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Nondestructive amino acid analysis at the picomole level of proline-containing peptides using aminopeptidase M and prolidase: application to peptides containing tyrosine sulfate.

A micromethod for nondestructive amino acid analysis was developed. Peptides were digested with amino-peptidase M followed by prolidase. The whole procedure was performed in a microvial for the autosampler of the analyzer. The resulting method has a working range from 10 to 600 pmol, at least, of sample taken into work. The usefulness of the method was demonstrated by its ability to determine the sulfation of tyrosine preceding proline in chicken gastrin.

Amino Acid Sequence↗

Synthesis of three tetrasaccharides containing 3-O-methyl-D-mannose, as model compounds for xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of methyl 3,6-di-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-beta-D-xylopyranosyl-be ta-D- mannopyranoside (2), methyl 6-O-alpha-D-mannopyranosyl-3-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (3), and methyl 3-O-alpha-D-mannopyranosyl-6-O-(3-O-methyl-alpha-D-mannopyranosyl)-2-O-b eta-D- xylopyranosyl-beta-D-mannopyranoside (4). The various methyl beta-D-Manp acceptor derivatives were prepared from the corresponding methyl beta-D-Glcp derivatives via oxidation-reduction. All glycosyl donors were coupled using the trichloroacetimidate method at -40 degrees C in dichloromethane with trimethylsilyl triflate as a catalyst. Methyl-3-O-benzyl-4,6-O-benzylidene-beta-D-mannopyranoside (7) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-benzylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and hydrogenation gave methyl 4-O-acetyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta-D-mannopyr anoside (12). Coupling of 12 with 2,4,6-tri-O-acetyl-3-O-methyl-alpha-D-mannopyranosyl trichloroacetimidate (18) afforded tetrasaccharide derivative 19, and subsequent O-deacetylation gave 2. Methyl 3-O-benzyl-4,6-O-prop-2-enylidene-beta-D-mannopyranoside (22) was condensed with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl trichloroacetimidate (8). Regioselective reductive 4,6-O-prop-2-enylidene ring-opening on the resulting disaccharide derivative, followed by acetylation, and deallylation at O-6 gave methyl 4-O-acetyl-3-O-benzyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranoside (26-a), which was either condensed with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (27) or 18, to give trisaccharide derivatives 28 or 31, respectively. Debenzylation of 28 followed by condensation with 18 gave, after O-deacetylation, 3, whereas debenzylation of 31 followed by condensation with 27 gave, after O-deacetylation, 4.

Carbohydrate Conformation↗