Enantio- and regioselective synthesis of organic compounds using enol esters as irreversible transacylation reagents

A process for irreversible regio- and stereoselective enzyme catalyzed acylation of alcohols using enol esters as acylating reagents is disclosed. The present invention permits the selective modification of hydroxyl group(s) of chiral and meso alcohols, including sugars, organometallics, and glycosides. The enol freed upon transesterification rapidly tautomerizes to the corresponding volatile aldehyde or ketone thereby preventing the reverse reaction from occurring.

BACKGROUND OF THE INVENTION 
The present invention relates to enantio- and regioselective synthesis of 
esters of alcohols, sugars, organometallics and glycosides and to their 
preparation using enzyme mediated transesterification. More particularly, 
the present invention relates to enzyme catalyzed irreversible 
transesterification using enol esters as transacylation reagents. 
Hydrolytic enzymes such as lipases, esterases, and proteases have been used 
extensively as catalysts in enantioselective syntheses. Whitesides, G. M., 
Wong, C-H. Angew. Chem. Int. Ed. Engl. 24 (1985) 617; Jones, J. B. 
Tetrahedron 42 (1986) 3351; Roberts, S. M. Chem. Br. (1987) 127; Akiyama, 
A., Bednarski, M., Kim, M. J., Simon, E. S., Waldmann, H. I., Whitesides, 
G. M. Ibid. (1987) 645. Because of their relatively high stability in 
organic media, many hydrolyric enzymes also can be used in organic 
solvents for certain types of transformations which are difficult to do in 
water. The most common reactions are esterase and lipase-catalyzed 
stereoselective esterifications and transesterifications. Klibanov, A. M. 
CHEMTECH (1986) 354-9; Klibanov, A. M., Cambou, B. J. Am. Chem. Soc. 106 
(1984) 2687-92. Chen, C-S., Wu, S-H., Girdaukas, G., Sih, C. J. J. Am. 
Chem. Soc. 109 (1987) 2812-17; Guo, Z. W., Sih, C. J. Ibid. 110 (1988) 
1999-2001; Gil, G., Ferre, E., Meou, A., Petit, J. L., Triantaphylides, C. 
Tetrahedron Lett. 28 (1987) 1647; Yokozeki, K., Yamanaka, S., Takinami, 
K., Hirose, Y., Tanaka, A., Sonomoto, K., Fukui, S. Eur. J. Appl. 
Microbiol. Biotechnicol 14 (1982) 1; Tambo, G. M. R., Schar, H-P., 
Busquets, X. F., Ghisalba, O. Tetrahedron Lett. 27 (1986) 5705-10; Belan, 
A., Bolte, J., Fauve, A., Gourey, J. G., Veschambre, H. J. Org. Chem. 52, 
256-60. Langrand, G., Baratti, J., Buono, G., Triantaphylides, C. 
Tetrahedron Lett. 27 (1986) 29-32. 
One disadvantage of enzyme catalyzed hydrolytic reactions is that they are 
very slow compared to simple hydrolyses. Langrand, G., Baratti, J., Buono, 
G., Triantaphylides, C. Tetrahedron Lett. 27 (1986) 29-32. In addition, 
the products produced by enzymatic hydrolyses very often have to be 
separated from other by-products (particularly alcohol generated from the 
acylating reagent). Due to the reversible nature of these reactions, and 
due to the same stereoselectivity of the enzyme catalysis in both 
directions, the optical purity of the product obtained decreases as the 
reverse reaction proceeds. This situation is illustrated in FORMULA 1 
where a racemic alcohol is to be resolved via an enzymatic esterification 
(R".dbd.H) or transesterification. 
##STR1## 
As shown in FORMULA 1, if the D-isomer is a better substrate than the 
L-isomer for the enzyme, accumulation of the D-ester and the unreactive 
L-alcohol will be observed. In the reverse reaction, however, the D-ester 
is a better substrate and will be converted to the D-alcohol. The 
enantiomeric excess of both the D-ester and the L-alcohol therefore will 
decrease progressively as the extent of the reverse reaction increases. 
This reverse reaction problem clearly has been illustrated in the kinetic 
resolution of menthol, Chen, C-S., Wu, S-H., Girdaukas, G., Sih, C. J. J. 
Am. Chem. Soc. 109 (1987) 2812-17; Guo, Z. W., Sih, C. J. Ibid. 110 (1988) 
1999-2001, and can be seen in the enantioselective esterification or 
transesterification of meso compounds. 
SUMMARY OF THE INVENTION 
The method of the present invention blocks the progress of the reverse 
reaction. The present invention is a process for irreversible regio- and 
stereoselective enzyme catalyzed acylation of alcohols using enol esters 
as acylating reagents. The present invention permits the selective 
modification of hydroxyl group(s) of chiral and meso alcohols, including 
sugars, organometallics and glycosides. The enol freed upon 
transesterification rapidly tautomerizes to the corresponding volatile 
aldehyde or ketone thereby preventing the reverse reaction from occurring.

DETAILED DESCRIPTION OF THE INVENTION 
Nuclear magnetic resonance (NMR) spectra were recorded on a Varian XL-200E 
spectrometer. All chemical shifts were reported in ppm using 
tetramethylsilane as an internal standard unless otherwise indicated. 
Rotations were determined on a Perkin Elmer 240 polarimeter. Gas 
chromatographic (GC) analyses were performed on a Hewlett-Packard 5890 
instrument with a 20-m DB-5 megabore column. The lipases from PSEUDOMONAS 
species (PSL, Type XIII), porcine pancreas (PPL, Type II), and CANDIDA 
CYLINDRACEA (CCL, Type VII) were obtained from Sigma Chemical Company. 
Cholesterol esterase was obtained from Amano Pharmaceutical Company. Vinyl 
acetate ($5/Kg, bp 72.degree. C.) and isopropenyl acetate ($25/Kg, bp 
94.degree. C.) were from Aldrich Chemical Co. Vinyl propionate ($25/25 g, 
bp 93.degree.-94.degree. C.) was from Pfaltz and Bauer, Inc. Some 
experimental protocols are described in Tables 1, 3 and 4. 
The procedure for preparation of isopropenyl valerate (1b of FORMULA 2) was 
similar to that reported for the preparation of other isopropenyl esters, 
with some modifications. Rothman, E. S., Serota, S., Peristein, T., Swern, 
D. J. Org. Chem. 27 (1962) 3123-27. To a 250 mL round bottom flask was 
added 10 mL of valetic acid (91.9 mmol) which had been freshly distilled, 
and 20 mL of valetic anhydride. Then, 200 mL of freshly distilled 
isopropenyl acetate was added followed by 2 drops of concentrated sulfuric 
acid. The mixture then was heated to reflux under an atmosphere of argon 
for 10 h, after which time all of the valetic acid had been consumed as 
evidenced by capillary GC. The reaction mixture was allowed to cool to 
room temperature and 0.5 g of sodium bicarbonate was added to quench the 
acid catalyst. The isopropenyl acetate then was removed by evaporation 
under reduced pressure. The orange liquid remaining was poured into 300 mL 
of 0.degree. C. saturated sodium bicarbonate which was overlayed with 100 
mL of diethyl ether. The mixture was stirred vigorously and the ether 
layer was analyzed by GC for the disappearance of the mixed valetic acetic 
anhydride. After all of the anhydride was consumed (6 h) the ether layer 
was separated and the aqueous layer was washed with 100 mL of ether. The 
combined ether layers were washed with 5.times.25 mL portions of saturated 
sodium bicarbonate to remove the valetic acid. The ether layer then was 
washed with saturated brine (30 mL) and the ether was then dried over 
sodium sulfate. The ether was removed under reduced pressure and the 
isopropenyl ester was purified by vacuum distillation 
(bp=50.degree.-52.degree. C., 8 mm Hg). 7.85 g of a clear colorless liquid 
(1b) was obtained (60.1% yield). .sup.1 H-NMR (CDCl.sub.3) 4.65 (m, 2H), 
2.35 (t, 2H) 1.90 (s, 3H), 1.65 (m, 2H), 1.35 (m, 2H), 0.90 (s, 3H). 
.sup.13 C-NMR 171.89, 153.00, 101.87, 34.02, 26.92, 22.16, 19.52, 13.16. 
In a similar manner isopropenyl butyrate was prepared from burytic acid in 
54% yield. 3.68 g of isopropenyl butyrate were prepared from 4.85 mL of 
butyric acid and 10 mL of burytic anhydride. .sup.1 H-NMR 4.60 (m, 2H), 
2.30 (t, 2H), L.85 (s, 3H), 1.60 (m, 2H), 0.90 (t, 3H). 
The method of Swern and Jordan was used to prepare vinyl valerate (1e of 
FORMULA 2). Swern, D., Jordan, E. F. Organic Synthesis, Coll. Vol. IV 
(1963) 977-80, incorporated herein by reference. Freshly distilled valeric 
acid (40 mL, 0.37 mol) and vinyl acetate (300 mL) were placed in a 
3-necked 500 mL round bottomed flask fitted with a reflux condenser, a gas 
inlet tube and a thermometer. The solution was stirred under argon and 
mercuric acetate (1.2 g, 0.37 mmol) was added. The reaction mixture was 
stirred under argon for 30 min, after which time 10 drops of 100% sulfuric 
acid was added. The solution was heated to reflux for 6 h and then was 
allowed to cool to room temperature. Sodium acetate (1.0 g) was added to 
quench the acid catalyst. The excess vinyl acetate was removed by 
distillation under argon. The product (vinyl valerate) 1e was isolated by 
distillation (bp=135.degree.-145.degree. C.) as a clear colorless liquid 
(29.4 g, 62% yield). .sup.1 H-NMR 7.24 (m, 1H), 4.80 (m, 1H), 4.48 (m, 
1H), 2.32 (t, 2H), 1.60 (m, 2H), 1.30 (m, 2H), 0.85 (t, 3H). .sup.13 C-NMR 
170.69, 141.11, 97.22, 33.54, 26.57, 22.10, 13.57. 
Any chiral or meso alcohol having no excessive steric hindrance can be used 
in the present method. Structures 15 and 16 of Table 2 represent compounds 
wherein excessive steric hindrance is present. 
Lipase-Catalyzed Reactions 
A number of lipase-catalyzed irreversible transesterifications using enol 
esters as acylating reagents were performed in a manner cutlined generally 
in FORMULA 2. 
______________________________________ 
##STR2## 
R.sub.1 R.sub.2 
______________________________________ 
1a CH.sub.3 CH.sub.3 isopropenyl acetate 
1b CH.sub.3 (CH.sub.2).sub.3 
CH.sub.3 isopropenyl valerate 
1c CH.sub.3 H vinyl acetate 
1d CH.sub.3 CH.sub.2 
H vinyl propionate 
1e CH.sub.3 (CH.sub.2).sub.3 
H vinyl valerate 
FORMULA 2 
______________________________________ 
The reactions produced optically active esters from several alcohols 
including those from glycerol and serinol derivatives, organometallics, 
nucleoside derivatives, sugars, and other chiral and racemic alcohols. The 
results are capsulized in Table 1. 
TABLE 1 
______________________________________ 
Lipase-catalyzed transesterifications with enol esters as 
acylating agents. 
% ee % ee 
enol % alcohol 
ester 
Entry Substrate 
Enz ester 
conversion 
(Config.) 
(Config.) 
______________________________________ 
1 2 PSL 1a 72 -- 96 (S) 
2 5 PPL 1e 60 -- 97 (R) 
3 8a PSL 1a 32 29 (S) 67 (R) 
4 8b.sup.a 
PSL H.sub.2 O 
50 92 (R) -- 
5 8a PSL 1b 25 21 (S) 64 (R) 
6 8a ChE 1a 33 22 (S) 54 (R) 
7 8a CCL 1a 21 14 (S) 50 (R) 
8 9a PPL 1d 43 -- 54 (S) 
9 9a PPL 1e 45 -- 39 (S) 
10 9a PSL 1d 50 -- 18 (S) 
11 10a PPL 1d 40 -- 33 (S) 
12 10a PPL 1c 40 -- 42 (S) 
13 10b.sup.a 
PPL H.sub.2 O 
30 82 (S) -- 
14 10a PPL 1c 80 65 (R) -- 
15 10a PPL 1e 40 -- 30 (S) 
16 10a ChE 1a 31 4 (R) 10 (S) 
17 11a PSL 1c 30 -- 70 (R) 
18 11b.sup.a 
PSL H.sub.2 O 
60 94 (R) -- 
19 12a CCL 1e 30 -- 37 (R) 
20 13a PPL 1c 37 56 (S) 98 (R) 
21 13a PPL 1c 58 &gt;98 (S) 
71 (R) 
22 14a PPL 1c 27 37 (S) 98 (R) 
23 14a PPL 1c 62 `98 (S) 
61 (R) 
24 15 CCL 1c -- -- -- 
25 16 CCL 1c -- -- -- 
26 17a PPL 1d 40 -- 84 (R) 
27 17a PPL 1d 60 84 (S) -- 
______________________________________ 
.sup.a The obtained optically active ester was used as substrate in 
hydrolysis in 0.1 M phosphate buffer (pH 7) at 28.degree. C. The pH was 
controlled at 7.0 during the reaction by addition of 1 N NaOH. Monitoring 
of the reaction progress and isolation of the products were the same as 
that in transesterification reactions. 
The reaction schemes for enantioselective acylation of 2-O-benzylgiycerol 
(2) and N-carbobenzoxy serinol (5) are shown in FORMULAE 3 and 4, 
respectively. The calculated kinetic parameters .alpha. and E are also 
listed. 
##STR3## 
Table 2 diagrams the starting materials and products formed from the 
reactions listed in Table 1, entries 3-27. 
TABLE 2 
__________________________________________________________________________ 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
__________________________________________________________________________ 
The general procedure used in the following lipase catalyzed 
transesterifications was as follows: 
The alcohol substrate and an excess of enol ester were dissolved in an 
organic solvent, such as pyridine or a less polar solvent. After a 
catalytic amount of enzyme was added, the suspension was stirred at 
28.degree. C. and the reaction was monitored by GC for conversion. Once 
the required extent of conversion was reached, the enzyme was filtered off 
and the solvent was removed by evaporation in a vacuum. The ester product 
and the unreacted alcohol were separated by chromatography on a silica gel 
column. 
Some of the esters (e.g, acyl sugars) that were prepared can be obtained 
only in nearly anhydrous solvents due to thermodynamic reasons, or because 
of the lack of appropriate esterases to use in obtaining such esters via 
hydrolysis (e.g., (S)-3 in FORMULA 3 and (R)-6 in FORMULA 4). For example, 
in the kinetic resolution of ferrocenylethanol, the (R)-propionate ester 
obtained in toluene is stable towards solvolysis, while in ethanol or 
water, the ester decomposes to ferrocenylethylether or ferrocenylethanol. 
Transesterification of symmetrically prochiral diols 
EXAMPLE 1 
PSL-catalyzed transesterification of 2-0-benzylglycerol (2 of FORMULA 3) 
with isopropenyl acetate (1a of FORMULA 2) 
Chiral 3-0-acetyl-2-0-benzylglycerol ((R)- or (S)-3 of FIG. 3), and 
3-0-acetyl-2-N-benzyloxycarbonyl serinol ((R) or (S) -6 of FORMULA 5), are 
considered to be useful building blocks for the preparation of 
enantiomerically pure, biologically active molecules such as 
phospholipids, PAF (platelet-activating factor), phospholipase A2 
inhibitors, sphingoglycolipids and many others. To prepare these chiral 
synthons, the prochiral diols, 2-0-benzylglycerol (2 of FORMULA 3) and 
2-N-benzyloxycarbonyl (Z) serinol (5 of FORMULA 4) were chosen as 
substrates, respectively. 
(a) A solution of 2-0-benzylglycerol (2 of FORMULA 3) (300 mg, 1.65 mmol) 
and isopropenyl acetate (1a of FORMULA 2) (0.73 mL, 6.6 mmol) in 4 mL of 
chloroform was mixed with 10 mg of PSL. After 27 h, the amounts of 
diacetate, monoacetate and diol were quantitatively determined to be 
43:57:0 by GC analysis. The reaction was terminated and worked up as 
described in the general procedure. The products were separated by column 
chromatography (ethyl acetate:n-hexane--1:3) on silica gel to give 196 mg 
(53%) of monacetate ((S)-3 in FORMULA 3), [.alpha.].sup.23.sub.D-- 20.1 (c 
1, CHCl.sub.3), and 175 mg of diacetate (4 in FORMULA 3). Monoacetate 
(S)-3: .sup.1 H-NMR 2.08 (3H, s), 3.60-3.78 (3H, m), 4.23 (2H, d, J=4.8 
Hz), 4.61 (1H, d, J=11.8 Hz), 4.72 (1H, d, J=11.8 Hz), 7.35 (5H, s). 
Diacetate 4 in FORMULA 3: .sup.1 H-NMR 2.06 (6H, s), 3.81 (1H, tt, J=5.2 
Hz), 4.15 (2H, dd, J=5.2 Hz and 11.8 Hz), 4.25 (2H, dd, J=5.2 Hz and 11.8 
Hz), 4.66 (2H, s), 7.34 (5H, s). The optical purity of monoacetate (15 mg) 
was determined to be 96% by .sup.1 H-NMR spectroscopy in the presence of 
Eu(hfc).sub.3 (30 mg). The relative intensities of the acetoxy group at 
3.05 (major) and 2.90 (minor) were used for ee determination. 
(b) A solution of 2-0-benzylglycerol (2 of FORMULA 3) (3 mmol) and 
isopropenyl acetate (1a of FORMULA 2) (12 mmol) in 6 mL of chloroform was 
mixed with 12 mg of the lipase from PSEUDOMONAS (PSL) species at 
28.degree. C. with stirring. After 24 h, the amounts of diacetate, 
monoacetate and diol were quantitatively determined to be 10.0:82.6:7.4. 
The products were separated by column chromatography on silica gel to 
afford 538 mg (80%) of monoacetate ((S)-3 of FORMULA 3), the optical 
purity of which was determined to be 75.5% by .sup.1 H-NMR spectroscopy in 
the presence of tris 
(3-(hepta-fluoropropylhydroxymethylene)-(+)-camphorato]europium (III) 
derivative (Eu(hfc).sub.3). The monoacetates produced in the 
transesterification reaction were expected to undergo further acetylation 
to yield the diacetate (4 of FORMULA 3) and the enzyme was expected to 
show the same stereo-chemical preference in the second step (i.e. k.sub.4 
&gt;k.sub.3) as in the hydrolysis of meso diacetate compounds, so that the 
optical purity of monoacetate (S)-3 could be enhanced by increasing the 
conversion. Wang, Y. F., Chen, C. S., Girdaukas, G., Sih, C. J. J. Am. 
Chem Soc. 106 (1984) 3695; Wang, Y. F., Sih, C. J. Tetrahedron Lett. 25 
(1985) 4999. 
To determine the constants, the diols, monoesters, and diesters were 
determined by GC analysis at a certain degree of conversion. The 
enantiomeric compositions of monoesters were determined by NMR analysis. 
As predicted, when the reaction was terminated at 71.5% conversion (a 50% 
conversion corresponds to the hydrolysis of one acetate group), the 
optical purity of the monacetate (S)-3 obtained was 96% (the isolated 
chemical yield was 53%). 
The reported rotations of ((R)-3 of FORMULA 3) are not in agreement with 
our values. The reported rotation of (R)-3 prepared through a lipoprotein 
lipase-catayzed hydrolysis of diacetate (4 of FORMULA 3) was 
[.alpha.].sub.D.sup.20 -13.2 (c 3, EtOH), 91% ee. Breitgoff, D., Laumen, 
K., Schneider, M. P., JCS Chem. Comm. (1986) 1523. Another reported value 
was [.alpha.].sub.D.sup.25 +15.0 (c 2, CHCl.sub.3) or -12.3 (c 1.8, EtOH); 
Kerscher, V., Kreiser, W., Tetrahedron Lett. 28 (1987) 531. Based on the 
rotation of the enantiomer prepared in the present reaction, the specific 
rotation of R-3 corresponds to 77% ee. 
The kinetics of these irreversible transesterifications can be treated as 
similar to the kinetics Of hydrolysis, and the equation developed by Sih 
et al. for use in prediction of ee rs. conversion in hydrolysis should be 
applicable here. Wang, Y. F., Chen, C. S., Girdaukas, G., Sih, C. J. J. 
Am. Chem. Soc. 106 (1984) 3695; Wang, Y. F., Sih, C. J. Tetrahedron Lett. 
25 (1985) 4999. To determine the constants, the diols, monoesters, and 
diesters were determined by GC analysis at certain degree of conversion. 
The enantiomeric compositions of monoesters were determined by NMR 
analysis. Indeed, the kinetic constants for the transesterification of 
2-O-benzylglycerol (2 of FORMULA 3) using PSL were determined to be 
.alpha.=k.sub.1 /k.sub.2 =5.6, E.sub.1 =k.sub.3 /(k.sub.1 +k.sub.2)=0.02, 
E2=k.sub.4 (k.sub.1 +k.sub.2)=0.33. Wang, Y. F., Chen, C. S., Girdaukas, 
G., Sih, C. J. J. Am. Chem Soc. 106 (1984) 3695; Wang, Y. F., Sih, C. J. 
Tetrahedron Lett. 25 (1985) 4999. 
To determine the absolute stereochemistry of the monoacetate, it was 
converted to 2,2-dimethyl-1,3-dioxolane-4-methanol (glycerol acetonide) 
according to the procedures of Suemune, H., Mizuhara, Y., Akita, H., 
Sakai, K. Chem. Pharm. Bull, 34 (1986) 3440-44; Hirth, G., Barner, R. 
Helv. Chim. Acta 65 (1982) 1059 (platelet-activating factor). The 
resulting glycerol acetonide was the "R" configuration based on rotation, 
indicating that the monacetate obtained had the "S" configuration. It has 
been reported that (R)-3 can be prepared from 2-0-benzylglycerol diacetate 
via a lipoprotein lipase-catalyzed hydrolysis. The same enantioselectivity 
in the hydrolysis of the diacetate (4 of FORMULA 3) was observed with PSL 
and (R)-3 was obtained in 52% yield with 71% ee. When (S)-3 (91% ee) was 
suspended in phosphate buffer (0.1M, pH 7) at 28.degree. C. without 
enzyme, the optical purity was found to decrease 2-2.5% per hour. These 
two irreversible enzymatic processes thus provide a new route to (R)- and 
(S)-3. 
EXAMPLE 2 
PPL-catalyzed transesterification of 2-N-benzyloxycarbonyl (Z) serinol (5 
of FORMULA 4) with vinyl valerate )1e of FORMULA 2) 
A solution of 2-N-benzyloxycarbonyl (Z) serinol (5 of FORMULA 4) (225 mg, 1 
mmol) and vinyl valerate (1e of FORMULA 2) (512 mg, 4 mmol) in 22.5 mL of 
THF was incubated with 900 mg of PPL at 28.degree. C. with stirring. After 
11 hours, the reaction was terminated. The products were separated by 
silica gel column chromatography (ethyl acetate: n-hexane=1:4.fwdarw.1:1) 
to afford 238 mg (77%) of monovalerate (R)-6 of FORMULA 4, 
[.alpha.].sup.23.sub.D +3.2 (c 1.0, CHCl.sub.3), and 75 mg of divalerate 7 
of FORMULA 4. Monovalerate (R)-6 of FORMULA 4: .sup.1 H-NMR 0.91 (3H, t, 
J=7.2 Hz), 1.34 (2H, tq, J=7.2, 7.2 Hz), 1.60 (2H, tt, J=7.2, 7.2 Hz), 
2.33 (2H, t, J=7.2 hz), 3.65 (2H, m), 3.94 (1H, m), 4.23 (2H, d, J=5.6 
Hz), 5.11 (2H, s), 5.2 (1H, br), 7.36 (5H, s). Divalerate (7 of FORMULA 
5): .sup.1 H-NMR 0.91 (6H, t, J 7.2 Hz), 1.33 (4H, tq, J=7.2, 7.2 Hz), 
1.59 (4H, tt, J=7.2, 7.2 Hz), 2.31 (4H, t, J=7.2, 7.2 Hz), 
.delta.4.02.congruent.4.30 (5H, m), 5.11 (7H, s), 5.05.congruent.5.20 (1H, 
br), 7.36 (5H, s). To determine the optical purity of monovalerate R-6, 
R-6 was treated with (+)-2-methoxy-2-(trifluoromethyl)phenylacetyl 
chloride [(+)-MTPA chloride] and the resulting (+)-MTPA ester (20 mg), 
which was analyzed by .sup.1 H-NMR spectroscopy in the presence of 
Eu(hfc).sub.3 (80 mg) to establish an enantiomeric excess (ee) greater 
than 97%. The relative intensities of benzylic methylene group at 4.8 
(major) and 4.6 (minor) were measured for ee determination. 
EXAMPLE 3 
PSL-catalyzed transesterification of seudenol (8a in Table 2) with 
isopropenyl acetate (1a of FORMULA 2) 
Compound 8a has been used in natural product synthesis via radical-mediated 
cyclization. Stork, G., Sofia, M. J. J. Am. Chem. Soc. 108 (1986) 6826-28. 
A solution of isopropenyl acetate (0.44 mL, 4 mmol) and seudenol 8a (224 
mg, 2 mmol) in 2 mL of n-hexane was mixed with 3 mg of PSL at 28.degree. 
C. with stirring. After 20 hours, the amounts of seudenol acetate 8b and 
seudenol 8a were quantitatively determined to be 32:68 by GC analysis. The 
reaction mixture was worked up as usual and the products were separated by 
silica gel column chromatography (dichloromethane: 
n-hexane=1:3.fwdarw.1:0) to afford 91 mg (29.5%) of acetate 8b, 
[.alpha.].sup.23.sub.D +138.3 (c 0.8, CHCl.sub.3) and 138 mg (61.8%) of 
alcohol 8a, [.alpha.].sup.23.sub.D -26.7 (c 1.5, CHCl.sub.3). Acetate 8b: 
.sup.1 H-NMR 1.6.congruent.2.0 (6H, m), 1.71 (3H, s), 2.03 (3H, s), 5.24 
(1H, m), 5.47 (1H, m). The optical purity of monoacetate (+) 8b (9 mg) was 
determined to be 67% ee by .sup.1 H-NMR spectroscopy in the presence of 
Eu(hfc).sub.3 (57 mg). The relative intensities of the methyl group in 
double bond at 2.27 (major) and 2.31 (minor) were measured for ee 
determination. The alcohol (-)-8a was converted to the corresponding 
acetate by treatment of acetic anhydride in pyridine and then analyzed by 
the same procedure: ee=29%. 8a and 8b were assigned the designations of 
"S" and "R", respectively, based on their rotations compared to the 
reported values. Mori, K., Hazra, B. G., Pfeiffer, R. J., Gupta, A. K., 
Lindgren, B. S. Tetrahedron Lett. 43 (1987) 2249-54. 
EXAMPLE 4 
PPL-catalyzed transesterification of glycidol (9a in Table 2) with vinyl 
propionate (1d in FORMULA 2) 
To a 100 mL round bottomed flask was added glycidol (9a in Table 2) (2.3 g, 
31 mmol), vinyl propionate (1d in FIG. 2) (7.0 g, 70 mmol), toluene (0.61 
g) as an internal standard, and 80 mL of chloroform. The enzyme (PPL, 5 g) 
was suspended in the reaction mixture and the suspension was stirred. At 
43% conversion (3.5 h), 5 g of celite was added to the suspension and the 
mixture was filtered. The filtrate was extracted with three 15 mL portions 
of distilled water and then washed once with 15 mL of saturated brine. The 
solvent was removed under reduced pressure and a yellow oil was obtained 
corresponding to pure glycidol propionate (9b in Table 4) (0.95 g, 23.2% 
yield from racemic glycidol). The optical purity as determined by 
Eu(hfc).sub.3 was 54%, while the optical rotation was found to be +15.2 (c 
4, chloroform) corresponding to an optical purity of 53.5% (lit. for R 
ester is -28.4.degree. C.).sup.25. .sup.1 H-NMR 4.05 (dd, 1H), 3.90 (dd, 
1H), 3.24 (m, 1H), 2.85 (m, 1H), 2.65 (m, 1H), 2.35 (q, 3H), 1.15 (t, 3H), 
.sup.13 C-NMR 174.19, 64.83, 49.38, 44.65, 27.33, 9.01. 
EXAMPLE 5 
PPL-catalyzed transesterification of solketal (10a in Table 2) with vinyl 
esters 
In a representative procedure, 1 g of solketal (10a in Table 2) (7.5 mmol) 
and 2.3 g of vinyl acetate (1c in FIG. 2) (26.7 mmol) in 50 mL of 
chloroform was incubated with PPL (2 g) along with 0.5 g of hexane as an 
internal standard. After the reaction had proceeded to 40% conversion, the 
mixture was worked up as usual to give the ester with an optical purity of 
42% by analysis with Eu(hfc).sub.3. The relative intensities of the methyl 
group of the isopropyl group at 2.63 (major) and 2.57 (minor) were 
measured to determine ee. In a similar manner the esterificiation was 
allowed to proceed to 80% conversion and the unreacted alcohol was 
isolated as described above. solketal acetate 10b was found to have an 
optical purity of 65% ee. Solketal acetate 10b: .sup.1 H-NMR 4.40 (m, 1H), 
4.05 (m, 3H), 2.72 (m, 1H), 2.07 (s, 3H), 1.35 (s,3H). 
EXAMPLE 6 
PSL-catalyzed transesterification of 2-hydroxypropanal dimethyl acetal (11a 
in Table 2) and 3-hydroxybutanal dimethyl acetal (12a in Table 2) 
Optically active 11a and 12a of Table 2 are useful as substrates in 
aldolase-catalyzed synthesis of novel sugars. Durrwachter, J. R., Wong., 
C-H. J. Org. Chem., submitted. To a stirred solution of 2-hydroxypropanal 
dimethyl acetal (11a of Table 2) (480 mg, 4 mmol) and vinyl acetate (1c of 
FIG. 2) (20 mmol) in petroleum ether (20 mL) was added 9.6 mg of PSL. 
After the reaction had proceeded to 30%, the reaction suspension was 
treated as described in the general procedure. The products were separated 
on a silica gel column (petroleum ether: ethyl acetate=9:1.fwdarw.3:1). 
2-acetoxy-propanal dimethyl acetal 11b: .sup.1 H-NMR (CDCl.sub.3); 1.16 
(3H d, J=5.5 Hz); 2.00 (3H, s); 3.33 (3H, s); 3.36 (3H, s); 4.20 (1H, d, 
J=5.5 Hz); 4.80.congruent.4.99 (1H, m). 
To determine the optical purity of 2-acetoxypropanal dimethyl acetal 11b it 
was transformed to (+)-MTPA ester by hydrolysis with NaOH followed by 
reaction with (+)-MTPA-Cl. The resulting (+)-MTPA ester was analyzed by 
.sup.1 H-NMR spectroscopy. The relative intensities of the methine group 
at 4.32 and 4.22 were used for ee determination. The same procedure was 
used for the resolution of 12a, except that vinyl valerate (1e of FIG. 2) 
and CCL were used. The methine group shifts of the MTPA ester at 4.38 and 
4.17 ppm were used for ee determination. 
EXAMPLE 7 
PPL catalyzed transesterification of (.+-.)-2-octanol (13a of Table 2) with 
vinyl acetate (1c in FORMULA 2) 
520 mg (4 mmol) of 2-octanol (13a of Table 2) was dissolved in 8 mL of 
benzene along with 240 .mu.L of dodecane as an internal standard. Two 
equivalents of vinyl acetate (1c in FORMULA 2) were added along with 520 
mg of PPL. The suspension was stirred at 28.degree. C. After the reaction 
had proceeded to 37%, the reaction suspension was worked as described in 
the general procedure. The products were separated on a silica gel column. 
The optical purities of isolated ester 2-octyl acetate (13b in Table 2) 
and 2-octanol (13a in Table 2) were determined by .sup.1 H-NMR 
spectroscopy in the presence of Eu(hfc).sub.3 (12 mg of acetate, or 
alcohol was added 84 mg or 72 mg of Eu(hfc).sub.3, respectively). The 
relative intensities of the methyl groups near the chiral center at 8.72 
(major) and 8.64 (minor) (alcohol) and 4.3 (major) and 4.42 (minor) 
(ester) were used for ee determination. The ester was found to have an 
optical purity of 98% ee. In a similar manner, the esterification was 
allowed to proceed at 58% conversion and the unreacted alcohol was 
isolated. The alcohol was found to have an optical purity of &gt;98% ee. The 
specific rotation of unreacted alcohol was +8.7.degree. (c 1.0, 
CDCl.sub.3) or +8.9.degree. (neat). Authentic (S)-2-octanol from Aldrich: 
[.alpha.].sup.17 +9.degree. (neat). This result confirms that the 
unreacted alcohol has the "S" configuration. (R)-2-octyl acetate 13b: 
.sup.1 H-NMR (CDCl.sub.3): 0.88 (3H, t, J=6.8 Hz); 1.20 (3H, d, J=6.2 Hz); 
1.27 (8H, s); 1.41.congruent.1.66 (2H, m), 2.02 (3H, s); 4.88 (1H, qt, 
J=6.2 Hz and 12.6 Hz). 
EXAMPLE 8 
PPL-catalyzed transesterification of sulcatol (14a in Table 2) with vinyl 
acetate (1c of FORMULA 2) 
Compound (S)-14a (Table 2) is a useful pheromone, which has been prepared 
via lipase-catalyzed transesterification of the racemic alcohol using 
trichloroethyl propionate and trifluoroethyl laurate. Tambo, G. M. R., 
Schar, H-P., Busquets, X. F., Ghisalba, O. Tetrahedron Lett. 27 (1986) 
5705-10; Belan, A., Bolte, J., Fauve, A., Gourey, J. G., Vaschambre, H. J. 
Org. Chem. 52, 256-60 (The latter is for synthesis of (S)-14a), Stokes, T. 
M., Oehlschlager, A. C. Tetrahedron Lett. 28 (1987) 2091-94 
(trifluoroethyl laurate), and via alcohol dehydrogenase-catalyzed 
reduction of the ketone precursor. Tambo, G. M. R., Schar, H-P., Busquets, 
X. F., Ghisalba, O. Tetrahedron Lett. 27 (1986) 5705-10; Belan, A., Bolte, 
J., Fauve, A., Gourey, J. G., Vaschambre, H. J. Org. Chem. 52, 256-60 (The 
latter is for synthesis of (S)-14a). All of these processes give (S)-14a 
with &gt;98% ee. The procedure described here using readily available vinyl 
acetate is faster and the product is easier to isolate. 
513 mg (4 mmol) of sulcatol (14a in Table 2) was dissolved in 8 mL of 
benzene along with 240 .mu.L of dodecane as an internal standard. Two eq 
(8 mmol) of vinyl acetate (1c of FORMULA 2) was added along with 512 mg of 
PPL. The suspension was stirred at 28.degree. C. After the reaction had 
proceeded to 27%, the reaction was terminated and treated by the general 
procedures already described. The products were separated by silica gel 
column chromatography (CH.sub.2 Cl.sub.2 : n-hexane=0:1.fwdarw.1:4) giving 
acetate ester (14b of Table 2) and unreacted alcohol 14a. The optical 
purities of isolated sulcatol acetate and sulcatol were determined by 
.sup.1 H-NMR spectroscopy in the presence of Eu(hfc).sub.3. The relative 
intensities of methyl group near chiral center at 9.75 (major) and 9.56 
(minor) (alcohol) and at 4.92 (major) and 5.02 (minor) (ester) were used 
for ee determination. The ester was found to have 98% ee. 
In a similar manner, the esterification was allowed to proceed to 62% 
conversion and the unreacted sulcatol was isolated. The ee of unreacted 
sulcatol was found to be &gt;98%. The specific rotation of unreacted alcohol 
was +15.1 (c 2, b, EtOH). [(S)-sulcatol; [.alpha.].sup.25.sub.D 
+15.6.degree. (c 0.015, EtOH)]. This result confirms that the unreacted 
alcohol had the spectrum "S" configuration. The .sup.1 H-NMR and the 
optical rotation of 14b corresponded to that reported for the "S" 
configuration of 14b. Tambo, G. M. R., Schar, H-P., Busquets, X. F., 
Ghisalba, O. Tetrahedron Lett. 27 (1986) 5705-10; Belan, A., Bolte, J., 
Fauve, A., Gourey, J. G., Vaschambre, H. J. Org. Chem. 52,256-60 (The 
latter is for synthesis of (S)-14a). 
EXAMPLE 9 
PPL-catalyzed transesterification of ferrocenylethanol (17a of Table 2) and 
vinyl propionate (1d of FORMULA 2) in toluene. 
The resolution of ferrocenylethanol (17a of Table 2) represents an 
interesting example of enzyme-catalyzed kinetic resolution of chiral 
organometallic compounds. The ester (17b of Table 2) in aqueous ethanol 
decomposes via solvolysis to ferrocenylethyl ethyl ether and 17a. Gokel, 
G. W., Marquarding, D., Ugi, I. K. J. Org. Chem. 37 (1972) 3052-58. The 
acetate was subjected to SN.sub.1 and SN.sub.2 displacement. When the 
enzymatic resolution was carried out in aqueous solution, racemic 17a and 
17b were obtained. The resolution therefore must be carried out in 
non-polar aprotic solvents such as toluene. 
A mixture of ferrocenylethanol (17a of Table 2) (1 g, 4.4 mmol), vinyl 
propionate (1d of FORMULA 2) (6 mL, 52.8 mmol), and PPL (3 g) in toluene 
(25 mL) was shaken for 6 days. The reaction was stopped at .congruent.40% 
conversion (determined by NMR, based on the ratio of the methyl doublet of 
the reactant alcohol to that of the product ester). The mixture then was 
filtered to remove the enzyme and the liltrate was evaporated to give a 
mixture of products (0.72 g) which were separated by silica gel 
chromatography with hexane: ethyl acetate=5:1 v/v as the solvent system to 
give ferrocenylethyl propionate 17b ([.alpha.].sub.D -11.2 (c 1, EtOH)). 
Ferrocenylethanol 17a (0.31 g, mp 70.degree.-71.degree. C., 
[.alpha.].sub.D.sup.25 +25.9 (c 1, benzene), lit.sup.21 +30.1) prepared by 
a similar reaction proceeded to 60% conversion. The enantiomeric excesses 
of ferrocenylethanol 17a and ferrocenylethyl propionate 17b were 
determined to be 84% and 84%, respectively, with H-NMR in the presence of 
Eu(hfc).sub.3 (the methyl doublet of ferrocenylethanol 17a at 3.35 ppm and 
the methyl triplet of the acyl portion of the ester 17b at 2.82 ppm were 
measured). The configurations were determined to be "S" for 
ferrocenylethanol 17a and "R" for ferrocenylethyl propionate 17b based on 
the rotation compared to the reported values. Gokel, G. W., Marquarding, 
D., Ugi, I. K. J. Org. Chem. 37 (1972) 3052-58. Ferrocenylethyl propionate 
17b: .sup.1 H-NMR 1.10 (t, 3H), 1.55 (d, 3H), 2.30 (q, 2H), 4.2-4.44 (m, 
9H), 5.80 (q, 1H). .sup.13 C-NMR (CDCl.sub.3)9.22, 20.12, 27.92, 65.95, 
67.92, 68.24, 68.70, 88.15, 173.89. The NMR data of ferrocenylethanol 17a 
were the same as reported. Gokel, G. W., Marquarding, D., Ugi, I. K. J. 
Org. Chem. 37 (1972) 3052-58. The acetate was subjected to SN.sub.1 and 
SN.sub.2 displacement. When the enzymatic resolution was carried out in 
aqueous solution, racemic 17a and 17b were obtained. 
Structural Effect of Enol Esters 
To compare the structural effect of different enol esters on the rate and 
stereoselectivity of the enzymatic transesterification, the resolution of 
solketal (10a of Table 2) using CCL as catalyst was performed. The results 
are shown in Table 3. 
TABLE 3 
______________________________________ 
Reaction rates and enantioselectivity of CCL-catalyzed trans- 
esterification of 10a with various acylating reagents.sup.a and of 
PPL-catalyzed reactions with 13.sup.b. 
sub- Rel. % 
Ester strate enzyme Rate conversion 
E.sup.c 
______________________________________ 
CH.sub.3 CO.sub.2 Et 
10a CCL 1.sup.d 
16 1.2.sup.e 
1a 10a CCL 20 42 1.4 
1b 10a CCL 8 44 2.7 
1c 10a CCL 62 37 1.4 
1d 10a CCL 122 53 2.0 
1e 10a CCL 13 23 3.1 
CH.sub.3 CO.sub.2 CH.sub.2 CF.sub.3 
10a CCL 1.5 34 1.4 
CH.sub.3 CO.sub.2 Et 
10a CCL 0.4 -- -- 
1a 10a CCL 1700.sup.g 
-- -- 
1c 13a PPL 5.5 58 96 
CH.sub.3 CO.sub.2 Et 
13a PPL 0.1 58 80 
H.sub.2 O 13b PPL 60 58 90 
______________________________________ 
.sup.a Reaction condition: the alcohol substrate (2 mmol) was dissolved i 
benzene (4 mL) along with 120 .mu.L of dodecane as an internal standard. 
The acylating agent (2 eq) and CCL (265 mg) were added and the suspension 
was stirred at 28.degree. C. At various intervals, the degree of 
conversion was determined by GC (20 m DB5 megabore column; initial 
temperature, 80.degree. C.; initial time, 1 minute; gradient, 10.degree. 
C./min; flow rate 15 mL/min). After a certain degree of conversion, the 
reaction was terminated by filtration and the filtrate was evaporated. Th 
residue was purified on a silica gel column (CH.sub.2 Cl.sub.2 : nhexane 
1:3.fwdarw.1:0) to obtain the ester product. The optical purity of the 
product was determined by .sup.1 HNMR in the presence of Eu(hfc).sub.3 (1 
mg of acetate, propionate or pentanoate was added 40 mg, 40 mg or 28 mg o 
the shift agent, respectively). 
.sup.b Conditions: for transesterification, PPL (520 mg), solvent (8 mL), 
substrate (4 mmol), acylating reagent (2 eq), temperature (28.degree. C.) 
For hydrolysis, the same as above except that phosphate (0.1M, pH 7) was 
used as solvent. 
.sup.c A measure of enantioselectivity determined by the method reported 
previously: Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. 
Chem. Soc. 1982, 104, 7194. 
.sup.d The initial rate was 0.8 .mu.mol ester product formed per min per 
of enzyme. 
.sup.e The E value was obtained without considering the reverse reaction. 
.sup.f The tributyl tin ether of glycerol acetonide was used as substrate 
.sup.g No transesterification was observed in 0.05M phosphate buffer at p 
7. The rate of hydrolysis of 1a was measured. 
The reaction rate of enol ester was 10-100 times faster than ethyl acetate. 
Among the enol esters, vinyl esters reacted faster than isopropenyl esters 
and vinyl propionate was faster than vinyl acetate, but enol valerates 
were slower than enol acetates. The reaction rate of transesterification 
using different acylating reagents was compared to that of hydrolysis. As 
indicated in the resolution of 13 using PPL as catalyst, the relative 
rates for the hydrolysis of 13b and transesterification of 13a with vinyl 
acetate and ethyl acetate were found to be 600:55:1. The longer enol 
esters gave higher enantioselectivity. The lower selectivity in the ethyl 
acetate reaction may be due to the reversible nature of the reaction. 
Effect of Organic Solvent 
The effect of organic solvent on the CCL-catalyzed transesterification also 
was examined. As shown in Table 4, the rate of transesterification of 
glycerol acetonide (solketal) and isopropenyl acetate was slower in more 
polar solvents than in less polar solvents. 
TABLE 4 
______________________________________ 
Effect of organic solvent on CCL-catalyzed 
transesterifications of glycerol acetonide and 1a 
Reaction Solvent.sup.a 
Relative Rate 
E 
______________________________________ 
Benzene 50 1.4 
Isopropanyl acetate 
37 1.6 
Chloroform 9 1.5 
Tetrahydrofuran 1.sup.b 1.5 
______________________________________ 
.sup.a Reaction conditions are the same as those described in Table 2. 
.sup.b The initial rate was 0.32 .mu.mol ester formed per min per gram of 
enzyme. 
All lipases tested were catalytically active in pyridine but inactive in 
DMF. In a study of the solvent effect on the activity of lipases in 
organic solvents, it was found that the rate of CCL-catalyzed acylation 
was enhanced in the presence of benzene. 
Many valuable chiral synthons were prepared in high optical purity via 
lipase-catalyzed transesterification. The combination of two irreversible 
enzymatic processes, ester hydrolysis and ester synthesis, enabled 
effective syntheses of a number of optically active monoesters and 
alcohols in both enantiomeric forms, even with a moderately 
enantioselective enzyme. The same procedures also can be applied to the 
resolution of chiral ferrocenylethanol to prepare both enantiomers, a 
process which is impossible to accomplish in aqueous solution. 
Lipases and cholesterol esterase were found to catalyze enantioselective 
ester syntheses in various organic media. The leaving groups (acetone and 
acetaldehyde) of enol esters used in the processes are volatile and easy 
to remove, making the product separation very simple. With regard to the 
rate of transesterification, vinyl esters were about 20-100 times faster 
than ethyl esters and about 5 times faster than isopropenyl esters, and 
generally the long chain esters were faster than short chain esters. As 
compared to lipase-catalyzed hydrolysis, vinyl esters reacted 10 times 
slower. Because the transesterification reaction is carried out in neutral 
apolar organic solvents, this procedure is suitable for acid-, base- or 
water sensitive substances. 
Regioselective Acylations of Sugars and Their Derivatives 
The methyl and higher glycosides of hexoses and pentoses are sufficiently 
soluble in pyridine or other less polar media such that the enzymatic 
acetylations of these compounds can be accomplished with lipase-catalysis. 
Stronger solvents such as N,N-dimethylformamide (DMF), dissolve many 
otherwise insoluble sugars but they also render the lipases inactive. 
Riva, S., Chapineau, J., Kieboom, A. P. G., Klibanov, A. M. J. Am. Chem. 
Soc. 110 (1988) 584-589. We have found that Protease N (neutral protease 
from Amano International Enzyme Company) will utilize enol esters as acyl 
donors. This enzyme also retains its catalytic activity in dry DMF. 
Summaries of some of the data obtained with hexoses (Table 5), furanosides 
(Table 6), and nucleosides (Table 7) are shown hereinafter. Selected 
specific as well as general procedures for acetylation of sugars and their 
derivatives also are disclosed. 
TABLE 5 
______________________________________ 
Enzyme-catalyzed acetylation of hexoses and 
their derivatives using enol esters. 
______________________________________ 
##STR14## 
Com- Enol Conversion 
Regioselectivity 
Isolated Yield 
pound ENZ ester (%) (%) (%) 
______________________________________ 
18a CCL 1c 30 &gt;98 23 
19a PN 1a 60 &gt;90 49 
20a PN 1a 85 &gt;90 73 
______________________________________ 
TABLE 6 
______________________________________ 
Lipase-catalyzed acetylation of furanosides. 
______________________________________ 
##STR15## 
R.sub.1 
R.sub.1' R.sub.2 
R.sub.2' 
R.sub.3 
R.sub.3' 
______________________________________ 
21 (H, OMe) H OH H OH 
22 H OMe OH H H OH 
23 (H, OMe) H OH OH H 
24 (H, OMe) H H H OH 
______________________________________ 
Parentheses indicate a mixture of anomers. 
______________________________________ 
Product Yields.sup.a (%) 
Regioselectivity 
Substrate 2-OAc 3-OAc 5-OAc % 
______________________________________ 
Methyl .alpha.,.beta.-D-ribo- 
0 0 75-80.sup.b 
100 
furanoside, 21 
Methyl .alpha.-D-arabino- 
0 0 75-80.sup.b 
100 
furanoside, 22 
Methyl .alpha.,.beta.-D-xylo- 
0 0 60-85.sup.b 
100 
furanoside, 23 
Methyl 2-deoxy-.alpha.,.beta.- 
-- 17.sup.c 
39.sup.d 
78-100.sup.e 
D-ribofuranoside, 24 
______________________________________ 
.sup.a Yields reported are for anomeric mixtures. 
.sup.b The anomeric products were separated to facilitate spectroscopic 
identification. 
.sup.c The product obtained was methyl 
3O-acetyl-2-deoxy-.beta.-D-ribofuranoside. 
.sup.d The product consisted of a 9:1 mixture of .alpha.:.beta. anomers. 
.sup.e Regioselectivity was calculated based upon the individual anomers. 
EXAMPLE 10 
CCL-catalyzed transesterification of methyl 8-D-glucopyranoside (18a of 
Table 5) with vinyl acetate (1c of FORMULA 2) 
Methyl .beta.-D-glucopyranoside (18a of Table 2) (388 mg, 2 mmol) and vinyl 
acetate (1c of FORMULA 2) (4 mmol) were dissolved in 12 mL of 
benzene--pyridine (2:1). Then 388 mg of CCL was added, and the suspension 
was stirred at 28.degree. C. After 24 hours, an additional 388 mg of CCL 
was added, and this was repeated after 48 hours. The suspension was 
stirred at 28.degree. C. for 5 days; then worked up as usual to afford 
methyl 6-O-.beta.-D-glucopyranoside 18b as a solid, which was crystallized 
from ethyl acetate-n-hexane; m.p. 129.degree..congruent.130.degree. C.; 
[.alpha.].sup.25.sub.D -27.1 (c 1.4, CH.sub.3 OH); .sup.1 H-NMR (CD.sub.3 
COCD.sub.3); 2.02 (3H, s); 2.98 (1H, s); 3.13.congruent.3.25 (1H, m), 
3.3.congruent.3.55 (3H, m), 3.45 (3H, s), 4.15.congruent.4.25 (2H, m); 
4.30.congruent.4.45 (3H, m); .sup.13 C-NMR (CD.sub.3 COCD.sub.3); 104.56 
(C1), 74.29 (C2), 77.36 (C3), 70.85 (C4), 74.33 (C5), 64.01 (C6), 20.42 
and 170.69; (acetyl), 56.39 (methoxy). 
Regioselective Acetylation of Methyl Pentofuranosides 
The following general procedure was used for the substrates listed in Table 
6. 
To a solution of 1.64 g (10 mmol) of methyl pentofuranoside in a 24 mL of 
dry tetrahydrofuran containing a trace of hydroquinone was added 4.7 mL 
(50 mmol) of vinyl acetate (1c) and 5.0 g of porcine pancreatic lipase 
(PPL). The mixture was shaken in the dark at 37.degree. C. on an orbital 
shaker at 250 rpm. The reaction was monitored by TLC. After 24-60 h the 
solution was filtered and the solids washed with fresh tetrahydrofuran. 
The liltrate and washings were evaporated in vacuo and the residue 
purified by silica gel chromatography using either chloroform-methanol or 
ethyl acetate-hexane mixtures as eluent. The regioselectivities and yield 
ranges for the specific reactions are listed in Table 6. 
Protease-Catalyzed Reactions 
Protease N obtained from Amano International Enzyme Co. was used on the 
following reactions. Other highly stable proteases, such as proteases 
obtained from thermophillic organisms or genetically engineered stable 
proteases, also could be used in the following reactions. The crude 
commercial preparation was dissolved in 0.1M phosphate buffer, pH 7.8 (2 
g/35 mL) and lyophilized. The dry powder that was obtained was pulverized 
with a mortar and pestle prior to use. 
Regioselective Acylation of Sugar 
EXAMPLE 11 
Preparation of 2-acetamido-6-O-acetyl-2-deoxy-D-mannopyranose 
Protease N from BACILLUS SUBTILIS (obtained from Amano) (2 g) was dissolved 
in 0.1 mol NaH.sub.2 PO.sub.4 (35 mL), and the resulting solution was 
stirred for 15 min. The pH was then adjusted to 7.8 with 8.0 NaOH and the 
solution was freeze-dried. This freeze-dried preparation was used in the 
synthetic procedure. N-acetyl-.beta.-D-mannosamine monohydrate (Sigma) 
(478 mg, 2 mmol) was suspended in anhydrous N,N-dimethylformamide (2 mL). 
Isopropenyl acetate (600 mg, 6 mmol) was added followed by the enzyme 
preparation (600 mg). The suspension was shaken at 45.degree. C. and 
monitored by TLC (silica gel; EtOAc:MeOH:H.sub.2 O=100:10:1). After 44 h, 
the suspension was filtered and the enzyme washed with methanol (2.times.3 
mL). The solvents were evaporated under vacuum at 40.degree. C. to give a 
yellow syrup. This syrup was fractionated on a silica gel column (45 g) 
eluted with EtOAc/MeOH/H.sub.2 O =100/10/1. Two products were obtained: 
the first with a higher R.sub.f corresponded to a triacetate compound (30 
mg, 10%). The second (major) product was obtained as an amorphous white 
solid which, upon analysis was revealed to be 
2-acetamido-6-O-acetyl-2-deoxy-D-mannopyranose. (384 mg, 73%): .sup.1 
H-NMR (D.sub.2 O/p-dioxane=3.57 ppm) .delta.4.93 (s, H 1 .alpha.), 4.84 
(s, H 1 .beta.), 4.29-4.02 (m, 5 H), 3.87 (dd, H 3 .alpha.), 3.70-3.27 (m, 
3 H), 1.95, 1.91, 1.88, and 1.87 (4 s, 6 H, acetal); .sup.13 C-NMR 
(D.sub.2 O/p-dioxane=67.46 ppm) .delta.176.60, 175.67, 174.96, and 174.92 
(all carbonyls), 94.05 (c 1 .beta.), 93.97 (C 1 .alpha.), 74.78 (c 
.beta.), 72.72 (C .beta.), 70.57 (C 5 .alpha.), 69.43 (C .sup..alpha.), 
67.94 (C .alpha.), 67.78 (C .beta.), 64.61 (C 6 .alpha.), 64.36 (C 6 
.beta.), 54.84 (C 2 .beta.), 54.18 (c 2 .alpha.); .alpha./.beta.=76/24; mp 
47.degree.-51.degree. C.; [.alpha.].sup.24.sub.D +15.9.degree. (c 1.13 
H.sub.2 O). Anal. Calcd for C.sub.10 H.sub.16 NO.sub.7 : C, 45.80; H, 
6.15; N, 5.34. Found: C, 45.89; H, 6.20; N, 4.95. 
Regioselective Acylation of Nucleosides 
EXAMPLE 12 
General Procedures 
The following general procedures were used performing the regioselective 
acylation of nucleosides listed in Table 7: 
1 mmol of nucleoside was dissolved in 2-4 mL of dry DMF and warmed. The 
solution was cooled to 45.degree. C. and 1.1 mL (10 eq) of isopropenyl 
acetate and 260 mg of pulverized protease N were added. The suspension was 
shaken at 45.degree. C. After the appropriate times, as indicated in Table 
7, the reaction mixture was filtered and the liltrate was evaporated to 
dryness. The residue was purified by silica gel chromatography using 
mixtures of ethyl acetate:ethanol:water as the eluent for the times 
indicated. The isolated products were obtained in the yields shown in 
Table 7. 
Table 7 indicates that, where acetylation occurred, the monoacetyl 
derivative was predominately formed. The preferential formation of the 
monoacetyl derivative indicates that the nucleoside was acetylated at the 
primary (5') hydroxyl group. 
TABLE 7 
______________________________________ 
Selective Enzymatic Acetylations of Nucleosides and Sugars 
in Anhydrous Dimethyl-Formamide 
Mono- Di- Starting 
Time acetyl 
acetyl 
Material 
Substrate Enzyme (days) (%) (%) (%) 
______________________________________ 
Guanosine PN 5 0 0 100 
Adenosine PN 1.75 40 0 60 
" PN 5 65 &lt;5 30 
2-Deoxy PN 2 50 -- -- 
adenosine 
2-Deoxy PN 4 80 -- -- 
adenosine 
Uridine PN 1.75 50 0 50 
" PN 5 80 &lt;5 15 
" PN(pyr) 5 60 0 40 
" PN(THF) 5 0 0 100 
" PPL(pyr) 3 &gt;95 0 &lt;5 
" PPL(THF) 3 &gt;95 0 &lt;5 
Cytidine PN 1.5 60 0 40 
" PN 3 80 &lt;5 15 
" S 3 0 0 100 
2-Deoxycytidine 
PN 2 60 -- -- 
" PN 4 80 -- -- 
Thymidine PN 1.5 90 0 10 
" S 1.5 0 0 100 
Methyl 2- PN 2 70 -- -- 
deoxy-D- 
ribofuranoside 
______________________________________ 
PN = protease N [Amano]- 
S = subtilisin BPN 
PPL = porcine pancreatic lipase 
The simplicity of this irreversible transesterification makes the operation 
useful for the preparation of chiral alcohols or esters that may be 
difficult to prepare by other means. 
The foregoing description has been for purposes of illustration. Those 
skilled in the art will appreciate a number of variations and 
modifications therefrom. The following claims are intended to cover all 
modifications and variations within the true spirit and scope of the 
present invention.