362
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[39]
T
~
p
,
5' ,~p
.__~~~
•.~i5'
(HIS-46 ~TRP-140J
FIG. 2. Schematic representation of the residues of staphylococcal nuclease that were found to react with the affinity labeling reagents depicted in Fig. 1.
and possibly with Met-26. Reagent IV reacts exclusively with Tyr-115, and reagent V with Tyr-85. Reagent VI reacts specifically with His-46 (55%) and Trp-140(45%). Such studies have been useful in determining the topography of the active site of this enzyme in solution and in relating catalytic and binding functions to specific amino acid residues within this region.
Comments Ribonuclease k also has been affinity labeled with the diazonium salt of adenosine 5'-p-aminophenylphosphate 2'(3')phosphate. Using methods similar to tha¢ described in this chapter, 1 mole of the reagent was found to be covalently bound to the enzyme. In this case Tyr-72 was modified. 1~ n M. Gorecki, M. Wilchek, and P. Patchornik, Biochim. Biophys. Acta 229, 590 (1971).
[39] Carbohydrate Binding Sites By E. W. THOMAS Several carbohydrate analogs incorporating haloacetyl TM or epoxideT M groups have been synthesized and evaluated as affinity labels for D. l:I. Buss and I. J. Goldstein, J. Chem. Soc. C p. 1457 (1968). =E. W. Thomas, J. Med. Chem. 13, 755 (1970). a F. Naider, Z. Bohak, and J. Yariv, Biochemistry 11, 3202 (1972). l S. Otieno, A. K. Bhargava, E. A. Barnard, and A. H. Ramel, Biochemistry 14, 2403 (1973). 5E. W. Thomas, Carbohydr. Res. 13, 225 (1970). 6 G. Legler and E. Bause, Carbohydr. Res. 28, 45 (1973). E. M. Bessel and J. H. Westwood, Carbohydr. Res. 25, 11 (1972). 8 G. Legler, Biochem. Soc. Trans. 3, 847 (1975). 9 M. L. Shulman, S. D. Shiyan, and A. Ya. Khorlin, Carbohydr. Res. 33, 229 (1974). ~oj. E. G. Barnett and A. Ralph, Carbohydr. Res. 17, 231 (1971).
[30]
CARBOHYDRATE BINDING SITES
363
carbohydrate binding sites in proteins. Analogs incorporating photosensitive functions (photoaffinity labels) have also been described. ~-16 This article deals with the synthesis and properties of representative compounds of the above type, in particular those in which the reactive function is attached to the anomeric carbon atom of the carbohydrate.
Bromoacetylglycosylamines The glycosylamines (1-amino sugars) are easily accessible by treatment of the appropriate aldose with anhydrous methanol saturated with ammonia. 17 The synthesis of fl-D-galactosylamine is given here, since a published method TM was not satisfactory. fl-D-Galactosylamine 19 ( I)
Methanol (250 ml) at 4 ° is saturated with gaseous ammonia from a cylinder. D-Galactose (15 g) in hot water (20 ml) is added, and the reaction vessel is stoppered. Compound (I) crystallizes after storage at 23 ° for about 14 days; yields may be increased by more prolonged storage at 4 °. The crystals are collected, washed with methanol, and recrystallized by dissolving water (2 parts), adding methanol (4 parts) followed by n-propanol to turbidity. The product has a melting point of 135°-136 °. The pH of an aqueous solution of (I) is about 9. The a-D-galactosylamine-ammonia complex, formed from D-galactose and ammonia in anhydrous methanol, TM gives an aqueous solution of pH 11. N-Bromoacetylation ~
The preferred reagent is bromoaeetic anhydride; the following procedure is convenient if radioactive reagent is required. Bromoacetic Anhydride. Dry bromoacetic acid (1 mM) in dry CC14 (3 ml) is treated with dicyclohexylcarbodiimide (0.5 mM) at 4 ° with 11E. W. Thomas, Carbohydr. Res. 31, 101 (1973). 1~M. B. Perry and L. W. Heung, Can. J. Biochem. 50, 510 (1972). 1, E. Saman, M. Claeyssens, H. Kersters-Hilderson, and C. K. de Bruyne, Carbohydr. Res. 30, 207 (1973). ~ A. E. Burkhardt, S. O. Russo, C. G. Rinehardt, and G. M. Loudon, Biochemistry 14, 5465 (1975). 15M. Beppu, T. Terao, and T. Osawa, J. Biochem. (Tokyo) 78, 1013 (1975). 1~G. Rudwick, H. R. Kaback, and R. Weil, J. Biol. Chem. 250, 6847 (1975). 17H. Isbell and H. L. Frush, J. Org. Chem. 23, 1309 (1958). is H. L. Frush and H. I. Isbell, J. Res. Natl. Bur. Stand. 47, 239 (1951). 19L. A. Lobry de Bruyn and F. H. Van Leent, Recl. Trav. Chim. Pays-Bas 14, 134 (1895).
364
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
N (I)
[39]
H(~//~\I~IHCOCHBr (II)
Fro. 1. F o r m a t i o n of N-bromoacetyl-fl-D-galactosglamine (II) from galactosylamine.
stirring. After 5 rain at 23 °, dicyclohexylurea is removed by centrifugation; the supernatant liquid either is used directly or concentrated under reduced pressure. N-Bromoacetyl-fl-D-Galactosylamine (H) (Fiq. 1). Galactosylamine (I) (2 raM), suspended in dry dimethylformamide (2 ml), is treated with bromoacetic anhydride (2.5 raM) with stirring at 23°: (I) dissolves as reaction proceeds. A large excess of anhydride should be avoided as O-acylation can occur. After 6 hr, crude (II) is precipitated by adding the reaction mixture dropwise with stirring to ice-cold diethyl ether (200 ml). The resultant solid is collected, washed well with ether, and crystallized by dissolving in methanol and adding ether to turbidity. Compound (II), formed in 80% yield, has m.p. 192 ° (dee) and is homogeneous by TLC (Silica gel G; methanol:acetone 1:10 v/v; detected by charring with H2S04). Other bromoacetylglycosylamines have the properties 2 in the tabulation. Melting N-Bromoacetyl-
point
{a}~)°(C = 1, H~O)
D-Glucosylamine ~Glucosylamine L-Fucosylamine a-Lactosylamine
187°-190 ° 178 ° 175 ° 158 °
- 13.5 ° -b 14 ° -b2.8 ° --
Compound (II) inhibits fl-galactosidase from Escherichia coli irreversibly; the inactivation shows saturation kinetics, with K~ = 1.13 mM and a first-order rate constant 0.063 min -1. Enzymic activity reappears on treating with fl-mercaptoethanol s and is ascribed to thiolysis of a sulfonium salt formed at a methionine in the active site. N-BromoaeetylL-fucosylamine reacts with fl-galactosidase, giving a covalent 1:1 complex which is enzymically active. 2° ~ 3 . Yariv, K. J. Wilson, J. Hfldesheim, and S. Blumberg, FEBS Lett. 15, 24 (1971).
[39]
CARBOHYDRATE BINDING SITES
OH
365
CHOH
>
(III)
2 ° y H2C'~,-CH2 (IV)
Fio. 2. Formation of 2,'3'-epoxypropyl-2-acetamido-2-deoxy-fl-D-glucopyranoside (IV) via 2',3'-epoxypropyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-~-D-glucopyranoside (III). fl-Galactosidase from E. coli is also inhibited in vivo by (II), but only if lac permeasc is functional. ~1 Presumably, (II) is actively accumulated by cells and is not acting as a site-directed inhibitor of permease in short-term experiments. Most of the bromoacetylglucosylamines show marked effects on glucose-stimulated insulin release from isolated pancreatic islet cells. 22
Epoxypropyl Glycosides These analogs are accessible ~ by epoxidation of the appropriate acetylated allyl glucoside (or vinyl C-glycoside'), followed by deacetylation. The following procedure for synthesis of epoxypropyl 2-acetamido fl-Dglycoside is applicable to the corresponding glucose, cellobiose, chitobiose, and chitotriose analogs. The allyl glycosides are themselves prepared from the appropriate glycosyl halide and allyl alcohol, e'g',5
2',3'-Epoxypropyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-fl-DgIucopyranoside (III) (Fig. 2) Allyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-fl-D-glucopyranoside (1 raM) in methylene chloride (10 ml) containing peroxyphthalic acid (2 mM; peroxybenzoic acid is also satisfactory) is refluxed gently for 3 hr. After cooling to 4 °, the solution is filtered (if peroxyphthalic acid is used), washed with cold aqueous potassium bicarbonate solution until free of peroxy acid (as tested with acidified KI), dried with Na~SO,, and evaporated to dryness. Compound (III) is crystallized from methanol-ether. Yield, 85%; m.p., 162°-163°; {a}~3, _34.4 ° (C = 0.4, CHC13). ~1W. Daring and E. W. Thomas, unpublished data (1974). 22B. Hellman, L-A Idahl, A. Lernmark, I-B Taljedal, and E. W. Thomas, Mol. Pharmacol. 12, 208 (1976).
366
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[39]
~',3P-Epoxypropyl-~-acetamido-~-deoxy-fl-D-gl~copyranoside (IV) Compound (III), suspended in dry methanol (final concentration 10~ w/v), is treated with methanolic barium methoxide solution to give a final concentration of 20 mM in base. After 12 hr at 4 °, the reaction mixture is partly neutralized with a few chips of solid C02 and evaporated to dryness. The residue is triturated with methanol and filtered, and dioxane is added dropwise to the filtrate until a permanent ttlrbidity obtains. Compound (IV) crystallizes as needles. Yield, 60%; m.p., 175 °178°; {a}~", --37.5 ° (C = 2, H20). Compound (IV) is homogeneous by thin-layer chromatography (TLC) under identical conditions used to evaluate (II). Solutions of (IV) in 0.1 N sodium thiosulfate become alkaline within 5 min at 23 °, indicating the presence of the epoxide group. Epoxypropyl-fl-D-glycosides of N-acetylglucosamine (Gle NAc), diN-acetylchitobiose (Glc NAc)2, and tri-N-acetylchitofriose (Glc NAc)3 irreversibly inhibit lysozyme from hen's egg-white. 2s Although the kinetics of inactivation have not been rigorously examined, inactivation has been shown to result from selective esterification 24 of Asp-52, in the case of the (Glc NAc)2 analog. The covalent enzyme inhibitor complex has been crystallized, and X-ray diffraction studies 25 show that the two pyranose rings of the analog occupy subsites B and C as originally predicted. The esterified enzyme cannot be reactivated with alkaline hydroxylamine. Successful cleavage of the complex with NaBH4 is possible only after prior chemical reduction at all disulfide linkages of the enzyme.26 Compound (IV) reacts with a-lactalbumin to give a 1:1 covalent complex.27 The site of attachment has not been determined, but the alactalbumin is not protected from (IV) by either Glc NAc or (Glc NAc) 2. The 3',4'-epoxybutyl-fl-D-glycoside of (Glc NAc)2 inhibits hen's eggwhite lysozyme at a similar rate to the epoxypropyl analog, but the 5',6'epoxyhexyl analog is much less effective.2s Photoaffinlty Labels The synthesis of a number of 2-nitro-4-azidophenyl lm6 and 4-azidophenyl-fl-D-glycosidesis has been reported. Nitro-substituted analogs 23E. W. Thomas, J. F. McKelvy, and N. Sharon, Nature (London) 222, 485 (1969). Y. Eshdat, J. F. McKelvy, and N. Sharon, J. Biol. Chem. 248, 5892 (1973). J. Moult, Y. Eshdat, and N. Sharon, J. Mol. Biol. 75, 1 (1973). ~6y . Eshdat, A. Dunn, and N. Sharon, Proc. Natl. Acad. Sci. U.S.A. 71, 1658 (1974). 2Ty . Eshdat and E. W. Thomas, unpublished data (1971). E. W. Thomas, unpublished data (1972).
[39]
CARBOHYDRATE BINDING SITES
367
have the advantage of being activable with light of wavelength >350 nm. Access to 2-nitro-4-azidophenyl-a-D-glycosides is not easy, except in the case of the mannoside, where reaction of a-acetochloromannose with the appropriate phenol proceeds with retention of configuration.11
4- (Benzyloxycarbonylamino ) -2-nitrophenyl-a-D-mannopyranoside Tetraacetate (V) (Fig. 3) 4-(Benzyloxycarbonylamino)2-nitrophenol (3.39) in 1 M sodium hydroxide (17 ml) and acetone (30 ml) at 5 ° is treated with tetra-O-acetyla-D-mannopyranosyl chloride (3.7 g) dissolved in acetone (20 ml). After 12 hr at 5 °, the mixture is poured into water (200 ml) and extracted five times with chloroform. Unreacted phenol is recovered from the chloroform extracts by six extractions with 1M sodium hydroxide. The chloroform layer is washed with dilute acid, dried, and evaporated to give a mixture of (V) and mannopyranosyl chloride. Elution of the mixture from neutral alumina (Woelm) with chloroform gives the unreacted mannosyl chloride. Compound (V), seen as a pale yellow band on the column, is eluted with chloroform-ethylacetate (1:1). Yield, 0.7 g (11%) ; m.p., 188 ° (from n-propanol) ; {a}~~, -~68 ° (C = 0.2, CHC13).
~-Azido-2-nitrophenyl-a-D-mannopyranoside (VI) Compound (V) (2.3 g) in dry chloroform (10 ml) is treated with a 45% solution (30 ml) of hydrogen bromide in glacial acetic acid with vigorous stirring. After 15 rain at 23 ° with stirring, the solution is poured into chloroform (200 ml), which is in turn poured onto crushed ice (200 ml). The chloroform layer is separated, freed from acid by several extractions with ice-cold aqueous sodium bicarbonate, dried, and evaporated. The residue obtained is washed with a small volume of ice-cold chloroform to remove benzyl bromide. In addition to removal of the benzyloxycarbonyl group, partial de-O-acetylation occurs during this step, the product being predominantly the tri-O-acetyl derivative. An
•~C
NHCO.OC , H2CGHs NO 2
(v)
~O ~Lmm~rC
~ NO
2
NH2
Ac
N3 NO2
(v~)
FIG. 3. A synthetic approach to 4-azido-2-nitrophenyl-a-D-mannopyranoside (VI).
368
ENZYMES~ ANTIBODIES, AND OTHER PROTEINS
[40]
improvement here would be to remove the benzyloxycarbonyl group by limited hydrogenolysis over palladium black. 12 To the amine triacetate (500 mg) in methanol-acetic acid (1:1 v/v, 10 ml) at 0 °, isopentylnitrite (0.5 ml) is added. After 30 rain, 1 M hydrochloric acid (2 ml) is added, followed by solid sodium azide (1 molar equivalent). After stirring for 60 min, the mixture is poured into excess water and extracted with chloroform. The extract is washed, dried, and evaporated to yield a cream colored solid (Va) having Vma,, = 2 1 2 0 cm -1 (Na). Conventional deacetylation (a small chip of sodium added to a solution in dry methanol) results in precipitation of (VI): 100 mg, 26% yield. After recrystallization from hot water, (VI) has m.p. 132°-135°; {~}~8, + 130 ° (C = 0.22, H20); .-max,~m°355 nm (d900); ~max, 2100 cm -1
(N~). Irradiation of 4-azido-2-nitrophenyl-~-D-mannopyranoside with light of wavelength >350 nm gives the corresponding azo compound in high yield. ~1 No evidence has been obtained ~9 for specific labeling of concanavalin A by irradiating mixtures of the protein and (VI), although one report ~5 claims specific labeling of concanavalin A with 4-azidophenyla-D-mannopyranoside. In the writer's opinion, the relatively long lifetimes of photogenerated nitrenes, and their propensity to undergo dimerization and intramolecular insertion reactions, make them unattractive canavalin A by irradiating mixtures of the protein and (Va), although one ing sites which do not contain nucleophilic side chains is still unsolved. E. W. Thomas, unpublished data (1973).
[40] Glucosidases By
G. LEGLER
Preparation of active site-directed inhibitors for glucosidases by the introduction of reactive groups into substrate analogs has been handicapped by the high specificity of these enzymes for the intact glucose molecule. 1,2 Modification of a hydroxyl group by a reactive acyl or alkyl residue usually prevents binding to the substrate binding site. Incorporation of such groups into the aglycon part is tolerated, but labeling will occur at functional groups of the enzyme not directly involved in cataly1 G. Legler, Mol. Cell. Biochem. 2, 31 (1973). 2 G. Legler, Biochem. Soc. Trans. 3, 847 (1975).
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[39]
T
~
p
,
5' ,~p
.__~~~
•.~i5'
(HIS-46 ~TRP-140J
FIG. 2. Schematic representation of the residues of staphylococcal nuclease that were found to react with the affinity labeling reagents depicted in Fig. 1.
and possibly with Met-26. Reagent IV reacts exclusively with Tyr-115, and reagent V with Tyr-85. Reagent VI reacts specifically with His-46 (55%) and Trp-140(45%). Such studies have been useful in determining the topography of the active site of this enzyme in solution and in relating catalytic and binding functions to specific amino acid residues within this region.
Comments Ribonuclease k also has been affinity labeled with the diazonium salt of adenosine 5'-p-aminophenylphosphate 2'(3')phosphate. Using methods similar to tha¢ described in this chapter, 1 mole of the reagent was found to be covalently bound to the enzyme. In this case Tyr-72 was modified. 1~ n M. Gorecki, M. Wilchek, and P. Patchornik, Biochim. Biophys. Acta 229, 590 (1971).
[39] Carbohydrate Binding Sites By E. W. THOMAS Several carbohydrate analogs incorporating haloacetyl TM or epoxideT M groups have been synthesized and evaluated as affinity labels for D. l:I. Buss and I. J. Goldstein, J. Chem. Soc. C p. 1457 (1968). =E. W. Thomas, J. Med. Chem. 13, 755 (1970). a F. Naider, Z. Bohak, and J. Yariv, Biochemistry 11, 3202 (1972). l S. Otieno, A. K. Bhargava, E. A. Barnard, and A. H. Ramel, Biochemistry 14, 2403 (1973). 5E. W. Thomas, Carbohydr. Res. 13, 225 (1970). 6 G. Legler and E. Bause, Carbohydr. Res. 28, 45 (1973). E. M. Bessel and J. H. Westwood, Carbohydr. Res. 25, 11 (1972). 8 G. Legler, Biochem. Soc. Trans. 3, 847 (1975). 9 M. L. Shulman, S. D. Shiyan, and A. Ya. Khorlin, Carbohydr. Res. 33, 229 (1974). ~oj. E. G. Barnett and A. Ralph, Carbohydr. Res. 17, 231 (1971).
[30]
CARBOHYDRATE BINDING SITES
363
carbohydrate binding sites in proteins. Analogs incorporating photosensitive functions (photoaffinity labels) have also been described. ~-16 This article deals with the synthesis and properties of representative compounds of the above type, in particular those in which the reactive function is attached to the anomeric carbon atom of the carbohydrate.
Bromoacetylglycosylamines The glycosylamines (1-amino sugars) are easily accessible by treatment of the appropriate aldose with anhydrous methanol saturated with ammonia. 17 The synthesis of fl-D-galactosylamine is given here, since a published method TM was not satisfactory. fl-D-Galactosylamine 19 ( I)
Methanol (250 ml) at 4 ° is saturated with gaseous ammonia from a cylinder. D-Galactose (15 g) in hot water (20 ml) is added, and the reaction vessel is stoppered. Compound (I) crystallizes after storage at 23 ° for about 14 days; yields may be increased by more prolonged storage at 4 °. The crystals are collected, washed with methanol, and recrystallized by dissolving water (2 parts), adding methanol (4 parts) followed by n-propanol to turbidity. The product has a melting point of 135°-136 °. The pH of an aqueous solution of (I) is about 9. The a-D-galactosylamine-ammonia complex, formed from D-galactose and ammonia in anhydrous methanol, TM gives an aqueous solution of pH 11. N-Bromoacetylation ~
The preferred reagent is bromoaeetic anhydride; the following procedure is convenient if radioactive reagent is required. Bromoacetic Anhydride. Dry bromoacetic acid (1 mM) in dry CC14 (3 ml) is treated with dicyclohexylcarbodiimide (0.5 mM) at 4 ° with 11E. W. Thomas, Carbohydr. Res. 31, 101 (1973). 1~M. B. Perry and L. W. Heung, Can. J. Biochem. 50, 510 (1972). 1, E. Saman, M. Claeyssens, H. Kersters-Hilderson, and C. K. de Bruyne, Carbohydr. Res. 30, 207 (1973). ~ A. E. Burkhardt, S. O. Russo, C. G. Rinehardt, and G. M. Loudon, Biochemistry 14, 5465 (1975). 15M. Beppu, T. Terao, and T. Osawa, J. Biochem. (Tokyo) 78, 1013 (1975). 1~G. Rudwick, H. R. Kaback, and R. Weil, J. Biol. Chem. 250, 6847 (1975). 17H. Isbell and H. L. Frush, J. Org. Chem. 23, 1309 (1958). is H. L. Frush and H. I. Isbell, J. Res. Natl. Bur. Stand. 47, 239 (1951). 19L. A. Lobry de Bruyn and F. H. Van Leent, Recl. Trav. Chim. Pays-Bas 14, 134 (1895).
364
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
N (I)
[39]
H(~//~\I~IHCOCHBr (II)
Fro. 1. F o r m a t i o n of N-bromoacetyl-fl-D-galactosglamine (II) from galactosylamine.
stirring. After 5 rain at 23 °, dicyclohexylurea is removed by centrifugation; the supernatant liquid either is used directly or concentrated under reduced pressure. N-Bromoacetyl-fl-D-Galactosylamine (H) (Fiq. 1). Galactosylamine (I) (2 raM), suspended in dry dimethylformamide (2 ml), is treated with bromoacetic anhydride (2.5 raM) with stirring at 23°: (I) dissolves as reaction proceeds. A large excess of anhydride should be avoided as O-acylation can occur. After 6 hr, crude (II) is precipitated by adding the reaction mixture dropwise with stirring to ice-cold diethyl ether (200 ml). The resultant solid is collected, washed well with ether, and crystallized by dissolving in methanol and adding ether to turbidity. Compound (II), formed in 80% yield, has m.p. 192 ° (dee) and is homogeneous by TLC (Silica gel G; methanol:acetone 1:10 v/v; detected by charring with H2S04). Other bromoacetylglycosylamines have the properties 2 in the tabulation. Melting N-Bromoacetyl-
point
{a}~)°(C = 1, H~O)
D-Glucosylamine ~Glucosylamine L-Fucosylamine a-Lactosylamine
187°-190 ° 178 ° 175 ° 158 °
- 13.5 ° -b 14 ° -b2.8 ° --
Compound (II) inhibits fl-galactosidase from Escherichia coli irreversibly; the inactivation shows saturation kinetics, with K~ = 1.13 mM and a first-order rate constant 0.063 min -1. Enzymic activity reappears on treating with fl-mercaptoethanol s and is ascribed to thiolysis of a sulfonium salt formed at a methionine in the active site. N-BromoaeetylL-fucosylamine reacts with fl-galactosidase, giving a covalent 1:1 complex which is enzymically active. 2° ~ 3 . Yariv, K. J. Wilson, J. Hfldesheim, and S. Blumberg, FEBS Lett. 15, 24 (1971).
[39]
CARBOHYDRATE BINDING SITES
OH
365
CHOH
>
(III)
2 ° y H2C'~,-CH2 (IV)
Fio. 2. Formation of 2,'3'-epoxypropyl-2-acetamido-2-deoxy-fl-D-glucopyranoside (IV) via 2',3'-epoxypropyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-~-D-glucopyranoside (III). fl-Galactosidase from E. coli is also inhibited in vivo by (II), but only if lac permeasc is functional. ~1 Presumably, (II) is actively accumulated by cells and is not acting as a site-directed inhibitor of permease in short-term experiments. Most of the bromoacetylglucosylamines show marked effects on glucose-stimulated insulin release from isolated pancreatic islet cells. 22
Epoxypropyl Glycosides These analogs are accessible ~ by epoxidation of the appropriate acetylated allyl glucoside (or vinyl C-glycoside'), followed by deacetylation. The following procedure for synthesis of epoxypropyl 2-acetamido fl-Dglycoside is applicable to the corresponding glucose, cellobiose, chitobiose, and chitotriose analogs. The allyl glycosides are themselves prepared from the appropriate glycosyl halide and allyl alcohol, e'g',5
2',3'-Epoxypropyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-fl-DgIucopyranoside (III) (Fig. 2) Allyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-fl-D-glucopyranoside (1 raM) in methylene chloride (10 ml) containing peroxyphthalic acid (2 mM; peroxybenzoic acid is also satisfactory) is refluxed gently for 3 hr. After cooling to 4 °, the solution is filtered (if peroxyphthalic acid is used), washed with cold aqueous potassium bicarbonate solution until free of peroxy acid (as tested with acidified KI), dried with Na~SO,, and evaporated to dryness. Compound (III) is crystallized from methanol-ether. Yield, 85%; m.p., 162°-163°; {a}~3, _34.4 ° (C = 0.4, CHC13). ~1W. Daring and E. W. Thomas, unpublished data (1974). 22B. Hellman, L-A Idahl, A. Lernmark, I-B Taljedal, and E. W. Thomas, Mol. Pharmacol. 12, 208 (1976).
366
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[39]
~',3P-Epoxypropyl-~-acetamido-~-deoxy-fl-D-gl~copyranoside (IV) Compound (III), suspended in dry methanol (final concentration 10~ w/v), is treated with methanolic barium methoxide solution to give a final concentration of 20 mM in base. After 12 hr at 4 °, the reaction mixture is partly neutralized with a few chips of solid C02 and evaporated to dryness. The residue is triturated with methanol and filtered, and dioxane is added dropwise to the filtrate until a permanent ttlrbidity obtains. Compound (IV) crystallizes as needles. Yield, 60%; m.p., 175 °178°; {a}~", --37.5 ° (C = 2, H20). Compound (IV) is homogeneous by thin-layer chromatography (TLC) under identical conditions used to evaluate (II). Solutions of (IV) in 0.1 N sodium thiosulfate become alkaline within 5 min at 23 °, indicating the presence of the epoxide group. Epoxypropyl-fl-D-glycosides of N-acetylglucosamine (Gle NAc), diN-acetylchitobiose (Glc NAc)2, and tri-N-acetylchitofriose (Glc NAc)3 irreversibly inhibit lysozyme from hen's egg-white. 2s Although the kinetics of inactivation have not been rigorously examined, inactivation has been shown to result from selective esterification 24 of Asp-52, in the case of the (Glc NAc)2 analog. The covalent enzyme inhibitor complex has been crystallized, and X-ray diffraction studies 25 show that the two pyranose rings of the analog occupy subsites B and C as originally predicted. The esterified enzyme cannot be reactivated with alkaline hydroxylamine. Successful cleavage of the complex with NaBH4 is possible only after prior chemical reduction at all disulfide linkages of the enzyme.26 Compound (IV) reacts with a-lactalbumin to give a 1:1 covalent complex.27 The site of attachment has not been determined, but the alactalbumin is not protected from (IV) by either Glc NAc or (Glc NAc) 2. The 3',4'-epoxybutyl-fl-D-glycoside of (Glc NAc)2 inhibits hen's eggwhite lysozyme at a similar rate to the epoxypropyl analog, but the 5',6'epoxyhexyl analog is much less effective.2s Photoaffinlty Labels The synthesis of a number of 2-nitro-4-azidophenyl lm6 and 4-azidophenyl-fl-D-glycosidesis has been reported. Nitro-substituted analogs 23E. W. Thomas, J. F. McKelvy, and N. Sharon, Nature (London) 222, 485 (1969). Y. Eshdat, J. F. McKelvy, and N. Sharon, J. Biol. Chem. 248, 5892 (1973). J. Moult, Y. Eshdat, and N. Sharon, J. Mol. Biol. 75, 1 (1973). ~6y . Eshdat, A. Dunn, and N. Sharon, Proc. Natl. Acad. Sci. U.S.A. 71, 1658 (1974). 2Ty . Eshdat and E. W. Thomas, unpublished data (1971). E. W. Thomas, unpublished data (1972).
[39]
CARBOHYDRATE BINDING SITES
367
have the advantage of being activable with light of wavelength >350 nm. Access to 2-nitro-4-azidophenyl-a-D-glycosides is not easy, except in the case of the mannoside, where reaction of a-acetochloromannose with the appropriate phenol proceeds with retention of configuration.11
4- (Benzyloxycarbonylamino ) -2-nitrophenyl-a-D-mannopyranoside Tetraacetate (V) (Fig. 3) 4-(Benzyloxycarbonylamino)2-nitrophenol (3.39) in 1 M sodium hydroxide (17 ml) and acetone (30 ml) at 5 ° is treated with tetra-O-acetyla-D-mannopyranosyl chloride (3.7 g) dissolved in acetone (20 ml). After 12 hr at 5 °, the mixture is poured into water (200 ml) and extracted five times with chloroform. Unreacted phenol is recovered from the chloroform extracts by six extractions with 1M sodium hydroxide. The chloroform layer is washed with dilute acid, dried, and evaporated to give a mixture of (V) and mannopyranosyl chloride. Elution of the mixture from neutral alumina (Woelm) with chloroform gives the unreacted mannosyl chloride. Compound (V), seen as a pale yellow band on the column, is eluted with chloroform-ethylacetate (1:1). Yield, 0.7 g (11%) ; m.p., 188 ° (from n-propanol) ; {a}~~, -~68 ° (C = 0.2, CHC13).
~-Azido-2-nitrophenyl-a-D-mannopyranoside (VI) Compound (V) (2.3 g) in dry chloroform (10 ml) is treated with a 45% solution (30 ml) of hydrogen bromide in glacial acetic acid with vigorous stirring. After 15 rain at 23 ° with stirring, the solution is poured into chloroform (200 ml), which is in turn poured onto crushed ice (200 ml). The chloroform layer is separated, freed from acid by several extractions with ice-cold aqueous sodium bicarbonate, dried, and evaporated. The residue obtained is washed with a small volume of ice-cold chloroform to remove benzyl bromide. In addition to removal of the benzyloxycarbonyl group, partial de-O-acetylation occurs during this step, the product being predominantly the tri-O-acetyl derivative. An
•~C
NHCO.OC , H2CGHs NO 2
(v)
~O ~Lmm~rC
~ NO
2
NH2
Ac
N3 NO2
(v~)
FIG. 3. A synthetic approach to 4-azido-2-nitrophenyl-a-D-mannopyranoside (VI).
368
ENZYMES~ ANTIBODIES, AND OTHER PROTEINS
[40]
improvement here would be to remove the benzyloxycarbonyl group by limited hydrogenolysis over palladium black. 12 To the amine triacetate (500 mg) in methanol-acetic acid (1:1 v/v, 10 ml) at 0 °, isopentylnitrite (0.5 ml) is added. After 30 rain, 1 M hydrochloric acid (2 ml) is added, followed by solid sodium azide (1 molar equivalent). After stirring for 60 min, the mixture is poured into excess water and extracted with chloroform. The extract is washed, dried, and evaporated to yield a cream colored solid (Va) having Vma,, = 2 1 2 0 cm -1 (Na). Conventional deacetylation (a small chip of sodium added to a solution in dry methanol) results in precipitation of (VI): 100 mg, 26% yield. After recrystallization from hot water, (VI) has m.p. 132°-135°; {~}~8, + 130 ° (C = 0.22, H20); .-max,~m°355 nm (d900); ~max, 2100 cm -1
(N~). Irradiation of 4-azido-2-nitrophenyl-~-D-mannopyranoside with light of wavelength >350 nm gives the corresponding azo compound in high yield. ~1 No evidence has been obtained ~9 for specific labeling of concanavalin A by irradiating mixtures of the protein and (VI), although one report ~5 claims specific labeling of concanavalin A with 4-azidophenyla-D-mannopyranoside. In the writer's opinion, the relatively long lifetimes of photogenerated nitrenes, and their propensity to undergo dimerization and intramolecular insertion reactions, make them unattractive canavalin A by irradiating mixtures of the protein and (Va), although one ing sites which do not contain nucleophilic side chains is still unsolved. E. W. Thomas, unpublished data (1973).
[40] Glucosidases By
G. LEGLER
Preparation of active site-directed inhibitors for glucosidases by the introduction of reactive groups into substrate analogs has been handicapped by the high specificity of these enzymes for the intact glucose molecule. 1,2 Modification of a hydroxyl group by a reactive acyl or alkyl residue usually prevents binding to the substrate binding site. Incorporation of such groups into the aglycon part is tolerated, but labeling will occur at functional groups of the enzyme not directly involved in cataly1 G. Legler, Mol. Cell. Biochem. 2, 31 (1973). 2 G. Legler, Biochem. Soc. Trans. 3, 847 (1975).
