Regioselective enzymatic acylation of methyl shikimate. Influence of acyl chain length and solvent polarity on enzyme specificity.

Article abstract of DOI:10.1021/jo025671p

Candida antarctica lipase A (CAL-A) selectively catalyzes the acylation at the secondary C-4 hydroxyl group of methyl shikimate (2), which possesses three secondary hydroxyl groups, the C-3 allylic one being chemically more reactive. The effect both of the acyl group of the acylating agents and of the solvent polarity has been studied. The selectivity of CAL-A is almost complete with acyl donors that possess short chains. However, when acyl donors have longer chains, better results are obtained by C. antarctica lipase B (CAL-B).

Full text of DOI:10.1021/jo025671p

Regioselective En zym a tic Acyla tion of
Meth yl Sh ik im a te. In flu en ce of Acyl Ch a in
Len gth a n d Solven t P ola r ity on En zym e
Sp ecificity
Nuria Armesto, Miguel Ferrero, Susana Ferna´ndez, and
Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica, Facultad
de Quı´mica, Universidad de Oviedo, 33071-Oviedo, Spain
F IGURE 1.
VGS@sauron.quimica.uniovi.es
Received March 1, 2002
ing steps. This methodology has been extensively used
with carbohydrates.5 Riva and co-workers6 have shown
that lipase from Chromobacterium viscosum (CVL) se-
lectively esterifies the hydroxyl group at the C-4 position
of methyl quinate (4) and benzyl quinate (5). However,
acylation of methyl shikimate (2) did not display regi-
oselectivity with any of the enzymes tested. To our
knowledge, regioselective enzymatic acylation of shikimic
acid derivatives has not been described, although satu-
rated and unsaturated esters (among them, cynnamoyl
esters) of quinic and shikimic acids were shown to be
plant metabolites,7 some of them with a broad spectrum
of biological activities. In our research program directed
toward the syntheses of quinic and shikimic acid deriva-
tives,8 we describe here the regioselective enzymatic
acylation of methyl shikimate and the influence of several
parameters such as enzyme, solvent, temperature, and
acyl chain length on the rate and regioselectivity.
The three hydroxyls present in the shikimic acid lead
to low solubility and reactivity, and therefore to increase
the reaction rate and shift the thermodynamic equilib-
rium toward acylation of the substrate, activated esters
were used. First, enzymatic acylation of methyl shikimate
(2) was done in vinyl acetate as both the solvent and the
acylating agent and in the presence of 4 Å molecular
sieves (Scheme 1).
Abstr a ct: Candida antarctica lipase A (CAL-A) selectively
catalyzes the acylation at the secondary C-4 hydroxyl group
of methyl shikimate (2), which possesses three secondary
hydroxyl groups, the C-3 allylic one being chemically more
reactive. The effect both of the acyl group of the acylating
agents and of the solvent polarity has been studied. The
selectivity of CAL-A is almost complete with acyl donors that
possess short chains. However, when acyl donors have longer
chains, better results are obtained by C. antarctica lipase B
(CAL-B).
Shikimic acid (1, Figure 1) is a key intermediate in the
biosynthesis of the amino acids L-phenylalanine, L-
tryptophan, and L-tyrosine, as well as being a precursor
to the folate coenzymes and various isoprenoid quinones.1
The enzymes in this pathway are unique to plants, fungi,
and microorganisms and are therefore important targets
as potential herbicidal, antifungal, and antibacterial
agents that do not affect mammals.2 As a result, increas-
ing effort has been directed toward the syntheses of
shikimate analogues.1c,3
This metabolite, together with quinic acid (3), an
alternate carbon source in the shikimate pathway, pos-
sesses several hydroxyl groups of similar reactivity in its
structure, and a clear discrimination between them still
remains a difficult task. Enzymatic modification4 offers
a highly efficient process compared to conventional
chemical syntheses using the process of blocking/deblock-
For the screening of suitable biocatalysts, lipases from
Candida antarctica A (CAL-A), C. antarctica B (CAL-
B), Pseudomonas cepacia (PSL-C), C. viscosum (CVL),
and Candida rugosa (CRL) were tested together with the
protease subtilisin (Table 1).
(5) (a) Davis, B. G. J . Chem. Soc., Perkin Trans. 1 2000, 14, 2137-
2160. (b) Ferna´ndez-Mayorales, A. Top. Curr. Chem. 1997, 186, 1-20.
(c) Gijsen, H. J . M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. Rev. 1996,
96, 443-473. (d) Drueckhammer, D. G.; Hennen, W. J .; Pederson, R.
L.; Barbas, C. F., III; Gautheron, C. M.; Krach, T.; Wong, C.-H.
Synthesis 1991, 499-525.
(1) (a) Knaggs, A. R. Nat. Prod. Rep. 2000, 17, 269-292. (b) Haslam,
E. Shikimic Acid Metabolism and Metabolites; Wiley and Sons:
Chichester, U.K., 1993. (c) Campbell, M. M.; Sainsbury, M.; Searle, P.
A. Synthesis 1993, 179-193. (d) Weiss, V.; Edwards, J . M. The
Biosynthesis of Aromatic Compounds; Wiley-Interscience: New York,
1987; pp 260-270.
(2) (a) Roberts, F.; Roberts, C. W.; J ohnson, J . J .; Kyle, D. E.; Krell,
T.; Coggins, J . R.; Coombs, G. H.; Milhous, W. K.; Tzipori, S.; Ferguson,
D. J . P.; Chakrabarti, D.; McLeod, R. Nature 1998, 393, 801-805. (b)
Sikorski, J . A.; Gruys, K. J . Acc. Chem. Res. 1997, 30, 2-8. (c)
Kozlowski, M. C.; Tom, N. J .; Seto, C. T.; Sefler, A. M.; Bartlett, P. A.
J . Am. Chem. Soc. 1995, 117, 2128-2140. (d) Grossbard, E.; Atkinson,
D. The Herbicide Glyphosate; Butteworths: Boston, 1985.
(6) Danielli, B.; De Bellis, P.; Barzhaghi, L.; Carrea, G.; Ottolina,
G.; Riva, S. Helv. Chim. Acta 1992, 75, 1297-1304.
(7) (a) Sefkow, M.; Kelling, A.; Schilde, U. Eur. J . Org. Chem. 2001,
2735-2742, and references therein. (b) Chen, T.; Li, J .; Cao, J .; Xu,
Q.; Komatsu, K.; Namba, T. Planta Med. 1999, 65, 56-59. (c) Saito,
T.; Yamane, H.; Murofushi, N.; Takahashi, N.; Phinney, B. O. Biosci.,
Biotechnol., Biochem. 1997, 61, 1397-1398. (d) Dupre, S.; Grenz, M.;
J akupovic, J .; Bohlmann, F.; Niemeyer, H. M. Phytochemistry 1991,
30, 1211-1220. (e) Barrero, A. F.; Sa´nchez, J . F.; Alvarez-Manzaneda,
E. J . R.; Alvarez-Manzaneda, R. R. Phytochemistry 1988, 27, 1191-
1194.
(8) (a) Oves, D.; D´ıaz, M.; Ferna´ndez, S.; Ferrero, M.; Gotor, V.
Synth. Commun. 2001, 31, 2335-2343. (b) D´ıaz, M.; Ferrero, M.;
Ferna´ndez, S.; Gotor, V. J . Org. Chem. 2000, 65, 5647-5652. (c)
Armesto, N.; Ferrero, M.; Ferna´ndez, S.; Gotor, V. Tetrahedron Lett.
2000, 41, 8759-8762. (d) D´ıaz, M.; Ferrero, M.; Ferna´ndez, S.; Gotor,
V. Tetrahedron Lett. 2000, 41, 775-779. (e) Ferna´ndez, S.; D´ıaz, M.;
Ferrero, M.; Gotor, V. Tetrahedron Lett. 1997, 38, 5225-5228.
(3) J iang, S.; Singh, G. Tetrahedron 1998, 54, 4697-4753.
(4) (a) Klibanov, A. M. Nature 2001, 409, 241-246. (b) Koeller, K.
M.; Wong, C.-H. Nature 2001, 409, 232-240. (c) Roberts, S. M.; Turner,
N. J .; Willetts, A. J .; Turner, M. K. Introduction to Biocatalysis Using
Enzymes and Microorganisms; Cambridge University Press: New
York, 2000. (d) Faber, K. Biotransformations in Organic Chemistry,
4th ed.; Springer-Verlag: Berlin, 2000. (e) Carrea, G.; Riva, S. Angew.
Chem., Int. Ed. 2000, 39, 2226-2254. (f) Bornscheuer, U. T.; Kazlaus-
kas, R. J . Hydrolases in Organic Synthesis: Regio- and Stereoselective
Biotransformations; Wiley-VCH: Weinheim, Germany, 1999.
10.1021/jo025671p CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/21/2002
4978
J . Org. Chem. 2002, 67, 4978-4981

SCHEME 1
SCHEME 2
TABLE 1. En zym a tic Acyla tion of 2 in Vin yl Aceta te a t
30 °C
TABLE 3. Acyla tion of 2 w ith Differ en t Acyla tin g
Agen ts in TBMEa Ca ta lyzed by CAL-A
entry enzyme
t (h) conv (%)a 7a (%)a,b 8a (%)a,b 9a (%)a,b
1
2
3
4
5
6
CAL-A
CAL-B
PSL-C
Subtilisin 24
CVL
CRL
0.75
24
4.5
100
55
70
25
25
45
17
45
41
28
63
30
83
18
59
19
37
38
acylating ratio of
agent
T
t
conv 7a
8a
diacyls
37
53
32
entry
2:10 (°C) (h) (%)b (%)b,c (%)b,c (%)b,c
1
2
3
4
5
6
10a
10b
10b
10c
10d
10d d
1:10
1:10
1:5
1:10
1:10
1:5
30
30 23
30 21
20
20
20
4.5
45
100
100
71
7
32
12
3
93
65
75
97
89
97
24
24
3
13
3.5
3
1
a
b
Based on 1H NMR signal integration. Percentage of regiose-
67
94
11
3
lectivity at C-3, C-4, and C-5.
a
tert-Butylmethyl ether. b Based on 1H NMR signal integration.
TABLE 2. Acyla tion of 2 w ith Vin yl Aceta te (r a tio )
1:10) Ca ta lyzed by CAL-A a t 20 °C
c Percentage of regioselectivity at C-3, C-4, and diacyl derivatives.
K2CO3 (0.25 equiv) was added.
d
entry
solvent
t (h)
conv (%)a 7a (%)a,b 8a (%)a,b
1
2
3
4
5
6
7
8
9
vinyl acetate
tBuOH
1,4-dioxane
toluene
TBMEc
CHCl3
0.75
22
22
3
2.5
22
22
22
22
22
100
37
90
85
100
85
74
70
52
70
10
42
7
90
58
93
95
97
90
88
75
93
77
for biocatalysis since they favor substrate solubility and
the formation of more polar products, in this case the
monoacylated species.11 Since CAL-A is immobilized, the
decrease in the catalytic activity of the enzyme in polar
solvents is less pronounced. Reactions were done at 20
°C using a ratio of triol to vinyl acetate of ∼1:10. This
lower temperature gave rise to an increase in the
selectivity shown by CAL-A in vinyl acetate (entry 1,
Table 2).
In general, high conversions and regioselectivities were
achieved with most of the solvents (Table 2). Excellent
results were observed with CAL-A in tert-butylmethyl
ether (TBME) and toluene (entries 4 and 5, Table 2).
Thus, if the process was done in TBME, after 2.5 h the
1H NMR spectrum of the reaction crude showed 97% of
C-4 monoacetate 8a and only traces of derivative 7a .
We subsequently evaluated a variety of acylating
agents in the process catalyzed by CAL-A in TBME as
the solvent (Scheme 2).
If isopropenyl acetate (10a ) was used instead of vinyl
acetate, the reaction was slower and gave rise to 45%
conversion after 4.5 h at 30 °C. However, the enzyme kept
the excellent selectivity shown in the formation of
compound 8a (entry 1, Table 3 vs entry 5, Table 2).
Other versatile acylating agents were oxime esters.12
In principle, acylation was run at 30 °C using a 1:10 ratio
of shikimate 2 to oxime ester 10b. In this case, total
conversion was obtained after 23 h with a moderate
selectivity toward the C-4 position (entry 2, Table 3).
Similar behavior was observed at 20 °C. When the
reaction took place in a 1:5 ratio of 2:10b at 30 °C, CAL-A
exhibited a moderate increase in the selectivity toward
5
traces
10
12
25
7
THF
CH2Cl2
acetone
MeCN
10
23
Based on 1H NMR signal integration. Percentage of regiose-
a
b
lectivity at C-3 and C-4. c tert-Butylmethyl ether.
Reactions were allowed to run at 30 °C until high or
total conversions were achieved or, in reactions failing
to achieve such conversions, for 24 h. Higher conversions
were obtained with immobilized9 biocatalysts (CAL-A,
CAL-B, and PSL-C). Of the enzymes studied, CAL-A gave
the best result. It catalyzed the acetylation of the C-4
hydroxyl group with high regioselectivity in less than 1
h, giving rise to the monoacetylated derivative 8a (entry
1, Table 1). PSL-C also revealed some preference for the
same hydroxyl, but the selectivity was lower (entry 3,
Table 2). When CAL-B or CVL was used, both enzymes
showed a slight preference for C-3 acetylation (entries 2
and 5, Table 1). However, protease subtilisin showed a
certain degree of selectivity toward the C-5 position
(entry 4, Table 1). In the case of CRL, an equimolecular
mixture of monoacetates 7a , 8a , and 9a was obtained at
a low conversion (entry 6, Table 1).
To increase the selectivity exhibited by CAL-A in the
enzymatic acylation and introduce acyl groups other than
acetyl, an adequate solvent should be chosen. Taking into
account the polar nature of the substrate, solvents of log
P10 < 2 (polar solvents) are considered to be most suitable
(11) (a) Bellot, J . C.; Choisnard, L.; Castillo, E.; Marty, A. Enzyme
Microb. Technol. 2001, 28, 362-369. (b) Fevrier, P.; Gue´gan, P.;
Yvergnaux, F.; Callegari, J . P.; Dufosse´, L.; Binet, A. J . Mol. Catal. B:
Enzym. 2001, 11, 445-453. (c) Kuo, S.-J .; Parkin, K. L. J . Am. Oil
Chem. Soc. 1996, 73, 1427-1433.
(9) (a) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. 2000, 39, 2226-
2254. (b) Reetz, M. T.; Zonta, A.; Simpelkamp, J . Biotechnol. Bioeng.
1996, 49, 527-534.
(10) (a) Chen, C.-S.; Sih, C. J . Angew. Chem., Int. Ed. Engl. 1989,
28, 695-707. (b) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol.
Bioeng. 1987, 30, 81-87. (c) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev.
1971, 71, 525-616.
(12) Ferrero, M.; Gotor, V. In Enzymes in Action. Green Solutions
for Chemical Problems; Zwanenburg, B., Mikolajczyk, M., Kielbasinski,
P., Eds.; Kluwer Academic Plublishers: Dordrecht, The Netherlands,
2000; Chapter 18, pp 347-363.
J . Org. Chem, Vol. 67, No. 14, 2002 4979

TABLE 5. Acyla tion of 2 w ith Vin yl Stea r a te (r a tio )
1:3) a t 30 °C over 24 h Ca ta lyzed by CAL-B
SCHEME 3
entry
solvent
conv (%)a 7f (%)a,b 8f (%)a,b 9f (%)a,b
1
2
3
4
5
6
7
8
9
acetone
1,4-dioxane
tBuOH
MeCN
CH2Cl2
CHCl3
THF
toluene
TBMEc
77
61
60
47
80
85
67
70
50
100
100
100
100
79
70
68
20
32
21
30
19
60
22
13
20
46
a
b
Based on 1H NMR signal integration. Percentage of regiose-
TABLE 4. Acyla tion of 2 w ith Vin yl Ester s in TBME a
Ca ta lyzed by CAL-Ab
lectivity at C-3, C-4, and C-5. c tert-Butylmethyl ether.
acylating ratio of
T
t
7
8
9
diacyls
flash chromatography purification. We previously ob-
served that in solution, the C-4 acetate (8a ) was trans-
formed little by little into the C-3 acetate (7a ) until an
equilibrium was achieved, in which 7a and 8a were the
major and minor compounds, respectively. To avoid this
migration, C-4 acyl derivatives should be handled very
carefully using neutral silica gel for chromatography and
their stay in solution should be shortened as much as
possible. In the case of the C-4 chloroacetate, this
migration was favored by the inductive effect of the
chlorine atom.
When the reaction was done with unsaturated esters
such as vinyl acrylate (6h ) and vinyl crotonate (6i),
CAL-A showed excellent regioselectivity (93-96%) to the
hydroxyl group at the C-4 position (entries 7 and 8, Table
4). The fact that the reaction using vinyl crotonate
resulted in a higher regioselectivity than that using vinyl
butyrate, which has the same chain length in the acyl
moiety, revealed that CAL-A is more specific with
unsaturated acyl chains.
Finally, we studied the reaction with vinyl benzoate
(6j). Due to the lower reactivity of this acylating agent,
the process did not evolve at 30 °C with either 1:5 or 1:10
ratios, and it was necessary to heat the mixture to 40 °C
to reach total conversion with high selectivity toward the
C-4 position (entry 9, Table 4).
Since the preference of CAL-B for long-chain acyl
donors has been described in the literature,13 and in view
of the poor selectivity exhibited by CAL-A with vinyl
decanoate, vinyl laurate, and vinyl stearate, the acylation
of 2 performed with vinyl stearate (C18) and catalyzed
by CAL-B was checked. Among the solvents tested,
acetone, 1,4-dioxane, tert-butyl alcohol, and acetonitrile
gave exclusively acylation at the C-4 hydroxyl group
(entries 1-4, Table 5).
entry
agent
2:6
(°C) (h) (%)c (%)c (%)c (%)c
1
2
3
4
5
6
7
8
9
6b
6c
6d
6e
6f
6g
6h
6i
1:5
1:5
15
15
30 24
30 24
30 24
15
20 11
20 10
0.5 10
0.66 11
90
70
12
7
1:10
1:10
1:10
1:5
1:10
1:10
1:10
complex mixture
complex mixture
31
8
52
83
93
96
90
17
9
0.7
7
4
6j
40
8
10
a
b
tert-Butylmethyl ether. Reactions were allowed to reach
100% conversion. c Percentage of regioselectivity at C-3, C-4, C-5,
and diacyl derivatives based on 1H NMR signal integration.
the C-4 position, leading to 75% yield of derivative 8a
(entry 3, Table 3). On the other hand, if 2,2,2-trifluoro-
ethyl acetate (10c) or acetic anhydride (10d ) was used
as the acylating agent, CAL-A catalyzed almost exclu-
sively the formation of C-4-monoacetate (entries 4-6,
Table 3). In the case of acetic anhydride, to remove the
generated acetic acid (which inhibited the enzyme),
K2CO3 was added to the reaction mixture. No changes
in the regioselectivity were observed with any of the
acylating agents tested.
To take advantage of the observation that CAL-A in
TBME exhibits excellent acetylation selectivity with vinyl
acetate, the reaction of 2 with other vinyl esters was
studied (Scheme 3).
Table 4 shows optimized conditions for each of the vinyl
esters. Enzymatic acylation with vinyl propionate (6b)
occurred with high selectivity (entry 1, Table 4) when the
reaction was done at 15 °C with a 1:5 ratio of 2:6b, 90%
of C-4 monoacyl derivative 8b being obtained.
If the acylating agent was vinyl butyrate (6c), the
process took place with
a moderate selectivity of
70% toward the same position mentioned above (entry
2, Table 4). However, CAL-A did not selectively catalyze
the acylation with vinyl decanoate (C10), vinyl laurate
(C12), or vinyl stearate (C18) (entries 3-5, Table 4). The
influence of the selectivity as a function of the acyl donor
chain length is noteworthy. CAL-A is more specific
toward short- rather than long-chain vinyl esters. This
enzyme preference decreases in the order vinyl acetate
∼ propionate > butyrate > decanoate ∼ laurate ∼
stearate.
Higher conversions were achieved with acetone > 1,4-
t
dioxane ∼ BuOH > MeCN. When the reaction took place
in methylene chloride, chloroform, or THF, CAL-B showed
moderate selectivities toward the same position (entries
5-7, Table 5). On the other hand, if toluene or TBME
was used as a solvent, the process was not selective
(entries 8 and 9, Table 5). The fact that only monoacy-
lated products were formed can be attributed to the
nature of the solvents (log P e 2), which are hydrophilic
Although vinyl chloroacetate (6g) gave preferably C-4
chloroacetate 8g (entry 6, Table 4), isolation from the
reaction crude was not possible due to an acyl migration
from the C-4 position to the C-3 or C-5 position during
(13) (a) Ottosson, J .; Hult, K. J . Mol. Catal. B: Enzym. 2001, 11,
1025-1028. (b) Otero, C.; Arcos, J . A.; Berrendero, M. A.; Torres, C.
J . Mol. Catal. B: Enzym. 2001, 11, 883-892. (c) Soultani, S.; Engasser,
J . M.; Ghoul, M. J . Mol. Catal. B: Enzym. 2001, 11, 725-731.
4980 J . Org. Chem., Vol. 67, No. 14, 2002

TABLE 6. En zym a tic Acyla tion of 2 w ith Vin yl Ester s
biocatalysts in organic solvents, novel saturated, unsat-
urated, and aromatic monoesters of shikimic acid have
been synthesized. These can be candidates for use as
potentially therapeutically useful derivatives.
(r a tio ) 1:10) in Aceton e a t 30 °C
acylating
agent
conv
entry enzyme
t (h) (%)a 7 (%)a,b 8 (%)a,b 9 (%)a,b
1
2
3
4
5
6
7
8
9
CAL-B
CAL-B
CAL-B
CAL-A
CAL-A
CAL-A
PSL-C
PSL-C
PSL-C
6d
6e
6f
6d
6e
6f
6d
6e
6f
24
24
24
18 100
18 100
18 100
21
21
21
53
74
80
46
39
47
54
100
40
44
88
44
60
94
7
7
Exp er im en ta l Section
Gen er a l. C. viscosum lipase (CVL, 3800 U/mg of solid), C.
antarctica lipase B (CAL-B, Novozym 435, 7300 PLU/g), im-
mobilized P. cepacia lipase (PSL-C, 783 U/g), C. rugosa lipase
(CRL, typeVII, 950 U/mg of solid), Subtilisin (type VIII, 12 u/mg
of solid), and C. antarctica lipase A (CAL-A, chirazyme L-5, c-f,
lyophilized, 25 U/g using 1-phenylethyl acetate) were obtained
commercially. Column chromatography was performed over
silica 60 Å (32-63 µm) pH 7. Analytical TLC was performed
using silica 60 F254 aluminum sheets. The chromatograms were
visualized by heating after spraying with a 5% aqueous sulfuric
acid solution containing cerium sulfate (1%) and molybdophos-
phoric acid (2.5%). Molecular sieves (4 Å) were dried at 180 °C
in a vacuum over 2 h. Methyl shikimate was dried in a high
vacuum (10-5 mbar) before use.
60
56
12
56
40
6
65
77
80
a
b
Based on 1H NMR signal integration. Percentage of regiose-
lectivity at C-3, C-4, and C-5.
solvents with an affinity for more polar molecules, in this
case the monostearyl derivative. Moreover, long-chain
vinyl esters are less polar than short- and medium-sized
chain esters, which favors the precipitation of the monoa-
cyl compound13b,14 in the polar medium and avoids
polyacylation and transposition reactions.
Next, we compared the specific activity of CAL-B with
medium-sized vinyl esters in acetone. As shown in Table
6, the nature and the structure of the acylating agent
have a strong influence on the selectivity of the acylation
reaction. CAL-B did not display any remarkable selectiv-
ity with vinyl decanoate (C10) or laurate (C12) (entries 1
and 2, Table 6).
Additionally, we performed the acylation catalyzed by
CAL-A in acetone with medium- and long-chain acyl
donors. The best regioselectivity was reached with vinyl
stearate, whereas the reactions with vinyl decanoate and
vinyl laurate took place without selectivity (entries 4-6,
Table 6). Similar results were obtained with PSL-C
(entries 7-9, Table 6), which catalyzed the acylation of
methyl shikimate (2) using vinyl stearate with excellent
selectivity.
In summary, an exhaustive study of the influence of
parameters such as enzyme, solvent, temperature, and
acyl chain length on the selective acylation of methyl
shikimate has been performed. Of note is the excellent
selectivity exhibited by CAL-A toward the acylation at
the most highly hindered C-4 hydroxyl group of methyl
shikimate (2) using vinyl esters as acylating agents in
tert-butylmethyl ether. This enzyme is more specific with
short-chain than with long-chain vinyl esters. The rate
of selectivity decreases in the order vinyl acetate ∼
propionate > butyrate > decanoate ∼ laurate ∼ stearate.
Excellent results were also obtained with vinyl acrylate,
vinyl crotonate, and vinyl benzoate. For long-chain vinyl
esters, the change from tert-butylmethyl ether to acetone
as the solvent gives rise to an increase in the regioselec-
tivity of the enzymatic acylation. In this case, CAL-B is
more specific, catalyzing the acylation of 2 with vinyl
stearate exclusively at the C-4 position. By means of
Meth yl Sh ik im a te (2). To a solution of shikimic acid (2 g,
11.48 mmol) in MeOH (25 mL) was added 10 drops of concen-
trated HCl. The mixture was heated at 60 °C for 4 h. The solvent
was evaporated to give 2.1 g (quantitative yield) of 2 as a white
solid: 1H NMR (MeOH-d6, 300) δ 2.38 (dddd, 1H, H6a′, | J HH| )
2
2
18.2 Hz, J HH ) 5.5, 1.5, 1,5 Hz), 2.88 (dddd, 1H, H6e′, | J HH| )
18.2 Hz, J HH ) 4.8, 2.0, 2.0 Hz), 3.88 (m, 1H, H5), 3.93 (s, 3H,
H8), 4.18 (m, 1H, H4), 4.56 (m, 1H, H3), 6.98 (ddd, 1H, H2, J HH
) 2.1, 1.2, 1,2 Hz); MS (ESI+, m/z) 211 [(M + Na)+, 100].
En zym a tic Acyla tion of Meth yl Sh ik im a te (2). In
a
standard procedure, the acylating agent, dissolved in the ap-
propriate solvent (750 µL), was added to an Erlenmeyer flask
that contained triol 2 (13 mg, 0.069 mmol), the enzyme [CAL-A
(13 mg), CAL-B (13 mg), PSL-C (26 mg), subtilisin (13 mg), CVL
(13 mg), or CRL (50 mg)], and 4 Å molecular sieves (13 mg) under
nitrogen. The suspension was shaken at 250 rpm (the ratio of
acylating agent, temperature, and time are indicated in Tables
1-6). The mixture was filtered, and the organic solvent was
evaporated. Then, the reaction crude was subjected to a careful
flash chromatographic column to avoid migrations from C-4 to
C-3, using silica 60 Å (32-63 µm) pH 7 (40% acetone/CH2Cl2
for 8a ; 20% acetone/CH2Cl2 for 8b and 8c; 15% acetone/CH2Cl2
for 8f; 25% acetone/CH2Cl2 for 8h and 8i; and 10% CH2Cl2/Et2O
for 8j). The operation should be done within just a few minutes
to minimize the contact with both the silica gel and the eluent.
Then, solvents were evaporated at 20 °C under reduced pressure.
Ack n ow led gm en t. We express our appreciation to
Novo Nordisk Co. and Genzyme for the generous gifts
of the lipases CAL-B and CVL, respectively. Financial
support from Principado de Asturias (Spain; Project GE-
EXP01-03) and MCYT (Spain; Project PPQ-2001-2683)
is gratefully acknowledged. S.F. also thanks MCYT
(Spain) for a personal grant (Ramo´n y Cajal Program).
We thank Robin Walker for his revision of the final
manuscript.
Su p p or tin g In for m a tion Ava ila ble: Complete 1H and
13C NMR spectral data in addition to mp, IR, microanalysis,
optical rotation, and MS data for major monoacylated deriva-
tives; the level of purity is indicated by the inclusion of
microanalysis and copies of 1H and 13C NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(14) (a) Arcos, J . A.; Hill, C. G., J r.; Otero, C. Biotechnol. Bioeng.
2001, 73, 104-110. (b) Cao, L.; Fischer, A.; Bornscheuer, U. T.; Schmid,
R. D. Biocatal. Biotransform. 1997, 14, 269-283.
J O025671P
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