Chemo-biological preparation of the chiral building block (R)-4-acetoxy-2-methyl-1-butanol using Pseudomonas putida

Article abstract of DOI:10.1016/j.tetasy.2011.09.019

In order to develop new methyl substituted chiral building blocks which are useful for the synthesis of methyl branched natural products, the enantioselective bioreduction of an exo-methylene to a methyl group was investigated. 4-Acetoxy-2-methylene-1-butanol 3 was prepared from itaconic acid over four steps and converted to the chiral alcohol (R)-4-acetoxy-2-methyl-1- butanol 4, by growing cells of Pseudomonas putida. The bioconversion achieved a high enantioselectivity (92% ee) and a high chemical yield (65%) within a relatively short reaction time (18-20 h). Copyright

Full text of DOI:10.1016/j.tetasy.2011.09.019

Tetrahedron: Asymmetry 22 (2011) 1658–1661
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemo-biological preparation of the chiral building block
(R)-4-acetoxy-2-methyl-1-butanol using Pseudomonas putida
Youngju Kim a,b, Hidenori Watanabe a, Bieong-Kil Kim b, Young-Bae Seu b,
⇑
a Department of Agricultural Chemistry, The University of Tokyo, Yayoi l-l-l, Bunkyo-ku, Tokyo 113-0032, Japan
b School of Life Sciences and Biotechnology, Kyungpook National University, 1370, Sangyeokdong, Buk-ku, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2011
Revised 16 September 2011
Accepted 16 September 2011
Available online 4 November 2011
In order to develop new methyl substituted chiral building blocks which are useful for the synthesis of
methyl branched natural products, the enantioselective bioreduction of an exo-methylene to a methyl
group was investigated. 4-Acetoxy-2-methylene-1-butanol 3 was prepared from itaconic acid over four
steps and converted to the chiral alcohol (R)-4-acetoxy-2-methyl-1-butanol 4, by growing cells of
Pseudomonas putida. The bioconversion achieved a high enantioselectivity (92% ee) and a high chemical
yield (65%) within a relatively short reaction time (18–20 h).
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
1. Introduction
the exo-methylene of 3 to yield 4-acetoxy-2-methyl-1-butanol 4
with good conversion.
Optically active alcohols play a very important role as chiral
building blocks.1 In particular, chiral alcohols with methyl
branches are widely used for the synthesis of antibiotics, such as
azithromycin,2 natural products such as muscone,3–6 and many in-
sect pheromones, for example, 5,9-dimethyl-heptadecane (Leucop-
tera scitella), 5,9-dimethyl pentadecane (Perileucoptera coffeella).
and 4-methyl-1-nonanol (Tenebrio molitor).7,8 Due to their high
enantio- and regio-selectivities,7 bioconversions are becoming
essential approaches to prepare chiral compounds.8 Many biocon-
version processes are studied for the enantioselective reduction of
carbon carbon double bonds (C@C).9–11 The C@C bonds need to be
activated by electron-withdrawing groups to cause the addition of
hydrogen to occur in a trans-fashion across the double bond.9 Since
the enantioselectivity of the bioreductions of endo C@C bonds is
influenced by the geometry of the substrate, the E:Z control of
the substrate is essential.12 From this point of view, the use of an
exo C@C bond as a substrate is more attractive, because it is free
from the E:Z control problem. There have been several attempts
to reduce exo-olefins biocatalytically.11–13 Even though high
enantioselectivity has been achieved, the conversions rate are
quite low.13 As a result, the development of a bioreduction of exo
C@C bonds with good conversion and high enantioselectivity is
highly desired.
2. Results and discussion
2.1. Synthesis of 4-acetoxy-2-methylene-1-butanol 3
The starting point of the chemo-biological synthesis was the
inexpensive itaconic acid. Substrate 3 was synthesized by the se-
quence outlined in Scheme 1. Briefly, itaconic acid 1 was esterified
with 2-propanol in the presence of sulfuric acid to produce diiso-
propyl ester. The reduction of the ester groups required a suitable
reducing reagent to avoid an over-reduced product, which would
result in low enantiomeric purity of the bioconversion product 4.
This was accomplished through the use of aluminum hydride,
which was prepared from AlCl3 and 3 equiv of lithium aluminum
hydride (LAH). Diol 2 was acetylated with acetic anhydride to give
the diacetate, and the allylic acetate was selectively hydrolyzed
with LPS in a phosphate buffer (pH 7.0) to give 3.
2.2. Screening of microorganisms for the synthesis of (R)-4-
acetoxy-2-methyl-1-butanol 4
The abilities of the following six candidate microorganisms to
perform the bioconversion were assessed: Candida rugosa KCTC
7282,14 P. putida KCTC 1644,15 Alcaligenes sp. KCTC 2338,16 Achro-
Recently, we have investigated the bioreduction of exo-methyl-
enes to chiral methyl groups and found that Pseudomonas putida
KCTC1644 has capabilities for the enantioselective reduction of
mobacter sp. KCTC 2757,17 Chromobacterium sp. KCTC 2896,18
,
and Kluyveromyces fragilis KCTC 7260.19 Substrate 3 was added to
their culture media, and the strains were cultured at the appropri-
ate temperatures (C. rugosa at 25 °C, P. putida at 27 °C, Alcaligenes
sp. at 30 °C, Achromobacter sp. at 30 °C, Chromobacterium sp. at
30 °C and K. fragilis at 25 °C). Cell suspensions were extracted with
⇑
Corresponding author.
E-mail address: ybseu@knu.ac.kr (Y.-B. Seu).
0957-4166/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.09.019

Y. Kim et al. / Tetrahedron: Asymmetry 22 (2011) 1658–1661
1659
CH2
O
CH2
a, b
HO
c, d
HO
OH
OH
O
1
2
CH2
CH3
bioconversion
HO
HO
OAc
OAc
4
3
hydrolase
hydrolase
CH3
CH2
HO
HO
+
OH
OH
by-products
Scheme 1. Preparation and bioconversion of 4-acetoxy-2-methylene-1-butanol 3. Reagents and conditions: (a) H2SO4, 2-propanol, reflux, 30 h, 98%; (b) AlH3, Et2O, ꢂ30 °C,
5 min; (c) Ac2O, pyridine, CH2Cl2, 5 h, two steps 98%; (d) LPS⁄, P-buffer pH 7.0, 5 min, rt, 71%, ⁄LPS = Lipase from Pseudomonas sp.
ethyl acetate (EtOAc) after 20 h, and the organic phases were ana-
lyzed by gas chromatography (GC). The results are summarized in
Table 1. Acetate hydrolysis to produce by-products occurred in the
cultures of P. putida and K. fragilis (Table 1, entries 2 and 6). The
highest conversion to 4 was achieved with P. putida (entry 2),
which was therefore chosen for further optimization.
the cultures at 0, 4, 6, and 10 h after inoculation. Conversion was
analyzed by GC after 20 h (Table 2). The best results were achieved
when the substrate was added immediately. In contrast, when the
substrate was added after 6 h, only hydrolysis occurred without
reduction of the C@C.
Table 2
Effect of substrate 3 addition time point on the bioconversion by P. putida
Table 1
Substrate addition timea (h)
Product compositionb (%)
Screening of microorganisms for bioconversion
3
4c
By-products
Entry
Microorganism
Product compositiona (%)
By–products
0
4
6
10
11
30
60
73
73
40
2
16
30
38
25
3
4
1
2
3
4
5
6
Candida rugosa
Pseudomonas putida
Alcaligenes sp.
Achromobacter sp.
Chromobacterium sp.
Kluyveromyces fragilis
95
12
93
68
75
20
5
73
7
0
15
0
2
a
b
c
Marked on Figure 1.
Determined by GC after culture for 20 h.
The enantiomeric purity of products 4 was determined by chiral GC analysis,
31
25
65
0
0
15
and all of them were found to be 92% ee.
a
Determined by GC.
The reaction conditions were further optimized with respect to
the seed culture volume used for the inoculation (1–5%), substrate
3 concentration (7ꢀ21 mmol/L), culture scale (5–100 mL), culture
media (Nutrient broth, Luria-Bertani (LB) broth, yeast malt (YM)
broth, YPD broth), pH of the media after adding substrate 3 (pH
6.5–8.1) and the cultivation temperature (27–35 °C). The best reac-
tion conditions were obtained using 3% of seed culture with 0.10 g
(0.70 mmol, 14 mmol/L) of substrate in 50 mL (in 500 mL Erlen-
meyer flask) of YPD media (pH 7.1) at 30 °C. The time course of
the biotransformation under these optimal conditions is shown
in Figure 2. The formation of undesirable by-products gradually in-
creased after 15 h of cultivation.
2.3. Optimization of the bioconversion reaction conditions
The growth curve of P. putida is shown in Figure 1. To determine
the optimal starting time for the biotransformation, 3 was added to
1.8
1.6
d
1.4
1.2
1
c
0.8
0.6
0.4
0.2
0
b
4
2.4. Determination of absolute configuration and enantiomeric
excess of 4
a
To determine the absolute configuration of 4, it was hydrolyzed
to the corresponding diol and compared with the commercial com-
pound (R)-2-methyl-1,4-butanediol (>98.0%, GC). The specific rota-
0
2
6
8
10 12 14 16 18 20
Culture time (h)
tion value of deacylated 4 was measured to be ½a D27
¼ þ13:1 (c 0.5,
ꢁ
Figure 1. Cell growth of P. putida. Yeast peptone dextrose (YPD) media, 30 °C, 180
rotation per minute (rpm); Arrows indicate the time point of substrate addition,
a = 0 h, b = 4 h, c = 6 h, d = 10 h; Optical density (OD) at 660 nm was measured.
MeOH) while that of the commercial product was found to be
½
a 2D7
ꢁ
¼ þ13:2 (c 1.0, MeOH)/½a D27
¼ þ13:6 (c 3.3, MeOH). Hence,
ꢁ

1660
Y. Kim et al. / Tetrahedron: Asymmetry 22 (2011) 1658–1661
give an opportunity for further applications of exo-methylene
bioreductions.
4. Experimental
4.1. General
Unless otherwise stated, all chemicals were of reagent grade
and purchased from Sigma–Aldrich (Yongin, Korea). Microorgan-
isms were obtained from KCTC (Korean Collection for Type
Cultures). All separations were conducted using open column
chromatography with Merck silica gel 60 (40–63 mm). NMR
spectra were recorded using a Varian 300 and 400 MHz FT-NMR.
Chemical shifts were reported relative to TMS (d 0.00), CHCl3 (d
7.26) was used as an internal standard and coupling constants (J)
are reported in Hz.
Figure 2. Time course of bioconversion of 5 by P. putida. YPD media, 30 °C, 180 rpm,
analyzed by GC, (a) cultivation time 0 h, 8.1 min 3; (b) cultivation time 10 h, 8.0 min
3, 8.4 min 4; (c) cultivation time 20 h, 8.0 min 3, 8.4 min 4.
the product of bioconversion 4 was determined to have an (R)-con-
figuration. The enantiomeric purity of 4 was assessed by chiral GC,
and it was found to be 92% ee [(S)-4, 4.1%, (R)-4, 95.9%].
4.2. Analytics
The bioconversion of 3 to 4 was determined by gas chromatog-
raphy (Shimadzu GC 10) on
a column code CBP1-M25-025
2.5. Bioreduction mechanism of P. putida
(Shimadzu). Method: column oven temperature 70–120 °C, 3 °C/
min, injection port temperature 150 °C, detector temperature
280 °C, carrier gas N2, and column flow rate 6.1 mL/min. The
enantiomeric excess of 4 was determined by gas chromatography
(Agilent 6890N) on a MOMTBDMSGCD column. Method: column
oven temperature 70–150 °C, 3 °C/min, injection port temperature
150 °C, detector temperature 280 °C, carrier gas He, and column
flow rate 0.7 mL/min. Optical rotation values were measured on
a Jasco p-1020 polarimeter in a 10 mL cuvette.
It is well known that the asymmetric reduction of the C@C bond
of allylic alcohol proceeds via three steps: oxidation of the primary
alcohol to an aldehyde, enantioselective hydrogenation (1,4-reduc-
tion) of the activated C@C bond by enoate-reductase, and reduction
of the aldehyde.9 The reported mechanism of enoate-reductase12
also satisfies the stereocontrol observed in the methylene
reduction of 3 (Fig. 3). However, over the course of the bioreduction
of 3 to 4 by P. putida, any peak of the possible intermediates (unsat-
urated and saturated aldehydes) was not detected in the GC (Fig. 2).
This result cannot exclude the possibility that the rate-determining
step is the first oxidation of 3 and that the intermediate aldehydes
are quickly reduced to 4. Therefore, further experiments for the
elucidation of a detailed reaction mechanism are currently in
progress and will be reported in due course.
4.3. 2-Methylene-1,4-butanediol 2
Concentrated H2SO4 (300
lL) was added to a stirred solution of
itaconic acid (130 g, 1.00 mol) and isopropanol (100 mL,
1
1.60 mol) in benzene (1 L). After stirring for 48 h at 70 °C, the solu-
tion was cooled to room temperature and extracted with EtOAc.
The organic phases were combined and washed with aq NaHCO3,
dried over MgSO4 and evaporated. The crude product was purified
by distillation to yield the diisopropyl ester (210 g, 98%); 1H NMR
spectrum was identical to that reported in the literature; 1H
NMR (400 MHz, CDCl3) d: 6.29 (s, 1H), 5.65 (s, 1H), 5.07 (dq,
J = 6.3, 6.3, 1H), 5.02 (dq, J = 6.3, 6.3, 1H), 3.29 (s, 2H), 1.27 (d,
J = 6.3, 6H), 1.24 (d, J = 6.3, 6H).20
A suspension of LAH (11.4 g, 0.30 mol) in Et2O (200 mL) at 0 °C
was added to a stirred solution of AlCl3 (13.4 g, 0.10 mol) in dried
Et2O (300 mL). After 30 min, the ester (32.1 g, 0.15 mol) dissolved
in Et2O (100 mL) was slowly added at ꢂ30 °C. The reaction was
immediately stopped after the addition of the ester, by adding
10% of aq H2SO4. The reaction mixture was extracted with EtOAc,
and the organic phases were combined and washed with aq NaH-
CO3, dried over MgSO4 and evaporated to yield diol 2 (15.3 g, 98%);
1H NMR (300 MHz, CDCl3) d: 5.07 (s, 1H), 4.92 (s, 1H), 4.03 (s, 2H),
3. Conclusion
The desired compound
4 is not commercially available,
although a similar but costly (5 g, 162.50 USD) compound, (R)-2-
methyl-1,4-butanediol (>98.0%, GC), is available from TCI (Tokyo
Chemical Industry, Co., LTD). As a chiral building block, compound
4 is even more attractive than (R)-2-methyl-1,4-butanediol be-
cause one of the two hydroxy groups is protected as an acetate
and thus individual transformations are possible.
Starting from the inexpensive itaconic acid, enantioenriched
(R)-4-acetoxy-2-methyl-1-butanol 4 was readily prepared by a
chemo-biological method. The bioconversion was achieved by
growing cells of P. putida, within a relatively short reaction time
(18–20 h), with high enantioselectivity (92% ee) and in high yield
(65%). This bioreduction method is a convenient method for pro-
ducing methyl-branched chiral building blocks. Our findings will
CH2
CH2
CH3
P. putida
H
O
HO
HO
OAc
OAc
OAc
3
4
H
H
H-
H+
EWG
R1
Figure 3. The plausible mechanism for the bioreduction of 3 to 4 by P. putida.

Y. Kim et al. / Tetrahedron: Asymmetry 22 (2011) 1658–1661
1661
3.69 (t, J = 6.0 Hz, 2H), 2.31 (t, J = 6.0 Hz, 2H); the 1H NMR spectrum
4.7. Assignment of the absolute configuration: preparation and
comparison of the specific rotation value of deacylated 4
was identical to that reported in the literature.21
4.4. 4-Acetoxy-2-methylene-1-butanol 3
The specific rotation value of 4 was compared with the value of
the commercially available compound (R)-2-methyl-butane-1,4-
diol (>98.0%, GC) by deacylation of 4. The deacylation of 4 was
achieved by adding K2CO3 (30 mg) to a stirred solution of 4
(60 mg, 0.4 mmol) in MeOH (1 mL). After stirring for 1 h, aq NH4Cl
was added, and the reaction mixture was evaporated to remove the
MeOH and extracted with EtOAc. The organic phases were com-
bined and washed with brine, dried over MgSO4, and evaporated.
The residue was purified by flash chromatography (petroleum
hexane/EtOAc 2:1) to give the diol (35 mg, 84%); (R)-2-methyl-
At first, Ac2O (25.0 g) was slowly added at 0 °C to a stirred solu-
tion of diol 3 (20.4 g, 0.20 mol) in pyridine (200 mL) and stirred for
5 h at room temperature. The solution was evaporated with MeOH
to remove pyridine and extracted with EtOAc. The organic phase
was washed with brine, dried over MgSO4, and evaporated. The
residue was purified by flash chromatography (petroleum hex-
ane/EtOAc 4:1) to yield the diacetate (36.5 g, 98%); 1H NMR
(300 MHz, CDCl3) d: 5.1 (s, 1H), 5.0 (s, 1H), 4.51 (s, 2H), 4.16 (t,
J = 6.6 Hz, 2H), 2.36 (t, J = 6.6 Hz, 2H), 2.06 (s, 3H), 2.01 (s, 3H);
the 1H NMR spectrum was identical to that reported in the
literature.22
The diacetate (200 mg, 1.10 mmol) was added to a stirred solu-
tion of LPS (Lipase PS, Amano, 200 mg) in a sodium phosphate buf-
fer (pH 7.0, 10 mL). Stirring was continued for 5 min at room
temperature. The reaction was filtered on Celite to remove the en-
zyme. The monoacetate was extracted with EtOAc. The organic
phase was washed with aq NaHCO3, dried over MgSO4, and evapo-
rated. The residue was purified by flash chromatography (petro-
leum hexane/EtOAc 4:1) to yield monoacetate 3 (113 mg, 71.4%);
1H NMR (300 MHz, CDCl3) d: 5.05 (s, 1H), 4.862 (s, 1H), 4.15 (t,
J = 6.6 Hz, 2H), 4.02 (s, 2H), 2.34 (t, J = 6.6 Hz, 2H), 1.98 (s, 3H);
1H NMR spectrum was identical to that reported in the literature.22
butane-1,4-diol, prepared from 4: ½a D27
¼ þ13:1 (c 0.5, MeOH),
ꢁ
(R)-2-methyl-butane-1,4-diol, purchased from TCI: ½a D27
¼ þ13:2
ꢁ
(c 1.0, MeOH)/½a D27
ꢁ
¼ þ13:6 (c 3.3, MeOH); 1H NMR (300 MHz,
CDCl3) d: 3.63 (t, 2H, J = 5.4 Hz), 3.66 (m, 1H), 3.57 (dd, 1 H,
J = 10.8, 4.8), 1.56–1.64 (m, 2H), 0.93 (d, J = 6.9, 3H); 1H NMR spec-
trum was identical to that reported in the literature.23
Acknowledgements
This research was supported by the Kyungpook National Uni-
versity Research Grant. We thank Mr. Yusuke Ogura and Dr. Sig-
eyuki Tamogami for their assistance with chiral GC analysis.
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The seed cultured medium (1.5 mL), which was grown in YPD at
30 °C, 180 rpm, for 20 h, was added to 50 ml of YPD broth (Difco™)
in a 500 ml Erlenmeyer flask. At the same time, substrate 3
(100 mg, 0.7 mmol) was also added, and the pH was adjusted to
7.1 with aq 1 M NaOH. Cultures were grown at 30 °C and
180 rpm. The bioconversion progress was checked every 5 h by
GC. After approximately 18–20 h, the cell suspensions were ex-
tracted with EtOAc and the organic phases were washed with
water, dried over MgSO4, and evaporated. The residue was purified
by flash chromatography (petroleum hexane/EtOAc 4:1) to yield
(R)-4 (66 mg, 65%, 92% ee, chiral GC on a MOMTBDMSGCD col-
umn). Method: column oven temperature 70–150 °C, 3 °C/min,
injection port temperature 150 °C, detector temperature 280 °C,
carrier gas He, and column flow rate 0.7 mL/min: 25.4 min [(S)-4,
4.1%], 25. 9 min [(R)-4, 95.9%]; 1H NMR (300 MHz, CDCl3) d:
4.03–4.13 (m, 2H), 3.4 (d, J = 5.6 Hz, 2H), 2.0 (s, 3H), 1.65–1.78
(m, 2H), 1.33–1.41 (m, 1H), 0.87 (d, J = 6.8 Hz, 3H); the 1H NMR
spectrum was identical to that reported in the literature.23
23. Grisenti, P.; Ferraboschi, P.; Casati, S.; Santaniello, E. Tetrahedron: Asymmetry
1993, 4, 997–1006.