Charles C. Sweeley

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion☆

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.

Triacontanol: A New Naturally Occurring Plant Growth Regulator

Alfalfa meal and chloroform extracts of the meal have increased the growth and yield of several plant species. A crystalline substance isolated from the active fraction of alfalfa meal increased the dry weight and water uptake of rice seedlings when sprayed on the foliage or applied in nutrient culture. The substance was identified as triacontanol by mass spectrometry. Sprays containing this compound also increased the growth of corn, and barley grown in soil. Authentic triacontanol produced a similar response over a wide range of concentrations on rice grown in nutrient cultures and tomatoes grown in soil.

O-methyl oximes of sugars. Analysis as O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry

Abstract The mass spectra of aldoses, partially methylated aldoses, deoxyaldoses, and ketoses containing 3–7 carbons, were recorded on the ethers of the trimethylsilyl O-methyl oxime derivatives. Each compound gave a distinctive spectrum indicating the carbon-chain length and the location of substituents. The gas-liquid chromatographic properties of most compounds in this study were also examined.

Enzyme replacement in Fabry's disease, an inborn error of metabolism.

Two patients with Fabrys disease were infused with normal plasma to provide active enzyme (ceramide trihexosidase) for hydrolysis of the plasma substrate, galactosylgalactosylglucosylceramide. Maximum ceramide trihexosidase activity occurred 6 hours after infusion of the plasma, attaining a level approximately 150 percent of that in normal plasma; enzymatic activity was detectable for 7 days. The amount of accumulated substrate in the plasma of these recipients decreased about 50 percent on day 10 after infusion. Thus, periodic replacement of ceramide trihexosidase activity in the plasma of patients with Fabrys disease might lead to consistently lower amounts of substrate in the plasma and a decrease in its rate of accumulation in tissues.

Characterization of diacylglycerylphosphocholine molecular species by FAB-CAD-MS/MS: A general method not sensitive to the nature of the fatty acyl groups

The use of negative ion mode fast-atom bombardment-collision-activated dissociation-tandem mass spectrometry (FAB-CAD-MS/MS) for diacylglycerylphosphocholine molecular species determinations was investigated for 24 naturally occurring and synthetic compounds. The previously proposed method of selecting [M-15]− as the parent ion and using the relative abundance of the carboxylate daughter ions to distinguish the positions of esterification was found to be unreliable in cases where the fatty acyl group at sn1 was much larger than that at sn2. The predicted greater abundance of the sn2 carboxylate daughter, relative to the sn1 carboxylate daughter, was also violated when polyunsaturated fatty acyl groups were esterifred at sn2. In addition, several marginal cases were found where the ratio of intensities of the sn2/sn1 carboxylate daughters followed the expected pattern (sn2 > sn1) initially, but reversed over extended scanning time. The use of an alternative FAB-CAD-MS/MS method is proposed where the [M-B6]− ion is selected as the precursor and the relative intensities of the daughters resulting from loss of the free fatty acids at snl and sn2 are determined. In every case examined to date, the ion formed by loss of the free acid from the sn2 position was always more abundant. Because the parent ion is equivalent to the phosphatidic acid ion, this technique should be equally applicable to all other phospholipid classes where this fragment ion is present in the spectrum.

Diagnosis of Glycosphingolipidoses by Urinary-Sediment Analysis

Abstract Biochemical diagnosis of patients with Krabbes leukodystrophy, Gauchers disease, lactosylceramidosis, Fabrys disease, Sandhoffs disease and metachromatic leukodystrophy can be made by ...

Analysis of trimethylsilyl O-methyloximes of carbohydrates by combined gas-liquid chromatography-mass spectrometry.

Abstract We have concluded that the O -methyloxime derivative effectively “labels” the carbonyl portion of the surgar, and probably contributes to the stability of ions containing that part of the molecule. Electron impact-induced cleavage of the carbon-carbon bonds in the sugar, with charge retention on either fragment, provides a mass spectrum that may be used to analyze substituents on any carbon. We have examined the mass spectra of several classes of sugars, as their - O -methyloxime trimethylsilyl ethers, and as their - O -methyloxime peracetates and in most cases ions are found corresponding to the fragmentation pattern proposed here (unpublished results). In addition, the gas-liquid chromatographic retention behavior of these compounds suggests that they may be useful for quantitative determination of carbohydrates.

Characterization of N-ethoxycarbonyl ethyl esters of amino acids by mass spectrometry

Derivatization of amino acids by using ethyl chloroformate-ethanol-pyridine provides volatile products, N-ethoxycarbonyl amino acid ethyl esters (ECEEs), which are easily amenable to GC or GC-MS analysis. MS behavior of these compounds under electron-impact has been studied. The fragments observed in the spectra facilitate recognition of commonly occurring protein amino acids and characterization of unknown analogues.

Structure of HC-toxin, a cyclic tetrapeptide from helminthosporium carbonum

Abstract Helminthosporium carbonum , which is pathogenic to maize, produces a metabolite, HC-toxin ( 1 ), with selective toxicity to susceptible host genotypes. Resistant genotypes and non host plants are tolerant. The structure of 1 (C21H32N4O6) was determined as cyclo [(2-amino-9,10-epoxy-8-oxodecanoyl)-alanyl-alanyl-prolyl] based upon spectral evidence.

[7] Gas chromatography of carbohydrates

Publisher Summary This chapter discusses the preparation of gas chromatography of carbohydrates. Analyses of carbohydrates by gas–liquid partition chromatography are carried out with volatile derivatives of the substances under investigation. Extensive studies are made of the chromatographic behavior of derivatives such as O -methyl ethers, O -acetyl esters, O -trimethylsilyl (TMS) ethers, and various acetals and ketals. The separations can be obtained with any of these modified forms of carbohydrates, the volatilities of which are generally sufficient for gas chromatography under a wide variety of operating conditions. The reaction yielding a derivative must be rapid and quantitative with any carbohydrate; preferably it should be carried out at room temperature, and must be suitable for use over a wide range of concentration of the starting material. Nonpolar derivatives are usually less reactive than polar ones and are preferred for quantitative determinations. Packed columns or open tubular columns may be operated isothermally or with temperature programming. A short packed column containing a low percentage of dimethylsilicone gum (SE-30) is the most useful, all-purpose column for the determination of carbohydrates by gas chromatography.