:1.24,0.89,1.83;三种产品的13C NMR基本相同,均表明硫酸根连接在C3、C5、C6上.DMSO不适宜用作硫酸化溶剂.三种硫酸化方法是成功的,但以SO3-Py法操作简便,最适于菊糖硫酸化.     

Acetylation and characterization of xylan from hardwood kraft pulp

Alkaline treatment of eucalyptus hardwood kraft pulp with 10% NaOH yielded 6-8% xylan. The acetylation of the extracted xylan was carried in DMAC/LiCl/pyridine system to obtain a series of xylan acetates with different degrees of substitution (DS). Structure elucidation of xylan and xylan acetate was obtained by ¹H and ¹³C NMR spectroscopy and other homonuclear and heteronuclear 2D-NMR techniques. Inverse-gated ¹³C NMR was employed to determine the DS of xylan acetate. Furthermore, results also revealed equal reactivities at the C-2 and C-3 positions of xylan towards acetylation. Thermal stability, solubility behavior and nanofiber formation of xylan acetate were influenced by its DS values. The mechanical properties of xylan acetate propionate were also investigated.     

TBAF and cellulose esters: unexpected deacylation with unexpected regioselectivity

Tetrabutylammonium fluoride has been found to catalyze the deacylation of cellulose esters. More surprisingly, the deacylation is highly regioselective. Even more remarkably, in contrast with the C-6 regioselectivity of other reactions of cellulose and its derivatives, this deacylation shows substantial selectivity for the removal of the acyl groups from the esters of the secondary alcohols at C-2 and C-3, affording cellulose-6-O-esters with high regioselectivity by a simple one-step process employing no protective groups.     

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.     

Homogeneous sulfation of bagasse cellulose in an ionic liquid and anticoagulation activity

Homogeneous sulfation of bagasse cellulose (BC) with chlorosulfonic acid-dimethylformamide was accomplished in an ionic liquid 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl). The BCS products from the sulfation had degrees of substitution (DS) in the range of 0.52-2.95 and a simultaneous substitution pattern at C-6, C-2 and C-3 positions. The sulfated BCS attained significant anticoagulation activity, causing a dose-dependent prolongation of coagulation time and inhibition of FIIa and FXa activities in human plasma. The anticoagulation activity of BCS showed a positive correlation with DS, and some of the activity indexes exceeded those of heparin.     

Synthesis of a [6-pyridinyl-18F]-labelled fluoro derivative of WAY-100635 as a candidate radioligand for brain 5-HT1A receptor imaging with PET

In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.     

Cyanolysis and azidolysis of epoxides by haloalcohol dehalogenase: theoretical study of the reaction mechanism and origins of regioselectivity

Haloalcohol dehalogenase HheC catalyzes the reversible dehalogenation of vicinal haloalcohols to form epoxides and free halides. In addition, HheC is able to catalyze the irreversible and highly regioselective ring-opening of epoxides with nonhalide nucleophiles, such as CN (-) and N 3 (-). For azidolysis of aromatic epoxides, the regioselectivity observed with HheC is opposite to the regioselectivity of the nonenzymatic epoxide-opening. This, together with a relatively broad substrate specificity, makes HheC a promising tool for biocatalytic applications. We have designed large quantum chemical models of the HheC active site and used density functional theory to study the reaction mechanism of the HheC-catalyzed ring-opening of ( R)-styrene oxide with the nucleophiles CN (-) and N 3 (-). Both the cyanolysis and the azidolysis reactions are shown to take place in a single concerted step. The results support the suggested role of the putative Ser132-Tyr145-Arg149 catalytic triad, where Tyr145 acts as a general acid, donating a proton to the substrate, and Arg149 interacts with Tyr145 and facilitates proton abstraction, while Ser132 positions the substrate and reduces the barrier for epoxide opening through interaction with the emerging oxyanion of the substrate. We have also studied the regioselectivity of ( R)-styrene oxide opening for both the cyanolysis and the azidolysis reactions. The employed active site model was shown to be able to reproduce the experimentally observed beta-regioselectivity of HheC. In silico mutations of various groups in the HheC active site model were performed to elucidate the important factors governing the regioselectivity.     

Synthesis and characterisation of dextran and pullulan sulphate

Dextrans and pullulans of different molar masses in the range of 10(4)-10(5) g/mol were sulphated via a SO3-pyridine complex. The degree of substitution achieved was DS = 2.4 and DS = 1.4 for dextran sulphate and DS = 2.0 and DS = 1.4 for pullulan sulphate, respectively. Confirmation of sulphation was given by FTIR spectroscopy. Asymmetrical S=O and symmetrical C-O-S stretching vibrations were detected at 1260 and 820 cm(-1). Reactivity of the polysaccharide C-atoms was determined by 13C NMR spectroscopy: For dextran this was C-3 > C-2 > C-4, while for pullulan it was C-6 > C-3 > C-2 > C-4.     

Enzymatic synthesis of inulin-containing hydrogels

The Bacillus subtilis protease Proleather FG-F catalyzed the transesterification of inulin with vinyl acrylate (VA) in dimethylformamide (DMF). The reaction conversion for different VA concentrations was greater than 57% after 96 h at 50 degrees C. The degree of substitution (DS, defined as the amount of acrylate groups per 100 inulin fructofuranoside residues) with acrylate moieties can be controlled by varying the molar ratio of VA to inulin. Reasonable yields were obtained (44-51%, 2 days) using a two-step purification methodology. Inulin derivatized with VA (Inul-VA) was characterized by gel permeation chromatography, and its structure was established by (1)H, (13)C, and (1)H-(1)H correlation spectroscopy and (1)H-(13)C heteronuclear multiple quantum coherence NMR. The main positional isomer was at the 6 position of the fructofuranoside residue and two other minor isomers were observed at the 3 and 4 positions. Thus, the enzymatic reaction was largely regioselective. Furthermore, the inulin fructose residues were monosubstituted. Gels with swelling ratios at equilibrium of up to ca. 20 were prepared by free radical polymerization of aqueous solutions of Inul-VA with different DS and monomer concentrations. Gel pore sizes were calculated from swelling experiments and range from 19 to 57 A. To our knowledge, this work reports the first successful enzymatic modification of a polysaccharide solubilized in 100% DMF solution.     

Overcoming limiting side reactions associated with an NHS-activated precursor of polymethacrylamide-based polymers

Combinatorial polymer libraries have recently gained popularity for the development of novel materials for a variety of biomedical applications including non-viral gene delivery systems and biodegradable polymers for tissue engineering. To streamline the nontrivial task of library synthesis, activated ester homopolymers have been used to serve as a backbone to which primary amine-containing functional groups (NH2-FGs) can be covalently bound at varying ratios. Polymethacryloxysuccinimide (poly(MAOS)) is one such homopolymer that was previously reported to be an attractive precursor for polymeric drug and gene delivery systems. The reported functionalization protocols entailed conjugating the precursor with 2 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/150 microL DMSO for either 5 h at 50 degrees C or 16 h at 25 degrees C. More recently, both protocols were revealed to be associated with ring-opening and glutarimide-forming side reactions that compromise the utility of the homopolymer. Using 1-dimensional and 2-dimensional NMR spectroscopy techniques, we have characterized the side product distributions that result from conjugations performed at 50 degrees C/5 h and 25 degrees C/16 h. Moreover, by systematically altering the equivalents of the NH2-FGs, polymer concentration, reaction time, and reaction temperature, we have established a protocol that overcomes these side reactions. Using a final reaction protocol of 5 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/600 microL DMSO for 24 h at 75 degrees C, we have obtained functionalized polymers with minimal side products. This protocol is applicable for polymers ranging from 5000 to 50,000 g/mol, compatible with a variety of functional groups, and amenable to conjugating combinations of functional groups.     

Structure and antioxidant activities of sulfated guar gum: homogeneous reaction using DMAP/DCC catalyst

It was essential to understand the chemical structure of polysaccharides for further research and biochemical or medical application of this natural biopolymer. In the present study, sulfated derivatives of guar gum with high degree of sulfation (DS) were synthesized using 4-dimethylaminopyridine (DMAP)/dimethylcyclohexylcarbodiimide (DCC) as catalyst in homogeneous conditions. The effects of the ratio of chlorosulfuric acid to pyridine, the content of catalyst and reaction temperature were investigated. Results of FT-IR, (1)H and (13)C NMR indicated that C-6 substitution was predominant in sulfated polysaccharide. In the sulfation reaction, a sharp decrease in M(W) was observed. The enhanced antioxidant activities of sulfated polysaccharides were not a function of a single factor but a combination of high DS and low molecule weight.     

Mutation of an atypical oxirane oxyanion hole improves regioselectivity of the α/β-fold epoxide hydrolase Alp1U

Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.     

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.     

6-Amino-6-deoxy-chitosan. Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier

The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80 degrees C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan.     

Esterification of beta-chitin via intercalation by carboxylic anhydrides

beta-chitin is known to form intercalation complexes with aliphatic alcohols and amines. We found that it also forms complexes with carboxylic anhydrides. When the beta-chitin-acetic anhydride complex was heated to 105 degrees C, the hydroxyl groups of chitin were acetylated by a host-guest reaction, maintaining the host's crystal structure. Structures of complex and acetylated products were analyzed by X-ray diffraction, (13)C CP/MAS NMR, and infrared spectroscopy. The maximum degree of substitution (DS) was close to 1.0, suggesting regioselective esterification at the C6 position of chitin. Partially acetylated beta-chitin with a DS of 0.4 could incorporate various guest species that are difficult to be incorporated by original beta-chitin. In contrast, beta-chitin acetate with a DS of 1 lost the ability to form a complex. Intercalation complexes of beta-chitin with cyclic anhydrides (succinic and maleic) also underwent esterification by heating, and the products with a DS of approximately 1 dissolved in aqueous alkali, apparently as the result of the dissociation of introduced carboxyl groups. These phenomena are potentially useful in controlling the complexation ability of beta-chitin and the preparation of regioselectively esterified chitin derivatives.     

MMG and EMG responses of the superficial quadriceps femoris muscles.

Eighteen adults performed isometric muscle actions of the leg extensors at 25, 50, 75, and 100% maximal voluntary contraction (%MVC) at leg flexion angles of 25, 50, and 75 degrees. The results indicated that isometric torque production increased as leg flexion angle increased (75 degrees > 50 degrees > 25 degrees). For each muscle tested (rectus femoris, vastus lateralis, and vastus medialis), the EMG amplitude increased up to 100%MVC at each leg flexion angle (25, 50, and 75 degrees). The MMG amplitude for each muscle, however, increased up to 100%MVC at 25 and 50 degrees of leg flexion, but plateaued from 75 to 100%MVC at 75 degrees of leg flexion. We hypothesize that the varied patterns for the MMG amplitude-isometric torque relationships were due to leg flexion angle differences in: (1) muscle stiffness, (2) intramuscular fluid pressure, or (3) motor unit firing frequency.     

Two-step sequential synthesis of pyrimidine derivatives containing a sugar branch via combining of enzymatic Michael addition/acylation

A new strategy for the enzymatic synthesis of pyrimidine derivatives containing a sugar branch was developed via combining of Michael addition and acylation. The first-step reaction of pyrimidines and vinyl 3-propionyloxy propionate was catalyzed by Amano lipase M from Mucor javanicus in DMSO. The initial reaction rates of different pyrimidines decreased in the order of fluorouracil, uracil, thymine, in agreement with their nucleophilicity. The succeeding regioselective acylation of d-glucose and d-mannose with the Michael adducts was catalyzed by alkaline protease from Bacillus subtilis in pyridine. The d-glucose and d-mannose were all acylated at C-6 position. Moderate yield was obtained for each step.