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.     

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.     

Characterization and anti-tumor activities of sulfated polysaccharide SRBPS2a obtained from defatted rice bran

A novel chemically sulfated polysaccharide SRBPS2a with potent anti-tumor activity was derived from defatted rice bran by chlorosulfonic acid–pyridine (CSA–Pyr) method. The average molecular weight of SRBPS2a was 3.5 × 10⁵ Da and the degree of sulfation (DS) was 1.29. The Fourier-transform infrared spectra (FT-IR) and ¹³C NMR spectroscopy analysis revealed that SRBPS2a was mainly consist of β-(1 → 3)-d-galactopyranosyl residues, the sulfate substitution site was on C-2 and C-4 while the side chains were cut off during the sulfated reaction. Furthermore, SRBPS2a exhibited evident growth inhibition on mouse mammary tumor EMT-6 cells both in vitro and in vivo.     

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.     

Preparation and anticoagulant activity of N-succinyl chitosan sulfates

In order to develop a promising substitute for heparin, N-succinyl chitosan (NSC) was chemically modified by sulfating agent N(SO(3)Na)(3), which were synthesized with sodium bisulfite and sodium nitrite in aqueous solution. The N-succinyl chitosan sulfates (NSCS) products were characterized by infrared spectroscopy (FT-IR) and (13)C NMR. The degree of substitution (DS) of NSCS depended on the ratio of sulfating agent to N-succinyl chitosan, reaction temperature, reaction time and pH of sulfation agent. N-succinyl chitosan sulfates with DS of 1.97 were obtained under optimal conditions. The in vitro coagulation assay of NSCS was determined by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) assays. The results showed that NSCS obviously prolonged APTT. The anticoagulant activity strongly depended on DS, molecular weight (M(w)) and concentration of NSCS. The anticoagulant activity of NSCS promoted with the increase of DS and concentration, and NSCS exhibited the best anticoagulant activity with the M(w) of 1.37×10(4).     

Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan

Synthesis of chitosan sulfates with low molecular weight (M_v 9000–35,000 Da) was carried out by sulfation of low molecular weight chitosan (M_v 10,000–50,000 Da). The oleum was used as sulfating agent and dimethylfornamide as medium. The chitosans were prepared by enzymatic and acidic hydrolysis of initial high molecular weight chitosan as well as by extrusion solid-state deacetylation of chitin. As was shown by FT-IR and NMR-methods and elemental analysis, the sulfation occurred at C-6 and C-3 positions and substitution degree is 1.10–1.63. The molecular weight sulfated chitosan was determined by viscometric method and the Mark–Houwink equation [η]=10⁻⁵ 4.97 M^0.77. Study of anticoagulant activity showed that chitosan sulfates with lowered molecular weight demonstrated a regular increase of anti-Xa activity like heparins.     

Interaction of chondroitin sulfate and dermatan sulfate from various biological sources with heparin-binding growth factors and cytokines

Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.     

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Preparation and anticoagulation activity of sodium cellulose sulfate

Semi-synthesis of cellulose sulfate sodium (Na-MCS) was carried out by sulfation of microcrystalline cellulose (MCC) with chlorosulfonic acid-dimethylformamide complex as sulfating agent. As shown by FT-IR, NMR spectroscopy, and elemental analysis, the sulfation occurred mainly at C6, partially at C2, and no substitution at C3. The substitution degree ranged from 1.10 to 1.70 and the average molecular weight is between 1.1 and 3.5 x 10(4)Da. The anticoagulant efficacy and its possible mechanism were investigated using in vitro, in vivo coagulation assays and amidolytic tests in comparison with heparin. Results indicated that Na-MCS exhibited higher anticoagulation activity based on activated partial thromboplastin time (APTT) assay and prolonged the thrombin time (TT) to a lesser extent than heparin. No effect was detected on the prothrombin time (PT). Subcutaneous administration of Na-MCS to mice increased the clotting time (CT) in a moderate dose-dependent manner with a longer duration. Na-MCS exhibited anticoagulation activity mainly by accelerating the inhibition of antithrombin III (AT-III) on coagulation factors FIIa and FXa in plasma.     

Synthesis and anticoagulant activity of the quaternary ammonium chitosan sulfates

Quaternary ammonium chitosan sulfates with diverse degrees of substitution (DS) ascribed to sulfate groups between 0.52 and 1.55 were synthesized by reacting quaternary ammonium chitosan with an uncommon sulfating agent (N(SO₃Na)₃) that was prepared from sodium bisulfite (NaHSO₃) through reaction with sodium nitrite (NaNO₂) in the aqueous system homogeneous. The structures of the derivatives were characterized by FTIR, ¹H NMR and ¹³C NMR. The factors affecting DS of quaternary ammonium chitosan sulfates which included the molar ratio of NaNO₂ to quaternary ammonium chitosan, sulfated temperature, sulfated time and pH of sulfated reaction solution were investigated in detail. Its anticoagulation activity in vitro was determined by an activated partial thromboplastin time (APTT) assay, a thrombin time (TT) assay and a prothrombin time (PT) assay. Results of anticoagulation assays showed quaternary ammonium chitosan sulfates significantly prolonged APTT and TT, but not PT, and demonstrated that the introduction of sulfate groups into the quaternary ammonium chitosan structure improved its anticoagulant activity obviously. The study showed its anticoagulant properties strongly depended on its DS, concentration and molecular weight.     

Structure and chain conformation of five water-soluble derivatives of a beta-D-glucan isolated from Ganoderma lucidum

A linear water-insoluble (1-->3)-beta-D-glucan, coded as GL-IV-I, was isolated from the fruit body of Ganoderma lucidum by extracting with NaOH solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethylation, hydroxypropylation, and methylation, respectively, and these were labeled as S-GL, CM-GL, HE-GL, HP-GL and M-GL. Five derivatives exhibited good water solubility. Their structures and chain conformations were investigated with infrared spectroscopy, elemental analysis (EA), one- and two-dimensional NMR spectroscopy, laser light scattering (LLS), and size-exclusion chromatography combined with LLS (SEC-LLS). The reactivity of the hydroxyl group of GL-IV-I was ordered as C-6>C-4>C-2 for the five derivatives. The degree of substitution (DS) of the derivatives was calculated from EA and NMR spectroscopy to be from 0.32 to 1.18. The weight-average molecular mass (M(w)) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL, and M-GL was 13.3 x 10(4), 10.1 x 10(4), 6.3 x 10(4), 7.2 x 10(4), 5.1 x 10(4), and 14.1 x 10(4), respectively. The conformation analysis studies revealed that GL-IV-I exists as a compact coil in dimethyl sulfoxide, whereas the five derivatives are slightly expanded flexible chains in 0.9% aqueous NaCl solution.     

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.     

Synthesis of potato starch sulfate and optimization of the reaction conditions

Potato starch sulfate was obtained by the reaction between potato starch and chlorosulfonic acid in pyridine. It was characterized by FT-IR and SEM. The reaction conditions were studied systematically, which included the volume ratio of pyridine to chlorosulfonic acid, reaction temperature and time in preparing sulfating agent process, and the ratio of starch mass to chlorosulfonic acid volume, reaction temperature and time in sulfation process. Meantime, the degree of substitution (DS) of each sample was determined via barium sulfate–glutin nephelometery method. By investigating the relationship between these conditions and DS, the optimal conditions were obtained with the maximum DS.     

Mitogenic activity of sulfated chitosan and cellulose derivatives is related to protection of FGF-2 from proteolytic cleavage

Novel chitosan (CHS) and cellulose sulfates (CSs) are studied regarding their mitogenic activity and their protective effect against proteolytic digestion of FGF-2. An intermediate degree of sulfation (DS(S) ) and lower concentration of CHS have superior effect on 3T3 cell growth while the mitogenic activity of CS increases with DS(S) and concentration. Experiments with trypsin as model proteinase show that protection of FGF-2 from proteolytic digestion depends on DS(S) and the concentration of derivatives in the same manner as cell growth. Studies on stability of FGF-2 added to cultures of 3T3 cells show that the FGF-2 concentration remains higher in the presence of derivatives. Results indicate that the mitogenic activity of CHS and CS is due to protection of FGF-2 from proteolytic cleavage.     

Determination of substituents distribution in carboxymethylpullulans by NMR spectroscopy

The distribution of carboxymethyl substituents in the alpha-(1 --> 6)-linked maltotriosyl repeating units of a carboxymethylpullulan (CMP) series was investigated by high resolution NMR spectroscopy on very short oligomers (DPn = 1.2-1.5) obtained by acid hydrolysis. A series of 2D NMR experiments on parent pullulan, hydrolysed pullulan and CMP was used to assign the proton and carbon chemical shifts of CMP acid hydrolysates. The degree of substitution (DS) and the relative distribution of -CH2COONa groups at OH-2, OH-3, OH-4 and OH-6 of glucose residues (DSi) were determined from 1H NMR measurements. From a set of CMP samples, widely different in degree of substitution, it was observed that the substitution at C-2 is predominant and decreases according to the order C-2 > C-3 > C-6 > C-4. Taking into account the availability of each OH group in the parent pullulan, an order of relative reactivity of hydroxyl groups is defined according to the relation: Ri = DSi/ni, where ni is the number of free OH groups in a maltotriose unit (MTU) for a given site C-i, the reactivity order was found to be OH-2 > OH-4 > OH-6 > OH-3.     

Rapid esterification of wheat straw hemicelluloses induced by microwave irradiation

Esterification of wheat straw hemicelluloses with acetyl chloride, propionyl chloride, n-octanoyl chloride, lauroyl chloride, palmitoyl chloride, stearoyl chloride, and oleoyl chloride, respectively, using N-bromosuccinimide (NBS) as a catalyst was achieved in DMF/LICl medium by microwave irradiation. The effects of various acyl chlorides and the molar ratios of xylose units in hemicelluloses/acyl chloride on the degree of substitution (DS) were investigated and DS reached up to 1.34 by a few minutes. ¹³C NMR studies showed that the esterification occurred preferentially at the C-3 and C-2 positions. On the other hand, microwave irradiation brought a partial degradation of the polymer, and therefore resulted in a slight decrease in thermal stability of the hemicellulosic derivatives in comparison with conventional heating technique.     

Sulfated modification, characterization and antitumor activities of Radix hedysari polysaccharide

Sulfated modification of a polysaccharide obtained from Radix hedysari (RHP) was studied. Four sulfated derivatives (RHPS) with variable degrees of substitution (DS) were obtained by the chlorosulfonic acid method with ionic liquids (ILs) as solvent and 4-dimethylaminopyridine (DMAP) as catalyst. The structures of RHPS were characterized by FT-IR spectra and (13)C NMR spectra, and the results indicated that the sulfated groups were modified mainly at the C-6 position and C-2 position. Four kinds of RHPS showed different DS ranging from 0.63 to 1.45, and different weight-average molecular mass (Mw) ranging from 60.8 to 71.1kDa with a little degradation. Compared with RHP, all of RHPS exhibited obvious antitumor activity on A549 cells and BGC-823 cells in vitro. However, they had no obvious influence on HEK293 cells, which indicated that they had low toxicity to normal cells. Flow cytometric studies indicated that the treatment of RHPS against A549 cells and BGC-823 cells could mediate the cell-cycle arrest in the G1 phase.     

Selective carboxypropionylation of chitosan: synthesis, characterization, blood compatibility, and degradation

Two water-soluble chitosan (WSC) derivatives of N-succinyl-chitosan (NSCS) and N,O-succinyl-chitosan (NOSCS) with a degree of substitution (DS) that ranged form 0.28 to 0.61 were selectively synthesized by varying the molar ration of succinic anhydride and chitosan. The chemical structure and physical properties of the chitosan derivatives were characterized by FT-IR, ¹H NMR, and XRD. XRD analysis showed that the derivatives were amorphous. The lysozyme enzymatic degradation results revealed that the NSCS was of higher susceptibility to lysozyme. The degradation rate and the solubility of the chitosan derivatives were strongly determined by the degree of substitution and the position of the substitution. The results of antithrombotic properties, hemolytic properties and anticoagulant properties of WSCs indicated that the blood compatibility was dramatically improved, and the carboxyl group introduced on the C-6 or C-2 hydroxyl group appeared to impact anticoagulant activity in different ways.     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.     

The structure-anticoagulant activity relationships of sulfated lacquer polysaccharide: effect of carboxyl group and position of sulfation

Regiospecific oxidation of the primary hydroxyl groups in lacquer polysaccharide (LPL, Mw 6.85 x 10(4)) and its NaIO4 oxidation derivatives (LPLde) to C-6 carboxy groups was achieved with NaOCl in the presence of Tempo and NaBr. Sulfate groups were incorporated into the oxidated polysaccharides using Py.SO3 complex as a reagent. Reactivity of polysaccharide hydroxyl group was C-6 > C-2 > C-4. Sulfate groups were mainly linked to the second hydroxy at C-2 in the products. The results of APTT assay showed after incorporation of carboxyl groups into lacquer polysaccharides, the intrinsic coagulation pathway was promoted, and all sulfated polysaccharides had very weak anticoagulant activity within the scope of studied DS (0.39-1.11). These indicated that carboxyl groups and sulfate groups had the synergistic action. At the same time, the anticoagulant activity increased very slowly with the DS in the second hydroxy. This indicated that 6-O-SO3- in the side chains took an important role in the anticoagulant activity.