John R. Vercellotti

A sensitive estimation of the percentage of guanine plus cytosine in deoxyribonucleic acid by high performance liquid chromatography.

Abstract A rapid high performance liquid chromatographic (HPLC) method is deseribed which offers a fast quantitative procedure for determining the percentage of guanine (G) plus cytosine (C) in DNA preparations after acid hydrolysis in 88% formic acid. As little as 10 μg of DNA is amenable to this analysis. Although thymine is sometimes poorly resolved from impurities at the solvent front, the percentage of G + C was found to be accurately determined using adenine (A) simply from the ratio of concentrations from C/(A + C) or (G + C)/(2 A + G + C). The HPLC method gave values for the percentage of G + C in close agreement with thermal melting point and buoyant density values determined for the DNA of ten bacterial species and two fungi.

Metabolites of Aspergillus versicolor: 6,8-di-O-methylnidurufin, griseofulvin, dechlorogriseofluvin, and 3,8-dihydroxy-6-methoxy-1-methylxanthone

Abstract The isolation of the four title compounds from cultures of Aspergillus versicolor is described and a modified biosynthetic pathway for the conversion of averufin to versicolorin A is proposed.

Chemical-ionization mass spectra of per-O-acetyl-aldononitriles and methylated aldononitrile acetates

Per-0-acetylaldononitriles have been used as derivatives for the separation and identification of mixture of aldoses by gas-liquid chromatography (~.I.c.)‘*~ and by gas-liquid chromatography--mass spectrometry3-‘. Electron-impact mass spectrometry (e.i.-m.s.) does provide characteristic peaks from the aldononitrile peracetates for ready interpretation, although it seldom yields the molecular ion. The research reported here describes analysis of per-0-acetylaldononitriles by chemicalionization mass spectrometry 6. In contrast to the electron-impact spectra, where molecular ions are not normally seen, chemical-ionization spectra show M+ 1 peaks in each instance. The base peak in each spectrum is at (M + 1) 60 and results from the loss of the elements of acetic acid from tie per-O-acetylaldononitriles. A mixture containing the aldononitrile peracetates from D-rhamnose (l), Lfucose (2), D-arabinose (3), D-xylose (d), D-mannose (3, D-gluCOSe (6), and D-galactose (7) was separated and analyzed by g.l.c.-c.i.-m.s. The chromatographic nonseparalion of 2 and D-ribose (8) is resolved by the m.s., which permits separation of the ions characteristic of each component (Table I). G.1.C. was performed on a mixed liquid-phase consisting of 3% of OV-225 and 2.5% of tetramethylcyclobutanediol succinate on SO-100 mesh Supelcoport, with helium (20 ml/min) as the carrier gas: this system provides two major benefits: short retention-times nnd minimal bleeding of the liquid phase into the detection system. For all of the foregoing aldononitrile peracetates, relative intensities of principal ions and their mass-to-charge ratios (in/e values) are reported in Table I. Methylation of laminaran according to Lindberg’ gave a mixture of partially methylated sugars that was converted by hydrolysis and derivatization into 3,5,6-tri0-acetyl-2,4-di-0-methyl-D-glucononitrile (9), 3,5-di-0-acetyl-2,4,6-tri-O-methyl-D-

Serum glycoproteins in schizophrenia

A comparative investigation of the individual protein-bound carbohydrate components and the distribution of carbohydrates in the electrophoretic glycoprotein fractions was carried out in sera from 20 normal subjects and 30 schizophrenic patients matched for age and sex. The mean concentration of each of the protein-bound carbohydrate components was significantly elevated in schizophrenics. The electrophoretic patterns for serum glycoprotein showed increases in alpha-2 and beta globulins in schizophrenics. The serum glycoproteins contained glucose and L-arabinose, in addition to mannose, galactose, fucose, sialic acid, and a trace of xylose. The identity of glucose and arabinose was confirmed by g.l.c.-electron-impact mass spectrometry and by specific enzymic reactions. The contents of glucose and arabinose were higher in serum glycoproteins from schizophrenic patients. This elevation of serum glycoprotein paralleled serum glycosaminoglycan elevation previously reported by us, but was opposite to decrease of urinary glycoprotein in schizophrenics.

On arabinose as a component of brain hyaluronate. Confirmation by chromatographic, enzymatic and chemical ionization-mass spectrometric analyses.

The controversy about the presence of the pentose arabinose in brain hyaluronate was reinvestigated using modern analytical tehcnics. The purified bovine brain hyaluronate contained the neutral sugars: arabinose, 0.18%; glucose, 0.05%; and fucose, 0.22%. The confirmation of the presence of arabinose was obtained by paper and thin layer chromatography of the neutral sugars in deionized hyaluronate hydrolysate. Gas-liquid chromatography of the aldononitrile peracetate of the pentose isolated by preparative paper chromatography gave a single distinct peak, corresponding to standard arabinose on three columns packed with three different phases. Chemical ionization data and mass spectrum of the aldononitrile peracetate derivative agreed with those of the authentic arabinonitrile tetracetate. Analysis of the isolated pentose with the help of the enzymes L-arabinose isomerase and L-ribulose kinase, which are specific for their substrates, further established its identity as L-arabinose.

Synthesis and acid-catalyzed hydrolysis of 3-O-glycosyl-l-serine and -threonine

Abstract The acid stability of the glycosidic linkage of 3-O-β- d -xylopyranosyl- l -serine and -threonine, of import for graded acid hydrolysis of glycopeptides, was assessed through model compounds. Crystalline 3-O-(2,3,4-tri-O-acetyl-β- d -xylopyranosyl)- l -serine and threonine were synthesized in 20% yield. Syrupy 3-O-(4,6-di-O-acetyl-2,3-dideoxy-α- d -erythro-hex-2-enopyranosyl)-N-benzyloxycarbonyl- l -serine and -threonine p-nitrobenzyl ester afforded upon hydrogenation crystalline 3-O-(4,6-di-O-acetyl-2,3-dideoxy-α- d -erythro-hexopyranosyl)- l -serine and -threonine in 50% yield. Extensive kinetic studies of the acid-catalyzed hydrolysis in 0.05 m sulfuric acid of these four crystalline 3-O-glycosyl derivatives of serine and threonine and comparisons of their kinetic rate-constants and thermodynamic parameters with those of methyl α- and β- d -xylopyranosides were made. Although a protonated amino group is vicinal to the glycosidic acetal linkage in the 3-O- l -serine and -threonine xylosides, the rates of their hydrolysis are only slightly more than an order of magnitude lower than those of ordinary alkyl glycosides. The rate constants for the 2,3-dideoxy-α- d -erythro-hexopyranosides of l -serine and -threonine are similar in magnitude to those for the methyl glycosides. The values for the 2,3-dideoxy-α- d -erythro-hexopyranoside derivatives are nearly the same as those of the alkyl glycosides, whereas the d -xylopyranosides of l -serine and -threonine exhibit values some 2 kcal. mole-1 greater. All of the values are positive, but show no order interpretable in terms of structure-function relationships.

Fractionation and characterization of glycosaminoglycans of mammalian origin.

Summary Glycosaminoglycans (GAG), occurring as complex mixtures in many animal tissues, can be separated into individual components taking advantage of differences in charge density or complexing properties. Each mixture represents a special problem because of different degrees of heterogeneity of components as regards the degree of sulfation and the molecular weight. The fractionation of GAG extracts from a typical source (pig duodenum), performed in order to make individual GAG available for pharmacological tests, is described. The composition of the extracts was monitored at various steps of fractionation by quantitative cellulose acetate electrophoresis and NMR spectroscopy. The purified isolated GAG (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfates and hyaluronic acid) were characterized by various physico-chemical and enzymic methods.

Stability of 3-glycosyloxyprolines in an alkaline medium. Synthesis of model compounds

Abstract Glycosylation of the hindered secondary alcohol groups in substituted 3- and 4-hydroxyproline by reaction with 3,4,6-tri-O-acetyl- d -glucal and boron trifluoride etherate afforded 40–80% yields of 3- and 4-glycosyloxypyrrolidine-2-carboxylic acids. Modification of the 2,3-unsaturated glycosides of 4-hydroxyproline was achieved by hydrogenation, by addition of acetyl hypobromite, or by epoxidation with hydrogen peroxide in benzonitrile and ring-opening of the resultant 2,3-anhydro derivatives. From these model compounds was selected a 3-glycosyloxyproline that was suitable for assessment of the stability of the glycosyl linkage to base-catalyzed elimination. The model compound was N-acetyl-3-O-(4,6-di-O-acetyl-2,3-dideoxy-α- d -erythro-hexopyranosyl)-cis- d , l -hydroxyproline ethyl ester (12). Homogeneity of the compounds was ascertained by high-resolution n.m.r. spectral and chromatographic methods. The glycoside (12) of 3-hydroxyproline is stable even in warm base at 50°. The 3- and 4-hydroxyproline glycosides are both more stable to acid hydrolysis than the analogous compounds of serine and threonine. A reliable synthesis of crystalline cis- d , l -3-hydroxyproline ethyl ester hydrochloride is reported that affords quantities of 250 mg or more in overall yield of 15% from inexpensive starting materials.