J. Biochem. 115, 429-434 (1994) Linkage Position Analysis of Pyridylamino-Disaccharides by HPLC of Fluorogenic Smith Degradation Products1 Kaoru Omichi and Sumihiro Hase Department of Chemistry, Osaka University College of Science, Toyonaka, Osaka 560 Received for publication, October 18, 1993 Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA- oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0°C for 15 min or at 4•Ž for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4•Ž for 40h were purified by reversed phase HPLC, and the fractions collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0•Ž for 15min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney ƒ¿-L-fucosidase. Key words: linkage position analysis, PA-disaccharide, Smith degradation. ƒ¿-D-mannopyranoside (Manƒ¿1-2Manƒ¿-Me) Pyridylamination, fluorescent tagging of the reducing-end , methyl 6-O- residues of sugars, has been performed to analyze sugar ƒ¿-D-mannopyranosyl-ƒ¿-D-mannopyranoside (Manƒ¿1- structures with high sensitivity (1, 2). The linkage posi 6Manƒ¿-Me), and methyl 3-O-ƒÀ-D-galactopyranosyl-ƒÀ- tions of glycosidic bonds are usually determined by meth D-galactopyranoside (GalƒÀ1-3GalƒÀ-Me) from Sigma; ylation analysis, which requires a nanomole order amount maltose (Glcƒ¿1-4Glc) and isomaltose (Glcƒ¿1-6Glc) from of a sample (3-5). A relatively larger amount of a PA- Wako Pure Chemicals (Osaka); L-threose and D-erythrose oligosaccharide, however, is required to determine the from Sigma; p-nitrophenyl ƒÀ-D-mannopyranoside (ManƒÀ- substitution position of a PA-reducing-end residue due to PNP) and p-nitrophenyl ƒ¿-L-fucopyranoside (Fucƒ¿-PNP) poor characteristic fragment ions (1). from Nacalai Tesque (Kyoto); and bovine kidney ƒ¿-L-fuc An alternative method for determination of the linkage o sidase from Boehringer Mannheim. One unit of the enzyme position should be developed taking advantage of fluores is defined as the amount of enzyme which hydrolyzes 1 cent tagging. Smith degradation including periodate oxida ƒÊ mol of Fucƒ¿-PNP per min at pH 6.0 and 37•Ž. tion of vicinal diol (6-8), as well as methylation analysis, Pyridylamination•\Pyridylamination of disaccharides, provided information on the linkage positions. glyceraldehyde, erythrose, and threose was carried out This paper describes the highly sensitive linkage position according to the reported methods (9). Excess reagents analysis of PA-disaccharides involving Smith degradation. were evaporated with a Palstation (Takara Biomedicals, Kyoto). The remaining reaction mixtures were chromato MATERIALS AND METHODS graphed on a Toyopearl HW-40F column (1.0•~112cm) equilibrated with 10mM ammonium acetate buffer, pH Materials•\Sophorose (GlcƒÀ1-2Glc) was purchased from 6.0. The effluent was monitored by measuring fluorescence Funakoshi (Tokyo); nigerose (Glcƒ¿1-3Glc), Manƒ¿1-3Man, (excitation at 320nm, emission at 410nm). The PA-disac Galƒ¿1-4Gal, GalƒÀ1-6Gal, GalƒÀ1-3GlcNAc, GalƒÀ1-4Glc charides obtained were further purified by HPLC on a NAc, GalƒÀ1-6GlcNAc, methyl 2-O-ƒ¿-D-mannopyranosyl- Cosmosil 5C18 column (6•~150mm) (Nacalai Tesque). The elution buffer was 50mM ammonium acetate buffer, 1 This work was supported in part by the Japan Health Science pH 5.5, and the flow rate was 2.0ml/min. Foundation. Manƒ¿1-2Manƒ¿-Me, GalƒÀ1-3GalƒÀ-Me, and Manƒ¿1- Abbreviations: Ac, acetyl; Fuc, fucose; Gal, galactose; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; Me, methyl; PA, 6Manƒ¿-Me were partially hydrolyzed with 0.3 M trifluoro pyridylamino; PNP, p-nitrophenyl. acetic acid at 90°C for 160, 70, and 130min, respectively, Vol. 115_ No_ 3. 1994 429 430 K . Omichi and S. Hase and then each hydrolysate was lyophilized and pyridyl on a Toyopearl HW-40F column as described above. aminated as described above.PA-disaccharides were puri Fuc-Man-PA (a) and Fuc-Man-PA (b) were separated from fied by gel filtration, followed by HPLC as described above. the reaction mixtures of Fucƒ¿-ManƒÀ-PNP (a) and Fucƒ¿- PA-glycolaldehyde was prepared by reaction of 2-chloro ManƒÀ-PNP (b), respectively. The PA-disaccharides were pyridine and ethanolamine as reported (10). further purified by HPLC on a Wakosil-II 5C18 HG column Standard Linkage Position Analysis-Smith degrada (6 x 150mm). The elution buffer was 50mM ammonium tion of a PA-disaccharide was carried out by modifying the acetate, pH 5.5, and the flow rate was 2.0ml/min. conditions reported (11). A PA-disaccharide (50-500pmol) in 15 p 1 of 0.1M sodium acetate buffer, pH 4.0, was mixed RESULTS AND DISCUSSION with 15,u l of a 0.1M sodium metaperiodate solution, and then the mixture was kept at 0•Ž for 15min in the dark. Fluorogenic Smith Degradation Products of PA-Disac Ten microliters of the reaction mixture was added to 20 charides with PA-Hexose-PA-disaccharides, of which the pl of 0.26M sodium borohydride. After standing for 1 h at reducing-ends were PA-hexose, were oxidized with sodium 25•Ž, 5 pl of acetic acid was added to the reaction mixture metaperiodate at pH 4.0 and 4°C for 40h. After reduction to decompose residual sodium borohydride. The pH of the with sodium borohydride, the fluorogenic products were reaction mixture was adjusted to 5.0 by adding 25,u l of 2.2 analyzed by HPLC as described under "MATERIALS AND M sodium hydroxide. The mixture was frozen and stored METHODS" (Fig. 1). The products derived from 4- or at -60•Ž or below till HPLC analysis of the fluorogenic 6-substituted PA-disaccharides were eluted at the position periodate oxidation-reduction product at an early stage of periodate oxidation. Oxidation of the remaining solution was continued for 40 h at 4°C. After treatment with sodium borohydride (40," 1), followed by the addition of acetic acid (10 pl) and pH adjustment to 4.5 with 30 pl of 2.2 M sodium hydroxide, the reaction mixture was applied to a Wakosil-II 5C18 HG column (6 x 150mm) to isolate the fluorogenic oxidation- reduction product. The elution buffer was 50mM ammo nium acetate buffer, pH4.5, containing 0.025% 1-butanol, and the flow rate was 2.0ml/min. A part (1ml) of the oxidation-reduction product collected was added to 100 p l of 1.1M sulfuric acid. The mixture was heated at 80°C for 20min. After adjusting the pH to 5.0 with 110,u l of 2.2M sodium hydroxide, a part (100-150 pl) of the hydrolysate (1.21ml) was injected into the reversed phase HPLC column. The HPLC conditions were the same as those used for the isolation of the oxidation-reduction product except that the pH of the elution buffer was 5.0. HPLC of the 15-min reaction mixture stored was carried out under the same conditions. Preparation of p-Nitrophenyl O-ƒ¿-L-Fucopyranosyl-ƒÀ- D-Mannopyranoside (Fucƒ¿-ManƒÀ-PNP)-A mixture of 100mg of ManƒÀ-PNP, 20mg of Fucƒ¿-PNP, and 1.4 units of bovine kidney ƒ¿-L-fucosidase in 10ml of 50mM ammo nium acetate buffer, pH6.0, was incubated at 37°C for 5 h. The enzymatic reaction was stopped by adding 0.5ml of acetic acid. The digest was chromatographed on a Toyo pearl HW-40S column (2.0 x 192cm) equilibrated with 50 mM ammonium acetate. The elution was monitored by measuring the absorbance at 310nm. Two Fucƒ¿-ManƒÀ- PNPs [4.7mg of Fucƒ¿-ManƒÀ-PNP (a) and 0.24 mg of Fucƒ¿-ManƒÀ-PNP (b)] were separated from p-nitrophenol, L-fucose, ManƒÀ-PNP, and Fucƒ¿-PNP. Fucƒ¿-ManƒÀ-PNP (a) was eluted faster than Fucƒ¿-ManƒÀ-PNP (b). The Fucƒ¿-ManƒÀ-PNPs obtained were further purified by HPLC on a Cosmosil 5C18P column (6 x 150mm). The elution buffer was 50mM ammonium acetate, pH4.5, Fig. 1. Isolation of the fluorogenic periodate oxidation-reduc containing 1.3% 1-butanol, and the flow rate was 2.0ml/ tion products of PA-disaccharides with PA-hexose by HPLC. min. Arrowheads indicate the elution positions of intact PA-disaccharides . The fractions indicated by bars were collected for further analysis Preparation of Fuc-Man-PA from Fucƒ¿-ManƒÀ-PNP . A, elution profile of the periodate oxidation-reduction product from •\ Each Fucƒ¿-ManƒÀ-PNP (0.2mg) in 0.3ml of 0.3M trifluo GlcƒÀ1-2Glc-PA; B, Manƒ¿1-2Man-PA; C, Glcƒ¿1-3Glc-PA; D, Man roacetic acid was heated at 90•Ž for 20min, and then the ƒ¿1-3Man-PA; E, GalƒÀ1-3Gal-PA; F, Glcƒ¿1-4Glc-PA; G , Galƒ¿1- hydrolysate was lyophilized. The residue was pyridyl 4Gal-PA; H, Glcƒ¿1-6Glc-PA; I, Manƒ¿1-6Man-PA; J, GalƒÀ1-6Gal- aminated and the reaction mixture was chromatographed PA.