Albin Otter

Synthesis and conformational studies of the tyvelose capped, Lewis-x like tetrasaccharide epitope of Trichinella spiralis

Chemical synthesis and high resolution NMR studies are reported for the tetrasaccharide epitope and constituent structures that occur as terminal elements of Trichinella spiralis cell surface glycans. The 2-(trimethylsilyl)ethyl and methyl glycosides of Lewis-x type trisaccharides, in which beta GalNAc replaces beta Gal were synthesized from monosaccharide synthons utilizing thioglycoside and halide-ion glycosylation methods. The unique 3,6-dideoxy-beta-D-arabinohexopyranosyl residue that caps the structure was introduced to selectively protected 2-(trimethylsilyl)ethyl and methyl trisaccharide glycosides by utilizing an insoluble silver zeolite catalyst and a glycosyl halide. In separate reactions not only were the principal targets obtained but also the corresponding alpha-linked tetrasaccharides. A comparison of the NMR spectra of the methyl tetrasaccharide glycosides showed that at the site of the alpha-linked tyvelose structure, specific GalNAc resonances (C-1, C-2, C-3) possess uncharacteristically wide 13C (8-21 Hz) and 1H lines. The beta-linked tetrasaccharide glycoside, that represents the native parasite epitope, exhibited only narrow line widths (3 Hz, 13C). Since NOE derived distance constraints for the alpha-linked tyvelose structure were not consistent with the existence of unusual glycosidic conformers, the origin of the limited number of wide lines was attributed to local rigidity in the GalNAc residue, at the site of tyvelose glycosylation.

Synthesis and 1H NMR characterization of the six isomeric mono-O-sulfates of 8-methoxycarbonyloct-1-yl O-β--galactopyranosyl-(1 → 4)-2-acetamido-2-deoxy-β--glucopyranoside

All six isomeric mono-O-sulfates of β-d-Galp-(1 → 4)-β-d-GlcpNAc-O-(CH2)8COOMe (LacNAc-MCO) have been chemically synthesized and characterized by high resolution 1H NMR spectroscopy. Sulfation causes characteristic substitution-site-specific downfield shifts of 1H NMR signals. The 4C1 chair conformation of both pyranose residues of LacNAc are unaffected by mono-O-sulfation, and, with the exception of the 3-O-sulfate derivative, glycosidic torsion angles are also unaffected.

A 1H and 13C NMR study on the role of salt-bridges in the formation of a type I beta-turn in N-acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2.

The conformation of the tetrapeptide N-Acetyl-Asp7-Glu8-Lys9-Ser10-NH2, a fragment of the type I collagen alpha-1 chain N-telopeptide, has been studied by 1H and 13C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD3OH/H2O (60/40) at ca. neutral pH the tetrapeptide forms a beta-turn, stabilized by a hydrogen bond between NH(S10) and CO(D7) and a strong salt-bridge between COO-(E8) and NH3+(K9). The beta-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H alpha (E8) coupling constant, NH(E8) low-field shift and the temperature coefficient of NH(S10), whereas the conclusion regarding the salt-bridge is based on 13C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E8-K9 charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.

An NMR study of the conformations of N-terminal substance P fragments and antagonists.

Three N-terminal fragments of the neurotransmitter Substance P as well as two antagonist heptapeptides containing D-amino-acid residues were studied using different 1D and 2D NMR techniques. Total nonexchangeable 1H-NMR assignments were carried out in D2O and the NH protons were assigned in H2O by means of COSY experiments. The spectral data indicates that there are no preferred conformations for the backbone. The N-terminal tetrapeptide SP1-4-OH exists as a mixture of cis/trans isomers and this effect was studied as a function of pH.

Processing alpha-glucosidase I is an inverting glycosidase.

Abstractα-Glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the α-glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(α1-2)Glc(α1-3)GlcαO(CH2)4COOCH3 was directly monitored by 1H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released β-glucose concomitant with the formation of the Glc(α1-3)GlcαO(CH2)8COOCH3 disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.

Conformational Analysis of the Type II and Type III Collagen α-1 Chain C-Telopeptides by 1H NMR and Circular Dichroism Spectroscopy

The type II and type III collagen alpha-1 chain C-telopeptides are a 27 mer with the sequence NAc-GPGIDMSAFAGLGPREKGPDPLQYMRA and a 22mer,NAc-GGGVASLGAGEKGPVGYGYEYR, respectively. Their conformations have been studied in CD3OH/H2O (80/20) solution by means of two-dimensional proton NMR and CD spectroscopy. Based on TOCSY and NOESY experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H alpha coupling constants, sequential and medium range NOEs and amide proton temperature coefficients. The conformation of the type II C-telopeptide is essentially extended. Evidence from CD spectroscopy suggests that a very minor proportion of the peptide might be helical (ca.8%), but the NMR data show no evidence for a non-linear structure. The observation of reduced amide proton temperature dependence coefficients in certain sections of the molecule can, in view of the absence of any other supporting evidence, only be interpreted in terms of local shielding from solvent for sterical reasons (large hydrophobic side-chains). The conformation of the type III C-telopeptide is mostly extended except for a beta-turn ranging from Gly8 to Glu11, which is stabilized by a hydrogen-bond between NH of Glu11 and the carbonyl group of Gly8. The low temperature coefficient of NH(Glu11) and, in particular, the observation of a medium range NOE between H alpha (A9) and NH(E11) corroborate the existence of a beta-turn in this region. Although spectral overlap prevents a precise conclusion with regard to the type of beta-turn present, there is some evidence that it might be type II.

Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective Method for Kd Determination and Screening of α‐Gal Derivatives Binding to Anti‐Gal Antibodies (IgG)

Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was developed as an effective method for rapid determination of Kd values for α‐Gal derivatives binding to human anti‐Gal IgG antibodies. Using this method, Kd values for 23 α‐Gal compounds were determined for the first time, including an α‐Gal terminated N‐linked oligosaccharide which mimics a single N‐glycoform present on the surface of animal cells. A mixture of eight α‐Gal derivatives, a model for an α‐Gal compound library, was successfully screened against this anti‐Gal IgG using FAC/MS. The analyte breakthrough sequence, indicated by the ion chromatogram, reflected the magnitude of the Kd values, confirming its potential application in the screening of new α‐Gal derivatives and mimetics. Ten α‐Gal derivatives were designed and synthesized chemically or enzymatically. Among the compounds analyzed, trivalent compound 26 demonstrated the strongest binding affinity to anti‐Gal IgG with a Kd value of 3.1 µM. The α‐Gal terminated N‐linked oligosaccharide 28 had a Kd value of 8.6 µM.

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg.

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pHs show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.

Two-dimensional 13C NMR study of the substance P fragment ArgProLysPro

Abstract The N-terminal Substance P fragment Arg1Pro2Lys3Pro4 (SP1–4) has been studied by means of natural abundance 13C NMR. By the combined application of various two-dimensional 13 C 1 H correlation techniques, all 13C resonances in both isomeric forms (cis and trans about the Lys3Pro4 peptide bond) including the quaternary carbons could be assigned unambiguously. The experimental details are outlined and the results are discussed in terms of the peptide conformation. From the data it can be concluded that the cis-SP1–4 is stabilized by a charge interaction between the end group of the Lys3 side chain and the deprotonated carboxyl group at the C-terminal end of the peptide. A ring-like interaction with the Arg1 residue can be excluded. Some 13C1H long-range coupling constant have also been measured.

Hydrogen-deuterium isotope effects in 13C NMR experiments of peptides

In our recent NMR studies of linear peptides (l-3), ranging from 4 to 10 amino acids, we observed an interesting and unexpected line-splitting effect in the backbone 13C resonances. This effect was observed first in NAc-D-E-K-S-NH2 ( I ) , a fragment of the type I collagen (L1 chain N-telopeptide (4). A very similar effect was also found in two decapeptides related to the luteinizing hormone-releasing hormone (LHRH), namely the LHRH anti-sense (5) and reversed anti-sense peptide (2). In all these cases the C,, the backbone carbonyl, and the N-acetyl signals were each recorded as two lines separated by approximately 0.1 ppm whereas the signals of the side-chain carbons appeared as sharp lines. As there was independent and unambiguous evidence from the ‘H data that at least two of the three peptides mentioned were neither completely random nor extended, but rather an equilibrium between a type I p-turn with extended conformers ( I ) and a 3 iO-helical turn together with extended conformers (LHRH anti-sense (2))) we first considered the possibility of the presence of two conformers being the reason for the line doubling. However, a more recent study of the Cand N-terminal telopeptides of the type I collagen (u-2 chain (a hexapeptide and a nonapeptide, respectively), which provided up to four lines per a-carbon, revealed that this phenomenon cannot be conformation-related, since for both peptides the proton NMR data (3) clearly confirmed completely extended conformations. This conclusion was based on the absence of any particularly small or large 3 J Nn na _ COUphUg COnStantS, the lack Of NH ( i) -NH ( i + 1) nuclear Overhauser effects, and the absence of reduced NH temperature coefficients, all of which are in general associated with nonextended structures (6, 7). In our quest for the correct explanation of the phenomenon, we found a close relationship between the proton:deuteron ratio in solution and the observed peak intensities. All our experiments were carried out in so-called mixed solvents, containing a mixture of water and an organic solvent, such as trifluoroethanol or methanol, to mimic the physiological, nonaqueous conditions as found, for example, in the growing collagen fibrils. For most of our studies water/methanol solutions were used, typically 60 v% CD30H and 40 v% HzO. Half of the latter was added as D@ to