Lactone synthesis via biotransformations of γ-hydroxyamides

Article abstract of DOI:10.1016/j.tetasy.2004.09.038

An enzyme was expressed in E. coli from a cloned amidase gene. When characterized, it was more enantioselective than commercial amidases. Three pheromones were made. An enzyme was expressed in Escherichia coli from a cloned amidase gene. When characterized, it was more enantioselective than commercial amidases. Three pheromones were made using this biotransformation chemistry.

Full text of DOI:10.1016/j.tetasy.2004.09.038

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 3819–3821
Lactone synthesis via biotransformations of c-hydroxyamides
a,
Stephen K. Taylor, Carrie R. Arnold, Aaron T. Gerds, Nathan D. Ide, Keri M. Law,
a
a
a
a
*
a
a
a
Dale L. Kling, Matthew G. Pridgeon, Lloyd J. Simons, James R. Vyvyan,
a
a
Jennifer S. Yamaoka, Min-Ken Liao and Thomas E. Goyne
b
c
a
Department of Chemistry, Hope College, 35 E. 12th St., Holland, MI 49422, USA
Department of Biology, Furman University, 3300 Pointsett Highway, Greenville, SC 29613, USA
Department of Chemistry, 1610 Campus Drive East, Valparaiso University, Valparaiso, IN 46383, USA
b
c
Received 13 August 2004; accepted 22 September 2004
Abstract—An enzyme was expressed in Escherichia coli from a cloned amidase gene. When characterized, it was more enantioselec-
tive than commercial amidases. Three pheromones were made using this biotransformation chemistry.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
pheromones by this method). We are pursuing both
approaches.
The use of biotransformations offers significant advan-
tages over chemical hydrolyses of compounds that nor-
1
,2
mally require harsh hydrolytic conditions (e.g., nitriles
2
and amides ). For example, the hydrolysis of the nitrile
2. Results and discussion
4
3
-hydroxydodecanenitrile required reflux for 12h in
3
c-Hydroxy amide hydrolysis was attempted with com-
mercially available amidases; five of the amidases from
.5M KOH (3:1 ethanol/H O). The same reaction
2
1
0
was completed via a biotransformation in just 2h at
1
two screening kits did indeed hydrolyze the amide,
but not very enantioselectively. For example, penicillin
acylase, subtilisin Carlsberg, pig kidney acylase, PGA
450, and SP409 all gave a hydrolysis product, but with
only 0–10% ee at 40–50% conversion.
pH6 and 37°C. Another attractive feature of these
reactions is that they are usually enantioselective.
4
,5
5
–7
The biotransformation routes to hydrolyze nitriles
are illustrated below. A direct hydrolysis to an acid is
possible, but the nitrile can also be partially hydrolyzed
to an amide, followed by hydrolysis of the amide to the
Consequently, a model amidase was sought through
cloning and expression experiments. Three microorgan-
isms were grown, and the amidase from one, the Brevi-
5
corresponding carboxylic acid.
6
,7
O
C
bacterium species, was cloned and expressed. This
was enantioselective, and the major products of the
nitrilase
+
NH3
R-C N
R
OH
nitrile
hydratase
O
C
amidase
The amidase gene of Brevibacterium R312 was amplified using
primers TGACCTTTTCTTATGTGGGCTGATCATGTGT-
GAATC and CGATCCGGAAACAGTACTTCGGCAGCTTGC-
CACGAC. The 1.8Kb PCR product was cloned into pGEM-T Easy
(Wisconsin, Promega, Inc.) and the recombinant plasmid electropo-
rated into Escherichia coli XL-1 Blue. The resulting recombinant
strain was used. The recombinant plasmid was subjected to restric-
tion enzyme digestion and sequencing to further confirm that the
amidase was successfully cloned. Subsequent bacterial cultures were
grown in a rich nutrient broth for 24h at 30°C or for 18h at 24°C.
Cells were centrifuged at 10,000rpm, and the bacterial pellet was
resuspended in phosphate buffer for use in subsequent reactions.
R
NH2
The hydrolyses of c-hydroxy amides and c-hydroxy nitr-
iles offer a significant attraction since if enantioselec-
tively hydrolyzed, the resulting c-hydroxy acids could
esterify to form a chiral c-lactone, many of which are in-
8
,9
sect pheromones (see Table 1 for the synthesis of three
*
Corresponding author. Tel.: +1 616 395 7637; fax: +1 616 395 7118;
e-mail: staylor@hope.edu
0
957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.09.038

3820
S. K. Taylor et al. / Tetrahedron: Asymmetry 15 (2004) 3819–3821
Table 1.
O
O
NH2
O
OH
HO
enzyme
H2O
HO
O
R
R
R
1
2
a
R:S ratio 2a–e
Entry
Substrate R=
Conversion (%)
Isolated yield (%), 2a–e
E
Pheromone
b
1a
1b
1c
1c
1d
1e
1e
Ethyl
Propyl
Butyl
Butyl
Pentyl
45
57
90:10
83:17
75:25
91:9
20 (46)
19
14
9
Trogoderma beetle
65
11
45
—
Fopius arisanaus wasp
Fopius arisanaus wasp
Rice weevil
11
—
4
No reaction
None
20
c
Octyl
38
13
75:25
90:10
Rove beetle
Rove beetle
c
Octyl
10
a
b
c
Which were determined by chiral GC on a b-cyclodextrin column. The (R)-enantiomer eluted first.
Only 43% of the product could be extracted from the medium, which indicates a 46% yield. Some product was lost during extraction.
Must be dissolved in MeOH before adding buffer.
8
,9
hydrolyses led to the pheromones (Table 1). For some
reason, hydrolysis of the pentyl-substituted compound
1
This enzyme yielded better results than the comm-
ercial amidases that were evaluated. This high-
lights the occasional necessity of seeking enzymes not
commercially available to effect desired enzymatic
transformations.
d did not occur to yield the fourth pheromone.
à
These reactions are kinetic resolutions. However, the
precursors to these reactions can be made in nearly
1
1
quantitative yields. We are now working on a method
to achieve ꢀ100% conversion and minimize the limita-
tion of this kinetic resolution. The precursor hydroxy-
amide is highly polar, and can easily be separated
from the less polar lactone product. For example, the
product mixture (lactone+hydroxyamide) can be passed
through a plug of silica gel with 1:3 hexane/ethyl acetate.
The lactone [the (R)-enantiomer] will come through
quickly, but the hydroxyamide stays on top of the silica
plug. The hydroxyamide [the (S)-enantiomer] can then
be easily washed off the plug with ethyl acetate contain-
ing 10–20% ethanol. It can be inverted through the Mits-
Acknowledgements
The Howard Hughes Medical Institute, the NSF REU
Program (CHE 8804803), and the Glaxo-Wellcome
Summer Fellowship Program are gratefully acknowl-
edged for their financial support of this work. Professor
Maria Burnatowska-Hledin and students Kristen E.
Stolle, Amy Zwart, and Jennifer C. Lawson (Hope Col-
lege summer intern) are gratefully acknowledged for
developing conditions to show the existence or reactivity
of an amidase. Valparaiso students Kasey Hammond,
Kevin McCusker, Kyle Miner, and Kate Reinicke also
contributed to the work.
1
2
unobu reaction and then transformed into the (R)-
lactone product.
The two reactions shown for 1e (Table 1) have different
E values. Higher E values in the reaction were obtained
when liquid cultures were grown from freshly prepared
bacterial plates. Freshly grown cultures are important
for consistency in reaction results. Specific rotations
References
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8
for all of the lactones have been reported, and our data
is based on chiral GC and spectral comparison to sam-
ples obtained by independent synthesis of the
8
,13
enantiomers.
3
. Pirkle, W. H.; Adams, P. E. J. Org. Chem. 1979, 44, 2169–
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. Conclusion
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. DeSantis, G.; Zhu, Z.; Greenberg, W. A.; Wong, K.;
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5
. Faber, K. Biotransformations in Organic Chemistry;
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à
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rotary shaking for 27h at 30°C, the mixture was adjusted to pH3
with 1M HCl, saturated with NaCl, and extracted with ethyl acetate
three times. The residue left after evaporation was run through a plug
of Florisil with excess ethyl acetate, concentrated by rotary evapo-
ration, and analyzed by NMR. The pure lactone could be obtained
by running the product through a plug of silica gel with 1:1 hexane/
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S. K. Taylor et al. / Tetrahedron: Asymmetry 15 (2004) 3819–3821
3821
3482. Refs. 8 and 9 report all the optical rotations of the
compounds described herein.
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1