A thioesterase for chemoselective hydrolysis of S-acyl sulfanylalkanoates.

Article abstract of DOI:10.1021/ol0069195

[figure: see text] A thioesterase, isolated from a strain of Alcaligenes sp. ISH108, chemoselectively hydrolyzes thiol esters. The application of the enzyme has been demonstrated in the preparation of the antihypertensive agent captopril.

Full text of DOI:10.1021/ol0069195

ORGANIC
LETTERS
2001
Vol. 3, No. 2
283-285
A Thioesterase for Chemoselective
Hydrolysis of S-Acyl Sulfanylalkanoates
Ish Kumar† and Ravinder S. Jolly*
Department of Chemistry, Institute of Microbial Technology,
Sector 39, Chandigarh 160 036, India
jolly@imtech.ernet.in
Received November 23, 2000
ABSTRACT
A thioesterase, isolated from a strain of Alcaligenes sp. ISH108, chemoselectively hydrolyzes thiol esters. The application of the enzyme has
been demonstrated in the preparation of the antihypertensive agent captopril.
Although thiol esters are thermodynamically less stable than
oxo esters, their rates of base-catalyzed hydrolysis in aqueous
solutions are virtually identical.1 The rates of ammonolysis
of thiol esters are, however, several orders of magnitude
higher than those of the corresponding oxo esters. Thus,
neutral hydroxylamine has been used for the chemoselective
hydrolysis of thiol esters, but it is not suitable for racem-
ization-prone compounds in which a mercapto-bearing asym-
metric carbon is adjacent to an electron-withdrawing ester,
amide, or nitrile group.2 Another disadvantage of using
neutral hydroxylamine is that the acid component of thiol
ester gets converted into an amide and cannot be recovered
as such. Enzymatic hydrolysis of thiol esters has attracted
very little attention compared to their oxygen or nitrogen
analogues. Lipases and esterases have been used, but they
are either not chemoselective or the chemoselectivity has
been enforced by the substituents.3,4
The enzyme is stable to a wide range of pH (6.0-10.5) and
temperature (25-65 °C). Thioesterases are ubiquitous hy-
drolytic enzymes with a wide occurrence in plants, animals,
and microbes, but they have not been utilized in organic
synthesis.6 We have evaluated the thioesterase for applica-
tions in organic synthesis. The results are presented in Table
1.
We started our investigations by studying the thioesterase-
catalyzed hydrolysis7,8 of methyl 3-acylsulfanyl-2-methyl-
propanoate (1a-c) in phosphate buffer at pH 6.8 and a
(5) Isolation, purification, and characterization of thioesterase is being
communicated separately.
(6) (a) Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C. Chem.
Biol. 1999, 6, 117-125. (b) Lawson, D. M.; Derewenda, U.; Serre, L.;
Ferri, S.; Szittner, R.; Wei, Y.; Meighen, E. A.; Derewanda, Z. S.
Biochemistry 1994, 33, 9382-9388 and references therein.
(7) In a representative experiment a suspension of 1a (0.440 g, 2.5
mM) in phosphate buffer (pH 7.00, 50 mL) was purged with a stream of
nitrogen for 5 min, thioesterase (aliquot containing 1 mg pure protein) added,
and the contents were stirred vigorously; the pH of the solution was kept
at 7.00 by continuos addition of 0.1 N aqueous NaOH. After 10 min, when
there was no further drop in pH, the reaction mixture was extracted with
ether (3 × 10 mL). The organic extracts were washed with brine, dried
(sodium sulfate), and then evaporated to give thiol 2. The purification of
We have recently purified a thioesterase from Alcaligenes
sp. ISH108, which chemoselectively hydrolyzed thiol esters.5
† Present address: Research Associate, SUNY, UpState Medical Uni-
versity, Syracuse, NY 13210.
1
(1) (a) Bruice, T. C.; Benkovic, S. J. Bioorganic mechanisms; W. A.
Benjamin: New York, 1966; pp 268-294. (b) Jancks, W. P.; Gilchirst, M.
J. Am. Chem. Soc. 1964, 86, 4651-4654. (c) Conners, K. A.; Bender, M.
L. J. Org. Chem. 1961, 26, 2498-2504.
(2) Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664-3671
and references therein.
(3) Kumar, I.; Jolly, R. S. Org. lett. 1999, 1, 596-610.
(4) (a) Sproull, K. C.; Bowman, G. T.; Carta, G.; Gainer, I. L. Biotechnol.
Prog. 1997, 13, 71-76. (b) Bianchi, D.; Cesti, P. J. J. Org. Chem. 1990,
55, 5657-5659.
thiol was effected by flash chromatography. H NMR (300 MHz, CDCl3)
δ: 1.23 (3H, d, J ) 6.85 Hz), 1.48 (1H, t, J ) 8.2 Hz), 2.55-2.83 (3H,
m), 2.87 (m, 1H), 3.69 (3H, s). On storage and partly during workup, thiol
gets converted into disulfide. 1H NMR (300 MHz, CDCl3) δ: 1.28 (6H, d,
J ) 6.8 Hz), 2.70 (1H, dd, J ) 6.6, 12.8 Hz), 2.72 (1H, dd, J ) 6.6, 13.2
Hz), 2.88 (2H, m), 3.03 (1H, dd, J ) 7.4, 12.8 Hz), 3.05 (1H, dd, J ) 6.9,
13.2 Hz), 3.71 (6H, s).
(8) Compounds 1 and 3-12 are known compounds2-4 and have been
characterized on the basis of IR and NMR spectral data. The ee of optically
active compounds was determined by methods described by Kellog.2
10.1021/ol0069195 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/04/2001

once the thiol is formed, overall affinity of the substrate
changes, making it a poor substrate for further reaction.
Therefore, the ester group remains unaffected. However, the
failure of the biocatalyst to hydrolyze the compounds
containing only the oxo ester groups, viz., butyl acetate or
dimethyl succinate, clearly demonstrate that the biocatalyst
has no hydrolytic activity toward oxo esters.
Table 1. Biocatalyzed Chemoselective Hydrolysis of S-Acyl
Sulfanylalkanoates
Although the thioesterase was found to have absolute
chemoselectivity, it failed to discriminate between enanti-
omers of any of the racemates studied above. However, the
enzyme can still be used to advantage for the preparation of
optically pure 2-sulfanylpropanoates such as 8, 9, and 12.
Such compounds are increasingly becoming important in
pharmaceutical and other applications.9 The ready availability
of the corresponding thiol esters in optically pure form from
the chiral pool of lactic acids and amino acids could provide
an attractive route to these thiols in optically pure form. But
the racemization-free deacylation of these thiol esters with
a variety of reagents such as alcoholic HCl, ammonia,
4-chloroaniline, Ti(OR)4, etc. has not been successful and
leads to a varying degree of racemization.2 We attempted
the biocatalyzed hydrolysis of optically pure thiol esters
R-(4), S-(5), and S-(11) under the reaction conditions
described above. As expected, the reaction proceeded
chemoselectively and without any racemization to give the
corresponding thiols R-(8), S-(9), and R-(12) in optically pure
form.
Finally, we report an application of the biocatalyst in the
preparation of captopril (16), an ACE inhibitor having
considerable pharmaceutical and commercial importance as
an antihypertensive agent.10 The steps involved in the
commercial preparation of 16 are depicted in Scheme 1. We
Scheme 1
temperature of 37 °C. A rapid reaction occurred as evidenced
by the drop in pH. The pH of the reaction mixture was
maintained by the addition of 0.1 N NaOH. The reaction
was stopped when there was no further drop in pH. The
workup7 followed by flash chromatography (silica gel; 1:10
ethyl acetate/hexane) gave the hydrolyzed product (2). The
1H NMR spectral data of 2 revealed a peak at δ 3.69,
indicating that the oxo ester group has remained intact,
whereas the resonance peak due to thiol ester group was
absent. In the absence of biocatalyst, 1a was hydrolyzed to
the extent of only 8% after 20 h.
Similarly, the biocatalyzed reaction of methyl 3-acetyl-
sulfanylbutyrate (3), ethyl 2-acetylsulfanylpropanoate (4),
methyl 2-acetylsulfanyl-2-phenylacetate (5), and methyl
3-acetylsulfanylpropanoate (6) under the above reaction
conditions gave the corresponding thiols, methyl 3-sulfa-
nylbutyrate (7), ethyl 2-sulfanylpropanoate (8), methyl
2-phenyl-2-sulfanyl acetate (9), and methyl 3-sulfanylpro-
panoate (10), in near quantitative yield.
The oxo ester group remained intact in all the examples
studied above, which demonstrates that the thioesterase is
chemoselective for thiol esters. It may be argued that there
is a kinetic bias of enzyme in favor of thiol ester and that
and others have already described a biocatalytic method for
the preparation of crucial intermediate 14 in optically pure
form.3,11 Now, we report that intermediate 15 can be
efficiently deacylated to captopril (16) in 97% yield by the
(9) For examples, see the following: (a) Analogue of Ala-82 in backbone
of T4 lysozyme: Ellman, J. A.; Mendel, D.; Schultz, P. G. Science 1992,
255, 197-200. (b) Ultra short acting ACE inhibitor: Baxter, A. J. G.; Carr,
A. D.; Eyley, S. C.; Fraser-Reid, L.; Hallam, C.; Herper, S. T.; Hurved, P.
A.; King S. J.; Megani, P. J. Med. Chem. 1992, 35, 3718-3720. (c) Platelet
activating factor receptor antagonists: Tanabe, Y. Japan Kokai Tokkyo,
Koho, JP 0495 092/1992; Chem. Abstr. 1993, 118, 234049e. Tanabe, Y.;
284
Org. Lett., Vol. 3, No. 2, 2001

biocatalyzed method described above. At present, methanolic
ammonia is used for this deacetylation step. Thus, the present
biocatalytic method can be used as a viable alternative to
the currently employed chemical method.
Acknowledgment. Communication number 4699. One
of us (I.K.) thanks CSIR, New Delhi for the award of Senior
Research Fellowship.
Suzukamo, G.; Komuro, S.; Imaishi, S.; Morooka, S.; Enomoto, M.; Kojima,
A.; Sanemitsu, Y.; Mizutani, M. Tetrahedron Lett. 1991, 32, 379-382. (d)
In anti ulcer agents: Masai, N.; Enomoto, M.; Kojima, A.; Masumori, H.;
Hara, N.; Hara, Y.; Morooka, S. EP Appl. 325496/1989; Chem Abstr. 1990,
113, 11529u.
OL0069195
(10) (10) (a) Ondetti, M. A.; Cushman, D. W. CRC Crit. ReV. Biochem.
1984, 16, 381-411. (b) Shimazaki, M.; Hasegawa, J.; Kan, K.; Nomura,
K.; Nose, Y.; Kondo, H.; Ohashi, T.; Watanabe, K. Chem. Pharm. Bull
1982, 30, 3139-3146. (c) Ondetti, M. A.; Rubin, B.; Cushman, D. W.
Science 1977, 196, 441-444.
(11) (a) Sakimae, A.; Hosoi, A.; Kobayashi, E.; Ohsuga, N.; Numazawa,
R.; Watanabe, I.; Ohnishi,H. Biosci. Biotech. Biochem. 1992, 56, 1252-
1256. (b) Sakimae, A.; Numazawa, R.; Ohnishi, H. Biosci. Biotech. Biochem.
1992, 56, 1341-1341.
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