Synthesis of enantiopure glycidol derivatives via a one-pot two-step enzymatic cascade

Article abstract of DOI:10.1039/c4ob02186j

Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed a one-pot two-step enzymatic cascade using prochiral α,β-unsaturated ketones as the substrates. An S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de > 99%) were achieved for the majority of the substrates, and product yields reached up to >99%. This journal is

Full text of DOI:10.1039/c4ob02186j

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of enantiopure glycidol derivatives via a
one-pot two-step enzymatic cascade†
Yu-Chang Liu,a,b,c Yan Liua,b,c and Zhong-Liu Wu*a,b
Cite this: Org. Biomol. Chem., 2015,
13, 2146
Styrene monooxygenase (SMO) can catalyze the kinetic resolution of secondary allylic alcohols to provide
enantiopure glycidol derivatives. To overcome the low theoretical yield of kinetic resolution, we designed
a one-pot two-step enzymatic cascade using prochiral α,β-unsaturated ketones as the substrates. An
S-specific ketoreductase ChKRED03 was screened for the efficient bioreduction of the substrates to
provide (S)-allylic alcohols, which underwent SMO-catalyzed epoxidation to achieve glycidol derivatives
with contiguous stereogenic centers. Excellent enantioselectivity (ee > 99%) and diastereoselectivity (de >
99%) were achieved for the majority of the substrates, and product yields reached up to >99%.
Received 15th October 2014,
Accepted 8th December 2014
DOI: 10.1039/c4ob02186j
www.rsc.org/obc
shows a clear advantage over previously established chemistry
methods, and provides a valuable eco-friendly approach that
Introduction
Enantiomerically enriched glycidol derivatives are important employs molecular oxygen as the oxidant.
building blocks in the synthesis of natural products and bio-
However, kinetic resolution only provides the desired glyci-
logically active compounds.1–6 A series of chiral compounds dol product with a maximum theoretical yield of 50%. To over-
can be derived by opening the reactive oxirane moiety with come such a limitation, in this study, we employed a two-step
diverse nucleophiles.7,8 The Sharpless–Katsuki asymmetric enzymatic cascade reaction involving an S-specific ketoreduc-
epoxidation represents a classic catalytic approach to prepare tase and SMO, and using an α,β-unsaturated ketone as the
enantiopure glycidol derivatives. A wide range of primary starting material, which allowed us to achieve high conver-
allylic alcohols can be epoxidized with high stereo- sions as well as enantiomeric and diastereomeric excesses.
selectivity.9,10 The reaction is also suitable for the kinetic
resolution of secondary allylic alcohols, but is sometimes sen-
sitive to steric hindrance.1,11,12 For the aromatic substrate
1-phenylprop-2-en-1-ol (1b), it required several days at−20 °C to
Results and discussion
Identification of S-specific ketoreductase ChKRED03
reach 50% conversion, and yielded an epoxide with 90% ee.11
Styrene monooxygenase (SMO) is an enzyme involved in the
upper catabolic pathway of styrene degradation.13,14 It shows
excellent enantioselectivity in the epoxidation of styrene and
its derivatives.15–19 In our previous work on the SMO from
Pseudomonas sp. LQ26, we have found that the racemic second-
ary allylic alcohol 1-phenylprop-2-en-1-ol (1b) could be epoxi-
SMO specifically catalyzes the epoxidation of (S)-1-phenylprop-
2-en-1-ol (1b), which could be produced by the bioreduction of
1a catalyzed with ketoreductases following the Prelog’s rule.
Therefore, six recombinant ketoreductases in our inventory
that follow the Prelog’s rule21 were assayed against substrate
1a. Three of them, namely ChKRED03, ChKRED11 and
ChKRED15, displayed excellent enantioselectivity (>99% ee),
yielding a single enantiomer of (S)-1b without any by-product.
ChKRED03 performed the best in terms of activity (Fig. 1).
ChKRED03 shared the maximal similarity of 43% with a
known alcohol dehydrogenase RasADH from Ralstonia sp.
DSM642822 (PDB: 4BMS). It also shared 90% similarity with
one predicted protein from Chryseobacterium hispalense (NCBI
accession no. WP_029296933). Protein sequence alignment of
ChKRED03 with the confirmed ketoreductases is shown in
Fig. 2. ChKRED03 contains the cofactor binding motif
(G12xxxG16xG18) and the essential segment (S137, Y150, K154) for
the catalytic activity of SDRs (Fig. 2), which are highly
dized within
2 h with excellent enantioselectivity and
diastereoselectivity (>99% ee, >99% de) using recombinant
Escherichia coli expressing the SMO,20 and the unreacted (R)-1b
could be recovered with >99% ee. The enzymatic method
aKey Laboratory of Environmental and Applied Microbiology, Chengdu Institute of
Biology, Chinese Academy of Sciences, Chengdu 610041, China.
E-mail: wuzhl@cib.ac.cn; Fax: +86-28-82890434; Tel: +86-28-82890434
bEnvironmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041,
China
cUniversity of the Chinese Academy of Sciences, Beijing 100049, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02186j
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due to the generation of gluconic acid. Considering the reac-
tion conditions of the subsequent SMO-catalyzed epoxidation
(optimal pH 6.5), the bioreduction was performed at pH 7.0,
which yielded the same reaction efficiency.
ChKRED03 could as well catalyze the bioreduction of a
series of α,β-unsaturated ketones, analogues of 1a (Table 1).
The reaction proceeded smoothly without any by-product
observed. All the tested substrates were converted to the corres-
ponding (S)-alcohols (b) with excellent activity and enantio-
selectivity. In the majority of cases, complete conversion was
observed, and products with >99% ee were achieved (Table 1).
Substrates 6a and 14a showed slightly lower reactivity, but still
yielded >80% conversion within 5 h with good to excellent
enantioselectivity (Table 1, entries 6 & 14). The results indi-
cated that ChKRED03 could serve as a suitable ketoreductase
Fig. 1 Bioreduction of 1-phenylprop-2-en-1-one (1a) with S-specific
ketoreductases. Shown are the conversion rate (white) and enantiomeric
excess of the product (black).
conserved residues in the short-chain dehydrogenase/
reductase (SDR) superfamily.23
Reaction conditions and substrate adaptability of ChKRED03
The purified ChKRED03 catalyzed the reduction of 1a to enan-
tiopure (S)-1b over the pH range of 4.0–9.0 and displayed the
maximum activity at pH 7.5 in either Tris-HCl or potassium
phosphate buffer (Fig. 3A). No significant change of activity
occurred over the pH range of 5.5–8.0 (>90% of the maximum
activity). In addition, the optimum reaction temperature of
ChKRED03 was determined to be 30 °C (Fig. 3B). Hence, the
pH- and temperature-dependence of activities was compatible
with that of the bioepoxidation reaction using whole cells of
recombinant E. coli producing SMO, which displayed the
maximum activity at 30 °C and pH 6.5 in potassium phosphate
buffer.24
ChKRED03 was an NADPH-dependent ketoreductase and
did not accept NADH as a cofactor. Therefore, a cofactor regen-
eration system based on GDH-catalyzed glucose oxidation was
applied to ensure a sufficient supply of NADPH. Complete con-
version was achieved within 1 h with excellent enantio-
selectivity (ee > 99%), which was higher than the 80%
conversion without using the NADPH-regeneration system.
After the reaction, the resulting mixture had a pH drop of 0.5
Fig. 3 Effect of (A) pH and (B) temperature on the activity of
●
( ), potassium
ChKRED03. Buffers used: sodium citrate–citric acid
○
▲
phosphate ( ), Tris-HCl ( ).
Fig. 2 Sequence alignment of ChKRED03 with members of the short chain dehydrogenase/reductase (SDR) family. ChKRED03, predicted keto-
reductase in this work (KC342003); RasADH, short chain alcohol dehydrogenases from Ralstonia sp. DSM6428 (4MBS); LBADH, alcohol dehydrogen-
ases from Lactobacillus brevis, (1NXQ); BKR, β-keto acyl carrier protein reductase from Brassica napus (1EDO). Residues that are highly conserved in
the SDR family are shaded in grey. The cofactor-binding motif (GxxxGxG sequence) is underlined. The catalytic triad, Ser-Tyr-Lys, is highlighted with
asterisks.
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when using the recombinant whole cells as the catalyst, result-
ing in 1-phenyl-1-propanol. E. coli is known to contain several
ene-reductases,25–27 which apparently could take 1a as a sub-
strate, and competed with SMO for the CvC double bond. To
avoid the side reaction, a one-pot enzymatic cascade was
designed that used ChKRED03 and SMO in a step-wise fashion.
In the first step, the substrate 1a was reduced to (S)-1b
using ChKRED03 as a free enzyme in the presence of a
GDH-catalyzed cofactor recycling system. After the reaction
reached completion, the whole cells of E. coli expressing SMO
were added into the reaction mixture. As expected, SMO
efficiently catalyzed the epoxidation of (S)-1b, yielding the
product (1R, 2R)-phenyl glycidol (1c) with excellent stereo-
selectivity (ee > 99%, de > 99%) at 100% conversion in 2 h
(Table 2, entry 1).
Encouraged by this result, we then extended the one-pot
two-step cascade to the panel of α,β-unsaturated ketones listed
in Table 1 that were accepted as substrates by ChKRED03.
Because substituents at the ortho-positions of the benzene ring
are known to strongly prevent the bioepoxidation reaction,20
only meta- and para-substituted substrates were tested
(Table 2). After the two-step cascade, all the substrates (2a–
14a) were transformed to the corresponding glycidol deriva-
tives with excellent enantioselectivity (ee > 99%) and diastereo-
selectivity (de 86–99%). The conversions of substrates a to the
final product c were generally over 50%, which was the
maximum theoretical yield of the kinetic resolution. A single
(1R,2R)-enantiomer was achieved in the majority of cases.
Compared with previous results using SMO as the only cata-
lyst,20 the two-enzyme cascade delivered much improved sub-
Table 1 Asymmetric bioreduction of α,β-unsaturated ketones to sec-
ondary allylic alcohols
Entry
R
t (h)
Conv.a (%)
ee (%)
1
2
3
4
5
6
7
8
Ph (1a)
m-FC6H4 (2a)
p-FC6H4 (3a)
1
1
1
5
5
5
5
5
5
1
1
5
5
5
>99
>99
>99
>99
>99
82
>99
>99
>99
>99
>99
>99
>99
86
>99
>99
>99
>99
>99
94
>99
>99
>99
>99
>99
>99
>99
>99
m-MeC6H4 (4a)
p-MeC6H4 (5a)
2-Thienyl (6a)
3-Thienyl (7a)
m-ClC6H4 (8a)
p-ClC6H4 (9a)
m-BrC6H4 (10a)
p-BrC6H4 (11a)
m-OMeC6H4 (12a)
p-OMeC6H4 (13a)
Benzyl (14a)
9
10
11
12
13
14
a Conversions were determined by HPLC. The unreacted substrates
were detected for substrates 6a and 14a.
to generate the corresponding (S)-alcohols for the sequential
epoxidation reaction.
One-pot two-step biotransformation of α,β-unsaturated
ketones
ChKRED03 and SMO could be coexpressed in E. coli, but strate conversion and product yield. The isolated yields
reached up to 93% (for 1c) in a 50 ml reaction system.
efficient reduction of the CvC double bond of 1a was observed
Table 2 One-pot two-step cascade for the synthesis of enantiopure glycidol derivatives
Entry
R
ta (h)
Conv.b (%)
eec (%)
de1 d (%)
de2 e (%)
1
2
3
4
5
6
7
8
Ph (1a)
m-FC6H4 (2a)
p-FC6H4 (3a)
2
15
15
15
15
15
15
24
24
36
36
36
36
36
>99
97
98
87
86
79
98
83
84
54
64
37
75
55
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
91
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
86
>99
>99
>99
>99
m-MeC6H4 (4a)
p-MeC6H4 (5a)
2-Thienyl (6a)
3-Thienyl (7a)
m-ClC6H4 (8a)
p-ClC6H4 (9a)
m-BrC6H4 (10a)
p-BrC6H4 (11a)
m-OMeC6H4 (12a)
p-OMeC6H4 (13a)
Benzyl (14a)
9
10
11
12
13
14
a The reaction time for the epoxidation step. b Conversions of a to c were determined by HPLC. c The enantiomeric excess of the major product:
ee = (|[1R,2R] − [1S,2S]|)/([1R,2R] + [1S,2S]). d The diastereomeric excess for the hydroxyl group: de1 = (|[1R,2R] − [1S,2R]|)/([1R,2R] + [1S,2R]). e The
diastereomeric excess for the oxiranyl group de2 = (|[1R,2R] − [1R,2S]|)/([1R,2R] + [1R,2S]).
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In the two-step cascade, the first step was generally more at 37 °C in LB media containing kanamycin (50 μg ml−1). Two
efficient than the second one. For substrates 2a–5a and 7a– ml of overnight culture was then inoculated into 200 ml of ter-
13a, which had a lower conversion than 1a, no by-product for- rific broth containing kanamycin (50 μg ml−1) in a 500 ml
mation or unreacted substrate was observed. The HPLC trace flask, and incubated at 37 °C for 3 h, followed by incubation at
showed a portion of intermediate b, indicating incomplete 20 °C for another 20 h with gyratory shaking at 220 rpm. Cells
epoxidation of b to c. For substrates 6a and 14a, due to the were harvested by centrifugation, washed twice with potassium
incomplete bioreduction, the remaining substrate underwent phosphate buffer (0.1 M, pH 7.0) and stored at 4 °C.
CvC double bond reduction catalyzed with the ene-reductases
in the E. coli cells, yielding by-products 1-(thiophen-2-yl)-
propan-1-one (6d) and 1-phenylbutan-2-one (14d), respectively.
Cloning, expression and purification of ChKRED03
The DNA fragment encoding ChKRED03 (NCBI accession no.
KC342002) was amplified from the genomic DNA of Chryseo-
bacterium sp. CA49 with primers 5′-CATATGATGAATTT
CACAGATAAAAATGTAATC-3′ (forward) and 5′-CTCGAGTTA
TCTGCGGATCGTTACTC-3′ (reverse), and cloned into the
pMD-19 T vector (Novagen, Madison, WI, USA). The resulting
product was digested with EcoRI and HindIII, and subcloned
into the pET28a(+) plasmid (Novagen) digested with the same
restriction enzymes. The resulting plasmid encoding
ChKRED03 was transformed into E. coli BL21(DE3) for protein
expression.
Single colonies were grown overnight at 37 °C in Luria–
Bertani (LB) medium containing 50 μg kanamycin ml−1. Then
2 ml of the starter culture was transferred into 200 ml of terri-
fic broth containing 50 μg kanamycin ml−1, and incubated at
37 °C. The induction of protein expression was initiated with
the addition of 0.5 mM IPTG when OD600 reached 0.6–0.8, and
the incubation was continued at 30 °C for 16 h.
Conclusions
In summary, an enzymatic cascade reaction employing an
S-specific ketoreductase ChKRED03 and a styrene monooxy-
genase has been accomplished for the stereoselective synthesis
of glycidol derivatives with excellent diastereomeric and enan-
tiomeric excess. This convenient one-pot two-step approach
shows significant improvement over our previously established
process based on SMO alone, and provides a valuable alterna-
tive to classic chemo-catalyzed kinetic resolution for the epoxi-
dation of secondary allylic alcohols.
Experimental
Materials and methods
Cell pellets were collected, resuspended in buffer
A
Materials and general methods. Substrate 1a was syn-
thesized from 3-chloro-1-phenylpropan-1-one (Alfa-Aesar,
Tianjin, China).28 Other substrates were synthesized using
Jones oxidation from alcohols.29 Racemic 1b was purchased
from Sigma-Aldrich (St. Louis, MO, USA), and other α-substi-
tuted secondary allylic alcohols (b) were synthesized using
Grignard reaction at −10 °C.30 Racemic epoxides (c) were syn-
thesized according to a previous method.20 Glucose dehydro-
genase (GDH) was purchased from Sigma-Aldrich (St. Louis,
USA). Restriction enzymes and T4 DNA ligase were purchased
from New England Biolabs (Beverly, MA, USA).
(100 mM potassium phosphate, 300 mM NaCl and 10 mM imi-
dazole, pH 8.0), and disrupted using a high-pressure hom-
ogenizer (ATS-AH100B, ATS Engineering Inc., Canada) at
60 MPa. After removing the cell debris, the resulting supernatant
was loaded onto a Ni2+-nitrilotriacetic acid column (Qiagen,
Valencia, CA, USA). The His-tagged enzyme was eluted with
buffer A containing 250 mM imidazole, and dialyzed against pot-
assium phosphate buffer (20 mM, pH 7.5). Protein analysis was
done using SDS-PAGE and the BCA Protein Assay Kit (Beyotime,
China) with bovine serum albumin as the standard.
1H NMR spectra were recorded on a Bruker-600 (600/
150 MHz) spectrometer in CDCl3. All signals are expressed in
ppm downfield from tetramethylsilane. Optical rotations were
measured with a Perkin Elmer 341 polarimeter. Chiral HPLC
was conducted with a Shimadzu Prominence LC-20AD system
connected to a PDA-detector using Daicel OJ-H (2a–13a, 2b–
13b), Chiralcel OD-H (14a, 14b) and Chiralpak AS-H (1c–14c)
columns. The conversion rate and enantiomeric excess of (S)-
1-phenylprop-2-en-1-ol (1b) were determined by chiral GC ana-
lysis using a CP-Chirasil-DEX column (25 m × 0.25 mm,
Varian, USA), and nitrogen as the carrier gas on a Fuli 9790 II
GC system connected to a flame ionization detector.
Measurement of the enzyme activity of ChKRED03
The assay was performed by adding 0.2 mg of the enzyme to
1 ml potassium phosphate buffer (100 mM, pH 7.5) containing
7.5 mM 1a and 1 mM NADPH. Continuous spectrophotometric
measurements were then performed to monitor the oxidation
of NADPH at 340 nm for 2 min at 25 °C on a Shimadzu
UV-1800 spectrophotometer. One unit of activity was defined
as the amount of the enzyme catalyzing the oxidation of
1 µmol of NADPH per min.
To determine the pH-dependence of the activity of
ChKRED03, sodium citrate (pH 4.0–6.0), potassium phosphate
(pH 6.0–8.0) and Tris-HCl (pH 7.0–9.0) were used in different
pH ranges. To determine the temperature optima, the reac-
Heterologous expression of SMO
Recombinant Escherichia coli BL21 harboring the plasmid tions were performed under the standard conditions for
pET28a(+) encoding the styAB gene from Pseudomonas sp. 20 min, and terminated by extraction with ethyl acetate. The
LQ26 (NCBI accession no. GU593979) was cultivated overnight conversion rate was measured using GC.
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General procedure for biotransformation
OH). Retention times: tR (S) 9.7 min, tR (R) 10.3 min; 3a,
tR 8.1 min.
The bioreduction of ketones (a) was performed in 10 ml pot-
assium phosphate buffer (100 mM, pH 7.0) containing 7.5 mM
substrate, 50 mM glucose, 25 µM NADP+, 3U ChKRED03 and
3U GDH. The reaction was conducted at 30 °C, and the
process was monitored using HPLC or GC.
For the one-pot two-step biotransformation, the second
catalyst was added when the bioreduction of the ketone
reached an end. Freshly harvested whole cells of recombinant
E. coli expressing SMO (0.4 g dry cell weight) were added into
the reaction mixture, and then the incubation was continued
at 30 °C for 2–36 h with shaking at 240 rpm. The reaction was
terminated by extraction with diethyl ether (3 × 20 ml). The
organic layers were dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. All the samples were analyzed
by chiral HPLC to determine the yield and enantiomeric purity
of products.
(1R,2R)-(4-Fluorophenyl)-2,3-glycidol (3c). Elute: petroleum
ether–ethyl acetate 7 : 1, Rf: 0.25, 88% yield; 1H NMR(600 MHz,
CDCl3): δ 7.36–7.41 (m, 2H, Ar–H), 7.05–7.09 (m, 2H, Ar–H),
4.48 (d, J = 6 Hz, 1H, –CH), 3.19–3.21 (m, 1H, –CH), 2.87–2.88
(m, 1H, –CH2), 2.77–2.78 (m, 1H, –CH2), 2.24 (br, 1H, OH).
Retention times: tR1 13.6 min, tR2 18.1 min, tR3 21.4 min; 3b,
tR 7.2 min.
(S)-1-m-Tolylprop-2-en-1-ol (4b). Elute: petroleum ether–
ethyl acetate 15 : 1, Rf: 0.35, 91% yield; [α]2D0 −8.4 (c 0.57 in
1
CHCl3); H NMR(600 MHz, CDCl3): δ 7.23–7.25 (m, 1H, Ar–H),
7.19 (s, 1H, Ar–H), 7.16 (d, J = 12 Hz, 1H, Ar–H), 7.10 (d, J = 12
Hz, 1H, Ar–H), 6.02–6.08 (m, 1H, vCH), 5.35 (d, J = 12 Hz, 1H,
–CH), 5.16–5.20 (m, 2H, vCH2), 2.36 (m, 3H, –CH3). Retention
times: tR (S) 8.9 min, tR (R) 9.6 min; 4a, tR 7.4 min.
(1R,2R)-(m-Tolylprop)-2,3-glycidol (4c). Elute: petroleum
ether–ethyl acetate 5 : 1, Rf: 0.35, 76% yield; 1H NMR(600 MHz,
CDCl3): δ 7.14–7.29 (m, 4H, Ar–H), 4.45 (d, J = 6 Hz, 1H, –CH),
3.21–3.23 (m, 1H, –CH), 2.83–2.86 (m, 1H, –CH2), 2.76–2.78
(m, 1H, –CH2), 2.37 (s, 3H, –CH3), 2.34 (br, 1H, OH). Retention
times: tR1 11.4 min, tR2 12.8 min, tR3 13.4 min, tR4 14.1 min;
4b, tR 6.3 min.
Preparative biotransformations were performed at 30 °C in
50 ml potassium phosphate buffer (100 mM, pH 7.0) following
the same procedure. The extracted and concentrated products
were purified by silica gel column chromatography, and identi-
fied by NMR analysis.
(S)-1-p-Tolylprop-2-en-1-ol (5b). Elute: petroleum ether–ethyl
Spectral data for biotransformation products
acetate 15 : 1, Rf: 0.3, 91% yield; [α]2D0 −5.7 (c 0.63 in CHCl3);
1
(S)-1-Phenylprop-2-en-1-ol (1b). Elute: petroleum ether–ethyl {lit.32 [α]D20 −3.8 (c 0.8 in CHCl3)}; H NMR(600 MHz, CDCl3):
acetate 15 : 1, Rf: 0.4, 93% yield; [α]2D0–2.5 (c 1.0 in CHCl3); δ 7.26 (d, J = 12 Hz, 2H, Ar–H), 7.17 (d, J = 6 Hz, 2H, Ar–H),
{lit.31 [α]D25 −5.9 (c 1.73 in C6H6)}; H NMR(600 MHz, CDCl3): 6.02–6.07 (m, 1H, vCH), 5.24 (d, J = 18 Hz, 1H, –CH),
1
δ 7.25–7.36 (m, 5H, Ar–H), 6.04–6.06 (m, 1H, vCH), 5.32–5.35 5.16–5.19 (m, 2H, vCH2), 2.35 (m, 3H, –CH3), 1.98 (br, 1H,
(d, J = 18 Hz, 1H, –CH), 5.17–5.19 (m, 1H, vCH2), 2.09 (br, 1H, OH). Retention times: tR (S) 10.5 min, tR (R) 12.3 min; 5a,
OH). Retention times: tR (R) 12.5 min, tR (S) 13.3 min; 1a, tR tR 12.3 min.
5.9 min.
(1R,2R)-(p-Tolylprop)-2,3-glycidol (5c). Elute: petroleum
1
(1R,2R)-Phenyl-2,3-glycidol (1c). Elute: petroleum ether– ether–ethyl acetate 5 : 1, Rf: 0.3, 78% yield; H NMR(600 MHz,
ethyl acetate 7 : 1, Rf: 0.3, 93% yield; 1H NMR(600 MHz, CDCl3): δ 7.28–7.32 (m, 2H, Ar–H), 7.19–7.20 (m, 2H, Ar–H),
CDCl3): δ 7.37–7.43 (m, 5H, Ar–H), 4.94 (s, 1H, –CH), 3.22–3.25 4.45 (d, J = 6 Hz, 1H, –CH), 3.21–3.23 (m, 1H, –CH), 2.84–2.86
(m, 1H, –CH), 2.85–2.87 (m, 1H, –CH2), 2.76–2.77 (m, 1H, (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.36 (s, 3H, –CH3),
–CH2), 2.23 (br, 1H, OH). Retention times: tR1 12.5 min, 2.25 (br, 1H, OH). Retention times: tR1 12.5 min, tR2 15.1 min,
tR2 14.8 min, tR3 15.7 min, tR4 17.7 min; 1b, tR min.
tR3 16.8 min, tR4 19.6 min; 5b, tR 7.1 min.
(S)-1-(3-Fluorophenyl)prop-2-en-1-ol (2b). Elute: petroleum
(S)-1-(Thiophen-2-yl)prop-2-en-1-ol (6b). Elute: petroleum
ether–ethyl acetate 15 : 1, Rf: 0.4, 90% yield; [α]2D0 + 7.6 (c 0.83 ether–ethyl acetate 15 : 1, Rf: 0.3, 74% yield; [α]2D0 +13.1 (c 0.42
in CHCl3);{lit.31 [α]D35 + 12.1 (c 0.56, CHCl3)}; 1H NMR in CHCl3); {lit.31 [α]2D6 +18.4 (c 0.5, CHCl3)}; H NMR(600 MHz,
1
(600 MHz, CDCl3): δ 6.96–7.34 (m, 4H, Ar–H), 5.99–6.03 (m, CDCl3): δ 7.25–7.27 (m,1H, Ar–H), 6.97–6.99 (m, 2H, Ar–H),
1H, vCH), 5.38(d, J = 18 Hz, 1H, –CH), 5.24 (d, J = 12 Hz, 1H, 6.09–6.15 (m, 1H, vCH), 5.39–5.42 (br, 2H, vCH2, –CH), 5.25
vCH2), 5.20 (d, J = 6 Hz, 1H, vCH2), 1.98(br, 1H, OH). Reten- (d, J = 6 Hz, 1H, vCH2), 2.03 (br, 1H, OH). Retention times:
tion times: tR (S) 8.9 min, tR (R) 9.3 min; 2a, tR 7.1 min.
tR (S) 11.6 min, tR (R) 14.7 min; 6a, tR 10.7 min.
(1R,2R)-(3-Fluorophenyl)-2,3-glycidol (2c). Elute: petroleum
(1R,2R)-(Thiophen-2-yl)-2,3-glycidol (6c). Elute: petroleum
1
1
ether–ethyl acetate 7 : 1, Rf: 0.3, 85% yield; H NMR(600 MHz, ether–ethyl acetate 5 : 1, Rf: 0.3, 70% yield; H NMR(600 MHz,
CDCl3): δ 7.01–7.37 (m, 4H, Ar–H), 4.94 (s, 1H, –CH), 3.19–3.21 CDCl3): δ 7.32–7.33 (m, 1H, Ar–H), 7.10–7.11 (m, 1H, Ar–H),
(m, 1H, –CH), 2.87–2.88 (m, 1H, –CH2), 2.76–2.77 (m, 1H, 7.02–7.03 (m, 1H, Ar–H), 4.79 (d, J = 6 Hz, 1H, –CH), 3.31–3.32
–CH2), 2.28 (br, 1H, OH). Retention times: tR1 12.1 min, (m, 1H, –CH), 2.90–2.92 (m, 1H, –CH2), 2.84–2.87 (m, 1H,
tR2 14.4 min, tR3 14.9 min, tR4 15.8 min; 2b, tR 6.5 min.
–CH2). Retention times: tR1 15.9 min, tR2 18.5 min,
(S)-1-(4-Fluorophenyl)prop-2-en-1-ol (3b). Elute: petroleum tR3 22.5 min, tR4 27.7 min; 6b, tR 7.6 min.
ether–ethyl acetate 15 : 1, Rf: 0.3, 92% yield; [α]2D0 +11.3 (c 0.81
(S)-1-(Thiophen-3-yl)prop-2-en-1-ol (7b). Elute: petroleum
1
in CHCl3); H NMR(600 MHz, CDCl3): δ 7.25–7.37 (m, 2H, Ar– ether–ethyl acetate 15 : 1, Rf: 0.25, 89% yield; [α]2D0 +9.8 (c 0.33,
H), 7.04–7.06 (m, 2H, Ar–H), 5.98–6.03 (m, 1H, vCH), 5.36 (d, CHCl3; {lit.31 [α]D35 +13.4 (c 0.5, CHCl3)}; 1H NMR(600 MHz,
J = 18 Hz, 1H, –CH), 5.21–5.23 (m, 2H, vCH2), 2.03 (br, 1H, CDCl3): δ 7.25–7.26 (m, 1H, Ar–H), 7.15–7.16 (m, 1H, Ar–H),
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7.03 (d, J = 6 Hz, 1H, Ar–H), 6.00–6.05 (m, 1H, vCH), 5.30 (d, 1H, OH). Retention times: tR (S) 9.6 min, tR (R) 10.4 min; 1a, tR
J = 18 Hz, 1H, –CH), 5.16–5.19 (m, 2H, vCH2). Retention 8.5 min.
times: tR (S) 11.3 min, tR (R) 14.2 min; 7a, tR 10.6 min.
(1R,2R)-(4-Bromophenyl)-2,3-glycidol
(11c). Elute:
pet-
(1R,2R)-(Thiophen-3-yl)-2,3-glycidol (7c). Elute: petroleum roleum ether–ethyl acetate 5 : 1, Rf: 0.3, 52% yield; 1H NMR
1
ether–ethyl acetate 2 : 1, Rf: 0.2, 85% yield; H NMR(600 MHz, (600 MHz, CDCl3): δ 7.52–7.54 (m, 2H, Ar–H), 7.29–7.32 (m,
CDCl3): δ 7.35–7.36 (m, 2H, Ar–H), 7.14–7.15 (m, 1H, Ar–H), 2H, Ar–H), 4.49 (d, J = 6 Hz, 1H, –CH), 3.18–3.22 (m, 1H, –CH),
4.61 (br, 1H, –CH), 3.26–3.28 (m, 1H, –CH), 2.88–2.90 (m, 1H, 2.87–2.89 (m, 1H, –CH2), 2.76–2.78 (m, 1H, –CH2), 2.24 (br,
–CH2), 2.84–2.86 (m, 1H, –CH2). Retention times: tR1 17.3 min, 1H, OH). Retention times: tR1 14.4 min, tR2 19.4 min,
tR2 19.7 min, tR3 27.2 min, tR4 38.90 min; 7b, tR 8.1 min.
(S)-1-(3-Chlorophenyl)prop-2-en-1-ol (8b). Elute: petroleum
tR3 22.5 min; 11b, tR 7.8 min.
(S)-1-(3-Methoxyphenyl)prop-2-en-1-ol (12b). Elute: pet-
ether–ethyl acetate 15 : 1, Rf: 0.35, 92% yield; [α]2D0 +10.4 (c 0.65 roleum ether–ethyl acetate 10 : 1, Rf: 0.3, 88% yield; [α]2D0 +20.7
in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.36 (s, 1H, Ar–H), (c 0.9 in CHCl3); {lit.34 [α]2D0 +23.1 (c 0.75, CHCl3)}; 1H NMR
7.22–7.28 (m, 3H, Ar–H), 5.96–6.01 (m, 1H, vCH), 5.34(d, J = (600 MHz, CDCl3): δ 7.25–7.28 (m, 1H, Ar–H), 6.93–6.95 (m,
18 Hz, 1H, –CH), 5.21 (d, J = 12 Hz, 1H, vCH2), 5.15 (d, J = 2H, Ar–H), 6.81–6.83 (m, 1H, Ar–H), 6.01–6.06 (m, 1H, vCH),
6 Hz, 1H, vCH2). Retention times: tR (S) 8.9 min, tR (R) 5.35 (d, J = 18 Hz, 2H, –CH), 5.16–5.20 (m, 2H, vCH2), 3.80 (s,
9.3 min; 1a, tR 7.0 min.
3H, –CH3). Retention times: tR (S) 14.5 min, tR (R) 15.7 min;
(1R,2R)-(3-Chlorophenyl)-2,3-glycidol (8c). Elute: petroleum 1a, tR 10.0 min.
ether–ethyl acetate 5 : 1, Rf: 0.25, 71% yield; 1H NMR(600 MHz,
(1R,2R)-(3-Methoxyphenyl)-2,3-glycidol (12c). Elute: pet-
CDCl3): δ 7.28–7.43 (m, 4H, Ar–H), 4.48 (s, 1H, –CH), 3.20–3.23 roleum ether–ethyl acetate 4 : 1, Rf: 0.25, 30% yield; 1H NMR
(m, 1H, –CH), 2.88–2.89 (m, 1H, –CH2), 2.76–2.78 (m, 1H, (600 MHz, CDCl3): δ 7.27–7.30 (m, 1H, Ar–H), 6.95–6.98 (m,
–CH2), 2.38 (br, 1H, OH). Retention times: tR1 13.3 min, 2H, Ar–H), 6.85–6.86 (m, 1H, Ar–H), 4.41 (d, J = 6 Hz, 1H,
tR2 14.5 min, tR3 14.9 min, tR4 15.8 min; 8b, tR 6.7 min.
–CH), 3.81 (s, 3H, –OCH3), 3.18–3.20 (m, 1H, –CH), 2.80–2.84
(S)-1-(4-Chlorophenyl)prop-2-en-1-ol (9b). Elute: petroleum (m, 2H, –CH). Retention times: tR1 20.6 min, tR2 21.9 min,
ether–ethyl acetate 15 : 1, Rf: 0.3, 90% yield; [α]2D0 +1.2 (c 0.81 in tR3 26.8 min, tR4 28.3 min; 12b, tR 9.5 min.
CHCl3); {lit.31 [α]D26 +15.3 (c 1.0, CHCl3)}; 1H NMR(600 MHz,
(S)-1-(4-Methoxyphenyl)prop-2-en-1-ol (13b). Elute: pet-
CDCl3): δ 7.29–7.33 (m, 4H, Ar–H), 5.97–6.02 (m, 1H, vCH), roleum ether–ethyl acetate 10 : 1, Rf: 0.2, 90% yield; [α]2D0−3.9
5.34 (d, J = 12 Hz, 1H, –CH), 5.21 (d, J = 6 Hz, 1H, vCH2), 5.17 (c 0.4 in CHCl3); {lit.31 [α]D26 −6.04 (c 1.0, CHCl3)}; 1H NMR
(br, 1H, vCH2), 2.03 (br, 1H, –OH). Retention times: tR (S) (600 MHz, CDCl3): δ 7.28 (d, J = 12 Hz, 2H, Ar–H), 6.88 (d, J =
9.1 min, tR (R) 9.8 min; 1a, tR 7.9 min.
6 Hz, 2H, Ar–H), 6.01–6.06 (m, 1H, vCH), 5.32 (d, J = 18 Hz,
(1R,2R)-(4-Chlorophenyl)-2,3-glycidol (9c). Elute: petroleum 2H, –CH), 5.14–5.18 (m, 2H, vCH2), 3.79 (s, 3H, –CH3), 2.03
1
ether–ethyl acetate 5 : 1, Rf: 0.3, 72% yield; H NMR(600 MHz, (br, 1H, OH). Retention times: tR (S) 21.4 min, tR (R) 23.0 min;
CDCl3): δ 7.31–7.35 (m, 2H, Ar–H), 4.45 (d, J = 6 Hz, 1H, –CH), 1a, tR 17.9 min.
3.19–3.21 (m, 1H, –CH), 2.85–2.86 (m, 1H, –CH2), 2.74–2.76
(m, 1H, –CH2), 2.51 (br, 1H, OH). Retention times: roleum ether–ethyl acetate 3 : 1, Rf: 0.2, 65% yield; 1H NMR
tR1 13.5 min, tR2 18.3 min, tR3 20.7 min; 9b, tR 7.4 min. (600 MHz, CDCl3): δ 7.31–7.36 (m, 2H, Ar–H), 6.91–6.92 (m,
(1R,2R)-(4-Methoxyphenyl)-2,3-glycidol (13c). Elute: pet-
(S)-1-(3-Bromophenyl)prop-2-en-1-ol (10b). Elute: petroleum 2H, Ar–H), 4.44 (d, J = 6 Hz, 1H, –CH), 3.83 (s, 3H, –OCH3),
ether–ethyl acetate 15 : 1, Rf: 0.35, 93% yield; [α]2D0 +7.6 (c 0.48 3.20–3.22 (m, 1H, –CH), 2.84–2.85 (m, 1H, –CH2), 2.77–2.78
in CHCl3); 1H NMR(600 MHz, CDCl3): δ 7.52 (s, 1H, Ar–H), (m, 1H, –CH2), 2.29 (br, 1H, OH). Retention times:
7.40 (d, J = 12 Hz,1H, Ar–H), 7.25–7.28 (m, 1H, Ar–H), tR1 25.9 min, tR2 30.2 min, tR3 33.0 min; 13b, tR 15.7 min.
7.19–7.22 (m, 1H, Ar–H), 5.95–6.00 (m, 1H, vCH), 5.33(d, J =
(S)-1-Phenylbut-3-en-2-ol (14b). Elute: petroleum ether–ethyl
12 Hz, 1H, –CH), 5.21 (d, J = 12 Hz, 1H, vCH2), 5.13 (d, J = acetate 20 : 1, Rf: 0.2, 78% yield; [α]2D0 +7.1 (c 0.38 in CHCl3)
6 Hz, 1H, vCH2). Retention times: tR (S) 9.2 min, tR (R) {lit.35 [α]D25 +12.7 (c 1, CHCl3)}; 1H NMR(600 MHz, CDCl3):
9.7 min; 1a, tR 7.4 min.
δ 7.22–7.32 (m, 5H, Ar–H), 5.90–59.6 (m, 1H, vCH), 5.24 (d,
(1R,2R)-(3-Bromophenyl)-2,3-glycidol
(10c). Elute:
pet- J = 18 Hz, 1H, vCH2), 5.13 (d, J = 6 Hz, 1H, vCH2), 4.33–4.36
roleum ether–ethyl acetate 5 : 1, Rf: 0.25, 40% yield; 1H NMR (m, 1H, –CH), 2.88 (dd, J = 6 Hz, J = 12 Hz, 1H, –CH2−), 2.79
(600 MHz, CDCl3): δ 7.46–7.47 (d, J = 6 Hz, 2H, Ar–H), (dd, J = 6 Hz, J = 12 Hz, 1H, –CH2−). Retention times: tR (S)
7.24–7.27 (m, 2H, Ar–H), 4.47 (br, 1H, –CH), 3.19–3.22 (m, 1H, 10.1 min, tR (R) 11.4 min; 1a, tR 12.1 min.
–CH), 2.87–2.89 (m, 1H, –CH2), 2.76–2.77 (m, 1H, –CH2), 2.46
(br, 1H, OH). Retention times: tR1 14.2 min, tR2 15.3 min, ether–ethyl acetate 5 : 1, Rf: 0.4, 44% yield; H NMR (600 MHz,
tR3 15.9 min, tR4 16.6 min; 10b, tR 7.1 min. CDCl3): δ 7.22–7.33 (m, 5H, Ar–H), 3.88–3.90 (m, 1H, –CH),
(1R,2R)-Phenylbut-2,3-glycidol
(14c). Elute:
petroleum
1
(S)-1-(4-Bromophenyl)prop-2-en-1-ol (11b). Elute: petroleum 3.02–3.05 (m, 1H, –CH), 2.87–2.92 (m, 1H, –CH2), 2.84–2.86(m,
ether–ethyl acetate 15 : 1, Rf: 0.3, 92% yield; [α]2D0 +8.4 (c 0.78 in 1H, –CH2), 2.76–2.79 (m, 1H, –CH2), 2.72–2.73 (m, 1H, –CH2).
CHCl3); {lit.33 [α]D +11.59 (c 3.28, PhH) }; 1H NMR(600 MHz, Retention times: tR1 13.9 min, tR2 16.2 min, tR3 23.5 min,
CDCl3): δ 7.45 (d, J = 12 Hz, 2H, Ar–H), 7.20 (d, J = 12 Hz, 2H, tR4 26.8 min; 14b, tR 6.4 min.
Ar–H), 5.93–5.99 (m, 1H, vCH), 5.30 (d, J = 18 Hz, 1H, –CH),
5.18 (d, J = 12 Hz, 1H, vCH2), 5.10 (br, 1H, vCH2), 2.01 (br, (600 MHz, CDCl3): δ 7.71 (d, J = 6 Hz, 1H, Ar–H), 7.61 (d, J =
1-(Thiophen-2-yl)propan-1-one (6d). 9% yield; 1H NMR
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6 Hz, 1H, Ar–H), 7.11–7.13 (m, 1H, Ar–H), 2.92–2.96 (m, 2H, 16 S. Bernasconi, F. Orsini, G. Sello, A. Colmegna, E. Galli and
–CH2), 1.23–1.25 (m, 3H, –CH3). G. Bestetti, Tetrahedron Lett., 2000, 41, 9157–9161.
Phenylbutan-2-one (14d). 6% yield; 1H NMR(600 MHz, 17 S. Bernasconi, F. Orsini, G. Sello and P. Di Gennaro, Tetra-
CDCl3): δ 7.20–7.34 (m, 5H, Ar–H), 3.69 (s, 2H, –CH2), 2.47 (dd,
J = 6 Hz, J = 12 Hz, 2H, –CH2), 1.01–1.04 (m, 3H, –CH3).
hedron: Asymmetry, 2004, 15, 1603–1606.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21072183 & 21372216), and the Open
Fund of Key Laboratory of Environmental and Applied Micro-
biology (KLCAS-2013-06) and the Key Research Program
(KGZD-EW-606-14) of the Chinese Academy of Sciences.
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This journal is © The Royal Society of Chemistry 2015