Enzymatic resolution of ethyl 3-hydroxy-2(1′substituted-methylidene)-butyrate by Pseudomonas cepacia lipase catalyzed acetylation

Article abstract of DOI:10.1016/j.tetasy.2007.09.002

Enzymatic resolution of a series of enantiomerically pure ethyl 3-hydroxy-2(1′substituted-methylidene)-butyrates was performed using Pseudomonas cepacia lipase (EC 3.1.1.3) as a catalyst. Optically active ethyl 3-hydroxy-2(1′substituted-methylidene)-butyr

Full text of DOI:10.1016/j.tetasy.2007.09.002

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 18 (2007) 2227–2232
Enzymatic resolution of ethyl 3-hydroxy-2(10substituted-methylidene)-
butyrate by Pseudomonas cepacia lipase catalyzed acetylation
Fides Benfatti,* Giuliana Cardillo,* Luca Gentilucci, Elisa Mosconi and Alessandra Tolomelli
Department of Chemistry ‘G.Ciamician’, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
Received 30 July 2007; accepted 5 September 2007
Abstract—Enzymatic resolution of a series of enantiomerically pure ethyl 3-hydroxy-2(10substituted-methylidene)-butyrates was
performed using Pseudomonas cepacia lipase (EC 3.1.1.3) as a catalyst. Optically active ethyl 3-hydroxy-2(10substituted-methylidene)-
butyrates, as well as their acetates, were obtained from this reaction in good yield and excellent enantiomeric excess.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mic alcohols by using an enzymatic acetylation reaction
(Scheme 1).
Enantiomerically pure allylic alcohols constitute versa-
tile starting materials in organic synthesis. The kinetic
enzymatic resolution of this class of compounds by lipase
catalyzed acetylation1 has been reported under different
conditions and in the presence of different lipases.2 The
resolution of polyfunctionalyzed derivatives such as c-
hydroxy-a,b-unsaturated esters, via irreversible acylations
in hexane mediated by Pseudomonas sp. Amano has been
already reported in the literature.3 The same enzyme
promoted enantioselective esterifications of b-hydroxy-a-
methylene esters (Fig. 1).
Pseudomonas Cepacia
Lipase
OAc
OH
OH
+
R
R
R
CO2Et
CO2Et
CO2Et
OAc
(3R)-2a-c
(3S)-1a-c
1a-c
a: R =
b: R =
c: R =
Over the course of our research, we required the title com-
pounds in an enantiomerically pure form. Our substrates
are particular for the presence of the double bond, whose
configuration could affect the enzymatic activity, and also
for the presence of the carboxylic function. We decided
to investigate the resolution of a series of substituted race-
OMe
Scheme 1. Ethyl 3-hydroxy-2(10substituted-methylidene)-butyrate enzy-
matic irreversible acylation reaction.
2. Results and discussion
OH
O
OH
OMe
The racemic alcohols were prepared in an extremely simple
manner by treating 10 equiv of aldehyde and 1 equiv of
ethyl acetoacetate in the presence of 0.15 equiv of piperi-
dine under microwave assisted conditions for 7.5 min
(Scheme 2). Microwave assisted organic synthesis4 is in fact
a rapidly expanding area of research that offers the oppor-
tunity to strongly reduce reaction times from hours to
minutes. After the usual work up, a 30/70 mixtures of
E and Z ketones were obtained in good yields.
R
R'
R
O
Figure 1. Polyfunctionalized allylic alcohols resolved via enzymatic
irreversible acylation.
*
Corresponding authors. Tel.: +39 051 2099570; fax: +39 051 2099456
(G.C.); e-mail addresses: fides.benfatti@studio.unibo.it; giuliana.
cardillo@unibo.it
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.09.002

2228
F. Benfatti et al. / Tetrahedron: Asymmetry 18 (2007) 2227–2232
K2CO3), good results could be obtained only when the
reaction was performed in the presence of 1.5 equiv of
LiHMDS (Scheme 3).
O
+
O
OH
R
H
O
NaBH4
Piperidine
R
R
.
MW
67-77%
CeCl3 7H2O
CO2Et
CO2Et
THF/MeOH
EtO2C
LiHMDS
OAc
1a-c
Z/E 70/30
OH
3a-c
Z/E 70/30
70-85%
acetyl chloride
R
R
THF
CO2Et
CO2Et
Scheme 2. Synthesis of racemic alcohols 1a–c.
0°C to r.t.
2a-c
1a-c
85-90%
The mixture of ketones was subjected to reduction in the
presence of 1 equiv of CeCl3 and 1.1 equiv of NaBH4 in
THF/MeOH 9:1 (Scheme 2).5 The corresponding Z/E alco-
hols 1a–c were obtained in excellent yields and were sepa-
rated by flash chromatography.
Scheme 3.
After the required conversion in the lipase catalyzed reac-
tion was achieved, the enzyme was filtered off and the
mixture subjected to the usual work up. The H NMR of
1
the crude material showed a 1:1 ratio of alcohol/acetate,
based on the peak area of the hydrogen adjacent to the
secondary alcohol and its corresponding acetate (a quartet
at 4.50–4.60 ppm for alcohols 1a–c and a quartet at 5.60–
5.70 ppm for acetate 2a–c, Fig. 2). The resulting acetates
and the remaining alcohols were separated by flash
chromatography.
In order to select the most useful enzyme for the kinetic reso-
lution of these compounds, a series of screening experi-
ments were carried out with various lipases. Therefore,
the lipases from Candida cylindracea, Mucor miehei, Asper-
gillus niger, and Rhizopus nivens were tested via irreversible
acylation by treating the substrates with 4 equiv of vinyl
acetate and 0.5 mass equiv of enzyme in diethyl ether at
rt for several days.7
Finally, the use of Pseudomonas cepacia proved to be very
successful for the resolution of Z-1a–c alcohols, affording
the acetylated compounds in good yields and high ee,
although with lengthy reaction times. However, any effort
to resolve the allylic alcohols E-1a–c under these conditions
failed.1 The lipase from P. cepacia (EC 3.1.1.3) is a well-
known catalyst in organic synthesis, used both for the
kinetic resolution of a racemic mixture of secondary alco-
hols, as well as in hydrolysis and transesterification.8 The
racemic alcohols 1a–c were subjected to catalyzed enzy-
matic acetylation employing vinyl acetate in diethyl ether
at 40 ꢁC (Table 1). No advantages could be observed under
different reaction conditions, by using vinyl acetate both as
the acyl donor and solvent or performing the resolution at
higher temperatures. Monitoring of the reaction progress
by GC–MS allowed for the determination of the conver-
sion. Samples of racemic acetates 2a–c were prepared as
reference compounds, via acetylation of racemic alcohols
1a–c. Although several different basic conditions were
tested for the reaction with acetyl chloride (TEA, NaH,
Figure 2. 1H NMR of the crude enzymatic reaction.
Table 1. Biocatalytic resolution of allylic alcoholsa 1a–c
Entry
1
Substrate
Products
Conversionb (%)
50
Isolated yieldc (%)
[a]D
eed
Ee
1a
(3S)-1a
(3R)-2a
45
34
ꢀ11.0
>99
>99
>1000
+36.5
2
3
1b
1c
(3S)-1b
(3R)-2b
47
50
45
42
ꢀ8.7
88
>99
37
+27.2
(3S)-1c
(3R)-2c
36
40
ꢀ4.0
>99
>99
>1000
+44.3
a The substrate at 0.2 M concentration in diethyl ether was stirred with 0.5 mass equiv of enzyme, and 5 equiv of vinyl acetate for five days at 40 ꢁC.
b As monitored by GC–MS and by 1H NMR integrals.
c Determined on isolated and purified products.
d Determined by HPLC with a chiral column (see Section 4).
e Values determined using Sih’s method, from the extent of conversion and enantiomeric excess of the recovered substrate as described and checked via
values determined from the enantiomeric excess of the product (Ref. 6).

F. Benfatti et al. / Tetrahedron: Asymmetry 18 (2007) 2227–2232
2229
The racemic alcohols were analytically resolved into their
enantiomers by chiral HPLC and were compared with
the alcohols that were derived from enzymatic resolution.
Representative chromatograms of racemic 1a and (ꢀ)-1a
are reported in Figure 3a and b.
alcohol reacts faster, has received reliable application. On
the basis of the data reported in the literature, we have
attributed the (S)-configuration to the allylic alcohols and
the (R)-configuration to their corresponding acetates. In
fact in our case, the methyl group is unequivocally identi-
fied as the smaller substituent in comparison to the large
one (Fig. 4).
Fast reacting enantiomer
OAc
HO
H
R
R
CO2Et
CO2Et
PCL
LARGE
SMALL
(3R)-2a-c
(3R)-1a-c
OH
HO
H
R
R
CO2Et
CO2Et
(3S)-1a-c
(3S)-1a-c
Figure 4. Stereochemical assignments on the basis of Kazlauskas’ model.
3. Conclusion
In conclusion, optically active ethyl 3-hydroxy-2(10substi-
tuted-methylidene)-butyrates, as well as their acetates, were
obtained in excellent enantiomeric excess via lipase cata-
lyzed enzymatic resolution. P. cepacia lipase was shown
to be the most efficient biocatalyst, allowing us to obtain
these useful intermediates in a high yield and in an enantio-
merically pure form.
Figure 3. HPLC chromatograms of the racemic Z-1a in comparison with
the enantiomerically pure alcohols obtained by enzymatic resolution.
4. Experimental
4.1. General methods
Since the analytical resolution of acetates could not be eas-
ily achieved, acetates (+)-2a–c were hydrolyzed to the cor-
responding alcohols. For this purpose, samples of the
optically active acetates 2a–c were treated with K2CO3 in
methanol (Scheme 4). The enatiomeric excesses of (+)-
1a–c were determined via HPLC by comparison with the
corresponding racemates (Fig. 3c).
All chemicals were purchased from commercial suppliers
and used without further purification. Flash chromatogra-
phy was performed on silica gel (230–400 mesh). NMR
Spectra were recorded with 200 or 300 MHz spectrometers.
Chemical shifts were reported as d values (ppm) relative to
the solvent peak of CDCl3 set at d = 7.27 (1H NMR) or
d = 77.0 (13C NMR). GC–MS analysis were performed
on HP-5 (crosslinked 5% Ph Me silicone, 30 m ·
0.32 mm · 0.25 lm thickness) using an injection program
(initial temperature 50 ꢁC for 20, then 10 ꢁC/min up to
280 ꢁC) in scan mode acquisition. Microwave assisted
reactions were performed with a Milestone Mycrosynth
multimode apparatus, keeping irradiation power fixed
and monitoring internal reaction temperature with a
Built-in ATC-FO advanced fiber optic automatic tempera-
ture control. The reactions were performed in an open ves-
sel, equipped with a refrigerator connected to a fume hood.
The enantiomeric excesses of alcohols were determined by
HPLC analyses performed an HP1100 instrument with
UV–vis detector and equipped with Chiralcel OD column
(25 · 0.46 cm) or Chiralcel OF column (25 · 0.46 cm),
eluted with n-hexane/2-propanol. Optical rotations were
OAc
K2CO3, MeOH
30 min, rt
OH
R
R
90%
CO2Et
CO2Et
(+)-1a-c
ee>99%
(+)-2a-c
Scheme 4.
The stereoselectivity of the lipase prepared from P. cepacia
have been widely studied via models based on a classifica-
tion of the relative size of the substituents (small and large)
at the stereocenter of the secondary alcohol.9 The Kazlaus-
kas’ rule to predict which enantiomer of the secondary

2230
F. Benfatti et al. / Tetrahedron: Asymmetry 18 (2007) 2227–2232
measured in a Perkin Elmer 343 polarimeter using a 1 dm
cuvette and are referenced to the Na-D line value.
1260, 1222, 1176, 1028. 1H NMR (300 MHz, CDCl3) d
1.33 (t, 3H, J = 7.2 Hz), 2.44 (s, 3H), 3.88 (s, 3H), 4.40
(q, 2H, J = 7.2 Hz), 6.93 (d, 2H, J = 7.2 Hz), 7.46 (d, 2H,
J = 7.2 Hz), 7.50 (s, 1H); 13C NMR (200 MHz, CDCl3) d
14.0, 26.2, 55.2, 61.4, 114.2(2), 125.0, 131.6(2), 132.1,
140.9, 161.6, 168.1, 194.6. GC–MS rt 20.30 min, m/z:
248(100), 233(80), 217(21), 203(30), 189(8), 174(24),
161(55), 137(18), 117(9), 89(12).
4.2. Synthesis of ketones 3a–c
Ethyl acetoacetate (10 mmol, 1.3 g, 1.26 mL) was diluted in
the aldehyde (10 equiv, 100 mmol) and piperidine
(0.15 equiv, 1.5 mmol, 0.15 mL) was added in one portion.
The mixture was subjected to microwave irradiation
(Power 500 W) for 7.5 min and then diluted with ethyl ace-
tate (20 mL) and washed twice with 0.1 M HCl (20 mL).
The two phases were separated, the organic layer dried
over Na2SO4, and the solvent removed under reduced pres-
sure. Compounds Z-3a–c and E-3a–c were separated by
flash chromatography on silica gel (cyclohexane/ethyl ace-
tate 90/10 as eluant).
4.3.6. E-3c. Yellow oil; isolated yield 21%; IR (film)
m/cmꢀ1 2979, 2935, 2841, 2739, 1700, 1601, 1577, 1512,
1315, 1258, 1174, 1026. 1H NMR (300 MHz, CDCl3) d
1.31 (t, 3H, J = 7.2 Hz), 2.39 (s, 3H), 3.89 (s, 3H), 4.27
(q, 2H, J = 7.2 Hz), 7.00 (d, 2H, J = 7.2 Hz), 7.61 (s,
1H), 7.83 (d, 2H, J = 7.2 Hz); 13C NMR (200 MHz,
CDCl3) d 14.1, 26.5, 55.4, 61.6, 114.4(2), 125.5, 131.7(2),
132.3, 140.3, 161.5, 168.3, 194.6; GC–MS rt 20.20 min,
m/z: 248(100), 233(88), 217(26), 203(35), 189(12), 174(29),
161(76), 137(31), 117(17), 103(10), 89(31).
4.3. Characterization of ketones 3a–c
4.3.1. Z-3a. Yellow oil; isolated yield 61%; IR (film)
m/cmꢀ1 2967, 2936, 2872, 1731, 1698, 1670, 1467, 1321,
1258, 1212, 1036. H NMR (200 MHz, CDCl3) d 1.07 (d,
4.4. Synthesis of racemic alcohols 1a–c
1
6H, J = 6.6 Hz), 1.31 (t, 3H, J = 7.4 Hz), 2.30 (s, 3H),
2.65 (m, 1H), 4.29 (q, 2H, J = 7.4 Hz), 6.60 (d, 1H,
J = 10.6 Hz); 13C NMR (200 MHz, CDCl3) d 14.1, 21.9
(2), 26.8, 29.6, 61.2, 134.9, 154.0, 166.6, 195.4; GC–MS rt
11.74 min, m/z: 184(2), 138(100), 123(60), 110(15), 96(57),
81(24), 67(32), 55(26).
To a stirred solution of compound Z-3a–c and E-3a–c
(5 mmol) in THF/MeOH 9:1 (10 mL) at room temperature,
CeCl3Æ7H2O (1 equiv, 5 mmol, 1.86 g) and NaBH4
(1.1 equiv, 5.5 mmol, 0.2 g) were added in one portion.
The solution was monitored by TLC and quenched after
disappearance of the starting ketone by the addition of
water (5 mL). After removal of THF and MeOH under
reduced pressure, the residue was diluted with ethyl acetate
(10 mL) and washed twice with water (5 mL). The two
phases were separated, the organic layer was dried over
Na2SO4, and the solvent was removed under reduced pres-
sure. Racemic alcohols Z-1a–c or E-1a–c were isolated by
flash chromatography on silica gel (cyclohexane/ethyl ace-
tate 80/20 as eluant).
4.3.2. E-3a. Yellow oil; isolated yield 26%; IR (film)
m/cmꢀ1 2965, 2933, 2872, 1705, 1638, 1467, 1367, 1239,
1
1200, 1051. H NMR (200 MHz, CDCl3) d 1.02 (d, 6H,
J = 6.6 Hz), 1.30 (t, 3H, J = 7.4 Hz), 2.37 (s, 3H), 2.62
(m, 1H), 4.24 (q, 2H, J = 7.4 Hz), 6.69 (d, 1H,
J = 10.6 Hz); 13C NMR (200 MHz, CDCl3) d 13.9, 21.6,
21.8, 26.5, 29.4, 61.1, 134.8, 154.0, 166.5, 197.9; GC–MS
rt 11.45 min, m/z: 184(2), 138(100), 123(55), 110(15),
96(64), 81(26), 67(31), 55(19).
4.5. Synthesis of racemic acetates 2a–c
4.3.3. Z-3b. Yellow oil; isolated yield 55%; IR (film)
m/cmꢀ1 2981, 2928, 2852, 1731, 1698, 1673, 1635, 1448,
1380, 1305, 1210, 1150, 1035. 1H NMR (200 MHz, CDCl3)
d 1.21–1.43 (m, 5H), 1.34 (t, 3H, J = 7.0 Hz), 1.60–1.77 (m,
5H), 2.31 (s, 3H), 2.37 (m, 1H), 4.30 (q, 2H, J = 7.0 Hz),
6.63 (d, 1H, J = 9.8 Hz); 13C NMR (200 MHz, CDCl3) d
14.1, 25.1(2), 25.3, 26.7, 31.7, 31.8, 39.1, 61.0, 135.2,
152.5, 166.6, 197.9. GC–MS rt 16.60 min, m/z: 224(2),
178(100), 163(60), 149(20), 135(90), 121(29), 107(33),
95(27), 79(30), 67(21), 55(25).
To a solution of compound Z-1 (1 mmol) in dry THF
(5 mL), under an inert atmosphere at 0 ꢁC, LiHMDS
(1.5 equiv, 1.5 mL 1 M solution in THF) was added drop-
wise. The solution was stirred for 30 min and then acetyl
chloride (1.5 equiv, 1.5 mmol, 0.1 mL) was added in one
portion and the ice bath was removed. After 1 h, the mix-
ture was quenched with water (5 mL) and THF removed
under reduced pressure. The residue was diluted with ethyl
acetate (10 mL) and washed twice with water (5 mL). The
two phases were separated, the organic layer was dried over
Na2SO4, and solvent was removed under reduced pressure.
Racemic acetates Z-2a–c were isolated by flash chromato-
graphy on silica gel (cyclohexane/ethyl acetate 90/10 as
eluant).
4.3.4. E-3b. Yellow oil; isolated yield 23%; IR (film)
m/cmꢀ1 2927, 2853, 2360, 2342, 1701, 1654, 1637, 1448,
1
1274, 1253. H NMR (200 MHz, CDCl3) d 1.14–1.26 (m,
5H), 1.27 (t, 3H, J = 7.0 Hz), 1.66–1.77 (m, 5H), 2.38 (s,
3H), 2.42 (m, 1H), 4.26 (q, 2H, J = 7.0 Hz), 6.72 (d, 1H,
J = 10.6 Hz); 13C NMR (200 MHz, CDCl3) d 14.1, 25.0
(2), 25.6, 31.3, 31.9 (2), 38.3, 61.0, 133.9, 152.8, 164.7,
201.3. GC–MS rt 16.40 min, m/z: 224(5), 182(100),
163(9), 135(27), 107(42), 79(27), 67(20), 55(12).
4.6. Lipase catalyzed resolution of alcohols 1a–c
To a solution of racemic alcohol 1 (5 mmol) in diethyl ether
(25 mL) at 40 ꢁC, vinyl acetate (4 equiv, 4 mmol, 0.37 mL)
and lipase from P. cepacia (46 U/mg, 0.2 mass equiv) were
added. The progress of the reaction was assessed every 12 h
by GC–MS. The reaction was stopped by filtration of
the enzyme and elimination of solvent and by-products
4.3.5. Z-3c. Yellow oil; isolated yield 50%; IR (film)
m/cmꢀ1 2980, 2936, 2841, 1726, 1655, 1601, 1513, 1452,

F. Benfatti et al. / Tetrahedron: Asymmetry 18 (2007) 2227–2232
2231
under reduced pressure. Enantiomerically pure alcohols
(3S)-Z-1a–c and acetates (3R)-Z-2a–c were separated by
flash chromatography on silica gel (cyclohexane/ethyl ace-
tate 90/10 then 80/20 as eluant).
(3S)-Z-1b: HPLC on Chiralcel OF (97.5/2.5 n-hexane/
25
2-propanol, flow 1.0 mL/min): rt 17.7 min, ½aꢁD ¼ ꢀ8:7 (c
1.0, CHCl3).
(3R)-Z-1b: HPLC on Chiralcel OF (97.5/2.5 n-hexane/
25
2-propanol, flow 1.0 mL/min): rt 19.3 min, ½aꢁD ¼ þ9:4 (c
4.7. Hydrolysis of enantiomerically pure acetates 2a–c to
enantiomerically pure alcohols 1a–c
1.0, CHCl3).
4.8.4. E-1b. Yellow oil; IR (film) m/cmꢀ1 3423, 2926, 2852,
1691, 1448, 1368, 1263. 1H NMR (200 MHz, CDCl3) d
1.11–1.25 (m, 5H), 1.29 (t, 3H, J = 7.4 Hz), 1.39 (d, 3H,
J = 6.6 Hz), 1.44–1.73 (m, 5H), 2.34 (m, 1H), 4.19 (q,
2H, J = 7.4 Hz), 4.70 (q, 1H, J = 6.6 Hz) 6.50 (d, 1H,
J = 9.8 Hz); 13C NMR (200 MHz, CDCl3) d 14.1, 23.9,
25.4 (2), 25.7, 32.1(2), 37.0, 60.6, 65.1, 132.1, 147.8,
167.7. GC–MS rt 16.81 min, m/z: 226(2), 211(100),
179(18), 165(68), 143(55), 129(38), 119(35), 105(19),
91(32), 81(70), 67(45), 55(43).
Acetate (3R)-Z-2 (0.5 mmol) was stirred in MeOH (5 mL)
in the presence of K2CO3 (1 equiv, 0.5 mmol, 70 mg) for
30 min at room temperature. The reaction was quenched
by the addition of 0.1 M HCl (5 mL). After removal of
MeOH under reduced pressure, the residue was diluted
with ethyl acetate (10 mL) and washed twice with water.
The two phases were separated, the organic layer was dried
over Na2SO4, and solvent was removed under reduced
pressure. Enantiomerically pure alcohols (3R)-Z-1a–c were
purified by flash chromatography on silica gel (cyclohex-
ane/ethyl acetate 80/20 as eluant).
4.8.5. Z-1c. Yellow oil; IR (film) m/cmꢀ1 3448, 2977, 2934,
1718, 1607, 1511, 1458, 1300, 1254, 1193, 1031. H NMR
4.8. Characterization of alcohols 1a–c
1
(200 MHz, CDCl3) d 1.15 (t, 3H, J = 7.0 Hz), 1.41 (d,
3H, J = 6.6 Hz), 3.78 (s, 3H), 4.17 (q, 2H, J = 7.0 Hz),
4.58 (q, 1H, J = 6.6 Hz), 6.84 (d, 2H, J = 8.8 Hz), 6.88 (s,
1H), 7.26 (d, 2H, J = 8.8 Hz); 13C NMR (200 MHz,
CDCl3) d 13.9, 22.4, 55.2, 60.8, 70.2, 113.6(2), 127.8,
129.9 (2), 132.2, 135.5, 159.6, 167.6. GC–MS rt
19.60 min, m/z: 250(21), 232(34), 203(25), 189(75),
159(100), 144(66), 128(21), 115(57), 89(20), 77(18).
4.8.1. Z-1a. Yellow oil; IR (film) m/cmꢀ1 3437, 2964,
2933, 2870, 1717, 1467, 1372, 1228, 1178, 1159. H NMR
1
(200 MHz, CDCl3) d 1.00 (d, 6H, J = 6.6 Hz), 1.24 (m,
6H), 3.10 (m, 1H), 4.23 (q, 2H, J = 6.6 Hz), 4.42 (q, 1H,
J = 6.2 Hz) 5.87 (d, 1H, J = 10.0 Hz); 13C NMR
(200 MHz, CDCl3) d 14.1, 22.4, 22.5, 28.2, 60.3, 69.2,
133.1, 146.9, 167.7. GC–MS rt 11.62 min, m/z: 186(2),
171(36), 144(21), 125(100), 115(20), 97(61), 79(35), 67(25),
55(24).
(3S)-Z-1c: HPLC on Chiralcel OD (98/2 n-hexane/2-pro-
25
panol, flow 1.5 mL/min): rt 24.8 min, ½aꢁD ¼ ꢀ4:0 (c 1.0,
(3S)-Z-1a: HPLC on Chiralcel OF (97.5/2.5 n-hexane/
CHCl3).
25
2-propanol, flow 1.0 mL/min): rt 14.5 min, ½aꢁD ¼ ꢀ11:0
(c 1.0, CHCl3).
(3R)-Z-1c: HPLC on Chiralcel OD (98/2 n-hexane/2-pro-
25
panol, flow 1.5 mL/min): rt 27.1 min, ½aꢁD ¼ þ4:8 (c 1.0,
(3R)-Z-1a: HPLC on Chiralcel OF (97.5/2.5 n-hexane/
CHCl3).
25
2-propanol, flow 1.0 mL/min): rt 16.4 min, ½aꢁD ¼ þ12:4
(c 1.0, CHCl3).
4.8.6. E-1c. Yellow oil; IR (film) m/cmꢀ1 3442, 2976, 2929,
1717, 1607, 1512, 1253, 1178. 1H NMR (200 MHz, CDCl3)
d 1.21 (t, 3H, J = 7.2 Hz), 1.48 (d, 3H, J = 7.2 Hz), 1.90 (br
s, 1H), 3.81 (s, 3H), 4.09 (q, 1H, J = 7.2 Hz), 4.23 (q, 1H,
J = 7.2 Hz), 6.91 (s, 1H), 6.93 (d, 2H, J = 8.4 Hz), 7.33
(d, 2H, J = 8.4 Hz); 13C NMR (200 MHz, CDCl3) d 13.8,
22.3, 55.2, 65.0, 70.2, 113.7(2), 127.6, 128.6, 130.0, 133.0,
135.4, 159.1, 168.0. GC–MS rt 19.70 min, m/z: 250(11),
232(32), 203(25), 189(42), 159(100), 144(68), 128(22),
121(25), 115(60), 89(17), 77(12).
4.8.2. E-1a. Yellow oil; IR (film) m/cmꢀ1 3452, 2963, 2871,
1693, 1641, 1466, 1368, 1261, 1176, 1190. 1H NMR
(200 MHz, CDCl3) d 1.03 (d, 3H, J = 6.6 Hz), 1.06 (d,
3H, J = 6.6 Hz), 1.24 (t, 3H, J = 7.2 Hz), 1.42 (d, 3H,
J = 6.6 Hz), 2.77 (m, 1H), 4.22 (q, 2H, J = 7.2 Hz), 4.72
(m, 1H), 6.52 (d, 1H, J = 10.2 Hz); 13C NMR (200 MHz,
CDCl3) d 14.1, 22.2(2), 23.8, 27.1, 60.4, 65.0, 131.7,
149.2, 167.5. GC–MS rt 11.80 min, m/z: 186(2), 171(64),
143(27), 125(100), 115(11), 97(46), 79(20), 67(18), 55(15).
4.8.3. Z-1b. Yellow oil; IR (film) m/cmꢀ1 3438, 2926,
2851, 1714, 1448, 1373, 1303, 1263, 1208, 1178, 1153. H
4.9. Characterization of acetates 2a–c
1
NMR (200 MHz, CDCl3) d 1.01–1.36 (m, 5H), 1.25 (t,
3H, J = 7.0 Hz), 1.25 (d, 3H, J = 6.6 Hz), 1.60–1.73 (m,
5H), 2.30 (br s, 1H), 2.75 (m, 1H), 4.24 (q, 2H,
J = 7.0 Hz), 4.41 (q, 1H, J = 6.6 Hz), 5.88 (d, 1H,
J = 10.0 Hz); 13C NMR (200 MHz, CDCl3) d 14.0, 22.5,
25.4, 25.7, 25.9, 32.4, 32.5, 37.9, 60.2, 69.2, 133.4, 145.6,
167.7. GC–MS rt 16.40 min, m/z: 226(2), 208(38),
179(33), 162(100), 147(28), 133(51), 119(37), 107(20),
99(29), 91(36), 81(87), 67(38), 55(34).
4.9.1. (3R)-Z-2a. Yellow oil; IR (film) m/cmꢀ1 2963, 1644,
1466, 1241, 1178, 1097. 1H NMR (300 MHz, CDCl3) d 1.05
(d, 6H, J = 6.6 Hz), 1.34 (m, 6H), 2.08 (s, 3H), 3.15 (m,
1H), 4.26 (q, 2H, J = 7.2 Hz), 5.65 (q, 1H, J = 6.6 Hz)
5.91 (d, 1H, J = 9.9 Hz); 13C NMR (300 MHz, CDCl3) d
14.1, 19.8, 21.2, 22.4, 22.5, 28.2, 60.4, 69.9, 130.5, 147.9,
169.9,170.5. GC–MS rt 12.89 min, m/z: 213(2), 185(24),
168(35), 143(100), 123(57), 95(83), 79(54), 67(35), 55(28).
25
½aꢁD ¼ þ36:5 (c 1.0, CHCl3).

2232
F. Benfatti et al. / Tetrahedron: Asymmetry 18 (2007) 2227–2232
4.9.2. (3R)-Z-2b. Yellow oil; IR (film) m/cmꢀ1 2926, 2852,
1690, 1448, 1368, 1303, 1263, 1222, 1155, 1064. H NMR
References
1
1. Morgan, B.; Oehlschlager, A. C.; Stokes, T. M. J. Org. Chem.
1992, 57, 3231–3236.
2. (a) Ghanem, A. Tetrahedron 2007, 63, 1721–1754; (b) Faber,
K. In Biotransformations in Organic Chemistry: A Textbook;
Springer: Berlin, 2004; (c) Schmidt, R. D.; Verger, R. Angew.
Chem., Int. Ed. 1998, 37, 1608–1633.
(200 MHz, CDCl3) d 0.89–1.25 (m, 5H), 1.26 (t, 3H,
J = 7.0 Hz), 1.33 (d, 3H, J = 6.6 Hz), 1.55–1.77 (m, 5H),
2.02 (s, 3H), 2.80 (m, 1H), 4.19 (q, 2H, J = 7.0 Hz), 5.60
(q, 1H, J = 6.6 Hz), 5.88 (d, 1H, J = 10.0 Hz); 13C NMR
(200 MHz, CDCl3) d 14.1, 19.9, 21.2(2), 25.5(2), 26.0,
32.4, 32.6, 37.8, 60.2, 69.9, 130.8, 146.7, 166.4, 170.1. GC–
MS rt 17.40 min, m/z: 268(2), 225(18), 208(55), 179(63),
3. (a) Burgess, K.; Jennings, L. D. J. Am. Chem. Soc. 1991, 113,
6129–6139; (b) Burgess, K.; Henderson, I. Tetrahedron:
Asymmetry 1990, 1, A7–A13; (c) Burgess, K.; Cassidy, J.;
Henderson, I. J. Org. Chem. 1991, 56, 2050–2058; (d) Burgess,
K.; Jennings, L. J. Org. Chem. 1990, 55, 1138–1139; (e)
Kalaritis, P.; Regenye, R. W.; Partridge, J. J.; Coffen, D. L. J.
Org. Chem. 1990, 55, 812–815; (f) Chadha, A.; Manohar, M.
Tetrahedron: Asymmetry 1995, 6, 651–652.
4. (a) Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250–6284;
(b) Kappe, C. O.; Stadler, A. In Microwaves in Organic and
Medicinal Chemistry; Mannhold, R., Kubinyi, H., Folkers, G.,
Eds.; Methods and Principles in Medicinal Chemistry; Wiley-
VCH: Weinheim, 2005; Vol. 25, (c) Hayes, B. L. Aldrichim.
Acta 2004, 37, 66–76; (d) Hayes, B. L.. Microwave Synthesis:
Chemistry at the Speed of Light, 1st ed.; CEM: Matthews, NC,
2002.
162(100), 151(10), 143(95), 133(57), 119(35), 105(33),
25
91(41), 81(84), 67(38), 55(31). ½aꢁD ¼ þ27:2 (c 1.0, CHCl3).
4.9.3. (3R)-Z-2c. Yellow oil; IR (film) m/cmꢀ1 2981, 2936,
1735, 1718, 1618, 1607, 1511, 1458, 1370, 1254, 1178, 1055,
1029. 1H NMR (200 MHz, CDCl3) d 1.17 (t, 3H,
J = 7.4 Hz), 1.48 (d, 3H, J = 6.6 Hz), 2.05 (s, 3H), 3.79
(s, 3H), 4.17 (q, 2H, J = 7.4 Hz), 5.68 (q, 1H,
J = 6.6 Hz), 6.77 (s, 1H), 6.82 (d, 2H, J = 9.2 Hz), 7.23
(d, 2H, J = 9.2 Hz); 13C NMR (200 MHz, CDCl3) d 14.2,
20.2, 21.5, 55.5, 61.1, 71.7, 113.9(2), 127.6, 129.8(2),
130.4, 133.6, 160.0, 168.2, 170.2 GC–MS rt 20.60 min,
5. Luche, J.-C. J. Am. Chem. Soc. 1978, 100, 2226–2227.
6. Chen, C.-S.; Wu, S.-H.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1987, 109, 2812–2817.
7. Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114–120.
8. Ferraboschi, P.; Santaniello, E. Meth. Biotech. 2001, 15, 291–
305.
m/z: 292(3), 249(6), 232(21), 203(49), 187(19), 159(100),
25
144(62), 128(22), 115(59), 89(14), 77(12). ½aꢁD ¼ þ44:3 (c
1.0, CHCl3).
9. (a) Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.;
Cuccia, L. A. J. Org. Chem. 1991, 56, 2656–2665; (b) Schulz,
T.; Pleiss, J.; Schmidt, R. D. Protein Sci. 2000, 9, 1053–1062;
(c) Zuegg, J.; Ho¨nig, H.; Schrag, J. D.; Cygler, M. J. Mol.
Catal. B: Enzym. 1997, 83–98; (d) Rotticci, D.; Orrenius, C.;
Hult, K.; Norin, T. Tetrahedron: Asymmetry 1997, 8, 359–362;
(e) Shimizu, M.; Kawanami, H.; Fujisawa, T. Chem. Lett.
1992, 107–110.
Acknowledgments
We thank MIUR (PRIN 2006), CNR-ISOF, and the Uni-
versity of Bologna (funds for selected topics) for financial
support.