Stereoselective chemoenzymatic synthesis of sitophilate: a natural pheromone

Article abstract of DOI:10.1016/j.tetasy.2007.10.008

The aggregation pheromone of the granary weevil Sitophilus granarius, (2S,3R)-1-ethylpropyl 3-hydroxy-2-methylpentanoate, has been synthesized in 63% total isolated yield and high chemical and enantiomeric purity (98% de, >99% ee) from readily available m

Full text of DOI:10.1016/j.tetasy.2007.10.008

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2418–2426
Stereoselective chemoenzymatic synthesis of sitophilate:
a natural pheromone
Dimitris Kalaitzakis,a Spiros Kambourakis,b David J. Rozzellb and Ioulia Smonoua,*
aDepartment of Chemistry, University of Crete, Iraklion-Voutes, 71003 Crete, Greece
bCodexis Inc., 129 North Hill Ave, Suite 103, Pasadena, CA 91106, USA
Received 24 July 2007; accepted 4 October 2007
Abstract—The aggregation pheromone of the granary weevil Sitophilus granarius, (2S,3R)-1-ethylpropyl 3-hydroxy-2-methylpentanoate,
has been synthesized in 63% total isolated yield and high chemical and enantiomeric purity (98% de, >99% ee) from readily available
methyl 3-oxopentanoate. A stereoselective ketone reduction followed by an ester hydrolysis, were the two key steps of the synthesis
and were both performed by commercially available enzymes.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
In an effort to identify the absolute configuration of the
active form of the pheromone, both syn-enantiomers
(Fig. 1) were synthesized.4 A comparison of spectral, chro-
matographic, and biological properties between the natural
product and both synthesized enantiomers, showed that
the active form of this compound is the (2S,3R)-configura-
tion (Fig. 1), which was named sitophilate. Numerous ap-
proaches have been published for the synthesis of racemic
or enantiomerically pure sitophilate, however, either low
enantiomeric purities were reported or multi step reaction
schemes were required to obtain enantiomerically pure
sitophilate.3,5–8 Clearly, a simple, scalable, and economic
method for the synthesis of this weevil attractant phero-
mone is still lacking.
Sitophilus granarius (L.), the granary weevil,1 is a major
cosmopolitan pest of stored cereal grains responsible for
serious economic losses annually. It is, therefore, critical
to monitor and control the pest’s presence in stored grain
to avoid further damages. Due to the dry conditions and
cool temperatures of stored grain, these insects have
evolved to alter these conditions by forming aggregation
spots and by producing their own suitable moisture for
warmth, through their metabolic activities.2 The aggrega-
tion spots of male and female adults are partly due to the
pheromone of S. granarius, which is male-produced and
attracts both the sexes.1 This pheromone was isolated
by Burkholder et al.3 and was identified as 1-ethylpropyl
3-hydroxy-2-methylpentanoate, with syn (erythro) relative
configuration between the hydroxy and the methyl group
(Fig. 1).
Since sitophilate is an optically active a-methyl-b-hydroxy
ester, it could be easily produced by the stereoselective
reduction of an a-methyl-b-keto ester by asymmetric
reduction via dynamic kinetic resolution (DKR)9 (Fig. 2).
Ketoreductases are very powerful tools for stereoselective
reduction via DKR. We have recently shown that a num-
ber of a-alkyl-1,3-diketones10 and a-alkyl-b-keto esters11
can be reduced to enantiomerically pure keto alcohols
OH
O
OH
O
O
O
(2S,3R)
(2R,3S)
O
O
OH
O
OH
O
Figure 1. syn-1-Ethylpropyl 3-hydroxy-2-methylpentanoate.
R
R
O
O
O
*
Corresponding author. Tel.: +30 2810 545010; fax: +30 2810 545001;
e-mail: smonou@chemistry.uoc.gr
Figure 2. Retrosynthesis of sitophilate.
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.10.008

D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
2419
and keto esters, respectively, using commercially available
ketoreductases. Enzymatic reductions provide a simple,
highly stereoselective, and quantitative method for the syn-
thesis of different diastereomers of these valuable chiral
synthons.
of a glucose/glucose dehydrogenase system13 for in situ
recycling of the nicotinamide cofactors (Table 1).
Since the reduction of 1-ethylpropyl 2-methyl-3-oxopent-
anoate 5 will give sitophilate in a single step, this substrate
was first screened for reduction using commercially avail-
able ketoreductases. Methyl 2-methyl-3-oxopentanoate 1
and ethyl 2-methyl-3-oxopentanoate 3 were also screened
because of their structural similarity to 4-methyl-3,5-hep-
tanedione, the substrate that was successfully reduced to
(4S,5R)-5-hydroxy-4-methyl-3-heptanone by KRED-A1B,
in our previous work.12 Both keto esters 3 and 5 have been
previously used in the synthesis of sitophilate by the micro-
bial reduction with whole cells of Geotrichum candidum6
and Pichia farinosa IAM 4682,7 respectively; however, the
wrong (2S,3S)-stereoisomer was the major product in both
cases.
Using a similar enzymatic reduction, we recently reported
the synthesis of another pheromone, (4S,5R)-5-hydroxy-
4-methyl-3-heptanone (sitophilure), which is produced
from the rice weevil (Sitophilus oryzae L.) and maize weevil
(Sitophilus zeamais M.).12
Herein, we report a facile chemoenzymatic synthesis of
sitophilate using two sequential enzymatic steps, a reduc-
tion followed by hydrolysis, as the key reactions.
Many enzymes showed activity toward the reduction of
keto esters 1, 3, and 5, and the results in Table 1 only show
the best and most representative of these reactions. Irre-
spective of the ester group, two out of the four stereoiso-
mers of each hydroxy ester were produced in high yield
by a number of enzymes, with one of them having the same
2. Results and discussion
Three different a-methyl-b-keto esters, 1, 3, and 5, were
screened for reduction using over 100 NADPH and
NADH-dependent commercially available ketoreductases
(KREDs). All reactions were performed in the presence
Table 1. Enzymatic reduction of a-methyl-b-keto esters 1, 3, and 5 by isolated ketoreductases
O
O
OH
O
OH
O
OH O
OH
O
KRED
R
R
R
R
R
O
O
O
O
O
NAD(P)H
NAD(P)+
A
D
B
C
R
R
1
3
5
Me
Et
2
4
6
Me
Et
Glucono-
lactone
Glucose
1-ethylpropyl
1-ethylpropyl
KRED
Substrate
Diastereomeric ratioa (%)
Conversion (%)
Time (h)
A (2S,3R)
B (2R,3S)
C (2S,3S)
D (2R,3R)
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
1
3
5
4
—
—
—
3
—
29
—
—
1
—
—
1
—
—
—
—
1
93
93
60
94
92
71
—
9
22
98
98
65
>98
>99
>99
>98
99
3
7
>99
>99
32
2
107
—
—
3
12
24
24
12
24
2
40
—
—
—
9
13
61
2
>99
>99
>99
>99
>99
>99
>99
>99
82
>99
>99
>99
90
118
8
—
91
78
16
—
—
—
—
—
—
—
—
—
—
—
—
A1Bb
A1F
B1B
B1C
B1E
2
24
24
24
24
24
24
24
24
24
24
24
24
2
2
34
—
—
—
—
—
—
2
90
90
>99
>99
22
1
99
98
>99
>99
—
—
—
—
—
a Diastereomeric ratio A/B/C/D is categorized in every reaction according to their increasing retention time on chiral GC, with isomer A eluting first. All
the enzymatic reactions were performed at pH 6.9, 37 ꢁC.
b KRED-A1C and KRED-A1D gave very similar yields and product ratios as KRED-A1B.

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D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
absolute configuration as sitophilate [2S,3R, A, Table 1].
Interestingly, this stereoisomer is the result of an enzymatic
reduction with anti-Prelog selectivity. These results suggest
that an enzymatic reduction might be a viable method for
producing sitophilate, however, medium to low enantio-
meric excesses of the desired (2S,3R)-hydroxy ester were
obtained in all enzymatic reductions that gave this isomer
as the major product (KRED-A1B, A1C, and A1D, Table
1). An inverse correlation of the stereoselectivity of the
enzymatic reduction with KRED-A1B (as well as KRED-
A1C and A1D) and the size of the ester group was
observed. Decreasing the size of the ester group from
1-ethylpropyl to ethyl to methyl gave yields of 16%, 78%,
and 91%, respectively, for the desired (2S,3R)-stereoisomer
(KRED-A1B, Table 1). In contrast, the yield and enantio-
meric purity of stereoisomer C (2S,3S) was not affected by
the size of the ester group in all enzymes that were selective
in its synthesis. It should be noted that the absolute config-
uration of every diastereomer was assigned by comparing
as an increase in the diastereomeric purity from
the 82% de that is currently obtained (Table 1, KRED-
A1B, substrate 1). For this reason, two different ap-
proaches were investigated to complete the synthesis. In
the first approach, we examined the effect of temperature
and pH in the reduction of 1 with KRED-A1B, A1C,
and A1D, hoping that a change in the conditions will
increase the diastereoselectivity of these transformations
(Table 2, Fig. 3, pathway 1). In the second approach, an
enzymatic hydrolysis of the hydroxy ester 2 was investi-
gated (Fig. 3, pathway 2).
Reaction temperature and pH changes affected both the
selectivity and the activity of KRED-A1B, A1C, and
A1D for the reduction of methyl 2-methyl-3-oxopentano-
ate 1. Representative results for the reductions with
KRED-A1B are shown in Table 2. The most dramatic
effect was observed when the pH was increased from 6.0
to 6.9 resulting in an increase of the diastereoselectivity
from 48% de to 82% de, respectively. A further increase
of the pH to 8.0 considerably reduced the activity of the
enzyme without a significant improvement in its stereo-
selectivity (Table 2). The enantiomeric purity was increased
even more to 92% de when the reaction temperature
dropped to 0 ꢁC (Table 2). At this temperature and pH,
however, the reaction was not completed even after 24 h
of incubation.
1
their H NMR spectrum with the literature data4,6,8,14
and by transforming it into its corresponding MPA [(R)-
or (S)-methoxy-phenylacetic acid] esters.15
The results so far indicate that the synthesis of sitophilate
using an enzymatic reduction is possible. Enzymes that give
the desired (2S,3R)-stereoisomer were identified, and the
methyl ester derivative gave high, though not excellent, dia-
stereomeric purity of the product. To complete the synthe-
sis of sitophilate using methyl 2-methyl-3-oxopentanoate 1
(Table 1), a transesterification step will be required, as well
To this end, we decided that 92% de was the best enantio-
meric purity we can achieve with the existing enzymes, so
Table 2. Reaction optimization of KRED-A1B, A1C, and A1D with methyl 2-methyl-3-oxopentanoate 1
KRED
Reaction conditions
Diastereomeric ratio (%)
Yield (%)
Time (h)
A (2S,3R)
B (2R,3S)
C (2S,3S)
D (2R,3R)
pH 6.0, 37 ꢁC
pH 6.9, 25 or 37 ꢁC
pH 8.0, 37 ꢁC
pH 8.0, 25 ꢁC
pH 8.0, 0 ꢁC
74
91
92
93
96
—
—
—
—
—
1
—
1
1
—
25
9
7
6
4
>99
>99
>99
>99
95
2
2
20
24
24
A1Ba
a Enzymes KRED-A1C and A1D showed very similar behavior.
OH
O
OH
O
ii
iii
O
O
pathway 1
sitophilate 6A
2A, 90%de
O
O
O
O
98%de, >99%ee
i
O
O
vi
1
OH
O
OH O
v
pathway 2
iv
O
OH
7A, 98%de
2A, 82%de
Figure 3. Chemoenzymatic total synthesis of sitophilate. Reagents and conditions: (i) K2CO3, MeI, dry acetone, reflux, >99% yield; (ii) KRED-EXP-A1B,
NADPH, pH 8.0, 0 ꢁC (12 h), 25 ꢁC (12 h), 90% yield, 90% de, >99% ee; (iii) (a) NaOH, MeOH/H2O, 1:1 v/v; (b) 3-bromopentane, dry DMF, 50 ꢁC, 65%
yield, 98% de, >99% ee; (iv) KRED-EXP-A1B, NADPH, pH 6.9, 25 ꢁC, 95% yield, 82% de, >99% ee; (v) ICR-112, 25 ꢁC, 83% yield, 98% de, >99% ee; (vi)
KOH, 3-bromopentane, dry DMF, 50 ꢁC, 80% yield, 99% de, >99% ee.

D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
2421
an overall synthesis based on this enzymatic step was
worked out (Fig. 3, pathway 1). Commercially available
methyl 3-oxopentanoate was transformed quantitatively
to methyl 2-methyl-3-oxopentanoate, 1, by treatment with
methyl iodide in the presence of potassium carbonate
(Fig. 3, step i). An enzymatic reduction using the condi-
tions that give the highest enantiomeric excess of the prod-
uct was then performed in larger scale to produce hydroxy
ester 2A. Under these conditions, the enzymatic reaction
was kept at pH 8.0, and 0 ꢁC for the first 12 h, before addi-
tional quantities of enzyme and coenzyme were added and
the temperature was raised to 25 ꢁC. After another 12 h of
incubation, the product was isolated in 90% yield, 90% de,
and >99% ee (Fig. 3, step ii). Transesterification of hydroxy
ester 2A by nucleophilic substitution of its sodium salt with
3-bromopentane followed by silica gel chromatographic
separation of the diastereomeric mixture gave sitophilate
6A in 65% isolated yield, 98% de, >99% ee (Fig. 3, step
iii). The overall chemical yield calculated from methyl 3-
oxopentanoate was 58.0% (Fig. 3, pathway 1).
egy in improving the enantiomeric and diastereomeric
purity of the product and eliminating the chromatographic
separations of diastereomers.
3. Conclusion
An isolated NADPH-dependent ketoreductase (KRED-
EXP-A1B) and an isolated hydrolase (ICR-112) were used
for the synthesis of sitophilate, the aggregation pheromone
of the granary weevil S. granarius, in high enantiomeric
(98% de, >99% ee), and chemical purity (>99%), with an
overall yield of 63%. The starting materials for this synthe-
sis involved achiral and readily commercially available
chemicals, such as methyl 3-oxopentanoate. Every reaction
in this synthesis can be easily scaled to larger amounts, thus
an efficient method for the synthesis of this natural phero-
mone is provided.
4. Experimental
Even though the previous synthesis (Fig. 3, pathway 1)
gave the target molecule in high purity, a silica gel chro-
matographic separation was required in addition to per-
forming the enzymatic reduction in sub-optimal (in terms
of enzyme activity) conditions. Such a process requires
longer reduction times and higher enzyme loadings to max-
imize the diastereomeric purity and make the final chro-
matographic separation possible. In a second approach,
we investigated the possibility of increasing the enantio-
meric purity by hydrolyzing hydroxy ester 2, with an
enzyme. For this purpose, 15 commercially available
hydrolases (ICR)13 were screened. Hydroxy ester 2 at
82% de was synthesized on a larger scale by KRED-
EXP-A1B under its optimal conditions for activity (pH
6.9, 25 ꢁC, Table 2).
4.1. Materials and general methods
Methyl 2-methyl-3-oxopentanoate, 1, and ethyl 2-methyl-
3-oxopentanoate, 3, were prepared from commercially
available methyl 3-oxopentanoate and ethyl 3-oxopentano-
ate, respectively, by alkylation with methyl iodide. 1-Ethyl-
propyl 2-methyl-3-oxopentanoate, 5, was prepared from
methyl 2-methyl-3-oxopentanoate with transesterification
reaction with 3-pentanol.
All enzymes utilized in this report are commercially avail-
able from Codexis Inc.
Racemic a-methyl-b-hydroxy esters were prepared as stan-
dards by the reduction of their corresponding a-methyl-b-
keto esters with sodium borohydride.
Three enzymes (ICR-110: Lipase B from Candida antarc-
tica: CALB, ICR-112: Lipase A from C. antarctica: CALA
and ICR-123: pig liver esterase PLE) that were able to
selectively hydrolyze (2S,3R)-methyl 3-hydroxy-2-methyl
pentanoate 2 were identified (Table 3). In particular,
ICR-112 gave a high yield (83% of the maximum 91%)
and enantiomeric purity for the hydrolysis of the sitophilat-
e precursor 7A (Table 3). With this enzyme in hand, an im-
proved synthesis of the natural product was worked out
(Fig. 3, pathway 2). The overall yield calculated from
methyl 3-oxopentanoate was 63%. Introducing two consec-
utive enzymatic steps proved to be a very successful strat-
The absolute configuration of the hydroxy esters was as-
1
signed by comparing the H NMR spectra with the litera-
ture data and by transforming them into their
corresponding MPA [(R) or (S)-methoxy-phenylacetic
acid] esters.15
The progress of the enzymatic reactions and the selectivi-
ties were determined by gas chromatography (HP5890II
gas chromatograph equipped with an FID detector; col-
umn: 30 m · 0.25 mm · 0.25lm chiral capillary column,
Table 3. Enzyme-catalyzed stereoselective hydrolysis of methyl 3-hydroxy-2-methyl entanoate
Diastereomeric ratio % of hydroxy ester 2
ICR
Reaction time (yield)
Product 7
OH OH
O
O
OH
O
OH
O
O
O
OH
OH
2A
2C
7A
7B
110
112
123
24 h (nd)
24 h (83%)
1 h (nd)
92
99
95
8
1
5
91
9

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D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
20% permethylated b-cyclodextrin, HP Part No 19091G-
8233). 1H NMR spectra were recorded on 500 MHz Bruker
spectrometer in CDCl3 solutions, by using Me4Si as the
internal standard. Chemical shifts are reported in ppm
downfield from Me4Si.
4.5. Racemic methyl 3-hydroxy-2-methylpentanoate 2
Under nitrogen atmosphere, sodium borohydride
a
(24.4 mg, 0.646 mmol) was added in dry ethanol (10 cm3)
and the mixture was cooled to 0 ꢁC. After stirring for
5 min, a solution of dry ethanol (5 cm3) containing methyl
2-methyl-3-oxopentanoate 1 (303 mg, 2.1 mmol) was added
dropwise. After stirring for 3 h at 0 ꢁC, the reaction was
quenched with saturated ammonium chloride and the eth-
anol was evaporated under reduced pressure. Distilled
water (15 cm3) was then added and extracted twice with
ethyl acetate (2 · 15 cm3). The organic layer was dried over
MgSO4, and concentrated in vacuo. The residue was
purified by flash column chromatography (silica gel,
hexane/EtOAc, v/v, 6:1), to afford 2 (246 mg, 80%). dH
(500 MHz; CDCl3; Me4Si) 3.79–3.83 (1H, m, CHOH),
3.71 (6H, s, 2 · CO2CH3), 3.56–3.61 (1H, m, CHOH),
2.51–2.58 (2H, m, 2 · CHCO2), 1.39–1.61 (4H, m,
2 · CH2CH3), 1.20 (3H, d, J = 7.5, CHCH3), 1.17 (3H, d,
J = 7.5, CHCH3), 0.98 (3H, t, J 7.0, CH2CH3), 0.96 (3H,
t, J = 7.5, CH2CH3).
Optical rotation values were measured on a DIP 360 Jasco
polarimeter at 360 mmHg.
MS were taken on a GC–MS (Shimadzu GCMS-QP5050
equipped with a SPB-5 column and CI mass detector).
4.2. Methyl 2-methyl-3-oxopentanoate 1
Keto ester 1 was prepared from commercially available
methyl 3-oxopentanoate according to the following proce-
dure: under a nitrogen atmosphere, methyl 3-oxopentano-
ate (1.88 cm3, 15 mmol) was dissolved in anhydrous
acetone (20 cm3) and pre-dried potassium carbonate
(1.932 g, 14 mmol) was added. After stirring the solution
at room temperature for 5 min, methyl iodide (1.146 cm3,
18.4 mmol) was added with a syringe and the reaction mix-
ture was refluxed for 6 h. After completion of the reaction,
diethyl ether was added (30 cm3), the mixture was filtered
and the solvent was evaporated under a reduced pressure
to afford 1 (2.16 g, >99%). Without any further purifica-
tion, keto ester 1 was subjected to enzymatic reduction.
dH (500 MHz; CDCl3; Me4Si) 3.72 (3H, s, CO2CH3), 3.53
(1H, q, J = 7.5, CHCH3), 2.46–2.64 (2H, m, COCH2CH3),
1.34 (3H, d, J = 7.0, CHCH3), 1.07 (3H, t, J = 7.5,
CH2CH3).
4.6. Racemic ethyl 3-hydroxy-2-methylpentanoate 4
The preparation of racemic ethyl 3-hydroxy-2-methylpent-
anoate 4 was accomplished by following the same proce-
dure as in the preparation of hydroxy ester 2, using ethyl
2-methyl-3-oxopentanoate
3 (316 mg, 2 mmol). Pure
product 4 was obtained after flash column chromatography
(silica gel, hexane/EtOAc, v/v, 6:1) (262 mg, 82%). dH
(500 MHz; CDCl3; Me4Si) 4.16 (4H, q, J = 7.0,
2 · CO2CH2CH3), 3.78–3.83 (1H, m, CHOH), 3.56–3.60
(1H, m, CHOH), 2.49–2.56 (2H, m, 2 · CHCO2), 1.40–
1.61 (4H, m, 2 · CH2CH3), 1.27 (6H, t, J = 7.0,
2 · CO2CH2CH3), 1.20 (3H, d, J = 7.5, CHCH3), 1.17
(3H, d, J = 7.0, CHCH3), 0.98 (3H, t, J = 7.5, CH2CH3),
0.96 (3H, t, J = 7.0, CH2CH3).
4.3. Ethyl 2-methyl-3-oxopentanoate 3
Keto ester 3 was prepared by following the same procedure
as in the preparation of keto ester 1, by alkylation of ethyl
3-oxopentanoate (2.16 g, 15 mmol) with methyl iodide. Iso-
lated yield 2.37 g, >99%. dH (500 MHz; CDCl3; Me4Si)
4.18 (2H, q, J = 7.0, CO2CH2), 3.51 (1H, q, J = 7.0,
CHCH3), 2.46–2.65 (2H, m, COCH2CH3), 1.34 (3H, d,
J = 7.0, CHCH3), 1.27 (3H, t, J = 7.0, CO2CH2CH3),
1.08 (3H, t, J = 7.5, CH2CH3).
4.7. Racemic 1-ethylpropyl 3-hydroxy-2-methylpentanoate 6
The preparation of racemic 1-ethylpropyl 3-hydroxy-2-
methylpentanoate 6 was accomplished by following the
same procedure as in the preparation of hydroxy ester 2,
using 1-ethylpropyl 2-methyl-3-oxopentanoate 5 (400 mg,
2 mmol). Pure product was obtained after flash column
chromatography (silica gel, hexane/EtOAc, v/v, 6:1)
(343 mg, 85%). dH (500 MHz; CDCl3; Me4Si) 4.76–4.83
(2H, m, 2 · CO2CH), 3.78–3.83 (1H, m, CHOH), 3.54–
3.60 (1H, m, CHOH), 2.71 (1H, d, J = 7.0, OH), 2.61
(1H, d, J = 4.0, OH), 2.49–2.56 (2H, m, 2 · CHCO2),
1.41–1.63 (12H, m, 6 · CH2CH3), 1.22 (3H, d, J = 7.5,
CHCH3), 1.18 (3H, d, J = 7.0, CHCH3), 0.98 (3H, t,
J = 7.5, CH2CH3), 0.97 (3H, t, J = 7.5, CH2CH3), 0.89
(12H, t, J = 7.5, 4 · CO2CHCH2CH3).
4.4. 1-Ethylpropyl 2-methyl-3-oxopentanoate 5
In a solution of 3-pentanol (184 mg, 225 lL, 2.084 mmol)
and DMAP (13 mg, 0.1 mmol) in dry toluene (8 cm3),
methyl 2-methyl-3-oxopentanoate, 1, was added (150 mg,
1.042 mmol) and the mixture was refluxed for four days.
Saturated NH4Cl (15 cm3) was then added and the solution
was extracted with EtOAc. The organic layer was washed
with 1 M HCl, dried over MgSO4, and concentrated in va-
cuo to give substrate 5 (176 mg, 85%). Without any further
purification the product was subjected to enzymatic reduc-
tion. dH (500 MHz; CDCl3; Me4Si) 4.76–4.82 (1H, m,
CO2CH), 3.53 (1H, q, J = 7.5, CHCH3), 2.47–2.67 (2H,
m, COCH2CH3), 1.53–1.62 (2H, m, CHCH2CH3), 1.34
(3H, d, J = 7.0, CHCH3), 1.08 (3H, t, J = 7.5, CH2CH3),
0.88 (3H, t, J = 7.5, CO2CHCH2CH3), 0.87 (3H, t, J =
7.5, CO2CHCH2CH3).
4.8. Enzymatic reductions
76 NADPH-dependent ketoreductases from KRED-7600
kit and 29 NADH-dependent ketoreductases from
KRED-NADH-2900 kit were screened for the selective
reduction of keto esters 1, 3, and 5. In addition to the keto-
reductases, both NAD(P)H and glucose dehydrogenase

D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
2423
(GDH) that were used for cofactor recycling are products
available from Codexis.
KRED-EXP-A1B (50 mg) was stirred at 0 ꢁC. After 12 h
were added KRED-EXP-A1B (20 mg), glucose dehydroge-
nase (5 mg), and NADPH (8 mg) and the mixture was stir-
red at 25 ꢁC for 12 h until GC analysis of the extracts
showed complete reaction. Periodically the pH was read-
justed to 7.7–8.0 with NaOH (2 M). The product was iso-
lated by extracting the crude reaction mixture with
EtOAc (2 · 20 cm3). The combined organic layers were
washed with saturated NaCl solution (15 cm3), dried over
MgSO4, and evaporated to dryness to provide pure
(2S,3R) methyl 3-hydroxy-2-methylpentanoate 2A
(183 mg, 90% yield, 90% de, >99% ee). dH (500 MHz;
CDCl3; Me4Si) 3.79–3.84 (1H, m, CHOH), 3.71 (3H, s,
CO2CH3), 2.54–2.59 (1H, m, CHCO2), 1.40–1.53 (2H, m,
CH2CH3), 1.18 (3H, d, J = 7.0, CHCH3), 0.97 (3H, t,
J = 7.5, CH2CH3) [lit. data in Ref. 5g].
4.9. Small-scale screening of enzymatic reductions
The screening was performed on small-scale reactions
where each substrate (keto esters 1, 3, 5) (25 mM) was
mixed with NAD(P)H (2 mg, 2.5 mM), each ketoreductase
(2–3 mg/cm3), glucose (18 mg, 100 mM), glucose dehydro-
genase (GDH, 2 mg/cm3) for cofactor recycling and
sodium phosphate buffer (1 cm3, 200 mM, pH 6.9). The
reactions were incubated at 37 ꢁC and reaction aliquots
taken every hour. After extraction with ethyl acetate the
crude extracts were analyzed by chiral GC chromatogra-
phy. In the case of anti-Prelog enzymes (KRED-EXP-
A1B, A1C, A1D) the small-scale enzymatic reductions of
keto ester 1 were repeated in two different pH values (pH
6.0 or pH 8.0) and at two different temperatures (0 ꢁC
and 25 ꢁC) with the same quantities.
4.14.2. (2S,3R)-Methyl 3-hydroxy-2-methylpentanoate 2A
(pH 6.9, 25 ꢁC). A phosphate-buffered solution (100 cm3,
pH 6.9, 200 mM) containing methyl 2-methyl-3-oxopent-
anoate 1 (62.5 mM, 900 mg, 6.25 mmol), glucose (2.16 g,
120 mM), NADPH (0.5 mM, 44 mg, 0.05 mmol), glucose
dehydrogenase (15 mg), and KRED-EXP-A1B (50 mg)
was stirred at 25 ꢁC until GC analysis of the extracts
showed complete reaction. Periodically the pH was read-
justed to 6.9 with NaOH (2 M). The product was isolated
by extracting the crude reaction mixture with EtOAc
(2 · 100 cm3). The combined organic layers were washed
with saturated NaCl solution (50 cm3), dried over MgSO4,
and evaporated to dryness to provide pure (2S,3R)-methyl
3-hydroxy-2-methylpentanoate (866 mg, 95% yield, 82%
de, >99% ee). dH (500 MHz; CDCl3; Me4Si) 3.79–3.84
(1H, m, CHOH), 3.71 (3H, s, CO2CH3), 2.54–2.59 (1H,
m, CHCO2), 1.40–1.53 (2H, m, CH2CH3), 1.18 (3H, d,
J = 7.0, CHCH3), 0.97 (3H, t, J = 7.5, CH2CH3) [lit. data
in Ref. 5g].
4.10. Hydroxy ester 2C
d
H (500 MHz; CDCl3; Me4Si) 3.71 (3H, s, CO2CH3), 3.57–
3.61 (1H, m, CHOH), 2.51–2.58 (1H, m, CHCO2), 1.53–
1.62 (1H, m, CH2CH3), 1.39–1.48 (1H, m, CH2CH3),
1.20 (3H, d, J = 7.0, CHCH3), 0.98 (3H, t, J = 7.5,
CH2CH3) [lit. data in Ref. 5g].
4.11. Hydroxy ester 4A
dH (500 MHz; CDCl3; Me4Si) 4.16 (2H, q, J = 7.0,
CO2CH2CH3), 3.78–3.83 (1H, m, CHOH), 2.50–2.56 (1H,
m, CHCO2), 1.40–1.53 (2H, m, CH2CH3), 1.27 (3H, t,
J = 7.0, CO2CH2CH3), 1.17 (3H, d, J = 7.0, CHCH3),
0.96 (3H, t, J = 7.5, CH2CH3) [lit. data in Ref. 6].
4.12. Hydroxy ester 4C
4.14.3. (2S,3R)-1-Ethylpropyl 3-hydroxy-2-methylpentano-
ate 6A, sitophilate. To a solution of (2S,3R)-methyl 3-hy-
droxy-2-methylpentanoate 2A (90% de, >99% ee), derived
from enzymatic reduction with KRED-EXP-A1B at pH
8.0 and 0 ꢁC–rt, (183 mg, 1.26 mmol,) in MeOH–H2O
(1:1 v/v, 15 cm3) was added sodium hydroxide (50 mg,
1.25 mmol) and the mixture was stirred overnight at room
temperature. After completion of the reaction, the solution
was concentrated under vacuum to give a white solid. After
desiccation to dryness, in the same flask were added 10 cm3
of dry DMF. At room temperature, the addition of 3-
bromopentane (172 lL, 1.39 mmol, in 5 cm3 dry DMF)
was followed and the solution was stirred at 50 ꢁC for
24 h. Next 20 cm3 of water were added to the reaction mix-
ture and the mixture was extracted three times with CHCl3
(3 · 20 cm3). The combined extracts were washed with
water twice (2 · 20 cm3) and dried over MgSO4. The
solvent was evaporated under reduced pressure and the
residue was purified by flash column chromatography
dH (500 MHz; CDCl3; Me4Si) 4.17 (2H, q, J = 7.5,
CO2CH2CH3), 3.55–3.61 (1H, m, CHOH), 2.59 (1H, d,
J = 7.0, OH), 2.49–2.55 (1H, m, CHCO2), 1.53–1.61 (1H,
m, CH2CH3), 1.40–1.49 (1H, m, CH2CH3), 1.27 (3H, t,
J = 7.0, CO2CH2CH3), 1.20 (3H, d, J = 7.0, CHCH3),
0.98 (3H, t, J = 7.5, CH2CH3) [lit. data in Ref. 6].
4.13. Hydroxy ester 6C
dH (500 MHz; CDCl3; Me4Si) 4.78–4.83 (1H, m, CO2CH),
3.54–3.60 (1H, m, CHOH), 2.69 (1H, d, J = 7.0, OH),
2.50–2.56 (1H, m, CHCO2), 1.42–1.64 (6H, m,
3 · CH2CH3), 1.23 (3H, d, J = 7.0, CHCH3), 0.99 (3H, t,
J = 7.5, CH2CH3), 0.89 (6H, t, J = 7.5, 2 · CO2CHCH2-
CH3) [lit. data in Ref. 5g].
4.14. Larger-scale enzymatic reductions
4.14.1. (2S,3R)-Methyl 3-hydroxy-2-methylpentanoate 2A
(silica gel, hexane/EtOAc, v/v, 10:1) to give pure sitophilate
25
(pH 8.0, 0–25 ꢁC).
A
phosphate-buffered solution
(156 mg, 65%, 98% de, >99% ee). ½aꢀD ¼ ꢁ3:1 (c 1.7
(20 cm3, pH 8.0, 200 mM) containing 69.5 mM (200 mg,
1.39 mmol) of methyl 2-methyl-3-oxopentanoate, 1, glu-
cose (432 mg, 120 mM), NADPH (12.3 mg, 0.7 mM,
0.014 mmol,), glucose dehydrogenase (10 mg) and
CHCl3) (it compares to the literature data).4 dH
(500 MHz; CDCl3; Me4Si) 4.76–4.82 (1H, m, CO2CH),
3.78–3.84 (1H, m, CHOH), 2.60 (1H, d, J = 4.0, OH),
2.51–2.57 (1H, m, CHCO2), 1.41–1.64 (6H, m,

2424
D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
3 · CH2CH3), 1.19 (3H, d, J = 7.0, CHCH3), 0.98 (3H, t,
J = 7.5, CH2CH3), 0.89 (3H, t, J = 7.5, CHCH2CH3),
0.88 (3H, t, J = 7.5, CHCH2CH3). 13C NMR (CDCl3
75 MHz, d ppm): 176.20 (CO2CH), 76.94 (CO2CH), 73.20
(CHOH), 44.08 (CHCO2), 26.69 (CH2CH3), 26.42
(CH2CH3), 26.37 (CH2CH3), 10.75 (CH3), 10.36 (CH3),
9.58 (CH3), 9.52 (CH3). MS (m/z): 173 (2), 144 (6), 115
(40), 103 (20), 97 (10), 85 (12), 74 (100).
was stirred at 80 ꢁC for 3 h. Then 3-bromo pentane
(78 mm3, 0.625 mmol) was added and the mixture stirred
overnight at 50 ꢁC. After the completion of the reaction,
water (20 cm3) was added to the reaction mixture and the
mixture was extracted three times with CHCl3
(3 · 20 cm3). The combined organic extracts were washed
with distilled water twice (2 · 20 cm3) and dried over
MgSO4. The solvent was evaporated under reduced pres-
sure and the residue was purified by flash column chroma-
tography (silica gel, hexane/EtOAc, v/v, 9:1) to afford pure
sitophilate 6A (92 mg, 80%, 98% de, >99% ee).
4.15. Enzymatic hydrolysis
Fifteen lipases and esterases from NZL and NZP kits were
screened for the selective hydrolysis of hydroxy ester 2.
4.19. Preparation of MPA esters
4.16. Small-scale enzymatic hydrolysis
4.19.1. General method for the synthesis of MPA esters of 3-
hydroxy-2-methyl pentanoate esters. To a solution of the
corresponding hydroxy ester (0.1 mmol) in dry CH2Cl2
were added 1.1 equiv of DCC (0.11 mmol) and 1.1 equiv
of the corresponding (R)- or (S)-MPA (0.11 mmol) and
the reaction mixture was stirred at 0 ꢁC for 4–6 h. After
completion of the reaction, the urea produced was filtered,
and the filtrate was evaporated and then chromatographed
with 5:1 Hex/EtOAc. The produced corresponding MPA
ester was isolated in 90% isolated yield.
In
a
phosphate-buffered solution (4,5 cm3, pH 7.0,
200 mM) were dissolved enzyme (ICR) (5 mg) and
(2S,3R)-methyl 3-hydroxy-2-methylpentanoate (25 mg,
82% de, >99% ee), derived from enzymatic reduction with
KRED-EXP-A1B at pH 6.9 and 25 ꢁC. The mixture was
stirred at 37 ꢁC. At certain time points (1 h, 2 h, 4 h, and
1 day), 1 cm3 of the reaction aliquots were treated with
NaOH 2 M and after extraction with EtOAc were analyzed
by GC, to determine the enantiomeric purity of the remain-
ing ester. If the enantiomeric purity of the ester was chan-
ged, the mixture was treated with HCl 1 M, and the
produced acid was isolated by the extraction of the aliquots
with EtOAc (2 · 5 cm3). The pure acid was esterified with
diazomethane to the corresponding methyl ester, which
was analyzed by GC and the diastereomeric excess was
determined.
4.19.2. (R)-MPA ester of hydroxy ester 2A.
dH (500 MHz;
CDCl3; Me4Si) 7.30–7.45 (5H, m, ArH), 5.11–5.16 (1H, m,
CHOCO), 4.74 (1H, s, PhH), 3.43 (3H, s, CO2CH3), 3.41
(3H, s, OCH3), 2.53–2.59 (1H, m, CHCO2), 1.55–1.66
(2H, m, CH2CH3), 0.92 (3H, d, J = 7.0, CHCH3), 0.86
(3H, t, J = 7.5, CH2CH3).
4.19.3. (S)-MPA ester of hydroxy ester 2A.
dH (500 MHz;
4.17. Larger-scale hydrolysis of (2S,3R)-methyl 3-hydroxy-
2-methylpentanoate with ICR-112
CDCl3; Me4Si) 7.30–7.45 (5H, m, ArH), 5.11–5.16 (1H, m,
CHOCO), 4.74 (1H, s, PhH), 3.63 (3H, s, CO2CH3), 3.42
(3H, s, OCH3), 2.65–2.70 (1H, m, CHCO2), 1.43–1.51
(2H, m, CH2CH3), 1.12 (3H, d, J = 7.0, CHCH3), 0.59
(3H, t, J = 7.5, CH2CH3).
In a phosphate-buffered solution (15 cm3, pH 7.0, 200 mM)
were dissolved ICR-112 (23 mg) and (2S,3R)-methyl 3-hy-
droxy-2-methylpentanoate (102 mg, 0,699 mmol, 82% de,
>99% ee), derived from enzymatic reduction with
KRED-EXP-A1B at pH 6.9 and 25 ꢁC. The mixture was
stirred at 25 ꢁC for 24 h. The product was isolated by
extracting the crude reaction mixture with Et2O (15 cm3),
after treatment with NaOH 2 M until basic pH. Then at
acidic pH (treatment with HCl 1 M), the aqueous phase
was extracted with EtOAc (3 · 15 cm3), the organic phase
was dried over MgSO4 and evaporated to dryness to pro-
vide pure (2S,3R) 3-hydroxy-2-methylpentanoic acid, 7A,
which is a known compound5h (77 mg, 83% yield, 98%
de, >99% ee). The diastereomeric excess was determined
by transforming a small quantity of the acid into its methyl
ester using diazomethane. dH (500 MHz; CDCl3; Me4Si)
3.84–3.91 (1H, m, CHOH), 2.59–2.65 (1H, m, CHCO2),
1.45–1.57 (2H, m, CH2CH3), 1.21 (3H, d, J = 7.2,
CHCH3), 0.99 (3H, t, J = 7.5, CH2CH3).
4.19.4. (R)-MPA ester of hydroxy ester 2C.
dH (500 MHz;
CDCl3; Me4Si) 7.30–7.45 (5H, m, ArH), 5.07–5.12 (1H, m,
CHOCO), 4.73 (1H, s, PhH), 3.61 (3H, s, CO2CH3), 3.42
(3H, s, OCH3), 2.72–2.79 (1H, m, CHCO2), 1.50–1.58
(1H, m, CH2CH3), 1.40–1.49 (1H, m, CH2CH3), 1.12
(3H, d, J = 7.0, CHCH3), 0.57 (3H, t, J = 7.5, CH2CH3).
4.19.5. (S)-MPA ester of hydroxy ester 2C.
dH (500 MHz;
CDCl3; Me4Si) 7.31–7.45 (5H, m, ArH), 5.08–5.12 (1H, m,
CHOCO), 4.73 (1H, s, PhH), 3.41 (3H, s, OCH3), 3.36 (3H,
s, CO2CH3), 2.62–2.68 (1H, m, CHCO2), 1.64–1.71 (1H, m,
CH2CH3), 1.54–1.61 (1H, m, CH2CH3), 0.95 (3H, d,
J = 7.0, CHCH3), 0.87 (3H, t, J = 7.5, CH2CH3).
4.19.6. (R)-MPA ester of hydroxy ester 4A.
dH (500 MHz;
CDCl3; Me4Si) 7.30–7.45 (5H, m, ArH), 5.13–5.17 (1H, m,
CHOCO), 4.74 (1H, s, PhH), 3.91–3.98 (1H, m, CO2CH2),
3.83–3.89 (1H, m, CO2CH2), 3.42 (3H, s, OCH3), 2.50–2.57
(1H, m, CHCO2), 1.67–1.75 (1H, m, CH2CH3), 1.55–1.62
(1H, m, CH2CH3), 1.12 (3H, t, J = 7.0, CO2CH2CH3),
0.91 (3H, d, J = 7.0, CHCH3), 0.86 (3H, t, J = 7.5,
CH2CH3).
4.18. Esterification of (2S,3R)-3-hydroxy-2-methylpentanoic
acid with 3-bromo pentane. Synthesis of sitophilate 6A
To a solution of (2S,3R)-3-hydroxy-2-methylpentanoic
acid (75 mg, 0.568 mmol) in anhydrous DMF (10 cm3)
was added KOH (32 mg, 0.568 mmol) and the mixture

D. Kalaitzakis et al. / Tetrahedron: Asymmetry 18 (2007) 2418–2426
2425
4.19.7. (S)-MPA ester of hydroxy ester 4A.
dH (500 MHz;
ΔδRS<0
CDCl3; Me4Si) 7.31–7.45 (5H, m, ArH), 5.12–5.16 (1H, m,
CHOCO), 4.74 (1H, s, PhH), 4.09 (2H, q, J = 7.0,
CO2CH2), 3.42 (3H, s, OCH3), 2.62–2.68 (1H, m, CHCO2),
1.52–1.60 (1H, m, CH2CH3), 1.43–1.49 (1H, m, CH2CH3),
1.23 (3H, t, J = 7.0, CO2CH2CH3), 1.12 (3H, d, J = 7.0,
CHCH3), 0.58 (3H, t, J = 7.5, CH2CH3).
H
OH
O
MPA O
R
O
H
(S)
O
Me
RS>0
Me
Δδ
O
R
4.19.8. (R)-MPA ester of hydroxy ester 4C.
dH (500 MHz;
ΔδRS>0
ΔδRS<0
H
CDCl3; Me4Si) 7.31–7.45 (5H, m, ArH), 5.08–5.13 (1H, m,
CHOCO), 4.72 (1H, s, PhH), 4.03–4.11 (2H, m, CO2CH2),
3.41 (3H, s, OCH3), 2.70–2.76 (1H, m, CHCO2), 1.52–1.59
(1H, m, CH2CH3), 1.40–1.48 (1H, m, CH2CH3), 1.22 (3H,
t, J = 7.0 Hz, CO2CH2CH3), 1.11 (3H, d, J = 7.5,
CHCH3), 0.56 (3H, t, J = 7.5, CH2CH3).
OH
O
MPA O
R
O
H
(R)
O
Me
Me
O
R
4.19.9. (S)-MPA ester of hydroxy ester 4C.
dH (500 MHz;
CDCl3; Me4Si) 7.31–7.45 (5H, m, ArH), 5.10–5.14 (1H, m,
CHOCO), 4.73 (1H, s, PhH), 3.82–3.88 (1H, m, CO2CH2),
3.74–3.80 (1H, m, CO2CH2), 3.41 (3H, s, OCH3), 2.61–2.67
(1H, m, CHCO2), 1.64–1.72 (1H, m, CH2CH3), 1.52–1.60
(1H, m, CH2CH3), 1.10 (3H, t, J = 7.0, CO2CH2CH3),
0.94 (3H, d, J = 7.0, CHCH3), 0.86 (3H, t, J = 7.5,
CH2CH3).
Acknowledgments
I.S. thanks Professor Frances H. Arnold for her generous
hospitality during her sabbatical stay at Caltech (2006).
Financial support for this work came from the European
Social Fund and the Greek Secretariat of Research and
Technology (Pythagoras program). D.K. thanks the Greek
National Scholarships Foundation (IKY) for providing a
three year fellowship.
4.19.10. (R)-MPA ester of hydroxy ester 6A. dH
(300 MHz; CDCl3; Me4Si) 7.29–7.48 (5H, m, ArH), 5.09–
5.16 (1H, m, CHOCO), 4.75 (1H, s, PhH), 4.58–4.67 (1H,
m, CO2CH), 3.42 (3H, s, OCH3), 2.48–2.58 (1H, m,
CHCO2), 1.55–1.67 (2H, m, CH2CH3), 1.36–1.55 (4H, m,
2 · CO2CHCH2CH3), 0.86 (3H, d, J = 7.2, CHCH3), 0.81
(3H, t, J = 7.5, CH2CH3), 0.79 (6H, t, J = 7.5, 2 ·
CO2CHCH2CH3).
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(500 MHz; CDCl3; Me4Si) 7.31–7.46 (5H, m, ArH), 5.11–
5.15 (1H, m, CHOCO), 4.75 (1H, s, PhH), 4.58–4.63 (1H,
m, CO2CH), 3.41 (3H, s, OCH3), 2.65–2.70 (1H, m,
CHCO2), 1.39–1.65 (6H, m, 3 · CH2CH3), 0.91 (3H, d,
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