C. Allen Bush

Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatography

Abstract High-pressure liquid chromatography (hplc) of N -acetylglucosamine (GlcNAc) and galactosamine (GalNAc) containing carbohydrates was performed on several reverse-phase silica columns. Nanomolar level detection was accomplished using far uv-absorbance monitoring. Baseline separations of the α and β anomers of GlcNAc, chitobiose, chitotriose, and chitotetraose were observed with water elution of the reverse-phase column. With the addition of up to 3% acetonitrile to the eluting solvent, similar resolution of chitin oligomers up to a chain length of seven was observed. Anomerization of the residue could be followed by isolation of either anomeric peak with subsequent rechromatography. Reduction of chitotriose with borohydride yielded a single sharp peak with a retention volume similar to that of the reducing trisaccharide. Semipreparative reverse-phase hplc allowed for the separation and identification by 13 C NMR of the GlcNAc-α-1→6 GlcNAc disaccharide from the β-1→6 isomer. Methyl glycosides of GalNAc and GlcNAc were shown to have retention times much longer than the free sugar.

The combination of normal-phase and reverse-phase high-pressure liquid chromatography with NMR for the isolation and characterization of oligosaccharide alditols from ovarian cyst mucins

Normal-phase high-pressure liquid chromatography (HPLC) on amino-bonded silica with elution by aqueous acetonitrile is shown to be an especially suitable complement to reverse-phase HPLC on octadecyl silica for the fractionation of oligosaccharide alditols produced by alkaline borohydride degradation of mucin glycoproteins. The former technique separates well on the basis of molecular size, while the latter method shows selectivity for stereoisomers. Stereoisomeric pairs of tetra-, penta- and hexasaccharide alditols show relative retention times ranging from 3 to 12, resulting in excellent preparative separations in reverse-phase chromatography. From a single ovarian cyst glycoprotein, H and Lewis b active, 13 oligosaccharides, representing essentially the entire carbohydrate content, have been isolated. The structures of 12 of the oligosaccharides have been determined by 1H-NMR spectroscopy. For those oligosaccharides which have been isolated from other sources and whose NMR spectra have been previously reported, unambiguous structural identification follows directly. Structures of oligosaccharides differing by only one or two residues from those whose NMR spectra are known may be deduced by a simple algorithm utilizing chemical shift analogies.

Charaterization of lacto-N-hexaose and two fucosylated derivatives from human milk by high-performance liquid chromatography and proton NMR spectroscopy

Abstract The branched hexasaccharide whose trivial name is lacto-N-hexaose (LNH) as well as a mono- and a difuco-derivative were isolated from human breast milk of a Lewis a positive donor by high-performance liquid chromatography (HPLC). Reversed-phase HPLC on C 18 columns which have been used previously in our laboratory for successful separations of the smaller milk oligosaccharides were not effective in this system. However, normal-phase HPLC on an amino-bonded silica column (Varian AX-5) gave both analytical and preparative separations. The identity of the LNH core structure was verified both HPLC and by NMR spectroscopy of the fucosidase digestion products of the fucosylated oligosaccharides. LNH was identified by NMR spectroscopy of the reduced and acetylated derivative whose high field spectrum has been previously reported. The positions of fucosylation of β-GlcNAc residues were determined by the characteristic chemical shifts of the resonances assigned to α-fucose H1 and H5. The assignment of many of the resonances to sugar ring protons was possible using differences decoupling methods. The observation of nuclear Overhauser effects between the anomeric protons and the aglycone protons of adjacent residues confirmed the assignment of the glycosidic linkages.

Circular dichroism of oligosaccharides containing N-acetyl amino sugars

Abstract The circular dichroism (CD) spectra of N-acetyl amino monosaccharides and some of their methoxy derivatives, N-acetyl disaccharides, the chitin series of oligosaccharides (disaccharide through hexasaccharide), and a glycopeptide linkage compound 2-acetamido-1-N- l -aspartyl-2-deoxy-β- d -glucopyranosyl-amine (GlcNAc-Asn) were measured in order to determine which structural features are influential in determining the CD in the 190–220 nm region. The CD of the α anomers of 2-acetamido-2-deoxy- d -glucose and -galactose (GlcNAc and GalNAc, respectively) are identical to those of the corresponding equilibrium mixture whereas, for 2-acetamido-2-deoxy- d -mannose, the α anomer has a significantly more positive CD in the n-π ∗ (208–210 nm) region than does the β anomer. The CD of the α- and β-methoxy glycosides of GlcNAc and GalNAc show negative CD bands of comparable magnitude in the n-π ∗ region (209 nm) and positive CD bands of differing magnitudes in the π-π ∗ (190 nm) region. The CD spectra of β-1–4 and β-1–6 linked 2,2′-diacetamido-2,2′-dideoxyglucosyl glucoses both show a negative CD band in the n-π ∗ region and a positive band in the π-π ∗ region. The CD curves of the chitin series of oligosaccharides all exhibit a negative CD band at about 210 nm, followed by a positive band at about 192 nm. A small progressive increase in molar ellipticity per sugar residue for both the positive and negative bands with an increase in chain length is observed. The CD spectra of GlcNAc-Asn at three different pHs differ greatly from those of N-acetyl mono- and disaccharides. The CD spectrum of GlcNAc-Asn indicates a strong interaction of the exciton type between chromophores. It is concluded from the CD data that the n -π ∗ band for these compounds is not very sensitive to anomeric configuration or to disaccharide linkage of the 1–4 and 1–6 types. In contrast, the CD in the π-π ∗ region does show variation with anomeric configuration, substituent position, and linkage position. In particular, substituents at the 3 position lead to a small negative CD at 190 nm while substituents at the 4 position lead to enhanced positive CD at 190 nm.

The complete structure of the capsular polysaccharide from Streptococcus sanguis 34

A complete structure for the capsular polysaccharide of Streptococcus sanguis 34, which is responsible for coaggregation of this bacterium with Actinomyces viscosus T14V, an important step in the formation of dental plaque, is proposed, based partly on the 1H-n.m.r. spectrum, which was assigned by 2-dimensional COSY, homonuclear Hartmann-Hahn spectroscopy, and nuclear Overhauser effects. A phosphoric diester linkage was identified from the 31P-n.m.r. spectrum, and the linkage was determined from long range 1H-31P correlation spectroscopy. The proposed structure is supported both by methylation analysis before and after dephosphorylation and by g.l.c.-m.s. of the phosphorylated monosaccharides as their trimethylsilyl derivatives, isolated by partial hydrolysis of the polysaccharide. The structure is composed of repeating linear hexasaccharide units joined by a phosphoric diester linkage, i.e., [----PO4(-)----6)-alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-be ta -D-Glcp- (1----6)-beta-D-Galf-(1----6)-beta-D-GalpNAc-(1----3)-alpha-D-Galp -(1----]n.

Resolution of some glycopeptides of hen ovalbumin by reverse-phase high-pressure liquid chromatography

Reverse-phase high-pressure liquid chromatography has been shown to be useful in fractionation of oligosaccharides containing N-acetyl amino sugars on the basis of stereochemical differences. Data in this paper show this same stereochemical selectivity can be obtained for glycopeptides. Using dilute aqueous phosphate for elution, three major high-mannose glycopeptides from ovalbumin can be separated on the analytical scale in a few minutes and the same method can also be used for preparation of the glycopeptides on the milligram scale. High-field proton NMR spectroscopy is used to identify two of the compounds as the mannose-5 and -6 glycopeptides (E3 and D3) and the third as the mannose-6 glycopeptide substituted by two N-acetyl glucosamine (GlcNAc) residues (C3B). The NMR spectra and analytical chromatograms of the glycopeptides show high purity and freedom from contaminants previously identified in ovalbumin glycopeptides.

Structure determination of N-acetyl amino sugar derivatives and disaccharides by gas chromatography and mass spectroscopy

Abstract Gas chromatography retention times and mass spectra fragmentation patterns were obtained for the trimethylsilyl ethers of 2-acetamindo-2-deoxy- d -glucose, -galactose, and -mannose, (GlcNAc, GalNAc, and ManNAc, respectively), β-1,3-dimethoxy-GlcNAc, the α- and β-methoxy glycosides of GlcNAc and GalNAc, the α- and β-methoxy furanosides of GalNAc, and β-1-4 and β-1-6 linked 2,2′-diacetamido-2,2-dideoxy-glucosylglucoses, and a glycopeptide linkage compound, 2-acetamido-1- N -β- l -aspartyl-2-deoxy-β- d -glycopyranosylamine (GlcNAc-Asn). The mass spectra of these derivatives are discussed in detail. From the mass spectra of the trimethylsilyl derivatives of the free and partially methylated monosaccharides it is possible to determine which and how many positions are methylated and whether the ring is in the pyranose or furanose form. Fragmentation patterns of trimethylsilyl disaccharide derivatives show that it is possible to determine linkage position. Anomeric configuration and other steriochemical features such as axial or equatorial configuration cannot be easily determined from fragmentation patterns but can be determined from gas chromatography retention times, optical rotatory dispersion, and circular dichroism (3). A combination of these methods provides a means of solving the structural and stereochemical problems of carbohydrates.

Structure of a new hexasaccharide from the coaggregation polysaccharide of Streptococcus sanguis 34

The major constituent of a coaggregation polysaccharide from Streptococcus sanguis 34 is a hexasaccharide, isolated as the alditol. The proposed structure is alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -beta-D-Galf- (1----6)-beta-D-GalpNAc-(1----3)-D-Galol, based upon g.l.c.-m.s. of alditol acetates and partially methylated alditol acetates, f.a.b.-m.s., 1H-n.m.r. spectroscopy, g.l.c.-m.s. of trimethylsilylated (+)- and (-)-2-butyl glycosides, and cleavage by alpha-N-acetylgalactosaminidase. The structural deduction was facilitated by cleavage of the hexasaccharide at the furanoside linkage by 48% hydrogen fluoride, and reduction of the product, to yield alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -D-Galol.

Enhanced spectral resolution in 2D NMR signal analysis using linear prediction extrapolation and apodization

Abstract An alternative method to FFT combining linear prediction extrapolation and linesharpening apodization has been applied to complex NMR signal analysis. This method, in common with other LP procedures with a matrix decomposition routine, avoids truncation artifacts and increases resolution significantly. The advantage is particularly important for the t, dimension in 2D spectra which are invariably severely truncated as a result of practical limits on data acquisition time and storage space. Because of the incorporation of an efficient autoregression algorithm, our procedure can be used for routine spectral analysis of complex 1 D and general 2D spectra of complex molecules with computational resources which are commonly available in NMR laboratories.

The determination of the structure of blood group oligosaccharides from fully assigned 1H-n.m.r. spectra for solutions in non-aqueous solvents

The fully assigned 1H-n.m.r. spectra of a blood group A tetrasaccharide and of a blood group H hexasaccharide in dimethyl sulfoxide and in pyridine by use of two-dimensional COSY and homonuclear Hartmann-Hann coherence transfer methods are reported. The 1H-n.m.r. spectra of both of these compounds in deuterium oxide had been previously assigned. Since the relative proton chemical shifts in the three solvents are quite different, resonances which overlap or are strongly coupled for one solvent may be well resolved for another, thus providing an extension of the method of complete proton assignments for determination of the structure of complex oligosaccharides. Although the rotational correlation times (tau c) of these oligosaccharides are similar to the reciprocal of the spectrometer frequency, either negative or positive n.O.e. values were measurable for both oligosaccharides in all three solvents in one-dimensional difference spectroscopy by taking advantage of the dependence of tau c on the solvent viscosity and, thus, on sample temperature. Whereas n.O.e. depend strongly on temperature and solvent viscosity, the ratios of the effects between protons on the same pyranoside ring and those on different rings were observed to be similar, suggesting that the oligosaccharide conformations are not strongly dependent on solvent or temperature.