Marsha E. Daman

13C-N.M.R.-Spectral study of the mode of binding of Mn2+ and Gd3+ to N-acetyl-α-neuraminic acid

Abstract Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd3+ and Mn2+ to N-acetyl-2-O-methyl-α-neuraminic acid (2) and to methyl N-acetyl-2-O-methyl-α-neuraminate (3). The results showed that Gd3+ and Mn2+ bind in the region of the glycerol-1-yl side-chain and the 5-acetamido group of compound 3. When the α-NeuAc derivative contains a carboxylate anion, as in compound 2, multiple, metal-ion-binding sites occur, involving the head (the carboxyl end) and the tail (the glycerol-1-yl and 5-acetamido groups) of the molecule.

13C-n.m.r.-spectral study of the binding of Gd3+ to glycophorin

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd3+ to glycophorin, and also to the tetrasaccharides isolated from glycophorin after treatment of the glycoprotein with NaOH-NaBH4. Gd3+ binds to the tetrasaccharide (both in the isolated, reduced form and when still attached to the native glycoprotein), and, especially, to the alpha-NeuAc residues. In order to cause severe line-broadening of the 13C resonances of alpha-NeuAc, the ratios of the alpha-NeuAc residues of glycophorin, and of the isolated, reduced tetrasaccharide, to Gd3+ were much higher than that needed for causing similar broadening for 2-O-methyl-alpha-NeuAc-Gd3+ solutions. These results indicate that the other carbohydrate residues of the tetrasaccharide may be involved in the binding of Gd3+, producing a stronger metal-ion-binding effect.

Possible role of the carbohydrate residues in the display of the MN blood group determinants by glycophorina

Heterozygous glycophorin AM,N and homozygous glycophorin AM were reductively methylated with 13C-enriched formaldehyde in the presence of cyanoborohydride. Total reductive methylation modified the five lysine residues, and the N-terminal amino acid residues (serine and leucine) of glycophorins AM and AN, respectively. The 13C resonances of the incorporated labels were monitored as a function of the degree of glycosylation of the glycoprotein. While minimal, if any, structural changes were observed near the N-terminal amino acid upon removal of alpha-D-N-acetylneuraminic acid residues, gross structural changes were observed when most of the oligosaccharide chains were removed. We also found that progressive methylation of the lysine residues of glycophorin AM may influence either the chemical shift of one of the nonequivalent methyl groups of the N alpha, N-[13C]dimethyl serine residue, or one of the two states of glycophorin AM.

13C-N.M.R.-spectral study of some mono- and di-O-galactosylated dipeptides. Possible structural perturbations due to O-glycosylation

Abstract Carbon-13 nuclear magnetic resonance data for mono- and di- O -α- and -β- D -galactosylated dipeptides composed of Thr and Gly are presented. The results conclusively show that peptide-bond formation does not affect the chemical shifts of the attached carbohydrate carbon atoms. In the case of the di- O -glycosylated threonyl-threonine, no carbohydrate-carbohydrate interactions could be observed. For some of the mono- O -glycosylated dipeptides, the attached glycosyl group appears to have a peculiar effect on the chemical shifts of some of the carbon resonances of the amino acids.

13C-n.m.r. spectral study of 13C reductively methylated glycopeptides derived from glycophorin A

Abstract 13C-n.m.r. spectral data for 13C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the 13C-n.m.r. spectral data for the 13C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins AM and AN at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin AM.

13C-n.m.r.-spectral study of the mode of interaction of Gd3+ and Mn2+ with two vicinally di-O-D-galactosylated tripeptides

Abstract Natural-abundance, 13 C-n.m.r. spectroscopy was used to study the binding of Gd 3+ and Mn 2+ to two vicinally di- O -α- and -β- d -galactosylated tripeptides composed of Gly and l -Thr. Gd 3+ and Mn 2+ appear to interact with the α- d -Gal groups of the di- O -α- d -galactosylated tripeptide at two sites: near O-6′, and in the vicinity of O-2′ and Thr O-3. The metal-ion-binding to the β- d -Gal groups of the di- O -β- d -galactosylated tripeptide indicates that a strong binding-site exists near O-6′ and, possibly, several weak ones near O-3′ and Thr O-3. In the case of the di- O -α- d -galactosylated tripeptides, vicinal glycosylation appears to have little effect on the metal-ion-binding of the α- d -Gal groups.

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

Abstract 13C nuclear magnetic resonance (n.m.r.) spectral data for 13C reductively methylated N-terminal tryptic glycopeptides and for 13C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins AM and AN are presented. Their 13C chemical shift data are compared with the previously published 13C n.m.r. data for 13C reductively methylated homozygous glycophorins AM and AN in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the 13C resonances of N α ,N-[ 13 C ] dimethyl leucine of glyco-octapeptide AN and of N α ,N-[ 13 C ] dimethyl serine of glyco-octapepti AM indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of 13C reductively methylated glycophorin AM is lost when the glyco-octapeptide AM is produced. The 13C resonance of N α ,N-[ 13 C ] dimethyl leucine of glycooctapeptide AN titrated with a p K a of 7.7 (Hill coefficient ∼ 1 ). The 13C resonance of N α ,N-[ 13 C ] dimethyl serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for 13C-labelled glycooctapeptide AM molecule, the pH dependence of the chemical shift of the 13C resonance of N α ,N-[ 13 C ] dimethyl serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.

Stereochemistry of Gd3+ and Mn2+ interactions with D-gluconamide derivatives by 13C NMR spectroscopy

Natural abundance 13C nuclear magnetic resonance spectroscopy (13C NMR) was used to study the mode of binding of Gd3+ and Mn2+ to the polyol portion of several synthetic D-gluconamides. The results indicate that Gd3+ forms a single, unique binding structure requiring three oxygen atoms. The binding of Mn2+ to the polyol portion of these compounds appears to be nonspecific. The carbohydrate containing model compounds studied may be used to design new metal-ion chelating agents.

13C-N.M.R.-spectral study of the pH behavior of reductively [13C]methylated, glycophorin a glyco-octapeptides and a related glycopentapeptide

Abstract The pH dependence of the labeled-carbon resonances of reductively [ 13 C] methylated compounds tri- l -Ser, glyco-octapeptide A M , asialoglyco-octapeptide A M , glyco-octapeptide A N , asialoglyco-octapeptide A N , and a glycopentapeptide was investigated. The results are discussed relative to those previously observed for reductively [ 13 C]methylated, intact glycophorins A M and A N , and in terms of the mode of display of the MN blood-group specificities by these related glycoproteins. The results indicated that the α- d -NeuAc groups appear to affect the pH-titration results of glyco-octapeptides A M and A N . Moreover, comparison of the pH-titration results for reductively [ 13 C]methylated glyco-octapeptide A M and reductively [ 13 C]methylated asialoglyco-octapeptide A M with those of a reductively [ 13 C]methylated glycopentapeptide and reductively [ 13 C]methylated tri- l -Ser indicated that the other carbohydrate residues present (α- d -GalNAc and β- d -Gal) may also affect the pH-titration results. The reductive-methylation modification appears to affect the chemical shifts of the carbohydrate and peptide carbon atoms of the glycopentapeptide minimally.

The reaction of amines with N,N′-ethylenebis(salicylideneiminato)(nitrato)-iron(III) and related complexes

Abstract The title compound, Fe(salen)NO 3 , was reacted with imidazole, 1-methylimidazole, piperidine, and morpholine in either chloroform or dichloromethane solution. The reactions were monitored with proton NMR and electronic spectra and conductance measurements. The imidazole bases appeared to react with the complex in a 2:1 fashion with displacement of the nitrate, producing a high-spin iron(III) complex. The secondary amines promoted hydrolysis with any trace water present to form [Fe(salen)] 2 O. The chloro complex, Fe(salen)Cl, did not react with the imidazole bases, but did form the μ-oxo complex when a large excess of piperidine was present. The N,N′-phenylenebis-(salicylideneimine) complex, Fe-(salphen)NO 3 , was found to precipitate from an imidazole (im) chloroform solution as the high-spin complex, Fe(salphen)NO 3 ·2im.