The activation of fibroblast growth factors by heparin: synthesis, structure, and biological activity of heparin-like oligosaccharides

An effective strategy has been designed for the synthesis of oligosaccharides of different sizes structurally related to the regular region of heparin; this is illustrated by the preparation of hexasaccharide 1 and octasaccharide 2. This synthetic strategy provides the oligosaccharide sequence containing a D-glucosamine unit at the nonreducing end that is not available either by enzymatic or chemical degradation of heparin. It may permit, after slight modifications, the preparation of oligosaccharide fragments with different charge distribution as well. NMR spectroscopy and molecular dynamics simulations have shown that the overall structure of 1 in solution is a stable right-hand helix with four residues per turn. Hexasaccharide 1 and, most likely, octasaccharide 2 are, therefore, chemically well-defined structural models of naturally occurring heparin-like oligosaccharides for use in binding and biological activity studies. Both compounds 1 and 2 induce the mitogenic activity of acid fibroblast growth factor (FGF1), with the half-maximum activating concentration of 2 being equivalent to that of heparin. Sedimentation equilibrium analysis with compound 2 suggests that heparin-induced FGF1 dimerization is not an absolute requirement for biological activity.     

Sulfation of silk fibroin by sulfuric acid and anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated with an aqueous sulfuric acid solution. The sulfated fibroin was characterized by Fourier transform infrared spectroscopy (FTIR), amino acid analysis, and gel filtration chromatography (GFC). Maximum yield was obtained at around 2–4 h, and it decreased at 6 h and more. The molecular size decreased and dispersed with sulfation, and the molecular weight was estimated at around 10,000 by GFC using protein standards. The amino acid composition indicated that the crystal region of the fibroin molecule remained in sulfated fibroin until a sulfation reaction time of 4 h. The incorporation of sulfate groups was confirmed by FTIR and the amount of sulfate groups introduced for 4 h sulfation was estimated in 0.3 mmol/g by acidimetric titration. The efficiency of sulfation was calculated at 15.7%. Blood coagulation was prevented by 20 mg of sulfated fibroin in 1 mL of blood, while original fibroin did not show the effect. This result indicates that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Although a variety of polymer backbones have been used for synthesis of sulfated polymers as anticoagulant materials, no reports are available concerning a sulfated polymer based on a protein backbone. Sulfated fibroin is a new type of anticoagulant material having a protein backbone. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 87: 2377–2382, 2003     

Variable temperature FTIR study on the surface acidity of variously treated sulfated zirconias

The acidity of three related sulfated zirconias has been compared by IR spectroscopy using CO and H₂ as probe molecules. The parent material (SZ) was obtained by calcination of a commercial sulfated zirconium hydroxide. The other two samples, SZ-WW and SZ-SO₃, were obtained from SZ by water washing and by sulfation with gaseous SO₃, respectively. The labile sulfate groups responsible for alkane activation and associated with an IR band at 1406 cm⁻¹ are removed by water washing and increased by SO₃-sulfation. No remarkable differences in the strength of either Lewis or Brønsted acid sites have been observed between SZ and SZ-SO₃. Water washing strongly weakens Brønsted acidity but only slightly weakens Lewis acidity.     

Sulfate metabolism in mycobacteria

Pathogenic bacteria have developed numerous mechanisms to survive inside a hostile host environment. The human pathogen Mycobacterium tuberculosis (M. tb) is thought to control the human immune response with diverse biomolecules, including a variety of exotic lipids. One prevalent M. tb-specific sulfated metabolite, termed sulfolipid-1 (SL-1), has been correlated with virulence though its specific biological function is not known. Recent advances in our understanding of SL-1 biosynthesis will help elucidate the role of this curious metabolite in M. tb infection. Furthermore, the study of SL-1 has led to questions regarding the significance of sulfation in mycobacteria. Examples of sulfated metabolites as mediators of interactions between bacteria and plants suggest that sulfation is a key modulator of extracellular signaling between prokaryotes and eukaryotes. The discovery of novel sulfated metabolites in M. tb and related mycobacteria strengthens this hypothesis. Finally, mechanistic and structural data from sulfate-assimilation enzymes have revealed how M. tb controls the flux of sulfate in the cell. Mutants with defects in sulfate assimilation indicate that the fate of sulfur in M. tb is a critical survival determinant for the bacteria during infection and suggest novel targets for tuberculosis drug therapy.     

Single-Step Per-O-Sulfonation of Sugar Oligomers with Concomitant 1,6-Anhydro Bridge Formation for Binding Fibroblast Growth Factors

Many circulating cancer-related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans—particularly heparan sulfate—at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF-1 and FGF-2. Taking note of the typical pose of l -iduronic acid, we conceived of per-O-sulfonated analogues of such disaccharides, and devised a single-step procedure for per-O-sulfonation of unprotected sugars with concomitant 1,6-anhydro bridge formation to achieve such compounds through direct use of SO₃ ⋅ Et₃N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF-1 and FGF-2 mostly at the sub-micromolar level, although the tetrasaccharide analogue achieved low-nanomolar binding with FGF-2.     

Influence of platinum on ceria sulfation

The influence of platinum on ceria sulfation has been studied using thermogravimetry and infrared spectroscopy. The same types of sulfate species — surface and bulk-like species — are evidenced with or without Pt. Pt does not modify the thermal stability of both types of sulfate. It favors sulfate formation on low-surface-area CeO₂ samples. The reducibility of sulfate by H₂ is promoted by Pt irrespective of the types of sulfate formed on ceria. H₂S, which is formed by H₂ reduction of sulfate, partly remains on the catalyst. It is adsorbed not only on the oxide but also on Pt. Sulfur poisoning of Pt has been shown by IR study of CO adsorption.     

Some new developments in surfactant analysis

Three new analytical methods for surfactants have been developed and evaluated, and the stoichiometry in cationic titration of a dianionic surfactant has been determined. The three methods include: (a) a rapid method for pyrolytic cleavage of hydrophobic groups from surfactants to hydrocarbons, which can be used for quantitative identification of the hydrophobic groups; (b) a molecular weight method for alkylphenoxy and alkoxy polyethoxylates based on sulfation followed by cationic titration; and (c) a microcationic titration method designed for low levels (1–10 microequivalents) of anionic surfactants, which possesses certain advantages over conventional methods. Previously stoichiometry in cationic titration has been reported only for monoanionic surfactants. In a study of a dianionic surfactant,n-octadecylbutane 1,4-disulfate, each sulfate group was found to react in a 1:1 equivalent ratio with a cationic titrant.     

Acidity of sulfated zirconia as studied by FTIR spectroscopy of adsorbed CO and NH3 as probe molecules

The acid properties of pure and sulfated zirconias were studied by FTIR spectroscopy of adsorbed CO and NH₃ as probe molecules. Whereas pure monoclinic zirconia shows only Lewis acidity, sulfation of tetragonal and monoclinic zirconia creates new bridging OH groups. Two types of Brønsted-acidic centers and two types of Lewis-acidic centers with enhanced acid strength were identified. A communication between the different types of Lewis-acidic sites and the related adsorbed sulfate molecules could be shown. The coordination of basic molecules such as CO onto Lewis-acid sites induces a decrease of the intrinsic Brønsted-acidity of the bridging OH groups. These effects are discussed on the basis of a model of the acidic centers that was previously proposed.On leave from Department of Chemistry, Faculty of Science, Minia University, El-Minia, Egypt.     

Regioselective one-pot protection and protection-glycosylation of carbohydrates

Deciphering the roles and structure-activity relationships of carbohydrates in biological processes requires access to sugar molecules of confirmed structure and high purity. Chemical synthesis is one of the best ways to obtain such access. However, the synthesis of carbohydrates has long been impeded by two major challenges--the regioselective protection of the polyol moiety of each monosaccharide building block and the stereoselective glycosylation to produce oligosaccharides of desired length. Here, we review the development of the first regioselective protection-glycosylation and a revolutionary regioselective combinatorial one-pot protection of monosaccharides that can be used to differentiate the various hydroxy groups of monosaccharides with a vast array of orthogonal protective groups in one-pot procedures.     

Design of fully active FGF-1 variants with increased stability

Fibroblast growth factor 1 is a powerful mitogen playing an important role in morphogenesis, angiogenesis and wound healing and is therefore of potential medical interest. Using homologous sequence and structure comparisons, we designed and constructed 16 mutants of FGF-1 with increased thermodynamic stability, as determined by chemical and heat denaturation. For multiple mutants, additive effects on stability were observed, providing mutants up to 7.8 degrees C more stable than the wild-type. None of the introduced mutations affected any FGF-1 biological activities, such as stimulation of DNA synthesis, MAP kinase activation and binding to the FGF receptor on the cell surface. Our study provides a good starting point to improve the stability of FGF-1 in the context of its wide potential therapeutic applications. We showed that a homology approach is an effective method to change the thermodynamic properties of the protein without altering its function.     

Investigating the Role of Sulfate Groups for the Binding of Gd3+ Ions to Glycosaminoglycans with NMR Relaxometry

Glycosaminoglycans (GAGs) are highly negatively charged macromolecules with a large cation binding capacity, but their interaction potential with exogeneous Gd³⁺ ions is under-investigated. These might be released from chelates used as Gadolinium-based contrast agents (GBCAs) for clinical MR imaging due to transmetallation with endogenous cations like Zn²⁺. Recent studies have quantified how an endogenous GAG sequesters released Gd³⁺ ions and impacts the thermodynamic and kinetic stability of some GBCAs. In this study, we investigate and compare the chelation ability of two important GAGs (heparin and chondroitin sulfate), as well as the homopolysaccharides dextran and dextran sulfate that are used as models for alternative macromolecular chelators. Our combined approach of MRI-based relaxometry and isothermal titration calorimetry shows that the chelation process of Gd³⁺ into GAGs is not just a long-range electrostatic interaction as proposed for the Manning model, but presumably a site-specific binding. Furthermore, our results highlight the crucial role of sulfate groups in this process and indicate that the potential of a specific GAG to engage in this mechanism increases with its degree of sulfation. The transchelation of Gd³⁺ ions from GBCAs to sulfated GAGs should thus be considered as one possible explanation for the observed long-term deposition of Gd³⁺ in vivo and related observations of long-term signal enhancements on T₁-weighted MR images.     

Modeling of ferric sulfate decomposition and sulfation of potassium chloride during grate‐firing of biomass

Ferric sulfate is used as an additive in biomass combustion to convert the released potassium chloride to the less harmful potassium sulfate. The decomposition of ferric sulfate is studied in a fast heating rate thermogravimetric analyzer and a volumetric reaction model is proposed to describe the process. The yields of sulfur oxides from ferric sulfate decomposition under boiler conditions are investigated experimentally, revealing a distribution of approximately 40% SO₃ and 60% SO₂. The ferric sulfate decomposition model is combined with a detailed kinetic model of gas‐phase KCl sulfation and a model of K₂SO₄ condensation to simulate the sulfation of KCl by ferric sulfate addition. The simulation results show good agreements with experiments conducted in a biomass grate‐firing reactor. The results indicate that the SO₃ released from ferric sulfate decomposition is the main contributor to KCl sulfation and that the effectiveness of ferric sulfate addition is sensitive to the applied temperature conditions. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4314–4324, 2013     

Investigation on the kinetics of the adsorption process of aliphatic amidpolyamine on cellulose

Investigation has been carried out on the kinetic dependences of the adsorption process of aliphatic amidpolyamine (lamide-1, L-1) on three kinds of bleached pulp (sulfate pulp of hard wood and sulfate and sulfite pulp of soft wood) at temperatures of 0, 20, 40, and 60°C within a concentration interval of 0.030 g/liter + 0.500 g/liter. It has been established that the kinetic process can be described by the Elovich–Tyomkin exponential kinetic equation. The activation energy is of the order of 4.12–4.42 kJ/mol and the entropy factors substantially affect the process speed. The coefficients of the correlations describing the effect of the L-1 solution concentrations on the coefficient of heterogeneity (α) and the initial adsorption speed (V₀) have been determined.     

Investigation on sulfation of modified Ca-based sorbent

When applied for desulfurization in coal combustion process, common Ca-based sorbents have very low Ca conversion and some additives were found can improve the sulfation effect of sorbents greatly. The sulfation characteristics and pore structure of Ca-based sorbent modified by some inorganic compound were investigated in this paper. Although the sulfur capture capability of M-CaO (calcined from modified limestone) greatly exceeds that of N-CaO (calcined from original limestone), the M-CaO sulfation phenomena cannot be explained well based only on pore structure, as has been suggested by some investigators. To find the sulfation mechanism, the crystal structure of M-CaO was measured. Based on experiments and solid-state ions diffusion theory, a new sulfation mechanism is proposed: lattice defects in M-CaO lead to higher calcium conversion. Based on this conclusion, a rule that estimates whether or not a compound can act as a Ca-based sorbent additive is suggested.     

NMR and FT Raman characterisation of regioselectively sulfated chitosan regarding the distribution of sulfate groups and the degree of substitution

Chitosan sulfates (CHS) were prepared with chlorosulfonic acid homogeneously and non-homogeneously. The total degrees of substitution (DS) ascribed to sulfate groups (DS_S) were determined with elemental analysis and the partial DS at 6-O-position was estimated via ¹³C NMR. CHS with diverse total DS_S and sulfation patterns were obtained according to the analysis. The effects of selected reaction parameters that can influence the distribution of sulfate groups were examined. The structure of CHS was then characterized with various NMR techniques, i.e. one- (1D-) and two-dimensional (2D-) NMR, and FT Raman spectroscopy. It was found that the primary hydroxyl groups were always predominantly sulfated for CHS prepared under homogeneous or non-homogeneous conditions and no sulfate groups at 2-N-position could be detected. Finally, the feasibility of using FT Raman spectroscopy as another alternative for determining the total DS_S of CHS was presented.     

纤维素硫酸钠的批量制备及其NaCS-PDMDAAC微胶囊固定化黄色短杆菌培养过程研究

在非均相反应制备纤维素硫酸钠方法的基础上,通过增加液固比,引入搅拌,使体系的温度分布和浓度分布都得到很大改进,由此可以保证产品质量的均一性。用正交试验进行了500 mL反应器制备条件的摸索,并将其逐步放大到1 L、5 L反应器中进行,确立了5 L规模的生产工艺条件。通过对不同规模下制得的硫酸纤维素钠产品在理化性质、制备微胶囊及在微囊化培养等方面对比较,表明研制过程有利于合成出高质量的硫酸纤维素钠,用于制备微胶囊。用NaCS-PDMDAAC(聚二甲基二烯丙基氯化铵—硫酸纤维素钠)微胶囊固定化黄色短杆菌,考察了葡萄糖浓度、摇床转速对黄色短杆菌生长和代谢谷氨酸的影响,确定了一个较优培养条件。胶囊平均生产能力达15.29 g /(Lh)(对微胶囊)。微胶囊固定化黄色短杆菌连续培养12批后,仍具有良好的代谢谷氨酸性能。     

Cooperative, heparan sulfate-dependent cellular uptake of dimeric guanidinoglycosides

Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin-phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.     

Beeinflussung des mechanisch-dynamischen Verhaltens von Polyamid 12 durch Wasser und Weichmacher

Similar to other polyamides, dry polyamide-12 shows two typical relaxation processes in the temperature range between—196°C and 150°C. The relaxation process at 50°C (γ-process, frequency 1 sec^-1) is related to a freezing process of the main chain mobility within the amorphous regions. The character of the molecular motions responsible for this process seems not to be influenced by water or plasticizer. The relaxation behaviour of the γ-process (approx. —150°C, freq. 1 sec^–1) in the presence of water or plasticizer indicates that the rotation of the methylene groups depends on the mobility of the carbonamide groups. A third relaxation process (γ′-process) appears between the β and γ region by adding water. This process is interpreted in a different manner as reported in the literature. Comparing the activation energy of the diffusion of water in polyamide-12 with that of the γ′-process it seems reasonable that above this transition temperature the water molecules are diffusing inter-or intramolecular between the carbonamide groups. At the temperature of the γ′-process, this diffusion process of the water molecules freezes in.     

硫酸酯基的位置与纤维素硫酸酯抗凝血活性关系

采用直接硫酸酯化法和定向硫酸酯化法制备硫酸酯基分别在C-6、C-2和C-6,2的3种纤维素硫酸酯CSC6、CSC2和CSC6,2,并分别通过凝血时间和凝血因子活性分析研究其抗凝血活性,以揭示硫酸酯基的位置与纤维素硫酸酯抗凝血活性之间的构效关系.结果表明,CSC6,2延长凝血酶时间（tTT）和抑制凝血因子Ⅱa的活性突出,CSC2具有较强延长部分凝血活酶时间（tAPTT）和抑制凝血因子Ⅹa的活性,CSC6的抗凝血效果和对凝血因子的抑制作用均不如前二者.硫酸酯基的位置影响纤维素硫酸酯糖链的伸展程度和空间构象,导致其与抗凝血酶ATⅢ的结合程度不同,从而产生不同的抑制Ⅱa、Ⅹa和延长tAPTT、tTT的效果.     

脂肪醇聚氧乙烯醚硫酸钠的制备及分析

介绍了脂肪醇聚氧乙烯醚硫酸钠(简称AES)的制备工艺及分析方法。重点阐述了硫酸化反应条件对产物的影响以及二噁烷的生成机理。