Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4

Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.     

Nickel(II) and copper(II) complexes of mixed benzene-triazolehemiporphyrazines

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Alkaline N-deacetylation of chitin enhanced by flash treatments. Reaction kinetics and structure modifications

A new method for N-deacetylation of chitin is proposed in which a polymer almost free of N-acetyl groups is obtained by flash treatment. The reaction is carried out in 40% NaOH solution for 30–270 s at 140–190°C, using saturated steam.

Flash treatment was found to proceed faster and with a higher activation energy for the deacetylation reaction (E_a = 36 kcal mol⁻¹) compared with the traditional treatment (E_a = 11 kcal mol⁻¹). X-Ray diffractometry, CP-MAS ¹³C-NMR and FTIR spectroscopy show that the flash treatment induces structure modifications; in particular, higher crystallinity indexes and specific area values are observed together with changes in the local and chain conformation.     

Liquid chromatography/mass spectrometry sequencing approach for highly sulfated heparin-derived oligosaccharides

Liquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of -32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10000 Da and a net charge of -60.     

Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33–39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean±S.E.M. plasma half-lives of recombinant hSHBG (t_1/2 0.11±0.03 h and t_1/2β 18.94±1.65 h) are shorter than previously measured for natural hSHBG (t_1/2 3.43±0.72 h and t_1/2β 38.18±7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr⁷ does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn³⁵¹ and Asn³⁶⁷. However, a 1.5–1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3–2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.     

Heats of reaction of HMo(CO)3(C5R5) (R = H, CH3) with diphenyldisulfide and of formation of the clusters [PhSMo(CO)x(C5H5)]2, X = 1,2. Thermodynamic study of molybdenum-sulfur bond strengths

The enthalpies of reaction of HMo(CO)₃C₅R₅ (R = H, CH₃) with diphenyldisulfide producing PhSMo(CO)₃C₅R₅ and PhSH have been measured in toluene and THF solution (R = H, ΔH= −8.5 ± 0.5 kcal mol⁻¹ (tol), −10.8 ± 0.7 kcal mol⁻¹ (THF); R = CH₃, ΔH = −11.3±0.3 kcal mol⁻¹ (tol), −13.2±0.7 kcal mol⁻¹ (THF)). These data are used to estimate the Mo---SPh bond strength to be on the order of 38–41 kcal mol⁻¹ for these complexes. The increased exothermicity of oxidative addition of disulfide in THF versus toluene is attributed to hydrogen bonding between thiophenol produced in the reaction and THF. This was confirmed by measurement of the heat of solution of thiophenol in toluene and THF. Differential scanning calorimetry as well as high temperature calorimetry have been performed on the dimerization and subsequent decarbonylation reactions of PhSMo(CO)₃Cp yielding [PhSMo(CO)₂Cp]₂ and [PhSMo(CO)Cp]₂. The enthalpies of reaction of PhSMo(CO)₃Cp and [PhSMo(CO)₂Cp]₂ with PPh₃, PPh₂Me and P(OMe)₃ have also been measured. The disproportionation reaction: 2[PhSMo(CO)₂Cp]₂ → 2PhSMo(CO)₃Cp + [PhSMP(CO)Cp]₂ is reported and its enthalpy has also been measured. These data allow determination of the enthalpy of formation of the metal-sulfur clusters [PhSMo(CO)_nC₅H₅]₂, N = 1,2.     

Dinuclear silver(I) complexes of 24-membered bibracchial tetraimine Schiff base macrocycles derived from 2,5-diformylthiophene

The cyclocondensation of 2,5-diformylthiophene and the amines N,N-bis-(2-aminoethyl)-2-phenylethylamine, N,N-bis-(2aminoethyl)-t-butyl-amine and N,N-bis-(2-aminoethyl)-t-butyl-amine in the presence of silver(I) salts yields homodinuclear bibracchial tetraimine Schiff base macrocyclic complexes. The structures of two such complexes are also reported. The complex Ag₂L⁴(NO₃)(PF₆) (2) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 12.834(6), B = 13.183(6), C = 14.588(7) Å, = 64.86(4), β = 79.77(4), γ = 69.44(3)° with Z = 2; there is a monodentate and singly bridging nitrate anion present and the Ag---Ag separation is 4.161 Å. The complex Ag₂L⁴(CH₃CN)₂(BF₄)₂·CH₃CN (9) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 9.297(4), B = 12.985(3), C = 21.770(5) Å, = 91.570(10), β = 92.33(3), γ = 97.92(3) ° with Z = 2; there is a strongly bonded acetonitrile molecule coordinated to each silver atom and the Ag---Ag separation is 4.920 Å.     

Studies of the cation complexing ability of crowns Part I. Synthesis, characterization and crystal structure of diazacrowns and their barium complexes

A number of N,N′-bis(4-substituted phenyl)-1,7-diaza-12-crown-4 and N,N′-bis(4-substituted phenyl)-1, 10-diaza-18-crown-6 (where the substituents are OCH₃, CH₃, H, Cl, respectively) have been prepared by cyclization reaction of a ditosylate with the appropriately substituted diol. These new macrocyclic ligands have been characterized by means of elemental analysis, IR, ¹H NMR and MS spectra. The crystal structures of N,N′-bis(4-chlorophenyl)-1,10-diaza-18-crown-6 (21) and its complex with barium thiocyanate Ba(SCN)₂ (22) have been determined by single crystal X-ray diffraction. The crystallographic data are as follows: 21: C₂₄H₃₂Cl₂N₂O₄, orthorhombic, P2₁2₁2₁, A=4.852(1), B=11.989(2), C=41.231(8) Å, V=2398.7(8) ų, Z=4; 22: C₂₆H₃₂Cl₂N₄O₄S₂Ba, monoclinic, P2₁/c, A=8.801(2), B=11.653(9), C=15.756(6) Å, ß=105.96(3)°, V=1553.7(14) ų, Z=2. In the complex, the Ba atom is eight-coordinate (O(1), O(2), O(1)′, O(2)′, N(1), N(1)′, N(21), N(21)′) to form a distorted D_6h geometry with the Ba atom at the center of crystallographic symmetry.     

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (, β₁, β₂, γ, δ and ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by , β₁, γ, δ and were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.     

Application of the hydro(solvo)thermal technique to the synthesis of metal carbonyl chalcogenide clusters Part 3. Synthesis, structural characterization, and spectroscopic studies of the clusters [{M(CO)4}n(MS4)] (M = Mo, W; N=1, 2)

The methanothermal reactions of M(CO)₆ (M = Mo, W) with Na₂S₂ gave a series of homonuclear clusters [{M(CO)₄}_n(MS₄)]²⁻ (M=Mo, W; N=1, 2), i.e. (Ph₄P)₂[(CO)₄Mo(MoS₄)] (I), (Ph₄P)₂[(CO)₄W(WS₄)] (II), (Ph₄P)₂[(CO)₄Mo(MoS₄)Mo(CO)₄] (III) and (Ph₄P)₂[(CO)₄W(WS₄)W(CO)₄] (IV). The two dimers, I and II, as well as the two trimers, III and IV, are isostructural to each other, respectively. All compounds crystallize in the triclinic space group with Z=2. The cell dimensions are: a=12.393(8), b=19.303(9), c=11.909(6) Å, =102.39(5), β=111.54(5), γ=73.61(5)°, V=2522(3) ų at T=23 °C for I; a=12.390(3), b=19.314(4), c=11.866(2) Å, =102.66(2), β=111.49(1), γ=73.40(2)°, V=2511(1) ų at T=23 °C for II; a=11.416(3), b=22.524(4), c=10.815(4) Å, =91.03(2), β=100.57(3), γ=88.96(2)°, V=2733(1) ų at T=−100 °C for III, a=11.498(1), b=22.600(4), c=10.864(3) Å, =90.92(2), β=100.85(1), γ=88.58(1)°, V=2771(2) ų at T=23 °C for IV. The dimers are each formed by the coordination of the tetrathiometalate as a bidentate chelating ligand to an M(CO)₄ fragment while addition of another M(CO)₄ fragment to the dimers results in the trimers. All compounds contain both tetrahedral and octahedral metal centers with the formal 6+ and 0 oxidation states, respectively.     

Ruthenium complexes containing tertiary phosphines and imidazole or 2,2′-bipyridine ligands

Complexes RuCl₃(PPh₃)L₂ (L = MeIm (1a, Im (1b)) and [RuCl₂(PPh₃)₂(bipy)]Cl·4H₂O (2) have been synthesized via the ruthenium(III) precursor RuCl₃(PPh₃)₂ (DMA), and characterized, including an X-ray structural analysis for 1a (MeIm = N-methylimidazole, Im = imidazole, bipy = 2,2′-bipyridyl, and DMA = N, N′-dimethylacetamide). Crystals of 1a are monoclinic, space group P2₁/n, A = 10.5491(5), B = 20.4934(9), C = 12.8285(4) Å, β = 90.166(4)°, Z = 4. The structure, which reveals a mer configuration for the chlorides, and cis-methylimidazoles, was solved by conventional heavy atom methods and was refined by full-matrix least-square procedures to R = 0.041 and R_w = 0.042 for 3328 reflections with I 3σ(I). From the RuCl₂(PPh₃)₃ precursor, the ruthenium(II) complexes RuCl₂(PPh₃)₂L₂ and [RuCl(PPh₃)L₄]Cl have been made (L = Im or MeIm), while [RuCl(dppb)Im₃]Cl has been made from [RuCl₂(dppb)]₂(μ-dppb) (dppb = Ph₂P(CH₂)₄PPh₂).     

π-Arene complexes.: Part 12. Synthesis of chromiumtricarbonyl complexes of quinoline and N-methylindoles and selected metallation reactions

The lithiation of indole, using a slight excess of n-butyl lithium in THF, followed by methylation and reaction with [Cr(CO)₆] in refluxing dibutyl ether, resulted in the formation of [Cr(η⁶-N-methylindole)(CO)₃] (1a) and [Cr(η⁶-N-methyl-2-methylindole)(CO)₃] (1b). In contrast, lithiation of quinoline in THF, silylation and the subsequent reaction with [Cr(CO)₆] under similar reaction conditions, afforded [Cr(η⁶-N-trimethylsilyl-2-butyl-1,2-dihydroquinoline)(CO)₃] (2) and [Cr(η⁶-{2-butyl-1,2,3,4-tetrahydroquinoline})(CO)₃] (3). The formation of [Cr(η⁶-2,2′-bis{N-methylindolyl})(CO)₃] (4) implied lithiation at the 2-position of 1a. However, metallation at the 7-position was also indicated during the same reaction. In the presence of [Mn(CO)₅Br], product 4 and the transmetallation product [Cr(η⁶-{7-(N-methylindolyl)Mn(CO)₅})(CO)₃] (5) were isolated. Reaction with titanocene dichloride gave [Cr(η⁶-{2-(N-methylindolyl)TiCp₂Cl})(CO)₃] (6), which slowly converted into [TiCp₂{Cr(η⁶-2-(N-methylindolyl)(CO)₃}₂] (7).     

Selective addressing of heteroditopic ligands by iron(II) and platinum(II)

The heteroditopic ligand 4′-(4,7,10-trioxadec-1-yn-10-yl)-2,2′:6′,2″-terpyridine, 2, contains an N,N′,N″-donor metal-binding domain that recognizes iron(II), and a terminal alkyne site that selectively couples to platinum(II). This selectivity has been used to investigate routes to the formation of heterometallic systems. The single crystal structures of ligand 2 and the complex [Fe(2)₂][PF₆]₂ are reported.     

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C(5)-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments.     

One-dimensional polymeric chlorocadmate(II) systems of N-methylpiperazinium and N,N′-dimethylpiperazinium compounds: synthesis, structural and thermal properties

The chlorocadmate(II) systems of (H₂me₂pipz)[Cd₂Cl₆(H₂O)₂] (1) and (H₂mepipz)₂[Cd₃Cl₁₀(H₂O)] (2) (L = me₂pipz = N,N′-dimethylpiperazine; L′ = mepipz = N-methylpiperazine) were prepared and their structural and thermal properties investigated. Compound 1 is monoclinic, space group P2₁/c, A = 7.664(1), B = 7.472(4), C = 15.347(1) Å, β = 99.468(7)°, Z = 2, R = 0.024. The crystal structure consists of organic cations and infinite one-dimensional chains of [CdCl₃(H₂O)]_n³⁻ anions. Each Cd atom is octahedrally surrounded by bridged and terminal chlorine atoms and by a water molecule, which is in trans position with respect to the terminal chlorine atom. Inter- and intrachain hydrogen bond interactions between the terminal chlorine atoms and the water molecules contribute to the crystal packing. Compound 2 is orthorhombic, space group Cmc2₁, A = 15.286(3), B = 13.354(3), C = 13.154(3) Å, R = 0.023. The crystal structure consists of organic dications and infinite chains of [Cd₂Cl₆(CdCl₄H₂O]_n⁴⁻ units running along the [001] axis. Each unit is formed of regularly alternate six-coordinated Cd atoms, one of them linking one pentacoordinated Cd atom which completes its coordination througha water molecule. A strong hydrogen bond interaction involving the organic dication and the inorganic chain contributes to the crystal packing. Differential hydrogen bond interaction involving the organic dication and the inorganic chain contributes to the crystal packing. Differential scanning calorimetry measurements did not show the presence of any structural phase transitions. The structures are compared with those of (H₂pipz)[Cd₂Cl₆(H₂O)₂] (3), (H₂mepipz)[Cd₂Cl₆(H₂O)₂]·H₂O (4) and (H₂mepipz)[Cd₂Cl₆] (5) (L = pipz = piperazine, L′ = mepipz = N-ethylpiperazine).     

Sulfated malto-oligosaccharides bind to basic FGF,inhibit endothelial cell proliferation,and disrupt endothelial cell tube formation

The interaction of basic FGF (bFGF) with heparin, heparan sulfate and related sugars can potentiate or antagonize bFGF activity, depending on the size of the saccharide used. Oligosaccharides based on heparin structures, as small as six sugar residues, have been demonstrated to bind to bFGF and block its activity, while larger structures (> 10 sugar residues) tend to potentiate bFGF. In this study we have synthesized a series of compounds designed to test the requirements of size and sulfation for binding of oligosaccharides to bFGF. These oligosaccharides are not derived from heparin, but rather, are linear chains of glucose linked α1–4 (malto-oligosaccharides) that have been chemically sulfated. In addition to bFGF binding, these compounds were tested for their ability to block basic functions of endothelial cells that are known to be mediated, at least in part, by bFGF. We report that the ability of sulfated malto-oligosaccharides to block binding of bFGF to heparan sulfate was dependent on the size (at least a tetrasaccharide is required), and the degree of sulfation. The activity profile in the bFGF ELISA closely correlated with the ability of these compounds to block REEC or HMVEC tube formation on Matrigel. There was a similar relationship of size and sulfation to the ability of the sulfated malto-oligosaccharides to inhibit endothelial cell growth for most human and rat EC types tested. The single exception was REEC cell growth. One isolate of these cells was stimulated by sulfated malto-oligosaccharides rather than inhibited by them, while a second isolate was neither stimulated nor inhibited. This stimulation showed no correlation with inhibition of bFGF binding in the ELISA assay, suggesting that growth of this cell type was probably not dependent on bFGF. Compounds derived from this series of sulfated, malto-oligosaccharides have the potential to function as bFGF antagonists, are relatively easy to produce, and possess relatively low anticoagulant properties. © 1996 Wiley-Liss, Inc.     

Studies on the reactivity of organometallic Ru-, Rh- and Os-pta complexes with DNA model compounds

The reactions of arene–metal complexes (arene = p-cymene, benzene or pentamethylcyclopentadienyl, metal = Ru, Rh or Os), including 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decanephosphine (pta) and chloro co-ligands, with 9-methylguanine, adenine, and a series of nucleosides were studied in water to ascertain the binding modes. The products were characterized by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Tandem mass spectrometry was found to provide excellent information on preferential binding sites. In general, the N7 position on guanine (the most basic site) was found to be the preferred donor atom for coordination to the metal complexes. The X-ray structures of the precursor complexes, [(η⁵-C₁₀H₁₅)RhCl(pta-Me)₂]Cl₂, [(η⁶-C₁₀H₁₄)OsCl(pta)₂]Cl, and [(η⁶-C₆H₆)OsCl₂(CH₃CN)], are also reported.     

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.     

N,N′-Ethylene-bis(3-methoxysalicylideneimine) mononuclear (4f) and heterodinuclear (3d–4f) metal complexes: Synthesis, crystal structure and luminescent properties

The stepwise synthesis of mononuclear (4f) and heterodinuclear (3d–4f) Salen-like complexes has been investigated through structural determination of the intermediate and final products occurring in the process. In the first step, reactions of ligand H₂L and Ln(NO₃)₃ · 6H₂O give rise to three mononuclear lanthanide complexes Ln(H₂L)(NO₃)₃ [H₂L = N,N′-ethylene-bis(3-methoxysalicylideneimine), Ln = Nd (1), Eu (2) and Tb (3)], in which N,N′-ethylene-bis(3-methoxysalicylideneimine) acts as tetradentate ligands with the O₂O₂ set of donor atoms capable of effective coordination. These species are fairly stable and have been isolated. Then, addition of Cu(Ac)₂ · H₂O to the mononuclear lanthanide complex yields expected heterodinuclear (3d–4f) complexes Cu(L)Ln(NO₃)₃ · H₂O [Ln = Nd (4) and Eu (5)] where the Cu(II) ion is inserted to the inner N₂O₂ cavity. Luminescent analysis reveals that complex 3 exhibits characteristic metal-centered fluorescence of Tb(III) ion. However, the characteristic luminescence of both Sm(III) and Eu(III) ions is not observed both in solution and solid state of the complexes.