A two-step, one-pot enzymatic synthesis of 2-substituted 1,3-diols

Article abstract of DOI:10.1021/jo101519t

A biocatalytic cascade reaction was designed for the stereoselective synthesis of optically pure 2-alkyl-1,3-diols employing two enzymes. The cascade process consists of two consecutive steps: a stereoselective diketone reduction and a hydroxy ketone reduction. Chiral diols were formed by the addition of ketoreductases in the same vessel, in high stereoselectivity and chemical yield, without the isolation of the intermediate β-hydroxy ketones.

Full text of DOI:10.1021/jo101519t

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significantly improved process economy as well as to more
A Two-Step, One-Pot Enzymatic Synthesis
of 2-Substituted 1,3-Diols
sustainable synthetic routes.3 Biotransformations can offer
great applications in cascade processes.4 Since enzymes
generally function under the same or similar conditions
(aqueous solution, pH ∼7, rt), several biocatalytic reactions
can be carried out in one pot. Thus, sequential reactions are
feasible by using multienzyme systems in order to facilitate
and simplify reaction processes but also to shift an unfavor-
able equilibrium to produce the desired product.5 A number
of examples have been published for the one-pot biocatalytic
synthesis of various compounds, combining the enzymatic
stereoselectivity with the simplicity of the cascade procedure.6
The compatibility of enzymes and chemical catalysts in or-
ganic or aqueous medium can also be very useful for one-pot
organic reactions.7
Dimitris Kalaitzakis and Ioulia Smonou*
Department of Chemistry, University of Crete,
Heraklion 71003, Crete, Greece
smonou@chemistry.uoc.gr
Received August 3, 2010
Consecutive reduction reactions can be applied for the
cascade one-pot synthesis of diols starting from the corre-
sponding diketones. More specifically, the 1,3-diols are im-
portant targets for many synthetic methodologies since they
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A biocatalytic cascade reaction was designed for the
stereoselective synthesis of optically pure 2-alkyl-1,3-
diols employing two enzymes. The cascade process con-
sists of two consecutive steps: a stereoselective diketone
reduction and a hydroxy ketone reduction. Chiral diols
were formed by the addition of ketoreductases in the same
vessel, in high stereoselectivity and chemical yield, with-
out the isolation of the intermediate β-hydroxy ketones.
€
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Published on Web 11/18/2010
DOI: 10.1021/jo101519t
r
2010 American Chemical Society

Kalaitzakis and Smonou
JOCNote
TABLE 1. Enzymatic Reduction of Diketones 1-5
FIGURE 1. 1,3-Diketones as substrates for cascade reductions.
are high value chiral synthons or building blocks for the
synthesis of many natural products and pharmaceuticals.8
Several synthetic methods have been developed for the one-
pot stereoselective synthesis of 1,3-diols. Chemoenzymatic,9
enzymatic,10 or simply chemical11 procedures have been
developed for the synthesis of 2-unsubstituted 1,3-diols.
However, very few examples have been reported for the one-
pot chemo- or chemoenzymatic formation of 2-monosub-
stituted 1,3-diols.12 In all reported cases, the substituent in
the 2-position was always a methyl group.
In this paper, we present the cascade enzymatic reduction
of 2-alkyl-1,3-diketones utilizing commercially available keto-
reductases. To the best of our knowledge, this is the first
report for the enzymatic, one-pot stereoselective synthesis
of optically pure 2-mono- and 2-disubstituted 1,3-diols,
starting from the corresponding chiral or achiral 2-alkyl-
1,3-diketones, by the consecutive addition of two reductive
enzymes in the same reaction medium.
aIsolated yield. bee (%) was measured by chiral GC analysis. cde (%)
was determined by chiral GC analysis and 1H NMR spectra.
In order to accomplish the consecutive reduction, we
utilized NADPH-dependent ketoreductase preparations
(Kred), combined with the well-established recycling system
for the regeneration of NADPH, glucose/glucose dehydro-
genase.13 In our previous studies, we have shown that keto-
reductases are remarkable catalysts for the stereoselective
formation of optically pure R-mono- and R-disubstituted
β-hydroxy ketones starting from the corresponding 1,3-dike-
tones. In most of the cases, the reaction was highly enantio-
and diastereoselective forming exclusively one of the four
possible stereoisomers.14 These reductions rarely led directly
to the formation of the corresponding diol.
It is possible to prepare single diastereomers of the product
diol by tailoring the choice of enzyme. The substrates for this
study are three monoalkyl- and two dialkyl-substituted 1,3-
diketones. Four of them were nonchiral compounds (1, 2, 4, 5),
and one was chiral (3) (Figure 1). The reductions were carried
out in a phosphate buffer (200 mM, pH 6.5-6.9) in the
presence of ketoreductases, glucose dehydrogenase, glucose,
and NADPH. In all cases, the first enzymatic reduction led
to the β-hydroxy ketone,14 and the corresponding diol was
formed by the addition of a second ketoreductase without
the isolation of the intermediate product. The results of the
enzymatic reductions are summarized in Table 1.
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In an achiral environment, the reduction of these dike-
tones would yield a mixture of all possible diastereomers.
Reductive enzymes, on the other hand, are both stereo- and
regioselective and can distinguish between the faces of pro-
chiral ketones reducing them in a highly stereoselective
fashion. It is clearly demonstrated that by the cascade addi-
tion of these biocatalysts, 2-substituted 1,3-diols were formed in
highopticalpurityandhighisolatedyield(Table 1). In all cases,
the chemical purity of the products was very high, and the
diols were isolated without any chromatographic purifica-
tion. The efficiency of this methodology is due to the com-
plete enzymatic reduction of starting material, with the
simultaneous absence of any detectable byproduct. The
enzymes chosen for the first reduction step led to quantita-
tive formation of hydroxy ketone14a,c with no further reduc-
tion to the 1,3-diol. This first step was highly stereoselective,
producing only one of the four possible stereoisomers, which
is crucial for the optical purity of the final product. It is also
important to note that the absolute configuration of the
intermediate product was already known.14c The completion
of the first step of this reaction (24 h for substrates 1, 3, and 5,
12 h for substrates 2 and 4) was followed by the addition of
ketoreductase no. 2. The reaction was monitored by gas
chromatography. In all cases, the second reduction was also
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2002, 828. (b) Cho, H. Y.; Ohtsuka, Y.; Kubota, T.; Ikeno, T.; Nagata, T.;
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J. Org. Chem. Vol. 75, No. 24, 2010 8659

Kalaitzakis and Smonou
JOCNote
SCHEME 1. Enzymatic Reduction through Dynamic Kinetic
Resolution
300 and 500 MHz spectrometers in CDCl3 solutions using Me4Si
as the internal standard. Chemical shifts are reported in ppm
downfield from Me4Si. Thermo LTQ-OrbitrapXL with an ETD
ion trap mass spectrometer was used for the high resolution
mass spectra.
Enzymatic Reduction of 1,3-Diketones 1-5. In every enzy-
matic reaction, diketones 1-5 were used as substrates.14a,c The
reduction was performed as follows. In a phosphate buffer
solution (23 mL, 200 mM, pH 6.5 for substrates 1 and 3 or 6.9
for substrates 2, 4, and 5), the 1,3-diketone (75 mM, 200 mg for
1, 270 mg for 2, 224 mg for 3, 245 mg for 4, and 242 mg for 5),
20 mg of the corresponding ketoreductase no. 1, 10 mg of glucose
dehydrogenase, 10 mg of NADPH, and 500 mg of glucose were
added. The reaction was performed at rt until GC analysis of the
crude extracts showed a complete reaction. Periodically, the pH
was readjusted accordingly to 6.5 or 6.9 with NaOH (2 M). After
completion of the first reductive step, the pH was readjusted to
6.9 and to the same buffer solution were added 20 mg of the
corresponding ketoreductase no. 2, 10 mg of glucose dehydro-
genase, 10 mg of NADPH, and 315 mg of glucose. The second
step was also performed at rt until completion of the reaction as
shown by GC analysis of the crude extracts. Periodically, the pH
was readjusted to 6.9 with NaOH (2 M). The product was
isolated by extracting the crude reaction mixture with EtOAc
(30 mLꢀ2). The combined organic layers were then extracted
with saturated NaCl solution, dried over MgSO4, and evapo-
rated to dryness. Pure, optically active diols were obtained in
88-92% yield. The products were analyzed by NMR spectro-
scopy. Their optical purity was determined by chiral GC chro-
matography using 20% permethylated cyclodextrin column.
(2S,4S)-3-Methyl-2,4-pentanediol (diol 1a): isolated yield
88% (182 mg); NMR δH (500 MHz; CDCl3; Me4Si) 4.08-4.14
(1H, m, CHOH), 3.83-3.89 (1H, m, CHOH), 2.81 (2H, bs, OH),
1.54-1.61 (1H, m, CH), 1.24 (3H, d, J=6.5 Hz, Me), 1.19 (3H,
d, J = 6.5 Hz, Me), 0.88 (3H, d, J = 7.0 Hz, Me); NMR δC
(75 MHz; CDCl3; Me4Si) 71.0, 69.7, 44.5, 22.1, 19.0, 12.2;
HRMS (ESI) calcd for C6H14O2 m/z (M þ H) 119.1072, obsd
119.1064; GC data (column: 30 mꢀ0.25 mmꢀ0.25 μm chiral
capillary column, 20% permethylated cyclodextrin 140 °C iso-
thermal; carrier gas: N2, press 85 kPa); tR=8.51 min.
highly stereoselective, producing only one stereoisomer as
the final product, in 24 h. The absolute configuration of the
1,3-diols 1a, 2a, 4a, and 5a was easily determined by 1H NMR
spectroscopy. Only the diol 3a was converted to its di-MPA
ester in order to determine its absolute configuration, and
therefore, the absolute configuration of 3ai was assigned
too.15
Because of symmetry reasons (plane of symmetry), in all
but one of the substrates shown in Table 1, both carbonyl
carbons are enantiotopic and chemically equivalent in a
nonchiral environment. However, in the asymmetric bio-
catalytic environment, a regio- and stereoselective reduction of
the keto group leads to the formation of a single diastereo-
mer of the corresponding hydroxy ketone. In the case of sub-
strate 3, the two keto groups are chemically different (dia-
stereotopic) because of the nonsymmetrical structure of the
molecule. In this case, two diastereomers would have been
expected to be formed. However, only one stereoisomer
(3a or 3ai) was formed with high chemical yield and optical
purity. This interesting result was rationalized by the equil-
ibrium between the two enantiomeric diketones 3S and 3R,
through their enolate tautomers, under the enzymatic reac-
tion conditions. This example illustrates impressively the
application of dynamic kinetic resolution (Scheme 1).16 In
this case, we were able to synthesize both possible stereo-
isomers of the final product under the proper selection of the
second enzyme (Kred-101 or Kred-A1B).
(2S,4S)-3-Allyl-3-methyl-2,4-pentanediol (diol 2a): isolated
yield 91% (252 mg); NMR δH (500 MHz; CDCl3; Me4Si)
5.87-5.96 (1H, m, CHdCH2), 5.08-5.16 (2H, m, CHdCH2),
3.94-4.00 (1H, m, CHOH), 3.85-3.92 (1H, m, CHOH), 3.16
(1H, bs, OH), 2.74 (1H, bs, OH), 2.41-2.47 (1H, m, CH2),
1.92-1.98 (1H, m, CH2), 1.21 (3H, d, J=6.5 Hz, Me), 1.17 (3H,
d, J = 6.5 Hz, Me), 0.82 (3H, s, CCH3); NMR δC (75 MHz;
CDCl3; Me4Si) 134.9, 117.7, 73.0, 72.6, 42.7, 36.9, 19.1, 17.5,
17.4; HRMS (ESI) calcd for C9H18O2 m/z (M þ H) 159.1385,
obsd 159.1377; GC data (column: 30 m ꢀ 0.25 mm ꢀ 0.25 μm
chiral capillary column, 20% permethylated cyclodextrin 160 °C
isothermal; carrier gas: N2, press 85 kPa); tR=9.08 min.
(2S,3R,4S)-3-Methyl-2,4-hexanediol (diol 3a): isolated yield
90% (208 mg); NMR δH (500 MHz; CDCl3; Me4Si) 4.09-4.17
(1H, m, CHOH), 3.55-3.63 (1H, m, CHOH), 2.84 (1H, bs, OH),
2.72 (1H, bs, OH), 1.48-1.66 (3H, m, CH3CH2, CH), 1.18 (3H,
d, J=7.0 Hz, Me), 0.96 (3H, t, J=7.5 Hz, Me), 0.91 (3H, d, J=
7.0 Hz, Me); NMR δC (75 MHz; CDCl3; Me4Si) 76.7, 69.2, 41.9,
28.1, 19.5, 11.9, 9.8; HRMS (ESI) calcd for C7H16O2 m/z (M þ
H) 133.1229, obsd 133.0758; GC data (column: 30 mꢀ0.25 mmꢀ
0.25 μm chiral capillary column, 20% permethylated cyclodextrin
130 °C isothermal; carrier gas: N2, press 85 kPa); tR=11.63 min.
(2S,3R,4R)-3-Methyl-2,4-hexanediol (diol 3ai): isolated yield
85% (193 mg); NMR δH (500 MHz; CDCl3; Me4Si) 4.06-4.13
(1H, m, CHOH), 3.75-3.80 (1H, m, CHOH), 2.55 (1H, bs, OH),
2.40 (1H, bs, OH), 1.41-1.60 (3H, m, CH3CH2, CH), 1.20 (3H,
d, J=6.5 Hz, Me), 0.93 (3H, t, J=7.5 Hz, Me), 0.91 (3H, d, J=
7.0 Hz, Me); NMR δC (75 MHz; CDCl3; Me4Si) 78.7, 72.9, 41.4,
In conclusion, a straightforward and quantitative method
has been demonstrated for the synthesis of high valuable
synthons, with less waste and greater economic benefits. This
is an environmentally benign process, where NADPH-
dependent ketoreductases were utilized for two-step, one-
pot enzymatic reduction of 2-alkyl 1,3-diketones. These
enzymes proved excellent catalysts for the stereoselective
preparation of optically pure 2-substituted 1,3-diols. They
were able to catalyze the formation of chiral diols by cascade
addition in the same reaction buffer without isolation of the
intermediate β-hydroxy ketones. More importantly, no by-
products were identified and the final products were isolated
easily without any chromatographic separation.
Experimental Section
General Methods. Enzymes were purchased from Biocataly-
tics-Codexis. The progress of the enzymatic reactions and the
selectivities were determined by gas chromatographical analysis
of crude EtOAc extracts and of isolated products, using a gas
chromatograph equipped with an FID detector (column: 30 mꢀ
0.25 mmꢀ0.25 μm chiral capillary column, 20% permethylated
1
(R)-cyclodextrin). H and 13C NMR spectra were recorded on
(15) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17.
(16) Pellissier, H. Tetrahedron 2008, 64, 1563.
8660 J. Org. Chem. Vol. 75, No. 24, 2010

Kalaitzakis and Smonou
JOCNote
28.2, 21.5, 10.4, 3.8; HRMS (ESI) calcd for C7H16O2 m/z (M þ H)
133.1229, obsd 133.1220; GC data (column: 30 m ꢀ 0.25 mm ꢀ
0.25 μm chiral capillary column, 20% permethylated cyclodextrin
130 °C isothermal; carrier gas: N2, press 85 kPa); tR=10.83 min.
(2S,4S)-3-Ethyl-3-methyl-2,4-pentanediol (diol 4a): isolated
yield 90% (224 mg); NMR δH (500 MHz; CDCl3; Me4Si)
3.95-3.99 (1H, m, CHOH), 3.90-3.94 (1H, m, CHOH), 3.02
(1H, bs, OH), 2.97 (1H, bs, OH), 1.59-1.66 (2H, m, CH2CH3),
1.18 (3H, d, J=6.5 Hz, Me), 1.16 (3H, d, J=6.5 Hz, Me), 0.88
(3H, t, J=7.5 Hz, CH2CH3), 0.80 (3H, s, CCH3); NMR δC(75
MHz; CDCl3; Me4Si) 73.0, 72.4, 41.9, 24.5, 18.2, 17.7, 17.6, 7.6;
HRMS (ESI) calcd for C8H18O2 m/z (M þ H) 147.1385, obsd
147.1378; GC data (column 30 m ꢀ 0.25 mm ꢀ 0.25 μm chiral
capillary column, 20% permethylated cyclodextrin 140 °C
isothermal; carrier gas: N2, press 85 kPa); tR=11.43 min.
(2S,4S)-3-Allyl-2,4-pentanediol (diol 5a): isolated yield 92%
(228 mg); NMR δH(500 MHz; CDCl3; Me4Si): 5.76-5.86
(1H, m, CH=CH2), 5.02-5.11 (2H, m, CHdCH2), 4.23-4.28
(1H, m, CHOH), 4.01-4.06 (1H, m, CHOH), 2.97 (1H, bs, OH),
2.67 (1H, bs, OH), 2.21-2.28 (1H, m, CH2), 2.07-2.14 (1H, m,
CH2), 1.43-1.49 (1H, m, CH), 1.27 (3H, d, J=6.5 Hz, Me), 1.22
(3H, d, J = 6.5 Hz, Me); NMR δC(75 MHz; CDCl3; Me4Si)
137.4, 116.3, 69.1, 67.7, 48.8, 30.8, 22.1, 19.4; HRMS (ESI) calcd
for C8H16O2 m/z (M þ H) 145.1229, obsd 145.1216; GC data
(column: 30 mꢀ0.25 mmꢀ0.25 μm chiral capillary column, 20%
permethylated cyclodextrin 140 °C isothermal; carrier gas: N2,
press 85 kPa); tR=12.10 min.
reaction mixture was stirred at 0 °C for 6 h and at rt for 12 h. After
completion of the reaction, the produced urea was filtered, and the
filtrate was evaporated and then chromatographed with 5/1 (v/v),
Hex/EtOAc, producing the corresponding MPA diester (38 mg,
90% isolated yield).
(R,R)-MPA diester of (2S,3R,4S)-3-Methyl-2,4-hexanediol:
NMR δH (500 MHz; CDCl3; Me4Si): 7.28-7.45 (10H, m, 2 ꢀ
CHC6H5), 5.04-5.09 (1H, m, CHO2C), 4.77-4.82 (1H, m,
CHO2C), 4.76 (1H, s, CHOMe), 4.75 (1H, s, CHOMe), 3.45
(3H, s, OMe), 3.44 (3H, s, OMe), 1.72-1.79 (1H, m, CHCH3),
1.44-1.53 (1H, m, CH2), 1.25-1.38 (1H, m, CH2), 1.02 (3H, d,
J=6.0 Hz, CH3CHO2C), 0.83 (3H, d, J=7.0 Hz, CH3CH), 0.43
(3H, t, J=7.5 Hz, CH3CH2).
(S,S)-MPA diester of (2S,3R,4S)-3-Methyl-2,4-hexanediol:
δ
H (500 MHz; CDCl3; Me4Si) 7.29-7.42 (10H, m, 2ꢀCHC6H5),
4.67-4.72 (1H, m, CHO2C), 4.69 (1H, s, CHOMe), 4.66 (1H, s,
CHOMe), 4.45-4.50 (1H, m, CHO2C), 3.40 (3H, s, OMe), 3.39
(3H, s, OMe), 1.59-1.65 (1H, m, CHCH3), 1.23-1.32 (2H, m,
CH2), 1.09 (3H, d, J=6.0 Hz, CH3CHO2C), 0.64 (3H, t, J=7.5
Hz, CH3CH2), 0.55 (3H, d, J=7.0 Hz, CH3CH).
Acknowledgment. Financial support from ELKE of Uni-
versity of Crete (RG2752) is acknowledged. D.K. thanks the
Greek National Scholarships Foundation (IKY). We are also
thankful to ProFI (Proteomics facility at IMBB-FORTH) for
performing all the HRMS analyses.
Preparation of MPA Diester of (2S,3R,4S)-3-Methyl-2,4-hex-
anediol, 3a. To a solution of the corresponding diol (0.1 mmol,
13 mg) in dry CH2Cl2 were added 2.2 equiv of DCC (0.22 mmol,
46 mg), 2.2 equiv of the corresponding (R)- or (S)-MPA ester
(0.22 mmol, 36 mg), and a catalytic amount of DMAP, and the
1
Supporting Information Available: Copies of H and 13C
NMR spectra of all compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 24, 2010 8661