Specific Tritium Labeling of β-d -Galactofuranosides at the 6-Position: A Tool for β-d -Galactofuranosidase Detection

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NOTES & TIPS Specific Tritium Labeling of ␤-DGalactofuranosides at the 6-Position: A Tool for ␤-D-Galactofuranosidase Detection Karina Marin˜o, Carla Marino, and Rosa M. de Lederkremer 1

Received October 1, 2001; published online January 15, 2002

The glycobiology of galactofuranose is attracting considerable interest from many groups, since ␤-D-galactofuranosyl residues are constituents of infectious microorganisms, but are absent in mammal glycoconjugates (1). The enzymes involved in the metabolism of the sugar are good targets for the development of antimicrobial agents. The best known is the specific exo-␤-Dgalactofuranosidase first purified from the culture medium of Penicillium fellutanum (2) and later described in Helminthosporium sacchari (3) and Penicillium and Aspergillius species (4). An affinity-purification method (5) using the inhibitor 4-aminophenyl 1-thio-␤D-galactofuranoside (6) as ligand for the preparation of the affinity phase was also described. Activity of the enzyme is usually measured (6) with the substrate 4-nitrophenyl ␤-D-galactofuranoside (7). On the other hand the processes involved in Galf incorporation in glycoconjugates have not been completely elucidated. A mutase, which converts UDP-Galp in UDP-Galf with low efficiency (5– 8%), has been described (8, 9). Detection of the galactofuranose intermediates and of the enzymes involved in the metabolism of galactofuranose using radiolabeled precursors and substrates is essential for such studies. Here we report for the first time a procedure for introducing tritium at the 6-position of galactofuranose derivatives, being the key step in oxidation with pyridinium chlorochromate of HO-6 of a convenient derivative and reduction with NaB 3H 4 (Scheme 1). Using the synthesized [ 3H]methyl-␤-D-galactofuranoside, the activity of ␤-D-galactofuranosidase

Analytical Biochemistry 301, 325–328 (2002) doi:10.1006/abio.2001.5508 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

Materials and Methods Preparation of [6- 3H]Methyl-␤-D-galactofuranoside ([ H]1). Compound 1 (0.07 g, 0.4 mmol) (10) was dissolved in pyridine (10.0 mL) and tritylchloride (0.12 g, 0.4 mmol) was added. The solution was kept in the dark for 48 h and then benzoyl chloride (1.0 mL) was added while cooling. After 2 h of stirring the reaction mixture was poured over ice/water and decanted, and the syrup obtained was dissolved in methylene chloride and washed with a saturated solution of sodium bicarbonate and water and dried (magnesium sulfate). Compound 2 (R f 0.64, solvent A) was detritylated with BF 3 䡠 Et 2O (34 ␮L 2 meq) in methanol (1 mL) at room temperature, until TLC examination showed total conversion into the lower moving product 3 (R f 0.21, solvent A). The reaction mixture was coevaporated several times with methanol in order to eliminate the excess of BF 3. Compound 3 was purified by column chromatography (silica gel, toluene:ethylacetate, 9:1). For oxidation, a mixture of 3 (0.02 g), PCC (0.027 g), 3 Å molecular sieves powder (0.1 g), and sodium acetate (1 mg) in anhydrous methylene chloride (2.0 mL) was stirred for 4 h at room temperature. The mixture was filtered, the solvent was evaporated, and product 5 was purified by column chromatography (toluene:ethylacetate, 9:1). Compound 5 (1.8 mg) was reduced in methanolic solution (0.3 mL) with 1 mCi of NaB 3H 4 (NEN Life Science Products, 222.30 mCi/mmol) in 5 ␮l of 0.1 M KOH, and after 3 h at room temperature, solid NaBH 4 (2.2 mg) was added. The mixture was left overnight at 4°C and the solution was deionized by subsequent elution through a column of Bio-Rad AG 50 W-X12 (H ⫹ form) resin (Bio-Rad) and an Amberlite MB-3A (mixed form) for elimination of the boric acid. Debenzoylation was performed with 0.5 M sodium methoxide in methanol (5 ␮L), and after 2 h of stirring the solution was neutralized with Amberlite IR-120. The identity of [6- 3H]methyl-␤-D-galactofuranoside was tested by TLC and fluorography. 3

CIHIDECAR, Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabello´n II, Ciudad Universitaria, 1428 Buenos Aires, Argentina

1 To whom correspondence should be addressed. [email protected]. Research member of CONICET.

can be followed by detection of radioactive galactose by HPAEC 2 or TLC.

E-mail:

2 Abbreviations used: HPAEC-PAD, high pH anion-exchange chromatography with pulse amperometric detection; TLC, thin-layer chromatography; trityl, triphenylmethyl; PCC, pyridinium chlorochromate.

325

326

NOTES & TIPS

SCHEME 1.

i, TriCl, Py; ii, BzCl, Py; iii, BF 3 䡠 Et 2O, MeOH; iv, PCC, CH 2Cl 2; v, NaB 3H 4, MeOH; vi, NaMeO/MeOH.

NMR spectroscopy. NMR spectra were recorded with a Bruker AC 200 or a AM 500 spectrometer. Thin-layer chromatography. TLC was performed using silica gel 60 (Merck) with the following systems: (A) toluene:ethylacetate (9:1); (B) n-propanol:NH 3:H 2O (7:1:2). When necessary, the products were eluted from silica gel with H 2O:methanol (7:3). Nonradioactive compounds were detected by spraying the plates with SO 4H 2 (10%) in ethanol. Radiolabeled compounds were detected by fluorography. The plates were sprayed with EN 3HANCE (New England Nuclear) and were exposed to Kodak X-Omat A-R5 films at ⫺70°C. HPAEC-PAD analysis. Analysis by HPAEC-PAD was performed using a Dionex DX 300 HPLC system with pulse amperometric detection (PAD), set at 30 nA and E 1 ⫽ ⫹0.05 V, E 2 ⫽ ⫹0.60 V, and E 3 ⫽ ⫺0.60 V. The column used was a CarboPac PA-10 anion-exchange analytical column (4 ⫻ 250 mm), equipped with a guard column PA-10 (5 ⫻ 50 mm). The following program was used: 18 mM NaOH, isocratically, at a flow rate 1 mL min ⫺1. Radioactivity in the fractions was determined in a Rack-beta Wallack liquid scintillation counter using a scintillation cocktail (Optiphase “Hisafe” 3, LKB). The amounts of nonradioactive compounds recovered in the peaks were estimated from the peak areas using mannose as standard. The response for galactose and compound 1 with respect to mannose was independently determined.

Enzymatic assay. Enzymatic assay was performed using the filtered medium of a stationary culture of P. fellutanum (2) as enzyme source (60 ␮L, 12 ␮g protein), radiolabeled compound [ 3H]1 as substrate (5 ␮L, 450,000 cpm), 15 ␮L of 66 mM sodium acetate buffer (pH 4.0), in a final volume of 80 ␮L, and incubated overnight at 37°C. The sample was centrifuged for 25 min at 10,000g, through an Ultrafree-MC centrifugal filter (MW 5000). The filtrate was analyzed by TLC and HPAEC-PAD. Results and Discussion The strategy for the introduction of a tritium radiolabel in a galactofuranose derivative was developed with the methylglycoside 1 (10). The reaction sequence used had the purpose of obtaining a derivative with free HO-6 (Scheme 1). Thus, the subsequent oxidation/ reduction with NaB 3H 4 would incorporate the label in that position. With this aim, we prepared compound 2 by tritylation followed by benzoylation of 1. The diagnostic NMR signal for this compound was observed at ␦ 86.9 in the 13C NMR spectrum (Table 1), corresponding to the tertiary carbon of the trityl group. The other signals and those of the 1H NMR spectrum (Table 2) were assigned by comparison to those of the analogous ␤-D-galactofuranosides (10). Detritylation was a critical step, because only mild conditions could be used in order to avoid cleavage of the glycosidic linkage. The

TABLE 1 13

Compound a

1 2b 3b 4b 5b a b c

D 2O. CDCl 3. Interchangeable.

C NMR (200 MHz) Chemical Shifts of Compounds 1–5

C-1

C-2

C-3

C-4

C-5

C-6

OCH 3

109.0 106.7 106.8 107.0 106.8

81.6 80.9 81.9 81.6 81.4

77.5 78.3 77.6 78.2 77.8 c

83.8 82.3 82.1 83.0 82.0

71.8 72.0 74.9 69.1 77.6 c

63.6 62.6 62.6 66.2 196.4

55.8 54.8 55.0 55.0 55.0

Others

CPh 3, 86.9

327

NOTES & TIPS TABLE 2 1

H NMR (500 MHz, Cl 3CD) Chemical Shifts (ppm) of Compounds 2–5

Compound

H-1 (J 1,2)

H-2 (J 2,3)

H-3 (J 3,4)

H-4 (J 4,5)

H-5 (J 5,6)

H-6 (J 5,6⬘)

2 3 4 5

5.12 (⬍0.5) 5.21 (⬍0.5) 5.16 (⬍0.5) 5.26 (⬍0.5)

5.40 (1.1) 5.47 (⬍0.5) 5.29 (1.4) 5.53 (1.1)

5.49 (5.5) 5.60 (5.3) 5.66 (5.0) 5.84 (2.5)

4.74 (4.0) 4.66 (4.0) 4.38 (2.3) 4.85 (5.5)

5.80 (6.3) 5.62 (5.6) 4.48 a (6.5) 5.60 —

3.60 (5.1)

a

H-6⬘ (J 6,6) 3.45 (9.5) 4.07

4.61 (4.5) 9.73

4.48 a (11.3)

Center of a complex multiplet.

use of CuSO 4 (anh) has been described as a mild procedure (11), but in our case partial migration of the benzoyl group from HO-5 to HO-6 was observed. Both, compounds 3 and 4 were isolated by column chromatography and characterized (Tables 1 and 2). Detritylation with BF 3 䡠 Et 2O in methanol at room temperature afforded 3 in good yields (92%). The difference

between compounds 3 and 4 was clearly observed in the 13C NMR spectra (Table 1). For compound 3 the signal corresponding to C-5 is shifted downfield to 74.9 ppm, in comparison with the same signal in 2 (72.0

FIG. 1. TLC and fluorography of the [6-3H]methyl-␤-D-galactofuranoside and its use for the detection of exo-␤-D-galactofuranosidase from P. fellutanum. Lane I, [6-3H]methyl-␤-D-galactofuranoside; lane II, enzymatic hydrolysis of [6-3H]methyl-␤-D-galactofuranoside. The migration of the nonradioactive standards is shown on the left: 1, methyl␤-D-galactofuranoside; 2, galactose. The origin is indicated with an arrow.

FIG. 2. HPAEC-PAD analysis of [6- 3H]methyl-␤-D-galactofuranoside and its enzymatic hydrolysis. (A) [ 3H]Methyl-␤-D-galactofuranoside; B, enzymatic hydrolysis of [ 3H]methyl-␤-D-galactofuranoside by ␤-D-galactofuranosidase from P. fellutanum. A CarboPac PA-10 column with the conditions indicated under Materials and Methods was used. The number correspond to the nonradioactive standard: 1, methyl-␤-D-galactofuranoside.

328

NOTES & TIPS

ppm), due to the ␤ effect. The same effect for compound 4 shifts the C-6 signal, which is observed at 66.2 ppm. Deprotection of HO-6 also affects the pattern of H-6,6⬘ in the 1H NMR spectrum of 3. In this case, the two protons appear as a doublet at the same chemical shift (4.07 ppm, Table 1). Oxidation of HO-6 was performed with PCC as described (12) affording 5 (88%). Compound 5 showed a singlet at 9.2 ppm in the 1H NMR spectrum, and a signal at ␦ 196.5 in the 13C NMR spectrum, both diagnostic signals for the presence of the aldehyde group at C-6. For the reduction and debenzoylation steps, standard procedures were used. Compound [ 3H]1 showed the same chromatographic properties by TLC (Fig. 1, lane I) and HPAEC-PAD (Fig. 2A) than the nonradioactive compound. The specific activity (13 ␮Ci/␮mol) was calculated from the ratio between the radioactivity of the compound eluted from the HPAEC and the amount of material calculated by using mannose as standard. Compound [ 3H]1 was used as substrate for the exo␤-D-galactofuranosidase from P. fellutanum, under the usual conditions (6). The enzymatic reaction could be followed by TLC (Fig. 1, lane II) and HPAEC-PAD analysis (Fig. 2B). This is the first report on the synthesis of a radioactive substrate for ␤-D-galactofuranosidase. The radiolabeled material will facilitate detection of the enzyme in cultures or cellular fractions of microorganisms. In addition to being more sensitive, it can be used with colored biological materials without the interference caused in the colorimetric assay. The method could be useful for labeling other substrates in studies on the biosynthesis of galactofuranose glycans, like the galactan of Mycobacterium tuberculosis (13), although this chemical-labeling procedure would not be specific when other monosaccharides are present.

5. Miletti, L., Marino, C., Marin˜o, K., Lederkremer, R. M., Colli, W., and Manso Alves, M. J. (1999) Immobilized 4-aminophenyl 1-thio␤-D-galactofuranoside as a matrix for affinity purification of an exo-␤-D-galactofuranosidase. Carbohydr. Res. 320, 176 –182. 6. Marino, C., Marin˜o, K., Miletti, L., Manso Alves, M. J., Colli, W., and Lederkremer, R. M. (1998) 1-Thio-␤-D-galactofuranosides. Synthesis and evaluation as ␤-D-galactofuranosidase inhibitors. Glycobiology 8, 901–904. 7. Varela, O., Marino, C., and Lederkremer, R. M. (1986) Synthesis of p-nitrophenyl ␤-D-galactofuranoside. A convenient substrate for ␤-D-galactofuranosidase. Carbohydr. Res. 155, 247–251. 8. Nassau, P. M., Martin, S. L., Brown, R. E., Weston, A., Monsey, D., McNeil, M. R., and Duncan, K. (1996) Galactofuranose biosynthesis in Escherichia coli K-12: Identification and cloning of UDP-galactopyranose mutase. J. Bacteriol. 178, 1047–1052. 9. Sanders, D. A. R., Staines, A. G., McMahon, S. A., McNeil, M. R., Whitfield, C., and Naismith, J. H. (2001) UDP-Galactopyranose mutase has a novel structure and mechanism. Nature Struct. Biol. 8, 858 – 863. 10. Marino, C., Varela, O., and Lederkremer, R. M. (1989) Synthesis of galactofuranose disaccharides of biological significance. Carbohydr. Res. 190, 65–76. 11. Randazzo, G., Capasso, R., Cicala, M. R., and Evidente, A. (1980) A simple method for detritylation of carbohydrate derivatives. Carbohydr. Res. 85, 298 –301. 12. Corey, E. J., and Suggs, J. W. (1975) Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds. Tetrahedron Lett. 31, 2647–2650. 13. Kremer, L., Dover, L. G., Morehouse, C., Hitchin, P., Everett, M., Morris, H. R., Dell, A., Brennan, P. J., McNeil, M. R., Flaherty, C., Duncan, K., and Besra, G. S. (2001) Galactan biosynthesis in Mycobacterium tuberculosis. Identification of a bifunctional UDP-galactofuranosyltransferase. J. Biol. Chem. 276, 26430 – 26440.

Acknowledgments. This work was supported by Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica, Argentina (ANPCYT, BID 802/OC-AR Pict 06-3036, and ANPCYT, BID 1201/OC-AR PICT 0605133), and University of Buenos Aires.

Mark A. Doll and David W. Hein 1

REFERENCES 1. Lederkremer, R. M., and Colli, W. (1995) Galactofuranose-containing glycoconjugates in trypanosomatids. Glycobiology 5, 547–552. 2. Rietschel-Berst, M., Jentoft, N. H., Rick, P. D., Pletcher, C., Fang, F., and Gander, J. E. (1977) Extracellular exo-␤-galactofuranosidase from Penicillium charlesii. J. Biol. Chem. 252, 3219 –3226. 3. Daley, L. S., and Stro¨bel, G. A. (1983) ␤-D-Galactofuranosidase activity in Helminthosporium sacchari and its relationship to the production of helminthosporoside. Plant Sci. Lett. 30, 145–154. 4. Cousin, M. A., Notermans, S., Hoogerhout, P., and Van Boom, J. H. (1989) Detection of ␤-galactofuranosidase production by Penicillium and Aspergillus species using 4-nitrophenyl ␤-D-galactofuranoside. J. Appl. Bacteriol. 66, 311–317.

Rapid Genotype Method to Distinguish Frequent and/or Functional Polymorphisms in Human N-Acetyltransferase-1

Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky 40292 Received July 20, 2001; published online January 8, 2002

Human N-acetyltransferase 1 (NAT1) 2 catalyzes the N-acetylation of arylamine and hydrazine drugs and the O-acetylation of N-hydroxylated metabolites of ar1 To whom correspondence should be addressed. Fax: (502) 8527868. E-mail: [email protected]. 2 Abbreviations used: NAT1, N-acetyltransferase 1; NAT2, N-acetyltransferase 2; PCR, polymerase chain reaction; FAM, 6-carboxyfluorescein; TET, tetrachloro-6-carboxyfluorescein; MGB, minor groove binder; RFLP, restriction fragment length polymorphism; SSCP, single-strand conformation polymorphism; UTR, untranslated region.

Analytical Biochemistry 301, 328 –332 (2002) doi:10.1006/abio.2001.5520 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.