Bioconjuflte Chem. 1882, 3, 230-233
230
Efficient Photoaffinity Labeling of Human &Hexosaminidase A. Synthesis and Application of S-Azi-l-[(2-acetamido-2-deoxy-1-@-~-glucopyranosyl)thio]and -galactopyranosyl)thi01butane Cai-Steffen Kuhn,t Jochen Lehmann,*pt and Konrad Sandhoff3 Institut fur Organische Chemie und Biochemie der Universitat Freiburg, Albertstrasse 21, D-7800 Freiburg i. Br., Germany, and Institut fiir Organische Chemie und Biochemie der Universitat Bonn, Gerhard-Domagk-Strasse, D-5300 Bonn, Germany. Received December 11, 1991
Two photolabile thioglycosides (8 and 9) were synthesized by Koenigs- Knorr type glycosylation. These compounds, being enzyme-resistant analogues of N-acetylhexosaminides, were shown to be good competitive inhibitors of lysosomal 6-hexosaminidase (2-acetamido-2-deoxy-6-~-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) action. For photoaffinity labeling 3H-labeled 8a was prepared by enzymatic oxidation with galactose oxidase followed by reduction with sodium I3H1borohydride. Compound 8a,when photolyzed in the presence of hexosaminidase, specifically labeled both subunits of the enzyme.
3.2.1.52) from lysosomes was purified by chromatography on concanavalin-A sepharose, ion-exchange chromatogPhotoaffinity labeling with suitable ligands has been raphy, and gel filtration (the specific activity was 40.5 successfully applied to probe receptor binding sites (1). units/mg) (6). Galactose oxidase (D-ga1actose:oxygen 6For probing binding sites of glycoside hydrolases, which oxidoreductase, EC 1.1.3.9) from Dactylium dendroides usually have a highglycon specificity,the photolabile group (87units/mg lyophilisate) was purchased from Sigma. Catmust be introduced into the aglycon of an enzyme-resistant alase (hydrogen-peroxide:hydrogen-peroxide oxidoreducglycoside in order to gain sufficient affinity as well as tase, EC 1.11.1.6) from bovine pancreas was purchased stability against enzyme-catalyzed hydrolysis (2). We from Boehringer Mannheim. recently published the synthesis of the "C-glycoside" 4O-acetyl-2-deoxy-lacetamido-3,7-anhydro-2-azi-l,2,4-trideoxy-~-glycero-~-Benzoyl 2-Acetamido-3,4,6-trithio-8-D-galactopyranoside (3). A solution of acetochlogulo-octitol as a potential photoaffinity reagent for human rogalactosamine 1 (0.5 g, 1.4 mmol) and potassium hexosaminidase (3). Although the Ki value (10 mM) of thiobenzoate (0.35 g, 2.0 mmol) in dry acetone (10 mL) this compound with human hexosaminidase A was acwas heated under reflux. After 1 h the mixture was ceptable, the efficiency of photoaffinity labeling was filtrated and concentrated to dryness; the residue was unsatisfactory (4). We now describe facile syntheses of subjected to flash chromatography (cyclohexane/ethyl two photolabile thiohexosaminides and their successful acetate 3:l) and recrystallized from ethanol to give 3 (0.46 application as photoaffinity reagents. g, 70%): mp 141 "C; TLC (cyclohexane/ethyl acetate 3:l) EXPERIMENTAL PROCEDURES Rf 0.21; [(r]D +6.1° (c 1,chloroform); 'H NMR (CDCl3) 6 1.89,2.03,2.05,2.18 (4 X 9, 12 H, OAC),4.10 (t, 1H, H-5), General Methods. All reactions were monitored by 4.12 (d, 2 H, H-Ga,b), 4.67 (dd, 1 H, J 2 , 3 = 10.5 Hz, H-2), TLC on silica gel 60 F254 (Merck), using the solvents 5.21 (dd, 1H,J3,4 = 3.75 Hz, H-3), 5.49 (d, 1H, J1,z = 10.35 indicated. Flash column chromatography (5) was perHz, H-l), 6.16 (d, 1H, J N H =, ~9.5 Hz, NH), 7.47-7.97 (m, formed on ICN silica gel (32-63,60A). Melting points are 7 H, Bz). Anal. Calcd for C ~ I H Z ~ N OC, ~ S53.95; : H, 5.39; uncorrected, and the optical rotations were measured with N, 2.99; S, 6.86. Found: C, 54.00; H, 5.38; N, 2.84; S, 6.65. a Polartronic I spectrometer (Schmidt and Haensch). 'H Benzoyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-lNMR spectra were recorded with a Bruker WM 250 t hio-8-D-glucopyranoside (4). Acetochloroglucosamine spectrometer on CDCl3solutions (internal Mersi). Kinetic 2 (4 g, 14 mmol) in dry acetone (20 mL) was treated with data were determined with a fluorimeter (Perkin-Elmer potassium thiobenzoate (2.5 g, 14 mmol) and chromato165/10S). Radioactive material was detected either graphed (cyclohexanefethyl acetate 3:1), as described for radioautographically (Agfa-Gevaert Curix X-ray film) or 3. Compound 4 crystallized from ethanol to give white with a Berthold Automatic TLC linear analyzer. Radiocrystals (3.75 g, 73.6%): mp 160 "C; TLC (cyclohexane/ active samples in solution were assayed in a Berthold BFethyl acetate 3:l) Rf0.25; [a]D-10.5' (c 1, chloroform); 'H 815 liquid scintillation counter, using the scintillator NMR (CDCl3) 6 1.91, 2.05, 2.07, 2.08 (4 X S, 12 H, OAC), indicated. Photolabile compounds were irradiated with 3.89 (ddd, 1H, J5,6* = 4.65 Hz, J5,6b = 2.35 Hz, H-5), 4.14 a Rayonet RPR 100reactor equipped with 16 lamps (RPR (dd, 1 H, H-6a), 4.29 (dd, 1 H, H-6b), 4.53 (m, 1H, H-21, 3500 A, emitting at ,A, = 350 nm) and a ventilator for 5.21 (m, 2 H, H-3,4), 5.43 (d, 1H,Jl,z = 10.5Hz, H-l), 6.01 cooling. NaB3H4 was purchased from Amersham-Buchler. (d, 1H, J N H J= 9.5 Hz, NH), 7.49-7.94 (m, 7 H, Bz). Anal. Enzymes. Human &hexosaminidase A (2-acetamidoCalcd for C21H25NOgS: C, 53.95; H, 5.39; N, 2.99; S, 6.86. 2-deoxy-6-11-hexosideacetamidodeoxyhexohydrolase,EC Found: C, 53.74; H, 5.36; N, 3.21; S, 6.89. 3-Azi-1-[(2-acetamido-3,4,6-triO-acetyl-2-deoxy-B* Author to whom correspondence should be addressed. D-galactopyranosyl)thio]butane (6). A solution of 3 + Universitat Freiburg. * Universitlit Bonn. (0.4 g, 0.85 mmol) and 3-azi-l-[@-tolylsulfonyl)oxylbuINTRODUCTION
0 1992 American Chemical Society
Photolabeling Lysosomal 8-Hexosaminidase A
Bioconjugate Chem., Vol. 3, No. 3, 1992 231
tane 5 (0.26 g, 1mmol) in dry methanol (5 mL) was treated radioactivity cochromatographed with unlabeled 8 on twowith 1 M methanolic sodium methylate (200 pL). The dimensional TLC (ethyl acetate/methanol/water 7:2:1). mixture was stirred 2 h at room temperature, deionized Determination of the Inhibition Constants (Ki). with Amberlite IR 120 H+ resin, evaporated to dryness, Fluorescent substrate umbelliferyl4-methyl-2-acetamidoand reacetylated in pyridine/acetic acid (5 mL, 2:l). The 2-deoxy-P-~-glucopyranoside (Fluka) was used as substrate usual workup and flash chromatography (cyclohexane/ (1-2.5 mM) in sodium citrate buffer (pH 4.5, 50 mM) at ethyl acetate 1:l) gave 6 as white solid (350 mg, 61%): 37 "C (7). Inhibitors were used in the following TLC (cyclohexane/ethyl acetate 1:l)Rf 0.25; [(YID +2.6" concentrations: 8, 0-6 mM; 9, 0-10 mM. Each assay ( c 1, chloroform); lH NMR (CDC13) 6 1.06 (s, 3 H, H-4'1, involved 0.25 mUnit/mL 8-hexosaminidase A. 1.65 (m, 2 H, H-2'a,b), 1.97, 2.00, 2.07, 2.17 (4 X s, 12 H, Irreversible Deactivation of @-Hexosaminidase OAc), 2.61 (m, 2 H, H-l'a,b), 3.93 (t, 1 H, H-5), 4.11 (d, with Compounds 8 and 9. Solutions of enzyme (0.1 mL, 2 H, H-Ga,b),4.21 (dd, 1H, J 2 , 3 = 10.65 Hz, H-2), 4.62 (d, 3 units/mL) with compounds 8 (18 mM) or 9 (49 mM) 1H, J 1 , 2 = 10.35 Hz, H-1), 5.39 (d, 1H, J 3 , 4 = 3.7 Hz, H-4), were each irradiated for 20 min with UV light (Amm = 350 5.77 (d, 1 H, J N H = ,9.5 ~ Hz, NH). nm). This experiment was repeated with solutions of the 3-Azi-1-[ (2-acetamido-3,4,6-tri-0-acetyl-%-deoxy-@- same compositions each containing additional compound 10 (155 mM). For reference a solution of the enzyme D-glucopyranosyl)thio]butane (7). A solution of 4 (1 without photolabile reagent was treated in the same g, 2.12 mmol) and 5 (0.65 g, 2.5 mmol) in anhydrous manner. After diluting with citrate buffer (l:lOO),enzyme methanol (10 mL) was treated with methanolic sodium activity was assayed in each mixture as described above. methoxide (0.5 mL) as described for compound 6. After evaporation and reacetylation the crude product was Reaction of 3-Azi-1-[( [6-3H]-2-acetamido-2-deoxypurified by flash chromatography (cyclohexane/ethyl B-~galactopyranosyl)thio]butane with 8-Hexosaminiacetate 2:l). Crystallization from ethyl acetate/ethanol dase A. Compound 8a (3.6 pmol) was dissolved in a gave white crystals of 7 (578 mg, 61 % 1: mp 153 "C; TLC solution of 8-hexosaminidase A (100 pg) in 200 pL sodium (cyclohexane/ethyl acetate 2:l) Rf 0.19; [(YID -41.5' (c 1, citrate buffer (solution A). Part (100 pL) of solution A chloroform); lH NMR (CDCl3) 6 1.04 (s, 3 H, H-4'1, 1.65 was incubated with an excess (155 mM) of compound 10 (m, 2 H, H-P'a,b), 1.97,2.03,2.05,2.11 (4 X s, 12 H, OAc), (solution B). Solutions A and B were deoxygenated by 2.58 (m, 2 H, H-l'a,b), 3.68 (ddd, 1€3, J5,6a = 2.4 Hz, J5,6b bubbling with nitrogen gas for 2 min, then irradiated at 350 nm for 20 min, and dialyzed against buffer and water, = 2.5 Hz, H-5), 4.07 (t,1H, J 2 , 3 = 9.35 Hz, H-2), 4.15 (d, until no more radioactivity was released into the dialy1H, &,6b = 2.5 Hz, H-6a), 4.22 (dd, 1 H, H-6b), 4.56 (d, sate. The radioactivity of the protein solutions was 1H, J 1 , 2 = 10.5 Hz, H-l), 5.25 (d, 1H, J 3 , 4 = 3.75 Hz, H-4), determined by liquid scintillation counting of an aliquot 5.67 (d, 1 H, J N H = ,9.5 ~ Hz, NH). 3-Azi-1-[ (2-acetamido-2-deoxy-@-~-galactopyrano- with Quickszint 1(Zinsser). For solution A 0.19 nmol and for B 0.02 nmol of incorporated radioactive ligand was syl)thio]butane (8). A solution of compound 6 (0.2 g, found per 0.416 nmol of @-hexosaminidaseA. 0.45 mmol) in dry methanol (5 mL) was stirred with 1M Reduction and Carboxymethylation of 3H-Labeled methanolic sodium methoxide (100 pL) for 0.5 h, when @-Hexosaminidase.The samples (A and B)were freezeTLC (ethyl acetate/methanol5:1) indicated the absence dried, reduced (50 mM dithiothreitol at 60 "C for 10 min), of 6. Filtration through a short column of silica gel, and then alkylated (100 mM sodium iodoacetate at room evaporation of the solvent, and lyophilization yielded 8 temperature for 1h) in 7 M guanidinium hydrochloride/ (138 mg, 96%): [(YID +5.6" ( c 1.0, water); TLC (ethyl 0.5 M Tris-HC1, pH 8.5 (0.2pL). The reaction was stopped acetate/methanol/water 7:2:1) Rj 0.46. Anal. Calcd for by adding an excess of 2-mercaptoethanol(lO pL). After C12H21N305S: C, 45.13; H, 6.63; N, 13.15; S, 10.04. deionization with Sephadex-G25 (PD 10 column, PharFound: C, 44.04; H, 6.43; N, 12.77; S, 10.14. 3-Azi-1-[ (2-acetamido-2-deoxy-@-~-glucopyrano- macia), the samples were dried in a Speed Vac concentrator. sy1)thiolbutane (9). Peracetate 7 (0.5g, 1.12 mmol) was Gel Electrophoresis. Sodium dodecyl sulfate polydeacetylated as described for compound 6. Crystallization acrylamide gel electrophoresis (SDS-PAGE) was perfrom ethanol/ethyl acetate gave 9 (325 mg, 91 % ; colorless formed according to the method of Laemmli (8). A crystals): mp 168-171 "C; TLC (ethyl acetate/methanol separation gel (12 cm long) containing 10% acrylamide 5:l) Rf 0.25; [ a ] D -38" ( c 1.0, water). Anal. Calcd for was used in combination with a stacking gel (2 cm long, CizH21N305S: C, 45.13; H, 6.63; N, 13.15; S, 10.04. 4.5 % acrylamide). The reduced and alkylated samples Found: C, 43.87; H, 6.38; N, 12.78; S, 9.88. 3-Azi-1-[([ 6-3H]-2-acetamido-2-deoxy-@-~-galacto-(50 pg) were dissolved in 0.1 mL of Tris-HC1 buffer (62.5 mM, pH 6.8) containing 2% SDS, 10% glycerol, 5% 2pyranosyl)thio]butane (sa). Compound 8 (30 mg, 94 mercaptoethanol, and 0.0013' % bromphenol blue. Elecpmol) was dissolved in sodium phosphate buffer (1mL, trophoresis was carried out until the bromphenol blue pH 7.2, 10 mM). D-Galactose oxidase (0.4 mg, 87 units/ marker reached the bottom of the gel. After electrophoremg) and catalase (30 pL, 1300 units/mL) were added at sis the protein bands were localized by staining with Serva 37 "C. Formation of the 6-aldehydo compound was Blue. The molecular weight was estimated by the standard indicated by TLC (ethyl acetate/methanol/water 17:2:1, calibration kit from Pharmacia Fine Chemicals (Uppsala, Rf0.24). After 11h, when no educt was detected by TLC, Sweden). For determination of the incorporated radiothe reaction mixture was passed through a column (silica activity the gel was cut into 2-mm slices, each of which gel, 0.5 X 3 cm) with water as eluent. The eluate was was submerged in Biolute-S (Zinsser, 0.5 mL) and left concentrated under diminished pressure to 200 pL, made overnight. Quickszint-501 (4 mL) was then added, the alkaline with aqueous sodium hydroxide (1M, pH 9), and mixture was kept for 1h in the cold, and the radioactivity added to an ampule containing NaB3H4(100 mCi, specific was then counted. activity 5 Ci/mmol). After 12 h, the mixture was neutralized (acetic acid) and concentrated to dryness. The RESULTS residue was subjected to column chromatography (ethyl acetate/methanol/water 17:2:1) to give 33 mCi of the 6Lysosomal &hexosaminidase (2-acetamido-2-deoxy-P3H-labeled Sa (specific activity, 1.25 Ci/mmol). The D-hexoside acetamidodeoxyhydrolase, EC 3.2.1.52) exists
Kuhn et al.
232 Blocon/ugate C'hem., Vol. 3, No. 3, 1992 100 --I
subunit, with significant differences in specificity for Nacetylhexosaminides (11). Elucidation of the structure and function of the enzyme is of general clinical interest, because genetic defects in the a- (Tay Sachs' disease) or @-subunit(Sandhoff s disease) cause fatal accumulation of the ganglioside G M(12). ~ Thioglycosides are resistant against enzyme-catalyzed hydrolytic cleavage and therefore useful and generally potent competitive inhibitors. Especially the P-thioglycosides are easily prepared by Koenigs-Knorr type glycosylation of a thionucleophile
975 95
94 8
(13).
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-a-~-galactopyranosyl chloride (1) (14) and 2-acetamido-3,4,6-tri-Oacetyl-2-deoxy-a-~-glucopyranosyl chloride (2) (15) were lo lb
;ssy
R W R'
R
Ilo Ilb
Figure 1. Deactivation of @-hexosaminidaseA by 3-azi-1-[(2acetamido-2-deoxy-l-~-~-glucopyranosyl)thio] - and -galactopyranosy1)thiolbutane (8 and 9). Each sample contained enzyme and had been irradiated for 20 min at A,, = 350 nmunder cooling by ventilation: R, reference without additive;Ia, with compound 8; Ib, with compounds 8 and 10; IIa, with compound 9; IIb, with compounds 9 and 10.
OAc
AcO HNAc CI 1: R = H, R'=OAc 2: R = OAC, R'= H
HNAc
N=N
6: R = H; R', R"= OAC 7: R = R" = OAC;R' = H 8: R = H; R'= R" = OH 9: R, Fi" = OH; R' = H
3H HO I HC -OH
HNAc
HNAc
3: R = H; R'= OAC 4: R = OAC,R' = H
N=N
Be O ,H HO=SHO
I N=N 5
a
t
Start
t
t
t
67 kDa 30 kDo BPB Figure 2. SDS-PAGE of radioactively labeled @-hexosaminidase A. Radioactivityis associatedwith both a-and @-subunits, the @-subunitbeing labeled more efficiently. The arrowsindicate the position of calibration proteins and the marker bromphenol blue (BPB). Radioactivity was determined using a liquid scintillation counter: (*) protein, photoaffinity labeled by 3azi-1-[( [6JH] -2-acetamidc-2-deoxy-l-j3-~-galactopyranosyl)thio] butane (8a), ( 0 )protein, photoaffinity labeled by compound 8a in the presence of the competitive inhibitor compound 10 (155 mM).
as two main oligomeric proteins, hexosaminidase A ( M = 120 000) and hexosaminidase B ( M = 130 000) composed of the subunits a (54 kDa) (9) and @ (56 kDa), where A is a@ and B is 28. The &subunit consists of two peptide chains of approximately 30 000 Da each, which are linked by disulfide bridges (10). Under denaturing conditions (reduction with dithiothreitol and carboxymethylation with sodium iodoacetate) the peptides come apart and show as a single band in SDS-PAGE (compare Figure 2). Hexosaminidase A has two active sites, one on each
OH
HNAc
H d
'0r
10
both converted into the corresponding benzoyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-l-thio-/3-~-hexosaminides (3 and 4) using potassium thiobenzoate as nucleophile. 3-Azi-l-(tosyloxy)butane(5) (16)is a versatile alkylating agent for the facile introduction of the photolabile 3-azibutyl group into thio derivatives of carbohydrates (17). Compounds 3 and 4 were deacylated in situ by dilute sodium methoxide, alkylated with compound 5 and acetylated to yield 3-azi-1-[(2-acetamido-3,4,6-tri-0acetyl-2-deoxy-j3-~-galactopyranosyl)thiol butane (6) and -~-~-glucopyranosyl)thio] butane (7). For the de-0-acetylated thioglycosides 8 and 9, the inhibition constants Ki were determined by Dixon plot to be 1.8 (8) and 4.9 mM (9). When a solution of p-hexosaminidase A in buffer was irradiated for 20 min in the presence of either 8 or 9, using 350-nm UV light, the enzyme became irreversibly deactivated to 50.1% and 31.39% ,respectively. In the absence of 8 or 9, irradiation had hardly any effect on enzyme activity. In the presence of the competitive nonreactive inhibitor 10 the enzyme could be protected against photodeactivation by compounds 8 or 9 (Figure 1). Because of its higher affinity, the N-acetylgalactosaminide was chosen for radioactive labeling with tritium. Compound 8 was oxidized by galactose oxidase and the resulting aldehyde reduced with sodium borohydride to give 3-mi1-([6-3Hl-2-acetamido-2-deoxy-/3-~-galactopyranosyl)thiolbutane (8a). This material having a specific radioactivity of 1.25 Ci/mmol, was used for radiolabeling hexosaminidase A. Irradiation of an incubation mixture in the presence of 8a gave radioactively labeled protein. Radiolabeling could almost totally be suppressed when the competitive inhibitor 10 (18) had been added before
Photolabeling Lysosomal &Hexosaminidase A
irradiation. Incorporation of covalently bound ligand was 45%/mol of protein as calculated from the measured radioactivity in the thoroughly dialyzed solution. Electrophoresis in polyacrylamide under denaturating conditions separates labeled hexosaminidase A into the unequally labeled subunits (Figure 2). The a-subunit carries 30 7% ,the P-subunit 70 7% ,of the total radioactivity. ACKNOWLEDGMENT
J.L. thanks the Deutschen Forschungsgemeinschaft (DFG) for financial support. LITERATURE CITED (1) Knowles, J. R., and Bayley, H. (1977) Methods Enzymol. 46, 69-114. (2) Kuhn, C.-S., and Lehmann, J. (1987) Carbohydr. Res. 160, C6-C8. (3) Ats, S. C.,Lausberg, E., Lehmann, J.,and Sandhoff, K. (1990) Liebigs Ann. Chem. 1261-1264. (4) Ats, S. C., Lausberg, E., Lehmann, J., and Sandhoff, K. Unpublished results. (5) Still, M. C., Kahn, M., and Mitra, A. (1978) J . Org. Chem. 43, 2923-2925.
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(6) Sandhoff, K., Conzelmann, E., and Nehrhorn, H. (1977) Hoppe-Seyler’s 2.Physiol. Chem. 358, 779-787. (7) Leaback, D. H., and Walker, P. G. (1961) Biochem. J . 78, 151-156. ( 8 ) Laemmli, U. K. (1970) Nature 227, 680-685. (9) Mahuran, D. L., and Lowden, J. A. (1980) Can. J . Biochem. 58, 287-294. (10) Mahuran, D. J., Tsui, F., Gravel, R. A., and Lowden, J. A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1602-1605. (11) Kytzia, H.-J., and Sandhoff, K. (1985) J . Biol. Chem. 260, 7568-7572. (12) Sandhoff, K., Conzelmann, E., Neufeld, E. F., Kaback, M. M., and Susuki, K. (1989) The Metabolic Basic of Inherited Disease, 6th ed., (C. R. Sriver, A. L. Beaudet, W. S. Sly, D. Valle, Eds.) pp 1807-1842, McGraw-Hill, New York. (13) Horton, D. (1963) Methods Carbohydr. Chem. 2,435-436. (14) Tarasiejska, Z., and Jeanloz, R. W. (1958) J . Am. Chem. SOC.80,6325-6327. (15) Horton, D. (1972) Methods Carbohydr. Chem. 6,282-285. (16) Church, R. F., and Weiss, M. J. (1970) J . Org. Chem. 35, 2465-247 1. (17) Kuhn, C.-S., Lehmann, J.,and Steck, J. (1990) Tetrahedron Lett. 46, 3129-3134. (18) Kuhn, C.-S., Lehmann, J., and Sandhoff, K. (1992) Liebigs Ann. Chem. In press.
