J. Org. Chem. 1988, 53, 4939-4945
4939
Enzymes in Carbohydrate Synthesis: Lipase-Catalyzed Selective Acylation and Deacylation of Furanose and Pyranose Derivatives William J. Hennen, H. Marcel Sweers, Yi-Fong Wang, and Chi-Huey Wong* Department of Chemistry, Texas A&M University, College Station, Texas 77843
Received M a y 31, 1988
A number of furanose and pyranose derivatives were selectively acylated and deacylated on a preparative scale in lipase-catalyzed reactions. The primary hydroxyl functions of the methyl furanosidesof D-ribose, D-arabinose, D-XylOSe, and 2-deoxy-~-ribosewere selectively acetylated by crude porcine pancreatic lipase in tetrahydrofuran by using 2,2,2-trifluoroethyl acetate as the acyl donor. Selective deacetylations of the primary hydroxyl functions
in the peracetylated methyl furanosides of D-ribose, D-arabinose, D-XylOSe, and 2-deoxy-~-ribosewere best accomplished in a 9:l solution of 0.1 N phosphate buffer (pH 7 ) and N,N-dimethylformamideusing Candida cylindracea lipase. Selective cleavage of the 1-0-acetyl groups from 1,2,3,5-tetra-O-acetyl-~-ribose and -D-XylOSe were similarly accomplished with Aspergillus niger lipase. Similar regioselectivites were observed in the pyranose series. The Candida lipase was found to be the best for selective deacylation of the primary position from the peracylated methyl pyranosides, and porcine pancreatic lipase was the best for selective hydrolysis of the 1-0-acetyl groups from peracetylated pyranoses.
Introduction Table I. Regioselective Acetylation of the Primary Hydroxyl Groups in Furanosides by Porcine Pancreatic Selective protection and deprotection of polyfunctional Lipase molecules is a critical problem in organic synthesis.l In reactn carbohydrate chemistry these problems are accentuated substrate time (h) yieldn (%) due to the presence of multiple hydroxyl functions of very methyl a,@-D-ribofuranoside(1) 60 7Ib similar reactivity.2 Reactions of carbohydrates with trimethyl a-D-arabinofuranoside (2) 48 71 phenylmethyl chloride, or one of its derivatives, has been methyl a,@-D-xylofuranoside (3) 24 84b a standard method of regioselectively incorporating acidmethyl 2-deoxy-a,@-~-ribofuranoside (4) 18 39°C sensitive blocking groups into the primary hydroxyl Yields reported are for anomeric mixtures. bThe anomers were functions.' Similar incorporation of base-sensitive (i.e. separated to facilitate spectroscopic identification of the products. acyl) blocking groups usually requires a series of steps The product obtained was methyl 5-0-acetyl-2-deoxy-a,@-~-riboinvolving protections and deprotections of the other hyfuranoside. In addition 17% of methyl 3-O-acetyl-2-deoxy-&~droxyl groups present in the molecule.2 Regioselective ribofuranoside was obtained. deprotection of polyacylated sugars is seldom o b ~ e r v e d . ~ ~ ~ Selective enzymatic acylation and deacylation of sugars were terminated after 40-50% conversion of the starting have only been developed recently."8 Acylation of the material, presumably to preserve the initial kinetic reprimary hydroxyl group with trichloroethyl carboxylates gioselectivity. The isolated yields were thus relatively low in some hexoses in pyridine was achieved with high re(19-35% with only one example of 57%). In hydrolysis, gio~electivity.~~ In this and the subsequent reAspergillus niger lipase had been reported7to catalyze the on acylation of n-octyl glucopyranoside, the reactions selective cleavage of the 1-0-acetyl group of glucose pentaacetate after 20% conversion. When all the starting material had been consumed the product composition (1) Greene, T. W. Protective Groups in Organic Chemistry; Wiley: New York, 1981. consisted of approximately 45% of the 2,3,4,6-tetraacetate (2) Haines, A. H. Adu. Carbohydr. Chem. Biochem. 1976,33,11-109. and 55% of the 2,4,6- and 3,4,6-triacetates. In our previous Kovac. P.: Sokoloski. E. A.: Claudemans. C. P. J. Carbohydr. Res. 1984. work,6 we found that certain peracylated methyl pyrano128, 101. 'For one-step acylation, see: Plusquellec, D.; Baczko, K. Tet: rahedron Lett. 1987, 28, 3809-12. Ogura, H.; Furuhata, K.; Sato, S.; sides can be selectively hydrolyzed a t the primary position Amazawa, K. Carbohydr. Res. 1987,167, 77-86. (C-6) with 60-100% regioselectivity. (3) Haines, A. H. Adu. Carbohydr. Chem. Biochem. 1981,39, 13-70. T o extend the utility of lipase-catalyzed acylation and (4) Wolfrom, M. L.; Szarek, W. A. In The Carbohydrates. Chemistry and Biochemistry, 2nd ed.; Pigman, W., Horton, D., Eds.; Academic deacylation to virtually all monosaccharides including fuPress: New York, 1972; Vol. IA, pp 217-51. Peracylated sugars with free ranose and pyranose series and their anomeric isomers, we anomeric hydroxyl groups are normally prepared through hydrolysis of report that the primary hydroxyl groups of the methyl glycosyl halides by using silver salts (Schmidt, 0. T.; Herok, J. Liebigs 1958,80, Ann. Chem. 1954,587,63. Bonner, W. A. J. Am. Chem. SOC. furanosides of D-ribose, D-arabinose, D-xylose, and 23372) or through acid hydrolysis of protected glycosylamines (Honeyman, deoxy-D-ribose have been selectively acetylated in tetraJ. Method. Carbohydr. Chem. 1963,2,95). Other methods include direct hydrofuran by employing a crude preparation of porcine deacylation with hydrazine (Excoffies, G.; Gagnaire, D.; Utille, J. P. pancreatic lipase and the more active acyl donor 2,2,2Carbohydr. Res. 1975,39,368),with benzylamine (Ferrer Salat,C.; Exero Agneseti, P.; Bemporad Caniato, M. Spanish Patent 430636 1976), with trifluoroethyl acetate. Good to excellent regioselectivity ammonia in organic solvent (Fiandor, J.; Garcia-Lopez, M. T.; de las has been achieved with these substrates even a t high Heras, F. G.; Mendez Castrillon, P. P. Synthesis 1985, 1121), or with conversion of the starting sugars. We also report that the stannic chloride (Banaszek, A.; Bordas, C. X.; Zamojski, A. Carbohydr. Res. 1985. 144. 342). selective deacetylations of the primary hydroxyl functions (5) (a) Thehod,' M.; Klibanov, A. M. J. Am. Chem. SOC.1986, 108, in peracetylated methyl furanosides have been accom5638-40; (b) Ibid. 1987,109, 3977. plished in a 10% N,N-dimethylformamide (DMF) solution (6) Sweers, H. M.; Wong, C.-H. J.Am. Chem. SOC.1986,108,6421-22. The methyl glycoside derivatives of glucose, galactose, and mannose were using the lipase from Candida cylindracea. The 3-0-acetyl selectively hydrolyzed at position 6. group, however, was selectively hydrolyzed in the 2(7) Shaw, J.-F.; Klibanov, A. M. Biotech. Bioeng. 1987, 29, 648-51. deoxyriboside series. In the pyranose case, we have also (8) Wong, C.-H.; Drueckhammer, D. G., Sweers, H. M. In Fluorocarbohydrates: Chemistry and Biochemistry; Taylor, N. F., Ed.; ACS found that the regioselectivity can be enhanced by DMF. Symposium Series; American Chemical Society: Washington, DC, in . . Procine pancreatic lipase in 10% DMF exclusively cleaved press. glucose pentaacetate ester at C-1 and the tetraacetate was (9) Heath, P.; Mann, J. Walsh, E. B.; Wadsworth, A. H. J.Chem. SOC., Perkin Trans. 1 1983, 2675-79. obtained in 70% isolated yield. Similar selectivities and 0022-3263/88/1953-4939$01.50/00 1988 American Chemical Society
Hennen et al.
4940 J . Org. Chem., Vol. 53, No. 21, 1988
yields were obtained with six other peracylated hexoses including amino sugars. Similarly, we obtained 50-63 % isolated yields in the cleavage of the 1-0-acetyl esters from tetra-0-acetyl-D-xylofuranose and tetra-O-acetyl-D-ribofuranose using the lipase from A. niger. Surprisingly, C. cylindracea cleaved both the 4-and 6-0-acetyl groups from a-D-glucose pentaacetate, giving the triacetate in 73% isolated yield. The most interesting findings in this study are that the regioselectivity can be enhanced by DMF and that regioselective transformations can still be accomplished in the furanose series with appropriate lipases despite the fact that furanoses have more flexible conformations (mainly in the E and T forms with close energy) than pyranoses in solution. Thus both furanose and pyranose sugars can be efficiently acetylated or deacetylated by suitable lipases under proper reaction conditions.
Results and Discussion Furanose Series. Four methyl furanosides and their peracetylated derivatives were used in this study. They were methyl a$-D-ribofuranoside, methyl a-D-arabinofuranoside, methyl a,P-D-xylofuranoside, and methyl 2deoxy-cu,p-D-ribofuranoside.Selective acetylations of these free methyl glycosides were accomplished in tetrahydrofuran solutions (16 mL/g containing 5 equiv of 2,2,2-trifluoroethyl acetate in the presence of crude porcine pancreatic lipase (3 g/g). The reactions were agitated on an orbital shaker (250 rpm) at 37 "C. The reaction times and yields are listed in Table I. No acetylations of these substrates were observed when the reactions were run in the absence of enzyme. Ethyl acetate was too slow as an acylating reagent to be of practical use. Isopropenyl acetate and trichloroethyl acetate were faster but five times slower than trifluoroethyl acetate; both reagents showed the same regioselectivity.
Table 11. Enzymatic Regioselective Hydrolysis Peracetylated Furanose Derivatives reactn substrate enzymeb time (h) methyl 2.25 CCL 2,3,5-tri-O-acetyl-P-D-ribofuranoside (5) methyl 3.0 CCL
1 2 3 4
,h OMe) OMe (H OMa
ti OH H
H OMei
H
ti
Oti
ti H OH
3H OH H
5 5 7
ohk
ti
ti
OH
8
(H O W ) ti OMa
OH H
P a w l h e s e s n d ~ i a i sa m i x l u e 01 anomers
9
H
ti
H
OMe OMe
i o
ow
13 12
OAC H [ti OAc,
ti
H OAc H OAc H OAc ti OAc H OAc OAc H ti H OAC OAc H H H OAc ti OAc H H H OAc H OAc H H OAC OAc
We also examined the above reactions using the lipase from Psuedomonas sp. and cholesterol esterase. P. sp. lipase was found to be very active with methyl D-ribofuranoside, methyl D-xylofuranoside, and methyl 2deoxy-D-ribofuranosideas substrates but weak with methyl a-D-arabinofuranoside (50% conversion in 60 h). Both methyl D-ribofuranoside and methyl 2-deoxy-~-ribofuranoside were good substrates for cholesterol esterase but only in the case of methyl D-ribofuranoside was good selectivity observed. With both methyl D-arabinofuranoside and methyl D-xylofuranoside, poor rates and poor selectivities were observed when cholesterol esterase was employed. Neither of the above enzymes appeared to offer any great advantages over crude porcine pancreatic lipase. Portions of each of the methyl furanosides were peracetylated in a mixture of acetic anhydride and pyridine containing a catalytic amount of 4-(dimethylamino)pyridine. The anomeric mixtures of methyl 2,3,5-tri-0acetyl-a,P-D-ribofuranoside and methyl 3,5-di-O-acetyl-2deoxy-a,P-D-ribofuranosides were separated by column
yeild" (%)
96
85
2,3,5-tri-O-acetyl-a-D-ribo-
furanoside (6) methyl CCL 2,3,5-tri-0-acety1-au-D-arabinofuranoside (7) methyl CCL 2,3,5-tri-0-acetyl-a,P-D-xylofuranoside (8) methyl CCL 3,5-di-O-acetyl-2-deoxy-a-~ribofuranoside (9) methyl cc1 3,5-di-O-acetyl-2-deoxy-P-~ribofuranoside (IO) 1,2,3,5-tetra-O-acetyl-@-D-ribo- ANL furanose (11) 1,2,3,5-tetra-O-acetyi-D-xyloANL furanose (12)
2.5
98
7
50'
2.0
40d
2.0
63
0.5
63'
0.5
50e
Yields are for the 5-hydroxy products except where otherwise noted. CCL = Candida cylindraceae lipase; ANL = Aspergillus niger lipase. 30% of methyl 2,5-di-O-acetyl-~-D-xylofuranoside was also obtained. d50% of methyl 5-O-acetyl-2-deoxy-or-~-ribofuranoside was also obtained. e The isolated product resulted from the hydrolysis of the 1-0-acetyl group.
chromatography on silica gel to facilitate reaction monitoring and product identification. Methods for the efficient separation of the anomers of methyl 2,3,5-tri-O-acetyl-~xylofuranoside were not found; therefore the 1:l anomeric mixture was used in all reactions employing this substrate. Five enzymes were examined for use in the hydrolysis reaction. They were Rhizopus japonics lipase, Mucor sp. lipase, crude porcine pancreatic lipase, Aspergillus niger lipase, and Candida cylindracea lipase. The M . sp. lipase was inactive with these substrates under the reaction conditions. The lipase from R. japonics provided only a very slow reaction which was not selective with certain substrates. Crude pancreatic lipase, A. niger lipase, and C. cylindracea lipase accepted all the substrates examined. A. niger lipase and C. cylindracea lipase provided high reaction rates leading to monodeacetylated products. However, only C. cylindracea lipase was found to react quickly with high selectivity. A. niger lipase was found to selectively remove the anomeric group from 1,2,3,5tetra-0-acetyl-P-D-ribofuranoseand 1,2,3,5-tetra-Oacetyl-D-xylofuranose. The eight substrates that were tested in this study are listed in Table I1 along with the calculated regioselectivities and the isolated yields of the products. The hydrolysis reactions were best conducted by first dissolving the peracetylated substrates in N,N-dimethylformamide, diluting the solutions with nine volumes of 0.1 N phosphate buffer (pH 7), adding the enzyme C. cylindracea lipase (1g/g), and agitating the mixture a t 37 "C. In this way homogeneous reactions were obtained. The reactions were monitored by thin layer chromatography. When optimal conversions were observed the products were isolated by extracting the reaction media with three portions of ethyl acetate, drying the extract, and evaporating the solvent. The products were further purified by column chromatography on silica gel. The hydrolysis of methyl 3,5-di-O-acetyl-2-deoxy-a-~ribofuranoside was not selective. Both methyl 3-0acetyl-2-deoxy-a-~-ribofuranoside and methyl 5-0-
AcovR' R1'
of
J. Org. Chem., Vol. 53, No. 21, 1988 4941
Enzymes in Carbohydrate Synthesis
Table 111. Lipase-Catalyzed Selective Hydrolysis of Peracylated Sugars regioselectivity (%)* (isolated producta a/P ratio yield %) substrate 6-OH 100 (78) methyl a-D-glucoside, tetraoctanoate (13) 6-OH 100 (77) methyl 0-D-glucoside,tetraoctanoate (14) 6-OH 100 (75) methyl a-D-glucoside,tetrapentanoate (15) 6-OHd 60 (29)d methyl a-D-galactoside, tetrapentanoate (16) 6-OHd 67 (33)d methyl a-D-mannoside, tetrapentanoate (17) methyl 2-acetamido-2-deoxy-~-mannoside, tripentanoate (18) 6-OHd 70 (50)d methyl 2-acetamido-2-deoxy-~-glucoside, tripentanoate (19) e a-D-glUCOSe, pentaacetate (20) 4,6-OHf 75 (73)f 1-OH 70130 100 (70) 1-OH g g fi-D-gluCOSe,pentaacetate (21) 1-OH 78/22 100 (75) a-D-galaCtOSe,pentaacetate (22) 1-OH 8/92 100 (95) a-D-mannOSe, pentaacetate (23) 100 (88) 2-acetamido-2-deoxy-/3-~-mannose, tetraacetate (24) 1-OH 66/34 100 (96) 2-acetamido-2-deoxy-~-~-glucose, tetraacetate (25) 1-OH 9317 L-rhamnose, tetraacetate (26) 1-OH 85/15 100 (54) 100 (71) 1-OH 66/34 L-fucose, tetraacetate (27) 1-COZH 30 (30) methyl N-acetylneuraminate, pentaacetate (28)
enzyme CCL CCL CCL CCL CCL CCL CCL CCL PPL PPL PPL PPL PPL PPL PPL PPL RJL, PPL
re1 activity 1.0 0.3 0.2 0.1 0.1 6.2
