The Dramatic Modulatory Role of the 2\u27N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

Dinuclear dinitrogen and mononuclear paramagnetic complexes of zirconium

The basic focus of this work is the use of the tridentate, mixed donor ligand, [N(SiMe₂CH₂PR₂)₂]⁻ abbreviated as PNP, to generate zirconium compounds that contain dinitrogen ligands in unusual bonding modes, or to allow the stabilization of the very rare zirconium(III) oxidation state. The approach that is used is to combine synthetic methods in organometallic chemistry with semi-empirical molecular orbital calculations as a means of designing new complexes and to rationalize bonding. Reduction of ZrCpCl₂[N(SiMe₂CH₂PPri₂)₂] 2.6, or ZrCl₂(OAr*)[N(SiMe₂CH₂ PPri₂)₂] (Ar* = (C₆H₃Me₂-2,6)) 2.11, under a dinitrogen atmosphere gave complexes { [(PPri₂CH₂SiMe₂)₂N] ZrCp } ₂(μ-N₂) 2.9, and ([(PPri₂CH₂SiMe₂)₂N] Zr(OAr*) }₂(μ-N₂) 2.12, respectively. The X-ray structure determination of these complexes confirmed the presence of an end-on bridging (μ-η¹:η¹-N₂) clinitrogen ligand in 2.9 whereas, 2.12 has a side-on bridging (μ- η²:η²-N₂) dinitrogen ligand. The observed nitrogen-nitrogen bond distances in 2.9 was 1.301 (3) Å and in 2.12, it was 1.528 (7) Å. The resonance Raman spectra of the solid and the solution state samples of 2.9 and 2.2 showed isotope sensitive peaks around 1200 and 730 cm⁻¹ respectively, assigned to the nitrogen-nitrogen stretching of these complexes. Also, the fact that these resonance Raman features are the same in the solid and in the solution states strongly suggest that the mode of bonding of the dinitrogen ligand are same for the respective complexes. The semi-empirical molecular orbital studies performed on the end-on derivative 2.9 and on the side-on derivatives 2.2 and 2.12 show that the π-acceptor interactions involving the π*orbitals of the dinitrogen ligand are significantly different. In the end-on case these interactions give rise to two π-MOs, whereas the side-on cases give rise to one δ -MO and one π-MO where the π-MO was found to be much lower in energy than the δ-MO. Mulliken population and Wiberg indices were calculated to show that in the side-on mode there is greater electron donation into the dinitrogen ligand than in the end-on cases and also the side-on bound N₂ ligand has a very weak nitrogen-nitrogen bond, which is corroborated by the bond length parameters and the resonance Raman data. An analysis of the frontier orbitals of the fragment [(H₃P)₂(H₂N)ZrX], where X = Cl, Cp or OH, shows that the metal-ligand dπ-pπ interactions influence the mode of dinitrogen coordination, i.e., end-on vs. side-on. The paramagnetic zirconium(ffl) complex, Zr(η⁵-C₅H₅)Cl[N(SiMe₂CH₂PPri₂)₂] 4.1, and the corresponding hafnium(III) derivative, Hf(η⁵-C₅H₅)Cl[N(SiMe₂CH₂PPri₂)₂] 4.1, were synthesized by the reduction of the respective dichioro precursors M(η⁵-C₅H₅)Cl₂- [N(SiMe₂-CH₂PPri₂)₂], where M = Zr or Hf. The results of this study show that complex 4.1 is a viable precursor to the synthesis of a variety of derivatives such as the first stable examples of alkyl, aryl and borohydride complexes of zirconium(III). X-ray structure elucidation has been carried out for an alkyl 4.8, phenyl 4.4 and borohydride complex, 4.17. Spectroscopic studies (IR and ESR) indicate that in the case of the alkyl derivatives a weak agostic type interaction may be present between one of the α-hydrogens and the metal. Hydrogenolysis of certain alkyl complexes shows a clean conversion to the mononuclear zirconium(Ill) hydride complex 4.14. This hydride complex has been shown to undergo an insertion reaction with ethylene. The hydride complex also undergoes hydrogen exchange reactions with H-C(sp²) and H-C(sp³) bonds, presumably by way of σ-bond metathesis. Complexes 4.1 and the zirconium(III) borohydride complex undergo reversible disproportionation reactions under a CO atmosphere to give zirconium(IV) and zirconium(ll) derivatives. It was also shown that CH₃CN reacts in a similar fashion with 4.1, however the reversibility of this reaction was not established. In the case of the borohydride complex the reaction with CO proceeds further to give a complex containing a “formyl-ylid” type ligand.Science, Faculty ofChemistry, Department ofGraduat

Addition-transfer reactions of zirconium alkyne complexes

A unique type of reaction, namely the addition-transfer process, has been developed. This reaction transforms the zirconium alkyne complexes, Cp2Zr(η²-alkyne)(PMe₃), to 2-diphenylphosphino and 2-trimethylstannyl alkenyl zirconium compounds by reaction with Ph₂PCI and Me₃SnCl respectively. In the former process, the Ph₂P group is found to be cis to the Cp₂ZrCl group whereas, in the latter case, the Me₃Sn and the Cp₂ZrCl moieties are trans to one another. This reaction was also used to synthesize dienyl zirconium compounds having Ph₂P substitutions on the diene. Preliminary mechanistic proposals suggest that the Ph₂PCI is reacting via a four-centre pathway involving the P-Cl bond and one of the Zr-C bonds of the zirconium alkyne complex; whereas Me₃SnCl reacts via a transition state similar to a π-complex.Science, Faculty ofChemistry, Department ofGraduat

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

Interaction of the verotoxin 1B subunit with soluble aminodeoxy analogues of globotriaosyl ceramides.

Specific hydroxy groups of the terminal disaccharide unit of globotriaosyl ceramide (Gb(3)Cer) were identified from binding studies with deoxyGb(3)Cer and verotoxins (VTs) [Nyholm, Magnusson, Zheng, Norel, Binnington-Boyd and Lingwood (1996) Chem. Biol. 3, 263-275]. Four such hydroxy groups (2", 4", 6" and 6') were each substituted with an amino group and the corresponding deoxyamino globotrioses were conjugated to a ceramide-like aglycone which contained an adamantyl group instead of an acyl chain. Such aglycone modification significantly enhanced the water-solubility of the glycoconjugates [Mylvaganam and Lingwood (1999) Biochem. Biophys. Res. Commun. 257, 391-394]. The inhibitory potential of these soluble aminodeoxy conjugates on the binding of VT(1) to Gb(3)Cer immobilized on an ELISA plate was evaluated. Only the 2" and the 6' deoxyamino conjugates were effective inhibitors (IC(50) 10 microM); the 4" and 6" conjugates were ineffective up to 10 mM. To evaluate the importance of incorporating a rigid adamantyl hydrocarbon group into the ceramide aglycone, globotriaose was conjugated to a t- butylacetamido or an adamantaneacetamido aglycone. By similar ELISAs, only the adamantaneacetamido conjugate inhibited the binding of VT(1) to Gb(3)Cer. When deoxyamino conjugates were adsorbed to silica on TLC plates, only the 2" and 6" conjugates bound VT(1) and VT(2). By a similar TLC assay, acetamido derivatives of 2" and 6' deoxyamino conjugates showed less binding to VT(1) and VT(2). Neither the crystallographically determined structure of the VT(1)-globotriaose complex nor modelling studies fully explain the binding patterns shown by these deoxyamino glycoconjugates. Enhanced solvation of the ammonium group of the deoxyamino conjugate could enforce greater constraints in the binding interactions

Adamantyl Glycosphingolipids Provide a New Approach to the Selective Regulation of Cellular Glycosphingolipid Metabolism*

Mammalian glycosphingolipid (GSL) precursor monohexosylceramides are either glucosyl- or galactosylceramide (GlcCer or GalCer). Most GSLs derive from GlcCer. Substitution of the GSL fatty acid with adamantane generates amphipathic mimics of increased water solubility, retaining receptor function. We have synthesized adamantyl GlcCer (adaGlcCer) and adamantyl GalCer (adaGalCer). AdaGlcCer and adaGalCer partition into cells to alter GSL metabolism. At low dose, adaGlcCer increased cellular GSLs by inhibition of glucocerebrosidase (GCC). Recombinant GCC was inhibited at pH 7 but not pH 5. In contrast, adaGalCer stimulated GCC at pH 5 but not pH 7 and, like adaGlcCer, corrected N370S mutant GCC traffic from the endoplasmic reticulum to lysosomes. AdaGalCer reduced GlcCer levels in normal and lysosomal storage disease (LSD) cells. At 40 μm adaGlcCer, lactosylceramide (LacCer) synthase inhibition depleted LacCer (and more complex GSLs), such that only GlcCer remained. In Vero cell microsomes, 40 μm adaGlcCer was converted to adaLacCer, and LacCer synthesis was inhibited. AdaGlcCer is the first cell LacCer synthase inhibitor. At 40 μm adaGalCer, cell synthesis of only Gb3 and Gb4 was significantly reduced, and a novel product, adamantyl digalactosylceramide (adaGb2), was generated, indicating substrate competition for Gb3 synthase. AdaGalCer also inhibited cell sulfatide synthesis. Microsomal Gb3 synthesis was inhibited by adaGalCer. Metabolic labeling of Gb3 in Fabry LSD cells was selectively reduced by adaGalCer, and adaGb2 was produced. AdaGb2 in cells was 10-fold more effectively shed into the medium than the more polar Gb3, providing an easily eliminated “safety valve” alternative to Gb3 accumulation. Adamantyl monohexosyl ceramides thus provide new tools to selectively manipulate normal cellular GSL metabolism and reduce GSL accumulation in cells from LSD patients