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Biomedical subjects

Richard R Schmidt

Publications and source records attributed to Richard R Schmidt.

At least 19 recordsLinked to original sources
PubMed2006

Synthesis of Staphylococcus aureus lipoteichoic acid derivatives for determining the minimal structural requirements for cytokine induction.

For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b-O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1-4) could be readily obtained and provided for biological studies.

Cytokines↗
PubMed2006

Potential sialyltransferase inhibitors based on neuraminyl substitution by hetaryl rings.

Replacement of the neuraminyl residue by a wide range of aryl rings in transition-state analogs of CMP-Neu5Ac led to readily accessible and potent inhibitors of alpha-(2-->6)- and alpha-(2-->3)-sialyltransferases. The synthesis of a series of potential sialyltransferase inhibitors in which the neuraminyl residue is replaced by hetaryl methylphosphonate residues (thiazole, benzothiazole, benzoxazole, benzothiophene and thiophene) is described in this paper.

Cytidine Monophosphate N-Acetylneuraminic Acid↗
PubMed2006

Lessons learned from clustering of fluorinated glycolipids on selectin ligand function in cell rolling.

Selectin-induced leukocyte rolling along the endothelium is an essential step in the cellular immune response. Since clustering of binding epitopes is thought to be crucial for selectin-ligand interaction, we focused on requirements of ligand clusters in a flow chamber study. Neoglycolipids bearing the binding epitope sialyl Lewis X (sLeX) were used as artificial ligands in model membranes. sLeX ligands or matrix lipids or both were applied with partially fluorinated alkyl chains to increase the ligand cluster separation tendency. Cluster size, their inner structure, and separation distance were evaluated with high resolution by scanning force microscopy (SFM) and correlated with binding or rolling of E-selectin-expressing cells. Fluorination of only one component, sLeX ligand or matrix lipid, leads to a very high separation tendency and impeded cell rolling, although firm cell adhesion could be observed down to 0.005 mol % ligand concentration. As a sign of total immiscibility, cluster size increased with ligand concentration, and resulting excessive ligand densities within the clusters prevent cell rolling. Fluorination of both sLeX ligands and matrix created small clusters which could serve as rolling patches. Our results confirmed that cluster size and separation distance controlled by a certain miscibility of ligand and matrix as well as a sufficiently diluted ligand concentration within the clusters are necessary for cell rolling. Within this work, selectin ligand clustering and its ability to mediate cell rolling are presented as a balance between multivalency of binding and sufficient flexibility of the single epitopes. This might be helpful for better understanding the function of the natural selectin ligands.

Animals↗
PubMed2006

Solid-phase oligosaccharide synthesis of a small library of N-glycans.

Solid-phase oligosaccharide synthesis is based on a hydroxymethylbenzyl benzoate spacer linker which is connected to the Merrifield resin (1 P). Glycosylation was performed with O-glycosyl trichloroacetimidates of glucosamine, mannose, and galactose permitting chain extension (2e, 5e), branching (4b, 7b, 8b), and chain termination (3t, 6t, 9t) with the use of O-benzyl, O-benzoyl, and N-dimethylmaleoyl as permanent and O-fluorenylmethoxycarbonyl (Fmoc) and O-phenoxyacetyl (PA) as temporary protecting groups. The steps required on solid phase are i) glycosylation under TMSOTf catalysis, ii) selective cleavage of the temporary protecting groups, Fmoc with NEt3 and PA with 0.5 equivalents of NaOMe in CH2Cl2/MeOH, and iii) product cleavage from the resin with 4.0 equivalents of NaOMe in CH2Cl2/MeOH and following O-acetylation for convenient product isolation. Thus a highly successful synthesis of a small library of seventeen N-glycan structures was made possible comprising the N-glycan pentasaccharide core structure 53 and two further chain extended hexa- and heptasaccharide N-glycans with a glucosamine or a lactosamine residue, respectively, which is attached to one of the mannose residues of the core structure (56 and 59).

Benzoates↗
PubMed2006

A model for cell-surface-exposed carbohydrate moieties suitable for structural studies by NMR spectroscopy.

In the present study a synthetic glycolipid system is presented that can be readily incorporated into phospholipid micelles and that allows the study of cell-surface-exposed carbohydrate units by high-resolution NMR techniques. Here, we present an efficient route for the synthesis of glycolipid compounds that contain mannose, mannobiose, or mannotriose coupled either directly to an alkyl chain or through a poly(ethylene glycol) linker. Furthermore, we have validated our model system by measuring the binding of cyanovirin N (CV-N), a cyanobacterial protein that binds with nanomolar affinity to the terminal arms of high-mannose structures of the HIV surface-envelope glycoprotein gp120, to glycolipids the carbohydrate portions of which comprise the corresponding high-mannose moieties. From the results of chemical-shift mapping with uniformly (15)N-labelled CV-N, we conclude that binding to the protein occurs at sites similar to those involved in binding the nonconjugated carbohydrates. We characterized the insertion of the glycolipids into dodecylphosphocholine (DPC) micelles by measuring translational diffusion, and we observed that the diffusion constants of the glycolipids were very similar to those of the DPC micelles themselves, but significantly deviated from those of the free glycolipids. We also present experimental proof that the glycolipids remain inserted in the micelles while binding to CV-N. Finally, by addition of a ligand that had a higher affinity to CV-N but which was not attached did not couple to a lipid anchor, CV-N could be released from the glycolipid and, hence, from the micelle-associated state.

Bacterial Proteins↗
PubMed2005

Regulation of hematopoiesis in vitro and in vivo by invariant NKT cells.

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF-secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell-deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid alpha-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.

Animals↗
PubMed2005

Human invariant NKT cells are required for effective in vitro alloresponses.

NKT cells are a small subset of regulatory T cells conserved in humans and mice. In humans they express the Valpha24Jalpha18 invariant chain (hence invariant NKT (iNKT) cells) and are restricted by the glycolipid-presenting molecule CD1d. In mice, iNKT cells may enhance or inhibit anti-infectious and antitumor T cell responses but suppress autoimmune and alloreactive responses. We postulated that iNKT cells might also modulate human alloreactive responses. Using MLR assays we demonstrate that in the presence of the CD1d-presented glycolipid alpha-galactosylceramide (alphaGC) alloreactivity is enhanced (37 +/- 12%; p < 0.001) in an iNKT cell-dependent manner. iNKT cells are activated early during the course of the MLR, presumably by natural ligands. In MLR performed without exogenous ligands, depletion of iNKT cells significantly diminished the alloresponse in terms of proliferation (58.8 +/- 24%; p < 0.001) and IFN-gamma secretion (43.2 +/- 15.2%; p < 0.001). Importantly, adding back fresh iNKT cells restored the reactivity of iNKT cell-depleted MLR to near baseline levels. CD1d-blocking mAbs equally reduced the reactivity of the iNKT cell-replete and -depleted MLR compared with IgG control, indicating that the effect of iNKT cells in the in vitro alloresponse is CD1d-dependent. These findings suggest that human iNKT cells, although not essential for its development, can enhance the alloreactive response.

Antigens, CD1↗
PubMed2005

Chemical synthesis of cholesteryl beta-D-galactofuranoside and -pyranoside.

Two isomeric cholesteryl galactosides, cholesteryl beta-D-galactofuranoside and -pyranoside, have been synthesized by the Koenigs-Knorr reaction. Glycosylation of cholesterol with 2,3,5,6-tetra-O-benzoyl-D-galactofuranosyl bromide, followed by Zemplén saponification with sodium methoxide, gave cholesteryl beta-D-galactofuranoside. By using 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl bromide as the glycosyl donor, followed by alkaline hydrolysis, cholesteryl beta-D-galactopyranoside was obtained. The title compounds were characterized by their IR spectra and by their (1)H and (13)C NMR spectra. Structure considerations of the two cholesteryl galactosides correlated with data in the literature, thus confirming that cholesteryl beta-D-galactopyranoside is an antigenic lipid of Lyme disease agent, Borrelia burgdorferi.

Antigens, Bacterial↗
PubMed2005

The crystal structure of human CD1d with and without alpha-galactosylceramide.

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.

Animals↗
PubMed2005

Reversed approach to S-farnesylation and S-palmitoylation: application to an efficient synthesis of the C-terminus of lipidated human N-ras hexapeptide.

[reaction: see text] A general reversed approach is described to synthesize S-palmitoylated and S-farnesylated peptides via S(N)2 displacement of bromide by reaction of a thiol group containing lipid as nucleophile with bromoalanine-containing peptides as electrophile. By employing this approach, lipidated peptides, including characteristic partial structures of human Ras peptides, were synthesized in good yields. This method gives access to farnesylated, palmitoylated, and doubly lipidated peptides.

Amino Acid Sequence↗
PubMed2005

New methods for the generation of carbohydrate arrays on glass slides and their evaluation.

Glycosides, having spacers functionalized with an aldehyde or a carboxylic group, were immobilized through reductive amination or amidation, respectively, onto amino-functionalized glass slides. Hybridization experiments with lectins exhibited very little nonspecific protein binding, hence precluding the necessity for the blocking of unreacted functional groups on the glass slide. The covalency and the concentration dependency of the sugar ligation to the glass slide were demonstrated; the reversibility and the selectivity of lectin-carbohydrate interactions were shown.

Amides↗
PubMed2005

A new strategy for the synthesis of dinucleotides loaded with glycosylated amino acids--investigations on in vitro non-natural amino acid mutagenesis for glycoprotein synthesis.

The in vitro non-natural amino acid mutagenesis method provides the opportunity to introduce non-natural amino acids site-specifically into proteins. To this end, a chemically synthesised aminoacylated dinucleotide is enzymatically ligated to a truncated suppressor transfer RNA. The loaded suppressor tRNA is then used in translation reactions to read an internal stop codon. Here we report an advanced and general strategy for the synthesis of the aminoacyl dinucleotide. The protecting group pattern developed for the dinucleotide facilitates highly efficient aminoacylation, followed by one-step global deprotection. The strategy was applied to the synthesis of dinucleotides loaded with 2-acetamido-2-deoxy-glycosylated amino acids, including N- and O-beta-glycosides and O- and C-alpha-glycosides of amino acids, thus enabling the extension of in vitro non-natural amino acid mutagenesis towards the synthesis of natural glycoproteins of high biological interest. We demonstrate the incorporation of the glycosylamino acids--although with low suppression efficiency--into the human interleukin granulocyte-colony stimulating factor (hG-CSF), as verified by the ELISA technique.

Amino Acids↗
PubMed2005

Oligomer-to-polymer transition in short ethylene glycol chains connected to mobile hydrophobic anchors.

We studied the structure of short ethylene glycol (EG) chains with N repeating units (EGN, N = 3, 6, 9, 12, and 15) connected to hydrophobic dihexadecyl chains by means of a combination of differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS/WAXS). These synthetic amphiphiles dispersed in water form planar lamellar stacks and hexagonal cylinders confining the EG chains to restricted geometries. Owing to the self-assembly of the anchoring points, the lateral density of EG chains in planar lamella can be quantitatively controlled. Furthermore, the chain-melting phase transition of the anchors enables us to "switch" the intermolecular distance reversibly. SAXS/WAXS results suggest that the shorter EG chains (N = 3, 6, and 9) assume a helical conformation in stacks of planar lamella. When the EG chains are further elongated (N = 12 and 15), the lamellar periodicities cannot be explained by a linear extrapolation of shorter oligomers, but can be interpreted well as polymer brushes following the scaling theorem. Such rich phase behaviors of EGN molecules can be used as a simple model of oligo/poly-saccharide chains on cell surfaces, which act not only as flexible repellers between neighboring cells but also as stable spacers for functional ligands.

Calorimetry, Differential Scanning↗
PubMed2004

In vitro non-natural amino acid mutagenesis using a suppressor tRNA generated by the cis-acting hepatitis delta virus ribozyme.

In vitro non-natural amino acid mutagenesis requires aminoacyl-charged suppressor transfer RNAs which read an internal stop codon. For the synthesis of aminoacyl-tRNAs loaded with non-natural amino acids, T4 RNA ligase is used to ligate a chemically synthesised aminoacyl-dinucleotide to a truncated 74mer tRNA(-CA) lacking the two 3' end nucleotides. The 74mer tRNA(-CA) in turn is generated by run-off transcription from a linearised plasmid encoding the tRNA sequence under control of the T7 promoter. Transcripts with heterogeneous ends are commonly obtained, which interfere with subsequent reactions such as ligation or translation. Here we report an improved procedure for the generation and chromatographic purification of large amounts of homogeneous 3' end tRNA(-CA) by hepatitis delta virus ribozyme cis-cleavage and the first application of this tRNA to in vitro non-natural amino acid mutagenesis. Stop codon suppression is increased compared to conventionally synthesised suppressor tRNA; 2.5 microg of mutated protein was synthesised in a 50 microl batch reaction.

Amino Acid Substitution↗
PubMed2004

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: I. Investigation of the UDP-GlcNAc 2-epimerase functionality.

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design.

Animals↗
PubMed2004

Characterization of ligand binding to the bifunctional key enzyme in the sialic acid biosynthesis by NMR: II. Investigation of the ManNAc kinase functionality.

N-Acetylmannosamine (ManNAc) is the physiological precursors to all sialic acids that occur in nature. As variations in the sialic acid decoration of cell surfaces can profoundly affect cell-cell, pathogen-cell, or drug-cell interactions, the enzymes that convert ManNAc into sialic acid are attractive targets for the development of drugs that specifically interrupt sialic acid biosynthesis or lead to modified sialic acids on the surface of cells. The first step in the enzymatic conversion of ManNAc into sialic acid is phosphorylation, yielding N-acetylmannosamine-6-phosphate. The enzyme that catalyzes this conversion is the N-acetylmannosamine kinase (ManNAc kinase) as part of the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Here, we employed saturation transfer difference (STD) NMR experiments to study the binding of ManNAc and related ligands to the ManNAc kinase. It is shown that the configuration of C1 and C4 of ManNAc is crucial for binding to the enzyme, whereas the C2 position not only accepts variations in the attached N-acyl side chain but also tolerates inversion of configuration. Our experiments also show that ManNAc kinase maintains its functionality, even in the absence of Mg(2+). From the analysis of the STD NMR-derived binding epitopes, it is concluded that the binding mode of the N-acylmannosamines critically depends on the N-acyl side chain. In conjunction with the relative binding affinities of the ligands obtained from STD NMR titrations, it is possible to derive a structure-binding affinity relationship. This provides a cornerstone for the rational design of drugs for novel therapeutic applications by altering the sialic acid decorations of cell walls.

Adenosine Triphosphate↗
PubMed2004

Synthesis of thioglycoside-based UDP-sugar analogues.

Arbuzov reaction of O-acetyl-protected glycosylthiomethyl chlorides with triethyl phosphite and then phosphonate ethyl ester cleavage with trimethylsilyl bromide afforded glycosylthiomethyl phosphonates 13, 18, 22, and 26. These intermediates could be readily transformed into the O-deprotected phosphonates 7-10 and into title compounds 1-4. Similarly, sulfonomethyl phosphonate moieties containing UDP-sugar analogues 5 and 6 were obtained.

Glycosides↗