J. Biochem. 115, 429-434 (1994)

Linkage Position Analysis of Pyridylamino- 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- (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- was formed from 2-substituted PA-disaccharides with PA-, PA- (or PA-) from 3-substituted ones, and PA- 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-, 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-, 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 (Glcƒ¿1-4Glc) and (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 , 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, ; Gal, ; Glc, ; GlcNAc, N-acetylglucosamine; Man, ; 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.

J. Biochem. Linkage Position Analysis of PA-Disaccharides 431

of PA-glycolaldehyde. The fluorogenic products collected were hydrolyzed with 0.1M sulfuric acid at 80°C for 20 min, and then the hydrolysates were analyzed by HPLC (Fig. 2). PA-glyceraldehyde was formed from 2-sub stituted PA-disaccharides, PA-threose from 3-substituted ones with a PA-glucose or PA-galactose residue, PA-ery throse from Manƒ¿1-3Man-PA, and PA-glycolaldehyde from 4- or 6-substituted ones (Fig. 3). The CHOH-CH2PA bond was stable enough under the periodate oxidation conditions used. The fluorogenic Smith degradation prod ucts are thought to be stable as to acid hydrolysis. The common peaks eluted at 4.1-4.3min in Fig. 2 may be formed from contaminating compounds on acid hydrolysis. In order to differentiate 4- and 6-substitution, the products at an early stage of periodate oxidation were examined. The first glycol cleavage should occur at the flexible glycols of the PA-reducing-end residues rather than the glycols of the rigid non-reducing-end ring residues. Theoretically, PA-glyceraldehyde and PA-thre ose (or PA-erythrose) are characteristic of 6-substituted ones and never produced from 4-substituted ones (Fig. 4). Thus, identification of one of them at an early stage of periodate oxidation implies a 6-substituted one. PA-maltose, Galƒ¿1-4Gal-PA, PA-isomaltose, Manƒ¿1- 6Man-PA, and GalƒÀ1-6Gal-PA were oxidized at 0°C for 15 or 30min, and then the oxidation-reduction products were analyzed by HPLC as described under "MATERIALS AND METHODS." Chromatograms of the 15-min reaction mix tures are shown in Fig. 5. All PA-disaccharides disappeared within 15 min. In the case of 4-substituted ones, only PA-glycolaldehyde was detected. The first cleavage might not occur between C5 and C6. Otherwise, the product Fig. 2. HPLC analysis of the hydrolysates of the fluorogenic oxidation-reduction products obtained in Fig. 1. S, elution formed would be further oxidized to the secondary product profile of the standard mixture (1, PA-threose; 2, PA-erythrose; 3, very rapidly. In the case of 6-substituted ones, PA- PA-glyceraldehyde; 4, PA-glycolaldehyde); A, the hydrolysate ob glyceraldehyde and PA-glycolaldehyde were produced, and tained from GlcƒÀ1-2Glc-PA; B, Manƒ¿1-2Man-PA; C, Glcƒ¿1-3Glc- PA-glyceraldehyde was converted to PA-glycolaldehyde PA; D, Manƒ¿1-3Man-PA; E, GalƒÀ1-3Gal-PA; F, Glcƒ¿1-4Glc-PA; G, with the lapse of time (the chromatograms of the 30-min Galƒ¿1-4Gal-PA; H, Glcƒ¿1-6Glc-PA; I, Manƒ¿1-6Man-PA; J, GalƒÀ1- reaction mixtures are not shown). PA-threose or PA-ery 6Gal-PA. throse was not detected, indicating that the first cleavage

Fig. 3. Structures of the fluorogenic Smith degradation products examined in the present paper.

Vol. 115, No. 3, 1994 432 K. Omichi and S. Hase

Fig. 4. Possible fluorogenic products de rived from 4- and 6-substituted PA-disac charides at an early stage of periodate oxidation.

hyde and PA-threose (or PA-erythrose) means 2- and 3-substitution, respectively. When PA-glycolaldehyde is detected, HPLC of the 15-min reaction mixture reveals 4- or 6-substitution . Fluorogenic Smith Degradation Products from PA-Disac charides with PA-N-Acetylglucosamine-In the case of PA-disaccharides with PA-N-acetylhexosamine, the lin kage positions should be more simply determined by identification of three different fluorogenic products. Model compounds, GalƒÀ1-3GlcNAc-PA, GaƒÀ1-4GlcNAc-PA, and GalƒÀ1-6GlcNAc-PA, were oxidized at 4°C for 40 h and reduced as described above. The fluorogenic oxidation- reduction products were purified by HPLC and their hydrolysates were analyzed by HPLC as shown in Fig. 6. GalƒÀ1-3GlcNAc-PA, GalƒÀ1-4GlcNAc-PA, and GalƒÀ1- 6GlcNAc-PA gave three different peaks, a, b, and c, respectively. Their molecular weights were determined to be 239, 269, and 209, respectively, by matrix-assisted Fig. 5. HPLC of the 15-min oxidation-reduction products. F, laser desorption ionization mass spectrometry (Shimadzu/ reaction mixture of Glcal-4Glc-PA; G, Galƒ¿1-4Gal-PA; H, Glcƒ¿1- Kratos Kompact Maldi III), indicating that they were 6Glc-PA; I, Manƒ¿1-Wan-PA; J, GalƒÀ1-6Gal-PA. Arrows indicate 2-acetamido-2-deoxy derivatives of PA-threose, PA-xy the elution positions of standards (1, PA-threose; 2, PA-erythrose; 3, PA-glyceraldehyde; 4, PA-glycolaldehyde). Arrowheads indicate the lose, and PA-glyceraldehyde, respectively (Fig. 3). The elution positions of intact PA-disaccharides. results demonstrated that the linkage positions to PA-N- acetylhexosamine could be easily determined. Application of Linkage Position Analysis to Fuc-Man- might occur between C2 and C3 or between C3 and C4, but PA•\The method was applied to determination of the not between C4 and C5. Otherwise, the product formed linkage positions of Fuc-Man-PA (a) and Fuc-Man-PA (b), through cleavage between C4 and C5 would be very rapidly which were prepared with bovine kidney ƒ¿-L-fucosidase. converted to the secondary products. The product formed Each Fuc-Man-PA (100pmol) was treated as described on cleavage between C3 and C4 was rather stable and was under "Standard Linkage Position Analysis." The fluoro actually detected in the 30-min reaction mixture as PA- genic products were analyzed by HPLC, as shown in Figs. 7 glyceraldehyde. It is likely that a CHO-CHOH bond is less and 8. PA-erythrose was detected in the hydrolysate of the susceptible to periodate oxidation than a CHOH-CHOH 40-h oxidation-reduction product of Fuc-Man-PA (a) (Fig. bond. 8A). This clearly shows that the fucosyl residue was linked The above results show that PA-glyceraldehyde was the to the 3-position of the PA-mannose residue. PA-glycolal characteristic product for 6-substituted PA-disaccharides dehyde was derived from Fuc-Man-PA (b), indicating 4- or at an early stage of periodate oxidation. 6-substitution (Fig. 8B). In the 15-min reaction mixture, The linkage position can be determined by combination PA-glycolaldehyde was detected, but PA-glyceraldehyde, of HPLC as described under "Standard Linkage Position characteristic of 6-substitution, was not detected (Fig. 9). Analysis." On HPLC of the hydrolysate of the 40-h oxida Thus, Fuc-Man-PA (b) was Fucƒ¿1-4Man-PA. The results tion-reduction product, the identification of PA-glyceralde indicate that bovine kidney ƒ¿-L-fucosidase transferred a

J. Biochem. Linkage Position Analysis of PA-Disaccharides 433

Fig. 7. Isolation of the fluorogenic periodate oxidation-reduc tion products derived from Fuc-Man-PAs by HPLC. Periodate oxidation of Fuc-Man-PAs was carried out at 4°C for 40 h. A, HPLC of the periodate oxidation-reduction product from Fuc-Man-PA (a); B, Fuc-Man-PA (b). The fractions indicated by bars were collected for further analysis. The arrow indicates the elution position of PA- glycolaldehyde.

Fig. 6. Isolation of fluorogenic periodate oxidation-reduction

products of PA-disaccharides with PA-GlcNAc and HPLC of their hydrolysates. (A) HPLC of the periodate oxidation-reduction mixture of PA-disaccharides with PA-GlcNAc. K, oxidation-reduc tion mixture of GalƒÀ1-3GlcNAc; L, GalƒÀ1-4GlcNAc; M, GalƒÀ1- 6GlcNAc. The fractions indicated by bars were collected for further analysis. Arrowheads indicate the elution positions of intact PA- Fig. 8. HPLC analysis of the hydrolysates of the fluorogenic disaccharides. (B) HPLC of the hydrolysates of the fluorogenic oxidation-reduction products derived from Fuc-Man-PAs. The periodate oxidation-reduction products isolated in (A). K, hydro products collected in Fig. 7 were hydrolyzed, and the hydrolysates lysate of the fluorogenic oxidation-reduction product from GalƒÀ1- were analyzed as described under `MATERIALS AND METHODS. 3GlcNAc; L, GalƒÀ1-4GlcNAc; M, GalƒÀ1-6GlcNAc. Peaks a, b, and c A, HPLC of the hydrolysate of the oxidation-reduction product from were collected for matrix-assisted laser desorption ionization mass Fuc-Man-PA (a); B, Fuc-Man-PA (b). For the arrows, see the legend spectrometry. to Fig. 5.

usefulness of the method for linkage position analysis. This method can be applied to disaccharides with PA- reducing-end residues derived from pyranose without any alteration. The linkage positions to mono-substituted PA- reducing-end residues in larger oligosaccharides can also be determined by hydrolyzing the oxidation-reduction prod ucts under conditions with which the glycosidic bonds are split.

The authors are very grateful to Mr. Shozo Onishi, Kyoto Analytical Applications Center, Shimadzu Co., Kyoto, for recording the matrix- assisted laser desorption ionization mass spectra.

Fig. 9. HPLC of the 15-min reaction mixture of Fuc-Man-PA (b). Fuc-Man-PA (b) was oxidized with periodate at 0°C for 15min REFERENCES followed by reduction, and then the reaction mixture was analyzed by HPLC. For the arrows, see the legend to Fig. 5. 1. Hase, S., Ikenaka, T., & Matsushima, Y. (1978) Biochem. Biophys. Res. Commun. 85, 257-263 2. Hase, S., lkenaka, T., & Matsushima, Y. (1981) J. Biochem. 90, 407-414 fucosyl residue to the 3- or 4-position of the mannose 3. Hakomori, S. (1964) J. Biochem. 55, 205-208 residue of ManƒÀl-PNP. The results demonstrated the 4. Stellner, K., Saito, H., & Hakomori, S. (1973) Arch. Biochem.

Vol. 115, No. 3, 1994 434 K. Omichi and S. Hase

Biophys. 155, 404-472 8. Lipniunus, P., Angel, A-.S., Erlansson, K., Lindh, F., & Nilsson, 5. Geyer, R., Geyer, H., Kuhnhardt, S., Mink, W., & Stirm, S. B. (1992) Anal. Biochem. 200, 58-67 (1983) Anal. Biochem. 133, 197-207 9. Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126 6. Abdel-Akher, M., Hamilton, J.K., Montgomery, R., & Smith, F. 10. Mega, T. & Hase, S. (1991) J. Biochem. 109, 600-603 (1952) J. Am. Chem. Soc. 74, 4970-4971 11. Irimura, T., Tsuji, T., Tagami, S., Yamamoto, K., & Osawa, T. 7. Smith, F. & Van-Cleve, J.W. (1955) J. Am. Chem. Soc. 77, (1981) Biochemistry 20, 560-566 3091-3096

J. Biochem.