A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Gal beta 1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Gal beta 1-3GalNAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40 degrees C for 350 h gave the best results for releasing O-linked sugar chains. The total yield of Gal beta 1-3GalNAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.

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The pyridylamination methodis useful for the structural analysis of sugar chains from small amountsof glycoproteins. About 60% of the sialyl linkage was, however hydrolyzed under the reaction conditions reported previously.1>2) The growing need for fluorescence labeling oi sialyl sugar chains prompted us to re-investigate the reaction conditions.Recently, we tried pyridylamination conditions under which sialic acid residues were not released, but the yield of pyridylamino (PA-) sugar chains was low.3 4) Here, an improved procedure for the pyridylamination of sialyl sugar chains is described. The yield of PA-sugar chains was almost quantitative, and desialylation was negligible.2-Aminopyridine was purchased from Wako Pure Chem. Ind. Ltd. (Osaka), and re-crystallized twice from hexane. Borane-dimethylamine complex was purchased from Aldrich Chem. Co. (Milwaukee), and a CosmosilNAc-PA (Bi-3-PA) were those reported previously.3)The amounts of DS-Bi and DS-Bi-PA were quantified by GLC after methanolysis and trimethylsilylation,5) and also by an automatic amino acid analyzer (Hitachi model 835) after acid hydrolysis (4n HC1, 100°C, 6hr).trated to dryness in a glass tube tapered at the bottom (0.9x10cm).The residue was mixed with 10/^1 of a coupling reagent (prepared by dissolving 552mg of 2-aminopyridine in 200^1 of glacial acetic acid; when the reagent was diluted with 9 volumes of water, the pH of the solution should be 6.8). The tube was sealed and heated at 90°C for 60 min, before being cooled to room temperature and opened. To the solution, 10//I of a reducing reagent (prepared just before use by dissolving 39 mg of boranedimethylamine complex in 200 //I of glacial acetic acid) was added and mixed well. The tube was re-sealed and then heated at 80°C for 50min. After the tube had been opened, the reaction mixture was gel-filtered as described previously .2) Reversed-phase HPLC of the PA-sugar chains was done on a Cosmosil 5C18-P column. Two solvents, A and B, were used, solvent A bing 0.1 m acetic acid and solvent B, 0.1 m acetic acid containing 0.5% 1-butanol. The column was equilibrated with a mixture of solvents A and B at the ratio of 95:5. After injecting a sample, the proportion of solvent B was increased linearly to 100%in 20min at a flow rate of 1.5ml/min at 25°C. To detect the PA-sugar chains, an excitation wavelength of 315 nm and an emission wavelength of 400nmwere used. A typical chromatogram is shown in Fig. 1

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The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.