Sulfation and preliminary biological evaluation of ethylene‐vinyl alcohol copolymers

In order to obtain polymers provided with improved haemocompatibility a commercial ethylene‐vinyl alcohol copolymer (EVAL) was chemically modified by introduction of different amounts of sulfate groups. The sulfation reaction was carried out by the reaction of hydroxy groups with two different sulfating reagents (ClSO₃H and PySO₃). The influence of the molar ratio of the reagents and the reaction time on the functionalization degree was evaluated also for elevated substitution degrees. No difference in the sulfation agents efficiency was noted. The new terpolymers (EVAL‐SO₃H) have been characterized by chemical and physico‐chemical techniques as: titration of the acidic groups, Fourier Transform IR reflectance spectroscopy (FTIR‐ATR), differential scanning calorimetry (DSC), X‐ray photoelectron spectroscopy (XPS) and ¹H‐NMR. The haemocompatibility was evaluated by in vitro plasma coagulation (APTT) and platelet aggregation tests and is proved to be excellent for sulfation degrees higher than 35% of the hydroxy groups of the polymer.     

A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans

Introduction Chondroitin and dermatan sulfate (CS/DS) show considerable species, tissue, age and pathology‐related structural heterogeneity. In addition within chains their sulfation patterns are not random. To elucidate their structure/function relationships, methods for complete characterization are required. A method for keratan sulfate (KS) fingerprinting ( Whitham et al. 1999 ) has been extended for the linkage, repeat and chain cap regions of CS/DS, including the acquisition of CS/DS ratios. Methods Chains were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or ACII lyase at 50 mg/ml in 0.1 m ammonium acetate, pH 8 and for 15 h at 37 °C. Alternatively, chains were de‐N‐acetylated by hydrazinolysis at 98 °C for 24 h at 10 mg/1 ml in anhydrous hydrazine with 100 mg/ml hydrazine sulfate. Then, they were depolymerized by 3.9 m sodium nitrite/0.28 m acetic acid at 0 °C for 4 h. Unreduced chains were released from their protein core in 0.5 m LiOH at 4 °C for 12 h. Materials were fluorescently labelled with 2‐AA as previously described (Whitham et al. 1999) and characterized by HPAEC using a Dionex AS4‐SC column at 50 °C and 2 ml/min with constant 15% 1 m NaOH. A 5‐min isocratic period of 85% H₂O/0% 2 m NaCl was followed by a linear gradient of 0–30% 2 m NaCl over 60 min. The oligosaccharides were monitored using a λ_ex of 315 nm and a λ_em of 400 nm. Results and discussion This method resolves repeat region di‐, tri‐ and tetrasaccharides, capping oligosaccharides and linkage regions and can be used to profile known and unknown oligosaccharides. Unsulfated oligosaccharides elute between 2 and 10 min, monosulfated between 7 and 30 min, disulfated between 25 and 40 min and trisulfated between 49 and 54 min. Allied with data on size, oligosaccharide identification is facilitated. Hydrazinolysis/nitrous acid depolymerization of CS/DS chains results in disaccharides from CS with 4‐ or 6‐sulfation and from DS with 4‐sulfation which retain IdoA and GlcA structures and which can be distinguished chromatographically. The methodology was used to examine CS/DS from shark, whale, bovine and human tracheal, articular and meniscal cartilage and cornea. Tracheal cartilages show predominantly 4‐sulfation with porcine sources being more highly 4‐sulfated (ca. 75%) than bovine (ca. 65%). Articular cartilage comprises mainly 6‐sulfated GalNAc (ca. 95% in the adult), while adult meniscal cartilage shows only ca. 85%. Tracheal and articular cartilage aggrecan showed no IdoA; however, it represented ca. 20% of the uronic acids of bovine meniscal aggrecan, showing the presence of DS. Corneal CS/DS has a very low level of 6‐sulfation (<ca. 5%) but shows an equal abundance of unsulfated and 4‐sulfated residues and contains high levels, ca. 50%, of IdoA residues. Shark cartilage shows ca. 75% 6‐sulfation with significant levels of uronic acid 2‐sulfation found only between a 4‐sulfated residue and a 6‐sulfated residue, reflecting sulfotransferase specificity. Shark cartilage contains modest (ca. 1–5%) levels of DS that may be contaminants of preliminary isolation. This method extends a previous method to now allow the complete examination of KS, CS and DS chains by a single rapid chromatographic method.     

Concepts for preparation of novel regioselective modified cellulose derivatives sulfated,aminated, carboxylated and acetylated for hemocompatible ultrathin coatings on biomaterials

Some regioselective cellulose derivatives were synthesized on the basis of the just started longterm concept by variation of O—SO₃, N—SO₃, N—Ac, and N‐carboxymethyl groups solely or in combination for the development of athrombogenic and antithrombogenic nanocoatings on cellulose membranes. Similar regioselectively arranged functional groups are known partially in type 1 glycosaminoglycane (dermatan sulfate DS, and chondroitin sulfate CS) and all the groups are present in type 2 GAG (heparin HE and heparansulfat HS). The goal of this concept was to use instead of the backbone structure β 1–> 4, β 1–>3 (DS, CS) or β 1–> 4, α 1–> 4 (HE, HS or regioselective desulfated HE), the β 1–> 4 backbone structure of cellulose or chitosan, with the same functional groups as in HE. Furthermore different regioselective sulfated cellulose derivatives, such as cellulose‐3‐sulfate, cellulose‐2,6‐disulfate, and cellulose‐2,3‐sulfates with varied sulfation degrees have been synthesized and immobilized ionically on cellulose membranes. They were tested concerning their reduction of platelet adhesion from citrated whole blood in a perfusion system as well for AT III affinity. The lowest platelet adhesion and AT III affinity was observed using cellulose‐2,3‐sulfates and cellulose‐3‐sulfate derivatives, whereas cellulose‐6‐O‐sulfate derivatives show high AT III affinity and high platelet adhesion. This means that a high 6‐O‐sulfate content seems to promote anticoagulant properties of the derivatives with high (AT III) affinity, whereas low concentrations of 2‐O‐ and 3‐O‐sulfate groups show the lowest platelet adhesion. The latter seems to be important for developing athrombogenic coatings for biomaterials. The starting material chitosan with molecular weight of 150 KD was used to synthesize additionally some of 2‐deoxyaminocellulose derivatives containing N—Ac or N—SO₃ or N—CH₂—COOH and O—SO₃ groups. These derivatives are structurally closer to HE and HS and should enable us to work out the influence of each heparin like functional group in cellulose derivative on the athrombogenic and antithrombogenic properties. We synthesized only some of the latter mentioned derivatives with a distribution of the functional groups according to a Bernoulli statistics. Totally sulfated 2‐deoxyaminocellulose‐3,6‐disulfate derivatives will be regioselectively desulfated by reactions known from heparin chemistry.     

Lyotropic Liquid Crystalline Polyamides Containing Aromatic,Heterocyclic Structures: Preparation and Properties

Summary: A series of novel lyotropic liquid crystalline polyamides derived from 1,2‐dihydro‐2‐(4‐aminophenyl)‐4‐[4‐(4‐(aminophenoxyl)phenyl)]‐phthalazinone ( DHPZ‐DA ), p‐phenylene diamine ( PPD ) and terephthaloyl dichloride ( TPC ) have been successfully synthesized by the low temperature solution polycondensation method. The copolymers obtained with inherent viscosity of ≈1.21 to 3.29 dl · g^?1, determined in NMP (1 wt.‐% LiCl) or concentrated H₂SO₄ at 25 °C, were confirmed to be amorphous by WAXD and differential scanning calorimetry (DSC). Their solubility was improved by the introduction of non‐coplanar, twisted phthalazinone moieties and ether linkages into the main chain with the result that they can be soluble in some polar solvents containing a small amount of LiCl. Their T_gs were all above 310 °C and 10% weight loss temperatures in nitrogen above 500 °C. Most of these polyamides can form an anisotropic phase in concentrated H₂SO₄, NMP (1 wt.‐% LiCl) and DMAc (1 wt.‐% LiCl) solutions, observed on a polarizing light microscope.

Optical micrograph (×40) of polyamide P‐4060 at 10 wt.‐% in NMP/LiCl (1% w/v) solution at room temperature.     

Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) ( Lyon et al. 1994 ) and dermatan sulfate (DS) ( Lyon et al. 1998 ) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor ( Lyon et al. 2002 ). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity. Materials and methods A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line. Results Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm . This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones. Discussion The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.     

The cellular site of sulfation of influenza viral glycoproteins.

The incorporation of ³⁵SO₄^2? into viral polypeptides in MDBK cells infected with influenza virus was analyzed by SDS-polyacrylamide gel electrophoresis. When infected cells were labeled in the absence of calf serum, three polypeptides, HA, NP, and M, were resolved in isolated plasma membranes, and HA was the only polypeptide in which ³⁵SO₄^2? incorporation was detected. Sulfation of HA was also demonstrated in both smooth and rough cytoplasmic membranes, whereas there was no detectable ³⁵SO₄^2? incorporation into unglycosylated proteins. These results indicate that at least partial sulfation of HA is already completed at rough membranes. However, when infected cells were doubly labeled with [³H]leucine and ³⁵SO₄^2?, the ${}^{\mathrm{<mtext>35</mtext><mtext>S</mtext>}}{}^3\text{H}$ ratio in the HA polypeptide was not uniform in virions and subcellular components. The ratio was highest in virions, and decreased in the order of virions, plasma membranes, smooth membranes, and rough membranes, suggesting that further sulfate incorporation may occur in smooth membranes and plasma membranes as well as in rough membranes. The 355/3H ratio of HA associated with plasma membranes varied with the length of labeling; higher ratios were observed with shorter labeling periods. This observation may be explained by sulfate incorporation into performed HA. Significant amounts of ³⁵SO₄^2? incorporation into HA were found in the presence of cycloheximide, at concentrations wich completely inhibited the synthesis of viral polypeptides. Further, pulse-labeling of infected cells with ³⁵SO₄^2? at various times after inhibition of protein sy thesis by cycloheximide showed that sulfation of HA polypeptides continues to occur as long as 30 min or more after synthesis, which also suggests that ³⁵SO₄^2? continues to be incorporated into HA polypeptides even after they migrate from rough membranes. The acceptors for sulfation appear to be oligosaccharide units of viral glycoproteins since almost all ³⁵S label was recovered in association with glycopeptides after exhaustive digestion of virions with Pronase followed by gel filtration. As was observed for HA, the incorporation of ³⁵SO₄^2? into cellular mucopolysaccharides was also observed in every subcellular fraction tested. Further, when either smooth or rough cytoplasmic membranes isolated from infected cells were incubated with 3′-phosphoadenosine-5′-phosphosulfate ([³⁵S]PAP) in vitro, sulfate incorporation into mucopolysaccharide was detected, which suggests that sulfation of mucopolysaccharide occurs in both smooth and rough membranes in vivo. Additionally, it was found that the rate of incorporation of ³⁵SO₄^2? into cellular mucopolysaccharide was markedly inhibited by influenza virus infection.     

Effect of lithium intake on sodium and lithium appetite in sodium deficient cattle

Sodium deficient, unlike normal calves, will push a panel vigorously for Li⁺ or Na⁺ salts. In 5 min (VI 20 sec) operant tests calves (n=5) pressed for 10 ml rewards of 0.3 M NaHCO₃, NaCl, Na₂SO₄, LiCl and Li₂SO₄. When the tests were extended to 20 min, LiCl responses were reduced significantly in a few days and the aversion transferred to NaCl, Na₂SO₄ and Li₂SO₄ but the NaHCO₃ response remained at a high level. No signs of lithium toxicity were detectable. Lithium naive calves (n=2) given a single LiCl IV infusion which raised the plasma lithium to 12.5±1.3 mEq/l did not exhibit any reduction in Li or Na appetite. In 3 of the 5 calves mentioned above which had ingested lithium salts an IV injection of LiCl reduced their appetite for all Na and Li salts tested except for NaHCO₃. This suggests that either there is some retention of lithium within the body to produce avoidance behaviour when plasma lithium is raised or a taste memory of the aversive effects produced by raised plasma Li⁺ exists. The Li⁺ aversion, which varies between calves, appears to be connected directly with intake and the effect fades in weeks or months. The distinct selection made for NaHCO₃ illustrates the importance of this salt for the survival of Na⁺ depleted calves.     

Analysis of Heparan Sulfate from the Engelbreth-Holm-Swarm (EHS) Tumor

The size of the heparan sulfate chains from the Engelbreth-Holm-Swarm (EHS) tumor heparan sulfate proteoglycan (PG) was measured by several techniques in order to resolve uncertainty about their size and the chains were chemically characterized for comparison with other basement membrane heparan sulfate PGs. Heparan sulfate size was determined by gel filtration (M_r = 5.5 – 6.0 X 10⁴), by equilibrium sedimentation centrif-ugation (M_w = 6.8 × 10⁴), and by end group analysis (M_n = 7.1 × 10⁴). A higher molecular weight (HMW) (M_w = 2.13 × 10⁵) calculated from scattering measurements may reflect chain-chain interactions. Forty percent of newly synthesized chains eluted on gel filtration as a lower molecular weight (LMW) shoulder and in vivo turned over faster than the larger species.

A large heparan sulfate PG was present after 4 hours of in vivo ³⁵SO₄ labeling in both a low density form and a high density, slightly smaller form with large heparan sulfate chains (M_r ～ 8.0 × 10⁴). Heparan sulfate PG of intermediate size (K_av = 0.3–0.65, Sepharose CL-4B) and of smaller size (K_av = 0.75, CL-4B) were found predominantly as high density species. These PGs contained chains (M_r = 3.5 × 10⁴ and M_r = 1.2 × 10⁴, respectively) which were partially sensitive to chondroitinase ABC (CABC) and may include a hybrid heparan sulfate/chondroitin sulfate PG. Heparan sulfate chains, possibly intracellular degradation products, were also found.

Heparan sulfate chains were normal in N-sulfation (58% of hexosamine residues) and in iduronate content (～ 30%). N-sulfation started within two disaccharides of the linkage region. The EHS heparan sulfate was unusually low in O-sulfation (10% of the total sulfation) and no 6–0 sulfated, N-acetylated glucosamine residues adjacent to N-sulfated block regions were found.     

Towards highly functionalized and semi‐rigid polyzwitterions, 2. Poly(zwitterionic isocyanides): synthesis and specific properties

Selective quaternization of the tertiary amino site of N,N‐dimethyl‐N‐(3‐isocyanopropyl)amine by 1,3‐propanesultone or 2,2‐dicyanoketene‐ethylene acetal quantitatively yields the zwitterionic isocyanides of the ammoniopropanesulfonate ( SI , —N⁺(CH₃)₂—(CH₂)₃—SO₃^–) or ammonioethoxydicyanoethenolate ( EI , —N⁺(CH₃)₂—(CH₂)₂—O—CO—C^–(CN)₂) type, respectively. Their polymerization initiated with NiCl₂·6 H₂O in methanol ( SI ) or in N,N‐dimethylformamide ( EI ) solution leads with high yields (>70%) to the corresponding poly(isocyanides) in a rather low molecular weight range (DP_w ˜ 80–600). PSI is soluble in a few organic protic solvents such as glycol or m‐cresol and displays typical UCST behavior in pure water (T_c ≈ 35°C) with “salting in” effects in the presence of LiClO₄. PEI is soluble in a few dipolar aprotic solvents such as DMF, NMP or DMSO with typical LCST behavior and “salting in” effects in the presence of LiCl; secondly, in fairly concentrated KSCN aqueous solutions (>1 M) with a typical UCST behavior strongly dependent on salt concentration (dT/d[KSCN] ≈ 252°C·L·mol^–1). Analysis of the [η] – M_w data for PSI (0.05 M LiClO₄ in H₂O) and PEI (0.05 M LiCl in DMF) according to the Bohdanecky's approximation of the Yamakawa‐Fujii theory for wormlike chains allows to estimate their persistence length to be about 60 Å for a hydrodynamic diameter of about 15 Å. For polymer volume fractions Φ_p > 0.30, the PSI /glycol gels display local nematic order as revealed by polarizing optical microscopy and small angle X‐ray scattering (Bragg spacing d ∝ Φ_p^–0.45). The two poly(isocyanides) are non‐crystalline but slightly birefringent polymers showing only local order (one SAXS peak). PSI dissolves stoichiometric amounts of NaB(C₆H₅)₄, LiClO₄, NaI and NaSCN to yield amorphous and birefringent blends which display optimized long range order in the latter case: a hexagonal bidimentional lattice of zwitterionic cylinders spreading over a coherence length of about 300 Å.     

Cerebral oxygenation during the Richalet hypoxia sensitivity test and cycling time-trial performance in severe hypoxia

Background

The Richalet hypoxia sensitivity test (RT), which quantifies the cardiorespiratory response to acute hypoxia during exercise at an intensity corresponding to a heart rate of ~130 bpm in normoxia, can predict susceptibility of altitude sickness. Its ability to predict exercise performance in hypoxia is unknown.

Objectives

Investigate: (1) whether cerebral blood flow (CBF) and cerebral tissue oxygenation (O₂Hb; oxygenated hemoglobin, HHb; deoxygenated hemoglobin) responses during RT predict time-trial cycling (TT) performance in severe hypoxia; (2) if subjects with blunted cardiorespiratory responses during RT show greater impairment of TT performance in severe hypoxia.

Study design

Thirteen men [27 ± 7 years (mean ± SD), Wmax: 385 ± 30 W] were evaluated with RT and the results related to two 15 km TT, in normoxia and severe hypoxia (FIO₂ = 0.11).

Results

During RT, mean middle cerebral artery blood velocity (MCAv: index of CBF) was unaltered with hypoxia at rest (p > 0.05), while it was increased during normoxic (+22 ± 12 %, p < 0.05) and hypoxic exercise (+33 ± 17 %, p < 0.05). Resting hypoxia lowered cerebral O₂Hb by 2.2 ± 1.2 μmol (p < 0.05 vs. resting normoxia); hypoxic exercise further lowered it to ?7.6 ± 3.1 μmol below baseline (p < 0.05). Cerebral HHb, increased by 3.5 ± 1.8 μmol in resting hypoxia (p < 0.05), and further to 8.5 ± 2.9 μmol in hypoxic exercise (p < 0.05). Changes in CBF and cerebral tissue oxygenation during RT did not correlate with TT performance loss (R = 0.4, p > 0.05 and R = 0.5, p > 0.05, respectively), while tissue oxygenation and SaO₂ changes during TT did (R = ?0.76, p < 0.05). Significant correlations were observed between SaO₂, MCAv and HHb during RT (R = ?0.77, ?0.76 and 0.84 respectively, p < 0.05 in all cases).

Conclusions

CBF and cerebral tissue oxygenation changes during RT do not predict performance impairment in hypoxia. Since the changes in SaO₂ and brain HHb during the TT correlated with performance impairment, the hypothesis that brain oxygenation plays a limiting role for global exercise in conditions of severe hypoxia remains to be tested further.     

Synthesis of poly(glutamic acid)-tyramine hydrogel by enzyme-mediated gelation for controlled release of proteins

An in situ-formed hydrogel was synthesized by enzymatic cross-linking of poly(γ-glutamic acid)–tyramine conjugates (PGA–Tyr) using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The gelation time ranged from 25 s to 5 min was accomplished by tuning the concentration of HRP, H₂O₂/Tyr molar ratio and the degree of substitution (DS) of Tyr groups. The storage modulus (G′), cross-link density, and mesh size can be tailored by controlling the H₂O₂/Tyr ratio and DS. The rheological analysis indicated that the storage modulus (G′) can be tailored from approximately 40 to over 1100 Pa with the increasing H₂O₂/Tyr ratio and DS. The bovine serum albumin (BSA) was used as model protein and encapsulated into the hydrogel during the enzyme-mediated cross-linking reaction. Controlled release of BSA in vitro from the PGA–Tyr hydrogel was obtained. The release rate and cumulative release amount of encapsulated BSA were manipulated by controlling the H₂O₂/Tyr ratio and DS. More than 90% of encapsulated BSA was released from the hydrogel with low cross-link density and lager mesh size in 60 h, while only 68% of BSA was released from the hydrogel with high cross-link density and small mesh size. The results indicated that the PGA–Tyr hydrogel is a promising material for the controlled release of therapeutic protein or peptides.     

Determination of the Binding Situation of Pyridine in Xylan Sulfates by Means of Detailed NMR Studies

Xylan sulfate is an important drug to treat interstitial cystitis. Production of the drug by sulfation of the polysaccharide with a sulfating agent like chlorosulfuric acid and pyridine–SO₃ complex in pyridine may lead to products containing pyridine‐based impurities. Xylan sulfate containing nitrogen is investigated by different NMR measurements in order to clarify the binding situation of pyridine. The detailed NMR studies allow the conclusion that the pyridine‐based impurities are covalently bonded to the reducing end group. Furthermore, the NMR spectroscopic investigation indicates that the side reactions occur at shorter polymer chains only.     

Reabsorption of inorganic sulfate in the rat kidney: Evidence for an adaptive depression of TmSO4during SO4 loading

Clearance methods were employed to study reabosorption of inorganic sulfate (SO₄) in the rat kidney. During stepwise elevation of the SO₄ concentration in plasma by sulfate infusions (SO₄ titration), the reabsorption of SO₄ increased and reached the maximal reabsorptive capacity for inorganic sulfate (Tm_SO4). During further SO₄ loading Tm_SO4 decreased by about 50%. At the same time GFR was stable, and the decline of the Tm_SO4 was not due to volume loading during infusion, since in time-controls with Ringer infusion alone the reabsorption of inorganic sulfate was almost unchanged. The decrease of the Tm_SO4 was also observed in thyroparathyroidectomized animals. It is concluded that the decline of Tm_SO4 was a result of the SO₄ load per se and may be mediated by an unknown adaptive process.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 239/9-1)     

Surface characterization and platelet compatibility evaluation of surface-sulfonated chitosan membrane

The effect of various sulfonated derivatives of chitosan on platelet activation and blood coagulation was examined. The surface properties of artificial biomaterials have been thought as the key factors which mediate the interactions between the biological environment and biomaterial itself. In this study, the sulfonation was directly performed on the chitosan membrane surface. The chitosan membrane was surface-sulfonated by reactions with sulfur-pyridine trioxide complex (SO₃/pyridine) in H₂O solution and N,N-sulfur-dimethylformamide trioxide complex (SO₃/DMF) in DMF.Blood compatibility was evaluated by an in vitro platelet adhesion assay. The surface reaction of SO₃/pyridine in aqueous acid medium yields N,O-sulfated chitosan with cationic NH⁺ ₃ groups. After neutralization, this surface has been shown to induce a low degree of platelet adhesion and activation. When the surface-sulfonation is performed in an aqueous alkaline medium, although the degree of sulfonation is lower than the samples above, the N-sulfated chitosan significantly reduced the adhesion and activation of platelets. For the acidic SO₃/DMF reaction system, N,O-sulfated chitosan was obtained with a high extent of sulfonation and cationic NH3+groups. On this surface fully spread platelets and some platelet aggregates were found instead. This may be attributed to the ionic interactions between the platelets membrane surface and the cationic groups on the modified chitosan membrane.     

A New Versatile Radical Addition on α, ω‐Dimethacrylate Poly(ethylene oxide)

Summary: The synthesis of α,ω‐di(2‐methyl‐6‐pyrenyl‐2‐succinimidylhexanoic ester)poly(ethylene oxide) (Py(S)‐EO_n‐(S)Py) was obtained by a new radical reaction between 4‐pyrenylbutanoate N‐hydroxysuccinimidyl ester and α,ω‐dimethacrylate poly(ethylene oxide). The reason for the choice of such bulky groups is a possible application of this reaction to the synthesis of polyrotaxane from polypseudorotaxane. Two reaction media were examined, heterogeneous in water at room temperature using persulfate as initiator, or homogeneous in DMSO at 60 °C using AIBN as initiator. Structural characterization of the functionalization products was carried out by SEC, MALDI TOF, and NMR spectroscopies. It was shown that two types of α,ω‐dipyrenyl PEO could be obtained, one corresponding to the expected product (Py(S)‐EO_n‐(S)Py), the other corresponding to the same structure but without the succinimidyl substituent (Py(H)‐EO_n‐(H)Py). It was also shown that in the soluble system macrocycles could be formed and this last aspect has been assigned to intramolecular interactions existing between the pyrenyl groups. An interesting aspect of this synthesis is the possibility to find conditions giving high yields without crosslinking and fairly reduced amount of coupling.

     

Effects of dextran sulfates on the acute infection and growth stages of Toxoplasma gondii

Toxoplasma gondii is one of the most prevalent parasites, causing toxoplasmosis in various warm-blooded animals, including humans. Because of the broad range of hosts susceptible to T. gondii, it had been postulated that a universal component of the host cell surface, such as glycosaminoglycans (GAGs), may act as a receptor for T. gondii infection. Carruthers et al. (Infect Immun 68:4005–4011, 2000) showed that soluble GAGs have also been shown to disrupt parasite binding to human fibroblasts. Therefore, we investigated the inhibitory effect of GAGs and their analogue dextran sulfate (DS) on T. gondii infection. For up to 24 h of incubation after inoculation of T. gondii, the inhibitory effect of GAGs on T. gondii infection and growth inside the host cell was weak. In contrast, DS markedly inhibited T. gondii infection. Moreover, low molecular weight DS particularly slowed the growth of T. gondii inside host cells. DS10 (dextran sulfate MW 10 kDa) was the most effective agent in these in vitro experiments and was therefore tested for its inhibitory effects in animal experiments; infection inhibition by DS10 was confirmed under these in vivo conditions. In this report, we showed that DSs, especially DS10, have the potential of a new type of drug for toxoplasmosis.     

Zum mechanismus der aktivierung der anionischen polymerisation polarer monomerer durch Lewis-Basen

The mechanism of the activating effect of LEWIS-bases (LB) on the anionic polymerization of polar monomers has been studied on the basis of the results obtained from different systems of the type acrylonitrile-RM_g X-LB-toluene at ?75°C. Catalytic amounts of dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and hexamethyl phosphoric acid triamide (HMPA), were used as LB. The relative variation of the number of active sites of the propagation reaction and their reactivity have been estimated. As far as DMF is concerned, both factors are important. DMSO mainly gives rise to an increase of the propgation constant k_w. The activation of the polymerization by HMPA is only caused by an increase in the number of active sites.     

High molecular weight chitosan 6-O-sulfate. Synthesis,ESR and NMR characterization

Selective sulfation of the primary hydroxyl group of chitosan was performed by means of the pyridine-SO₃ complex after the positions 2 and 3 of the aminoglucopyranosidic ring were protected by coordination with copper ions. The requirements for complexing copper ions were investigated as a function of the pH and Cu(II)/chitosan mole ratio of the starting solution. Cu(II)-chitosan complexes were characterized by ESR and ¹³C NMR CP-MAS investigations; two different paramagnetic Cu(II) species were observed, whose relative quantity changes by changing the pH of the starting solution. The g tensor values (species I: g_1‖ = 2,28, g_1‖ = 2,06, A_2‖ = 160 G; species II; g_2‖ = 2,24, g_2‖ = 2,06, A_2‖ = 160 G) are consistent with a CuN₂O₂ chromophore. The sulfation reaction was studied in the range of 25 to 75°C using 2 : 1 to 6 : 1 pyridine-SO₃/Cu(II)-chitosan mole ratios. Under optimized reaction conditions, a selective 6-O sulfation was obtained in more than 90% yield.     

Poly(propylene sulfoxide) — a first approach to a dipolar aprotic,water-soluble and reactive polymer

The oxidation of atactic poly(2-methylthiirane) (PPS, M?_w ≈ 1 – 2 · 10⁵) to its corresponding sulfoxide (PPSO) is studied under a variety of experimental conditions: m-chloroperbenzoic acid in homogeneous chloroform solution ( ? 7°C) and hydrogen peroxide or bromine in an interfacial chloroform-water system (20°C) lead to selective and quantitative oxidation under stoichiometric conditions with simultaneous chain degradation (M?_w (PPSO) ≈ 0,5 – 3 · 10⁴). The high density of sulfoxide functions affords to the PPSO chain a number of specific properties such as: (a) solubility in dipolar protic (CH₃OH, CHCHl₃ …) and aprotic (DMF, DMSO) solvents, the polymer coils being, however, strongly associated in aqueous solution, (b) strong polarity of the bulk polymer, as shown by an “apparent local dipole moment” of about 4,1 D, (c) poor thermal stability, degradation starting at about 130°C under nitrogen, with a weight loss of 50% at 160°C (apparent activation energy E_a = 100 kJ · mol^?1).     

A New Polycondensation Involving Dicarboxylic Acids with Differently Activated Carboxyl Groups by TsCl/DMF/Py

Aromatic dicarboxylic acids, such as terephthalic (TPA) and isophthalic (IPA) acids having different structures of the activated carboxyl groups were prepared in situ by the reaction of the diacids with TsCl/DMF/Py containing various amounts of DMF. The polycondensation involved by these carboxyl groups using 0.3 equivalents of DMF relative to dicarboxylic acids and by dropwise addition of bisphenols significantly improved the η_inh values of the IPA/TPA(50/50)‐2,2‐bis(4‐hydroxyphenyl)propane (BPA) and IPA/TPA (50/50)‐methylhydroquinone polymers. It was also the case for the polycondensations of BPA and various combinations of aromatic dicarboxylic acids other than IPA/TPA. It is likely that various structures of the aggregates between the activated IPA and TPA are involved in a mixture of them, and evaluated by melting points of mixtures of their dimethyl esters prepared by quenching the aggregates with methanol. Effect of the amounts of DMF and the dropwise addition of BPA upon the distribution of oligomers formed at an earlier stage was also examined.