Fotakopoulou et al.
TABLE 3. Recommendations for the Hydrolysis of Various Esters
by BS2
diphenylmethyl esters of various acids. The results of their
hydrolysis by BS2 are summarized in Table 2. 2,2,2-Trichlo-
roethyl and phenacyl esters were removed from Boc-GABA
within 1 and 1.5 h, respectively, in quantitative yields (entries
1 and 2, Table 2). Diphenylmethyl esters require longer reaction
time and higher enzyme/substrate ratios (entries 3-5, Table 2).
However, benzoic acid and Boc-GABA were isolated in almost
quantitative yields (entries 3 and 4, Table 2). 2,2,2-Trichloro-
ethyl and 2-chloroethyl esters were selectively removed in the
presence of ethyl ester, and the products were isolated in 99
and 65% yields, respectively, after 30 min (entries 6 and 7, Table
2). In addition, the benzyl ester was selectively removed leading
to the isolation of ethyl N-benzyloxycarbonyl-R-glutamate in
73% (entry 8, Table 2).
entry
ester
enzyme/substrate
time (h)
1
2
3
4
5
6
7
8
9
2-chloroethyl
2,2,2-trichloroethyl
allyl
benzyl
methyl
phenacyl
ethyl
diphenylmethyl
tert-butyl
1:4
1:4
1:4
1:4
1:4
1:4
1:4
1:1
1:1
0.25-1
0.25-1
0.5-3
1-24
1.5-24
1.5-24
1.5-24
g48
g48
presented here and our previous results,6,8 it is clear that BS2
is a very attractive enzyme for applications in protecting group
chemistry.
From the above results, it is clear that the chloroethyl group,
containing either one or three chlorine atoms, is easily hydro-
lyzed by BS2. The increased carbonyl reactivity of the ester
group due to the inductive effect of chlorine atoms is in
accordance with the ease hydrolysis of such esters. The high
reactivity of BS2 against allyl esters may be attributed to the
presence of the double bond. A similar increase in activity and
enantioselectivity was reported by us several years ago, in the
kinetic resolution of chiral carboxylic acids. In that case, the
use of vinyl esters was significantly better compared to the ethyl
ester, despite the fact that the ethyl and the vinyl ester have the
same carbon length and differed only in the absence or presence
of the double bond.18 The steric hindrance of diphenylmethyl
esters may explain their slow hydrolysis by BS2. However, these
esters are indeed hydrolyzed by BS2, indicating that esterases
bearing a GGG(A)X motif in their oxyanion binding pocket
are able to cleave esters of sterically hindered alcohols, not only
esters of tertiary alcohols.
The rate of the hydrolysis of esters depends on the nature of
both the alcohol and the carboxylic acid. In general, the
recommended conditions for the ratio BS2/substrate, as well as
the expected reaction times, are summarized in Table 3. A
mixture of phosphate buffer/hexane/methanol (7:1:0.1) may be
used as a solvent at 37 °C. To improve the solubility, the ratio
of phosphate buffer/hexane may be changed to 1:1 or a double
volume of methanol may be used. Toluene may be alternatively
used instead of hexane. For substrates containing long chains,
higher yields of products may be achieved employing CAL-A
instead of BS2.
In conclusion, we have demonstrated that allyl, 2-chloroethyl,
2,2,2-trichloroethyl, phenacyl, and diphenylmethyl protecting
groups can be efficiently cleaved by an esterase from Bacillus
subtilis (BS2). In particular, the enzymatic quick and selective
removal of allyl, 2-chloroethyl, and 2,2,2-trichloroethyl esters
under mild conditions in high yields makes them attractive
protecting groups for applications in organic synthesis of
sensitive molecules, where conventional chemical methods
cannot be applied because of incompatibility.
To our knowledge, there is no report on the removal of
phenacyl and diphenylmethyl esters by any enzyme. Although
allyl esters have been reported to be cleaved by papain,11 no
esterases or lipases have been studied up to now for applications
in deprotection. A few examples of employment of chloroethyl
esters for enzymatic resolution are known.19 The results of the
present work show that esterases and lipases, such as BS2 and
CAL-A, may find applications in deprotection chemistry of
various esters. Up to now, the use of CAL-A for synthetic
purposes, despite its unique properties, has attracted less interest,
in comparison to CAL-B, which is one of the mostly employed
hydrolases in biocatalysis.20 This may be attributed to the fact
that a relatively high CAL-A loading seems to be required, in
particular when compared with CAL-B. However, during the
last years, many remarkable synthetic applications of CAL-A
have been presented.20 BS2 is an enzyme very similar to a
p-nitrobenzyl esterase from Bacillus subtilis (BsubpNBE) dif-
fering only in 11 amino acid residues.21 According to our results
Experimental Section
General Method for Enzymatic Hydrolysis. To a stirred
solution of the substrate (0.15-0.20 mmol) in n-hexane (1 mL)
and CH3OH (100 µL) was added a solution of the enzyme (12-50
mg, as indicated in Tables 1 and 2) in phosphate buffer (7 mL,
100 mM, pH 7.4). The reaction mixture was stirred at 37 °C. After
acidification until pH 6 and extraction with EtOAc (3 × 5 mL),
the organic layers were combined and washed with 5% NaHCO3
(3 × 5 mL). The aqueous layer was acidified until pH 6 and
extracted with EtOAc (3 × 10 mL). The combined organic layers
were dried over Na2SO4, and the organic solvent was removed under
reduced pressure to give the product.
All products of the enzymatic hydrolysis were identified by their
analytical data in comparison with authentic samples.
(18) Yang, H.; Henke, E.; Bornscheuer, U. T. J. Org. Chem. 1999, 64,
1709-1712.
Acknowledgment. This work was supported in part by the
University of Athens (Special Account for Research Grants).
We thank the Deutsche Luft- und Raumfahrt (DLR, Bonn,
Germany) for a grant (GRC 01/011) as well as the Greek
Secretariat for Research and Technology (Athens, Greece).
(19) (a) Botta, M.; Cernia, E.; Corelli, F.; Manetti, F.; Soro, S. Biochim.
Biophys. Acta 1997, 1337, 302-310. (b) Miyazawa, T.; Iwanaga, H.; Ueji,
S.; Yamada, T. Biocatal. Biotransform. 2000, 17, 445-458. (c) Laumen,
K.; Ghisalba, O.; Auer, K. Biosci. Biotechnol. Biochem. 2001, 65, 1977-
1980. (d) Liu, Y.-Y.; Xu, J.-H.; Wu, H.-Y.; Shen, D. J. Biotechnol. 2004,
110, 209-217.
Supporting Information Available: Experimental procedures
for the synthesis of esters and characterization data. This material
(20) For a review, see: Dominguez de Maria, P.; Carboni-Oerlemans,
C.; Tuin, B.; Bargeman, G.; van der Meer, A.; van Gemert, R. J. Mol.
Catal. B 2005, 37, 36-46.
(21) Zock, J.; Cantwell, C.; Swartling, J.; Hodges, R.; Pohl, T.; Sutton,
K.; Rosteck, P.; McGilvray, D.; Queener, S. Gene 1994, 151, 37-43.
JO061871F
786 J. Org. Chem., Vol. 72, No. 3, 2007