Lipase behavior in the stereoselective transesterification of zingerol-like derivatives and related biphenyls

Article abstract of DOI:10.1016/j.molcatb.2013.01.007

Using a highly stereoselective enzymatic procedure based on the irreversible transesterification of alcoholic functions in organic solvent, both enantiomers of zingerol and dehydrozingerol O-methyl derivatives 1 and 2 were obtained in high optical purity. The biocatalytic method was then extended to the corresponding biphenyl derivatives 5-8 for which an one-pot resolution/desymmetrization was carried out on the whole racemic/meso mixtures to give single stereoisomers with very high enantiomeric and diastereoisomeric excesses. The comparison of kinetic and enantioselective behavior of the lipase AK from Pseudomonas fluorescens in the transesterification of monomer (±)-1 and the related (±)-5 and meso-6 biphenyl dimers was also made.

Full text of DOI:10.1016/j.molcatb.2013.01.007

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Lipase behavior in the stereoselective transesterification of zingerol-like
derivatives and related biphenyls
Claudia Sanfilippoa,∗, Angela Pattia, Maria Antonietta Dettorib, Davide Fabbrib, Giovanna Delogub
a CNR – Istituto di Chimica Biomolecolare, Via Paolo Gaifami 18, I-95127 Catania, Italy
b CNR – Istituto di Chimica Biomolecolare, Traversa la Crucca 3, Regione Baldinca, I-07100 Sassari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Using a highly stereoselective enzymatic procedure based on the irreversible transesterification of
alcoholic functions in organic solvent, both enantiomers of zingerol and dehydrozingerol O-methyl
derivatives 1 and 2 were obtained in high optical purity. The biocatalytic method was then extended
to the corresponding biphenyl derivatives 5–8 for which an one-pot resolution/desymmetrization was
carried out on the whole racemic/meso mixtures to give single stereoisomers with very high enantiomeric
and diastereoisomeric excesses. The comparison of kinetic and enantioselective behavior of the lipase
AK from Pseudomonas fluorescens in the transesterification of monomer ( )-1 and the related ( )-5 and
meso-6 biphenyl dimers was also made.
Received 28 November 2012
Received in revised form 9 January 2013
Accepted 10 January 2013
Available online 11 February 2013
Keywords:
Lipase
Kinetic resolution
Desymmetrization
Zingerol derivative
Biphenyl
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
from the needless of himalayan Taxus baccata [16] and, recently, as
phenolic glucoside from leaves of Staphylea bumalda [17].
Many natural phenols are good antioxidants and have
a
The bioactivity of natural and unnatural compounds is strongly
dependent from stereochemistry, thus justifying the interest in
the implementation of enantioselective preparation of optically
active products, which includes asymmetric synthesis and kinetic
resolution of racemates. In this context enzyme catalysis is an
useful methodology to achieve enantiopure chiral drugs and lipase-
catalyzed resolution of racemic mixtures is a attractive procedure
thanks to large substrate acceptance and chemo-, regio- and enan-
tioselectivity of the catalyst at mild conditions of temperature and
pressure.
By using anhydrous organic solvents as reaction medium the
“natural” activity of lipases can be reversed so allowing their appli-
cation in the stereoselective transesterification of alcohols or acids
and in addition, the modification of the solvent (solvent engineer-
ing) influences the enzyme performances [18–21].
wide spectrum of biological activity related with antibacte-
rial, anticancer, anti-inflammatory and antihypertensive effects.
Curcuminoids and their structural analogs show several pharma-
cological and nutritional properties [1–3] and many of them have
been isolated from the rhizome extracts of Zingiberaceae as Cur-
cuma longa and zingiber. Zingerone and dehydrozingerone, key
components of ginger, appeared to be useful as antioxidant in the
treatment of Parkinson’s disease [4], radioprotector against gamma
radiation-induced damages in the cancer treatment [5,6], antidiar-
rheal [7,8], anticancer [9,10] and anti-inflammatory agents [11,12].
Biphenyl analogs of zingerone and dehydrozingerone, 3 and 4
display protective action in the oxidative cells stress (unpublished
results) and a marked inhibition in the cell-growth of melanoma
and neuroblastoma has been reported for 4 [13].
The related alcohols, zingerol and dehydrozingerol, have been
less investigated in their pharmacological properties but therapeu-
tic effects similar to those of the zingerone in the diarrhea treatment
have been demonstrated for zingerol [7]. In their racemic form
these alcohols are easily obtainable by chemical reduction of the
parent keto derivatives [14,15], while only a case exists in the lit-
erature in which the optically active (R)-zingerol has been isolated
Although the most of applications of the lipase catalysis in asym-
metric synthesis are found in the kinetic resolution of racemic
mixtures, the desymmetrization of meso- or prochiral substrates
has become very common due to two main factors: (1) the theoret-
ical yield of these reactions is 100% and (2) the enantioselectivity of
the process is often high and independent from substrate conver-
sion. A large part of lipase-catalyzed desymmetrizations involves
the transesterification of meso-diols and effective protocols applied
to different families of substrates have been developed for the
preparation of optically active building blocks useful in medicinal
chemistry or asymmetric synthesis (Fig. 1) [22].
∗
Corresponding author. Tel.: +39 957338330.
E-mail address: claudia.sanfilippo@icb.cnr.it (C. Sanfilippo).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.007

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C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
Fig. 1. Families of meso-alcohols desymmetrized by lipase-catalyzed reactions.
In the course of our continuous research on the preparation of
detected by 254-nm UV light. Preparative liquid chromatography
was performed using LiChroprep® Si 60 (25–40 ␮m) from Merck.
1H and 13C NMR spectra were recorded in CDCl3, unless other-
wise specified, on a Bruker AvanceTM 400 instrument at 400.13 and
100.03 MHz respectively. Chemicals shifts (ı) are reported in ppm
relative to TMS and coupling constants (J) are given in Hz. Opti-
cal rotations were recorded on a DIP 370 JASCO instrument using
a ␾ 3.5 mm × 100 mm cell. The enantiomeric excesses were deter-
mined by chiral HPLC at 25 ◦C on Phenomenex Lux® Cellulose-1
or Phenomenex Lux® Cellulose-2 (250 × 4.60 mm) column using
n-hexane/2-propanol isocratic mixtures as eluting solvent and
simultaneous UV-detection at ꢀ 220, 254, 266 and 300 nm. ESI+-MS
spectra were acquired on Waters Micromass ZQ2000 instrument (5,
10 or 20 V cone voltage, 150 ◦C source temperature).
new chiral alcohols in high enantiomeric purity through biocat-
alyzed reactions, we have recently reported a one-pot protocol
for the simultaneous resolution and desymmetrization of a 1:1
dl:meso mixture of conformationally flexible 1,1ꢀ-bipyridine diol as
the first example of lipase-promoted desymmetrization of a biaryl
diol [23]. The success of such protocol was strictly related to the
high enantioselectivity of the employed lipase that allowed the
selective formation of three separable products each one as a single
stereoisomer.
Herein we describe the results obtained in the kinetic resolu-
tion of racemic zingerol ( )-1 and dehydrozingerol ( )-2 O-methyl
derivatives with lipase AK from Pseudomonas fluorescens and, in
light of the high stereoselectivity evidenced for the enzyme, the
extension of the process to the related racemic and meso biphenyl
dimers ( )-5/meso-6 and ( )-7/meso-8 respectively (Fig. 2). Addi-
tional experiments have been also performed to compare the
behavior of the lipase in the two processes of kinetic resolution
of ( )-1 and ( )-5 and desymmetrization of meso-6.
2.2. Synthesis of ( )-1
Methylation of commercial zingerone, 4-(4-hydroxy-3-metho-
xyphenyl)butan-2-one, was performed by addition of K2CO3
(0.5 mmol, 69 mg) and CH3I (0.186 ml, 3 mmol,) to a 5 ml ace-
tone solution of substrate (185 mg, 0.95 mmol). The reaction was
refluxed overnight, cooled and taken to dryness under vacuum.
The residue was dissolved in CH2Cl2 and extracted with water. The
organic phase was washed with a saturated solution of NH4Cl and
dried over sodium sulphate. Removal of the solvent gave a white
solid (95% yield, 0.94 mmol, 195 mg) that was dissolved in MeOH
(5 ml). To this solution NaBH4 (71 mg, 1.9 mmol) was added and the
mixture left to react at room temperature for 3 h under stirring.
The reaction was then quenched by addition of NH4Cl and
extracted with CH2Cl2. The organic phase was then washed with
brine and dried on anhydrous sodium sulfate. The solvent was evap-
orated under vacuum to give title compound as a transparent oil
(0.92 mmol, 193 mg, 97% yield). 1H NMR ı 1.15 (d, J = 6.4, 3H), 1.67
(m, 2H), 2.58 (m, 2H), 2.67 (d, J = 5.6, 1H), 3.74 (m, 7H), 6.68 (m, 3H);
2. Experimental
2.1. General information
Lipase PS-C I (lipase from Pseudomonas cepacia immobilized
on ceramic), Novozyme 435® (lipase from Candida antarctica
immobilized on acrylic resin) and Amano Lipase AK (crude lipase
from P. fluorescens) were purchased from Aldrich. Lipozyme®
(lipase from Mucor miehei immobilized on a macroporous ion-
exchange resin) was obtained from Fluka. Compounds 3 and
4 were prepared according to previously described procedures
[13,24].
Thin-layer chromatography (TLC) was carried out on Merck sil-
ica gel 60-F254 precoated glass plates and the compounds were
Fig. 2. Zingerol and dehydrozingerol derivatives.

C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
109
13C NMR ı 23.50, 31.70, 40.99, 55.74, 55.86, 67.21, 111.36, 111.82,
120.16, 134.87, 147.08, 148.79. ESI-MS (+) 20 V: m/z 211.7 [M+H]+;
233.6 [M+Na]+ (100); 442.9 [2M+Na]+.
J = 6.8 and 6.4), 6.04 (dd, 1H, J = 16.0 and 6.8), 6.53 (d, 1H, J = 16.0),
6.79 (d, 1H, J = 8.2), 6.90 (m, 2H); 13C NMR ı 20.70, 21.69, 56.09,
56.17, 71.44, 109.14, 111.36, 120.18, 127.09, 129.64, 131.78, 149.30,
149.36, 170.63. ESI-MS (+) 20 V: m/z 190.7 (100), 251.6 [M+H]+,
272.4 [M+Na]+, 312.4, 522.8 [2M+Na]+. Chiral HPLC conditions:
Phenomenex Lux® Cellulose-1, 85:15 hexane/2-propanol, flow rate
0.5 ml/min, T 23 ◦C, 13.19 min (R enantiomer) and 15.39 min (S
enantiomer).
2.3. Synthesis of ( )-2
Commercial dehydrozingerone, 4-(4-hydroxy-3-methoxyp-
henyl)-3-buten-2-one, (1.04 mmol, 200 mg) was subjected to
methylation and successive reduction using the same procedure
above described for ( )-1 to give ( )-2 as a colorless oil with a
82% global yield (0.86 mmol, 178 mg). 1H NMR ı 1.34 (d, J = 6.4,
3H), 1.97 (bs, 1H), 3.84 (s, 3H), 3.86 (s, 3H), 4.42 (dt, 1H, J = 6.5 and
6.4), 6.10 (dd, 1H, J = 15.8 and 6.5), 6.46 (d, 1H, J = 15.8), 6.78 (d, 1H,
J = 8.2), 6.88 (m, 2H); 13C NMR ı 23.7, 56.0, 56.1, 68.2, 109.0, 111.4,
119.9, 129.4, 130.0, 131.9, 149.0, 149.2. ESI-MS (+) 20 V: m/z 190.7
(100); 230.9 [M+Na]+, 420.7, 454.8.
Alcohol (−)-2: [␣]D25 = –30.53 (c = 1.6, CHCl3). Chiral HPLC con-
ditions: Phenomenex Lux® Cellulose-1, 85:15 hexane/2-propanol,
flow rate 0.5 ml/min, T 23 ◦C, 22.41 min (S enantiomer) and
24.07 min (R enantiomer).
2.7. Synthesis of ( )-5/meso-6
To a solution of biphenyl 3 (400 mg, 1 mmol) in acetone
(7 ml) K2CO3 (69 mg, 0.5 mmol) and CH3I (0.186 ml, 3 mmol) were
added and the mixture left to react overnight under reflux. After
addition of saturated NH4Cl solution and extraction with CH2Cl2
the organic phase was dried on anhydrous sodium sulfate and
evaporated under reduced pressure. The residue was dissolved in
methanol (5 ml) and to the solution an excess of NaBH4 was added.
The reaction was stirred for 3 h at room temperature and then,
after addition of saturated solution of NH4Cl, extracted with CH2Cl2
which was washed with brine and dried on anhydrous sodium sul-
fate. After removal of the solvent, the residue was purified on silica
gel column to give a 1:1 mixture of ( )-5 and meso-6 as pale yellow
oil (376 mg, 0.90 mmol, 96% overall yield). 1H NMR ı 1.18 (d, J = 6.0,
6H), 1.74 (m, 4H), 2.29 (bs, 2H), 2.64 (m, 4H), 3.58 (s, 6H), 3.79
(m, 2H), 3.84 (s, 6H), 6.67 (d, 2H, J = 1.6), 6.73 (d, 2H, J = 1.6); 13C
NMR ı 23.7, 32.1, 40.9, 55.9, 60.7, 67.5, 112.0, 123.1, 132.6, 137.4,
144.9, 152.6. ESI-MS (+) 20 V: m/z 440.0 (100) [M+Na]+, 490.1, 646.9,
859.3 [2M+Na]+. Chiral HPLC (Phenomenex Lux® Cellulose-1, 90:10
hexane/2-propanol, flow rate 0.5 ml/min, T 23 ◦C): 41.9 min (R,R
enantiomer), 43.2 min (R,S meso-isomer) and 44.7 min (S,S enan-
tiomer).
2.4. General procedure for enzymatic transesterification of ( )-1
and ( )-2
To a solution of the suitable substrate (0.05 mmol) in tert-butyl
methyl ether (t-BME) (2 ml) lipase of choice (20 mg) and vinyl
acetate (0.2 mmol) were added. The mixture was incubated in a
shaker (250 rpm) at 28 ◦C and the progress of reaction was mon-
itored by chiral HPLC analysis to check the substrate conversion
and enantiomeric purities of both unreacted alcohol and formed
acetate.
2.5. Preparative enzymatic resolution of ( )-1
Lipase AK (200 mg) and vinyl acetate (0.1 ml, 1.08 mmol) were
added to a t-BME solution (10 ml) of ( )-1 (100 mg, 0.48 mmol).
Reaction mixture was stirred for 2.5 h at 28 ◦C and 250 rpm until
50% of substrate conversion was reached, then the enzyme was fil-
tered and the organic solution taken to dryness. The alcohol residue
and the acetate were isolated by silica gel chromatography eluting
with hexane/ethyl acetate 60:40 vol/vol mixture.
Acetate (+)-1a was obtained as a colorless oil in 46% yield (52 mg,
0.22 mmol) and ee =98%: [␣]D25 = +10.46 (c = 1.4, CHCl3). 1H NMR ı
1.21 (d, J = 6.3, 3H), 1.74 (m, 1H), 1.89 (m, 1H), 2.01 (s, 3H), 2.56 (m,
2H), 3.81 (s, 3H), 3.84 (s, 3H), 4.90 (dt, J = 6.5 and 6.3 1H), 6.67 (m,
2H), 6.76 (d, 1H, J = 8.8); 13C NMR ı 20.25, 21.53, 31.61, 37.97, 56.01,
56.12, 70.65, 111.63, 111.92, 120.32, 134.35, 147.48, 149.09, 170.89.
ESI-MS (+) 20 V: m/z 232.6, 274.8 (100) [M+Na]+, 306.5. Chiral
HPLC conditions: Phenomenex Lux® Cellulose-2, 85:15 hexane/2-
propanol, flow rate 1 ml/min, T 23 ◦C, 8.30 min (R enantiomer) and
9.18 min (S enantiomer).
Alcohol (+)-1 was obtained in 47% yield (46 mg, 0.23 mmol) and
ee > 98%: [␣]D25 = +13.06 (c = 1.1, CHCl3). Chiral HPLC conditions:
Phenomenex Lux® Cellulose-2, 85:15 hexane/2-propanol, flow rate
1 ml/min, T 23 ◦C, 12.68 min (R enantiomer) and 13.52 min (S enan-
tiomer).
2.8. Synthesis of ( )-7/meso-8
Biphenyl 4 (340 mg, 0.89 mmol) was methylated and subse-
quently reduced using the same conditions above described for the
synthesis of ( )-5 and meso-6 from 3. A 1:1 mixture of ( )-7 and
meso-8 was obtained in 88% overall yield (327 mg, 0.79 mmol). 1
H
NMR ı 1.31 (d, J = 6.4, 6H), 3.61 (s, 6H), 3.87 (s, 6H), 4.43 (dq, 2H,
J = 6.4 and 6.4), 6.15 (dd, 2H, J = 16.0 and 6.4), 6.46 (d, 2H, J = 16.0),
6.83 (bs, 2H), 6.92 (bs, 2H); 13C NMR ı 23.3, 55.7, 60.6, 68.7, 109.3,
121.6, 128.8, 132.1, 132.4, 132.9, 146.4, 152.6. ESI-MS (+) 10 V: m/z
228.9 268.1, 288.9, 437.5 [M+Na]+. Chiral HPLC (Phenomenex Lux®
Cellulose-1, 85:15 n-hexane/2-propanol, flow rate 0.5 ml/min, T
23 ◦C): 39.4 min (R,R enantiomer), 43.0 min (R,S meso-isomer) and
49.1 min (S,S enantiomer).
2.9. Enzymatic resolution and desymmetryzation of ( )-5 and
meso-6 mixture.
2.6. Preparative enzymatic resolution of ( )-2
To a t-BME (10 ml) solution of ( )-2 (100 mg, 0.48 mmol) lipase
AK (200 mg) and vinyl acetate (0.1 ml, 1.08 mmol) were added. The
suspension was shaken at 250 rpm and 28 ◦C temperature for 10 h
and then the reaction was stopped at 50% of substrate conversion
by filtering off the lipase. After removal of the solvent the residue
was purified by chromagraphy on silica gel column eluting with
hexane/ethyl acetate 60:40 vol/vol mixture to give acetate (+)-2a
(55 mg, 0.22 mmol, 45% yield, ee = 97%) and alcohol (−)-2 (44 mg,
0.21 mmol, 45% yield, ee > 98%) as colorless oils.
To a solution of ( )-5 and meso-6 (150 mg, 0.36 mmol) in t-BME
(15 ml) lipase AK (300 mg) and vinyl acetate (0.15 ml, 1.6 mmol)
were added The reaction was kept under stirring at 280 rpm and
28 ◦C of temperature for 6 h when 76% of substrate conversion was
reached. After removal of the enzyme by filtration, the solvent was
evaporated and the residue chromatographed on silica gel eluting
with 60/40 vol/vol n-hexane/ethyl acetate mixture.
Diacetyl derivative (+)-5b was the first eluted product and was
obtained as pale yellow oil in a 22% yield (40 mg, 0.08 mmol),
ee > 98% and de = 97%: [␣]D25 = +13.2 (c = 1.5, CHCl3). 1H NMR ı 1.24
(d, J = 6.0, 6H), 1.82 (m, 2H), 1.92 (m, 2H), 2.02 (s, 6H), 2.60 (m, 4H),
Acetate (+)-2a: [␣]D25 = +127.65 (c = 1.4, CHCl3). 1H NMR ı 1.39
(d, J = 6.4, 3H), 2.05 (s, 3H), 3.85 (s, 3H), 3.88 (s, 3H), 5.49 (dq, 1H,

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C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
3.61 (s, 6H), 3.88 (s, 6H), 4.94 (m, 2H), 6.66 (d, 2H, J = 2.0), 6.71 (d,
2H, J= 2.0); 13C NMR ı 20.3, 21.6, 31.9, 37.8, 56.1, 60.9, 70.9, 112.1,
123.1, 132.8, 136.9, 145.2, 152.8, 171.1. ESI-MS (+) 5 V: m/z 525
[M+Na]+, 576 (100). Chiral HPLC (Phenomenex Lux® Cellulose-1,
90:10 hexane/2-propanol, flow rate 0.5 ml/min, T 23 ◦C): 16.9 min
(R,R enantiomer), 18.7 min (R,S meso-isomer) and 20.8 min (S,S
enantiomer).
Monoacetyl derivative (+)-6a was obtained as pale yellow oil
in 46% yield (74 mg, 0.16 mmol) and ee and de > 98%. [␣]D25 = +10.1
(c = 1.1, CHCl3). 1H NMR ı 1.20 (d, J = 6.2, 3H), 1.23 (d, J = 6.2, 3H,),
1.74 (m, 3H), 1.93 (m, 1H), 2.01 (s, 3H), 2.64 (m, 4H), 3.60 (s, 6H),
3.84 (m, 1H), 3.87 (s, 6H), 4.93 (m, 1H), 6.66 (d, 1H, J = 1.8), 6.69 (d,
1H, J = 1.8), 6.71 (d, 1H, J = 1.8), 6.74 (d, 1H, J = 1.8); 13C NMR ı 20.3,
21.6, 23.8, 31.9, 32. 3, 37.8, 41.1, 56.1, 60.7, 60.9, 67.7, 70.9, 112.0,
112.1, 123.1, 123.2, 132.7, 132.9, 136.8, 137.5, 145.1, 145.2, 152.8,
171.1. ESI-MS (+) 5 V: m/z 440 (100), 860. Chiral HPLC (Phenomenex
Lux® Cellulose-1, 90:10 hexane/2-propanol, flow rate 0.5 ml/min,
T 23 ◦C): 25.8 min (R-acetoxy-S-hydroxy enantiomer) and 29.2 min
(S-acetoxy-R-hydroxy enantiomer). To determine the diastereoiso-
meric excess of monoacetate (+)-6a, the isolated product was
acetylated by standard procedure using pyridine and acetic anhy-
dride, and the obtained diacetyl derivative was analyzed by chiral
HPLC.
The unreacted diol (−)-7 was recovered in 18% yield (37 mg,
0.09 mmol), ee = 93% and de = 97%. [␣]D25 = −14.6 (c = 0.4, CHCl3).
2.11. Kinetic measurements
Three separate solutions of ( )-1 (10 mg, 0.048 mmol), ( )-5
(10 mg, 0.024 mmol) and meso-6 (10 mg, 0.024 mmol) in t-BME
(2 ml) were prepared. To each solution lipase AK (40 mg) and vinyl
acetate (0.2 mmol) were added and the mixtures left under stirring
at 250 rpm and 28 ◦C. The progress of reactions was monitored in
parallel by chiral HPLC analysis to check the substrate conversion
and enantiomeric purity.
3. Results and discussion
3.1. Lipase-catalyzed kinetic resolution of zingerol ( )-1 and
dehydrozingerol ( )-2 derivatives
Racemic ( )-1 and ( )-2, respectively zingerol and dehy-
drozingerol O-methyl derivatives, were obtained from carbonyl
reduction of commercial zingerone and dehydrozingerone, whose
phenolic hydroxyl groups had been methylated to improve the
solubility and chemical stability. The reduction with NaBH4 in
methanol at room temperature allowed the selective formation of
alcohols in quantitative yield, the double bond of dehydrozingerone
being unaffected. Structures of ( )-1 and ( )-2 were confirmed by
1H-NMR analysis on the basis of methinic resonances at 3.74 and
4.42 ppm, respectively, and a double doublet at 6.10 ppm coupled
with a doublet at 6.46 ppm accounting for the ethylenic protons in
dehydrozingerol.
In a preliminary phase of the study a screening of some com-
mercially available lipases was performed to compare the degree of
selectivity and reactivity of the catalysts. Among the lipases tested
for transesterification of ( )-1 and ( )-2, PS-C I from P. cepacia,
AK from P. fluorescens, Lipozyme from M. miehei and Novozym 435
from C. antarctica were found more selective in the kinetic res-
olutions. All the reactions were carried out at 28 ◦C, selected as
optimal reaction temperature, in tert-butyl methyl ether (t-BME)
using vinyl acetate (VOAc) as irreversible acyl donor (Scheme 1).
From the obtained data (Table 1) it was evident that the immobi-
lized lipases from P. cepacia, M. miehei and C. antarctica were highly
sensitive, in terms of stereoselectivity as well as reaction rate, to the
presence of unsaturation in ˛ to the stereogenic center whereas the
crude lipase AK maintained excellent levels of stereoselectivity for
both substrates.
Taking into the account the good results obtained in the
screening, lipase AK was chosen as catalyst for kinetic resolution
of ( )-1 in preparative scale. The reaction was monitored by HPLC
and after 2.5 h was stopped when a 50% of substrate conversion
was measured. The alcohol (+)-1 and acetate (+)-1a were recovered
by silica gel chromatography in high yields and ee = 98% and >98%
respectively. The acetylation of substrate was confirmed because of
the presence, in the 1H-NMR spectrum of (+)-1a, of a singlet reso-
nance at 2.01 ppm associated with the acetyl group and a downfield
shifted resonance (4.90 ppm) for the methinic proton. Alcohol (+)-
1 was assigned to S absolute configuration by comparison of the
sign of its optical rotation with literature data previously reported
for the related natural zingerol [16] and further support came from
the observed reaction outcome in a lipase AK-catalyzed acetylation
of ( )-zingerol, that gave unreacted (+)-S-zingerol and the corre-
sponding R-ester both in optically active form.
Diol (+)-5, the last eluted product, was recovered in a 21% yield
(32 mg, 0.07 mmol) and ee > 98%. [␣]D25 = +10.2 (c = 1.2, CHCl3).
2.10. Enzymatic resolution and desymmetryzation of ( )-7 and
meso-8 mixture
To a solution of ( )-7 and meso-8 mixture (200 mg, 0.48 mmol)
in t-BME (20 ml) lipase AK (400 mg) and vinyl acetate (0.2 ml,
2 mmol) were added. The reaction was shaked at 28 ◦C and
250 rpm until the substrate conversion reached 79% after 34 h.
After lipase filtration and evaporation of the solvent, the residue
was chromatographed on silica gel, eluting with 60/40 vol/vol n-
hexane/ethyl acetate mixture. Diacetyl derivative (+)-7b, the first
eluted product, was recovered as pale yellow oil in 18% yield
(43 mg, 0.08 mmol) and 95% ee: [␣]D25 = +98.4 (c = 0.4, CHCl3). 1H
NMR ı 1.39 (d, J = 6.4, 6H), 2.06 (s, 6H), 3.64 (s, 6H), 3.91 (s,
6H), 5.50 (dq, J = 6.8 and 6.4, 2H), 6.10 (dd, J = 16.0 and 6.8, 2H),
6.55 (d, J = 16.0, 2H), 6.88 (d, J = 1.6, 2H), 6.94 (d, J = 1.6, 2H); 13C
NMR ı 20.7, 21.7, 56.2, 61.1, 71.3, 110.0, 122.1, 128.5, 131.6,
132.1, 132.9, 153.1, 170.7. ESI-MS (+) 10 V: m/z 437.3, 479.2, 521.4
[M+Na]+ (100). Chiral HPLC (Phenomenex Lux® Cellulose-1, 85:15
n-hexane/2-propanol, flow rate 0.5 ml/min, T 23 ◦C): 17.0 min (R,R
enantiomer), 19.9 min (R,S meso-isomer) and 23.9 min (S,S enan-
tiomer).
The monoacetyl derivative (+)-8a was obtained as yellow oil
in 38% yield (83 mg, 0.18 mmol) and 94% ee. [␣]D25 = +48.6 (c = 0.6,
CHCl3). 1H NMR ı 1.36 (d, J = 6.4, 3H), 1.39 (d, J = 6.4, 3H), 2.06 (s,
3H), 3.64 (s, 3H), 3.65 (s, 3H), 3.91 (s, 6H), 4.47 (dq, 1H, J = 6.8 and
6.4), 5.50 (dq, 1H, J = 6.8 and 6.4), 6.09 (dd, 1H, J = 15.8 and J = 6.8),
6.18 (dd, 1H, J = 15.8 and 6.8), 6.50 (d, 1H, J = 15.8), 6.55 (d, 1H,
J = 15.8), 6.86 (d, 1H, J = 1.9), 6.88 (d, 1H, J = 1.8), 6.94 (d, 1H, J = 1.9),
6.95 (d, 1H, J = 1.8); 13C NMR ı 20.7, 21.7, 23.8, 56.2, 61.11, 69.3,
71.37, 109.7, 109.8, 122.0, 122.1, 128.5, 129.4, 131.6, 132.0, 132.4,
132.8, 132.9, 133.2, 146.9, 147.2, 153.0, 170.7. ESI-MS (+) 10 V: m/z
229.8, 479.4 [M+Na]+ 935.6 [2M+Na]+ (100). Chiral HPLC (Phen-
omenex Lux® Cellulose-1, 85:15 n-hexane/2-propanol, flow rate
0.5 ml/min, T 23 ◦C): 29.2 min (R-acetoxy-S-hydroxy enantiomer)
and 35.1 min (S-acetoxy-R-hydroxy enantiomer). To determine the
diastereoisomeric excess of monoacetate (+)-8a, the isolated prod-
uct was acetylated by standard procedure using pyridine and acetic
anhydride, and the obtained diacetyl derivative was analyzed by
chiral HPLC.
Under the same experimental conditions, 50% conversion of ( )-
2 was reached after 11 h when the reaction was stopped by filtering
the enzyme and the mixture purified to give alcohol (−)-2 and
acetate (+)-2a in high yields and optical purities (ee > 98%). Also in

C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
111
Scheme 1. Enzymatic kinetic resolution of ( )-1 and ( )-2.
this case the ester structure of (+)-2a was confirmed by NMR anal-
ysis thank the presence of singlet at 2.05 ppm and the downfield
shift of the CHOH proton due to esterification. A S absolute config-
uration of (−)-2 was tentatively assigned on the basis of the sign of
its optical rotation that is the same as reported for the structurally
similar (S)-(−)-4-phenyl-3-buten-2-ol [26].
Although the acetylation of a 1:1 mixture of diastereoisomeric
diols could be expected to be severely complicated by the presence
of up to ten possible stereoisomers in partial or complete acylated
form, the enzymatic transesterifications were carried out using the
whole mixtures in the hope that the high stereoselectivity of AK
lipase could promote a one-pot resolution/meso-desymmetrization
process giving a reduced number of stereoisomers.
From such assignments it follows that AK lipase displays R stere-
opreference in the kinetic resolution of both ( )-1 and ( )-2, that
is in agreement with the site active model proposed by Burgess
et al. for this enzyme by analyzing the substrate-structure/enzyme-
activity relationships for different unsaturated alcohols [27].
Taking into the account the high selectivity of AK lipase in the
recognition of stereogenic alcoholic centers here evidenced, the
kinetic resolution protocol was then extended to compounds ( )-
5/meso-6 and ( )-7/meso-8 that can be viewed as the dimeric forms
of ( )-1 and ( )-2, respectively.
The lipase-catalyzed transesterification of ( )-5/meso-6 was
then carried out in the same experimental conditions adopted for
(
)-1 and monitored by chiral HPLC at various reaction times. After
3 h the HPLC chromatogram of the reaction mixture displayed 45%
of unreacted substrate in the presence of both monoacetate (43%)
and diacetate (12%). The reaction was stopped at 6 h when the the-
oretical composition of 25% of diacetate (+)-5b, 50% monoacetate
(+)-6a and 25% of diol (+)-5 was reached. Chromatographic purifi-
cation of the reaction mixture gave diacetate (+)-5b with ee > 98%
and de = 97% together with the enantiomerically and diastereoiso-
merically pure monoacetate (+)-6a and diol (+)-5 (Scheme 2).
High values of optical purity for all the three compounds con-
firm that the lipase retained its excellent stereorecognition ability
yet displayed in the kinetic resolution of the parent ( )-1. The
same procedure was also applied to prepare optically active dehy-
drozingerol biphenyl derivatives. Enzymatic esterification of 1:1
mixture of ( )-7/meso-8 was monitored by chiral HPLC analysis
until the presence of three main peaks in a ratio about 1:2:1 was
observed in the chromatogram (34 h). After quenching the reaction
by filtering the enzyme the products were isolated in high yields by
column chromatography. Diacetate (+)-7b and monoacetate (+)-8a
were obtained as single diastereoisomers with 95% ee and 94% ee
respectively, while diol (−)-7 was isolated in 93% ee and 97% de
(Scheme 2).
3.2. Enzymatic one pot resolution/desymmetryzation of biphenyl
mixtures ( )-5/meso-6 and ( )-7/meso-8
The biphenyl zingerone derivative 3 was obtained in 65% yield
using a direct C C coupling reaction [24] of commercial zingerone
in the presence of methyl-tri-butylammonium permanganate in
CH2Cl2 at room temperature. Since this procedure was unsuccess-
ful in the case of dehydrozingerone, the biphenyl 4 was obtained
in 50% yield by Claisen-Schmidt condensation of acetone with 5,5ꢀ-
bivanillin [13]. The obtained biphenyl ketones were O-methylated
and then reduced with NaBH4 in MeOH to give the corresponding
diols, in both cases isolated as a chromatographically inseparable
mixture of diastereoisomers. Also in the 1H- and 13C-NMR spectra
a single set of resonances was observed for each diol whereas the
HPLC chiral analysis clearly revealed the presence of three peaks
in 1:2:1 ratio for each diol, accounting for a 1:1 mixture of racemic
and meso diastereoisomers ( )-5/meso-6 and ( )-7/meso-8, respec-
tively.
In agreement with the empirical rules proposed for the enan-
tiorecognition of lipases in the transesterification reactions of
secondary alcohols [28] and taking into the account the struc-
tural similarity of the alcoholic groups in ( )-1 and ( )-2 with
Table 1
Screening of lipases for transesterificationa of ( )-1 and ( )-2.
Lipase
Time (h)
c (%)b
eeb (S)-alcohol
eeb (R)-acetate
Ec
PS-C
3
11
1
38
23
40
43
61
29
63
75
>98
98
94
>200
131
62
Lipozyme
Novozym
AK
3
>98
>200
PS-C
24
24
6
22
37
45
46
27
55
80
83
96
94
98
98
64
56
>200
>200
Lipozyme
Novozym
AK
18
a
b
c
Substrate 0.05 mmol, lipase 20 mg, t-BME 2 ml, VOAc 0.2 mmol, 28 ◦C, 250 rpm.
Substrate conversion and ee were determined by HPLC using a chiral column.
See Ref. [25].

112
C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
Scheme 2. Enzymatic resolution/desymmetrization of ( )-5/meso-6 and ( )-7/meso-8 mixtures.
80
respect to those in the corresponding dimers 5–8 it was assumed
that lipase AK maintained the same R enantiopreference also in
the esterification of the biphenyl diols. Therefore, diester deriva-
tives (+)-5b and (+)-7b were assigned to R,R-absolute configuration
and also for monoacetyl derivatives (+)-6a and (+)-8a it was
believed that the acetyl groups are located on the R-stereogenic
centers.
70
60
50
40
30
20
10
0
3.3. Lipase behavior in the kinetic resolution and
desymmetrization of biphenyls ( )-5 and meso-6
Despite of a large number of diols has been considered as sub-
strates for lipase-catalyzed transesterification, a direct comparison
of the enzyme activity in the recognition of a C2-symmetric racemic
diol and the corresponding meso-form has not yet been reported
and, in this context, parallel reactions on separate ( )-5 and meso-
6 biphenyls as well as the monomer ( )-1 as reference compound
were carried out for kinetic investigation.
0
10
20
30
40
50
60
70
80
90
Time (min)
Fig. 3. Enzymatic transesterification rate of alcoholic functions on R stereogenic
centers of meso-6 (− −), ( )-1 (—) and ( )-5 (. . .. . .).
Since the diastereoisomeric diols are obtained as an insepa-
rable mixture from the reduction of the parent ketone 3, they
were prepared in pure form using a different approach. So, meso-
6 was easily accessible by chemical hydrolysis of the enzymatic
monoacetate (+)-6a whereas racemic ( )-5 was prepared by mixing
equal amounts of (+)-5 and (−)-5, the latter obtained from alkaline
hydrolysis of enzymatic diacetate (+)-5b.
All the enzymatic transesterifications were carried out using a
concentration of substrate such that in each reaction an equivalent
number of R stereogenic centers was present. So, double molar con-
centration of ( )-1 with respect to ( )-5 and meso-6 was required
in order to account that two stereogenic centers are present for
each molecule of biphenyl diol. The experiments were performed
in t-BME in the presence of lipase AK and vinyl acetate, monitoring
the progress of reactions by chiral HPLC.
Due to the excellent stereoselectivity of lipase AK the measured
amount of formed products in the reaction was directly related
with the conversion of R stereogenic centers, that was plotted
against time (Fig. 3). The curves in Fig. 3 display a very similar
reaction rate for monomer ( )-1 and meso-6 indicating that the
enzyme takes the same time to select and transform the R stere-
ogenic centers discarding the S-ones in both cases, either when
the R/S centers are on separate molecules (( )-1) either when they
are located on the same molecule (meso-6). Although the curve of
4. Conclusions
In summary, a lipase catalyzed kinetic resolution in organic
solvent of O-methylated zingerol and dehydrozingerol ( )-1 and
(
)-2 has been successful carried out giving the products in excel-
lent yield and optical purity. The biocatalytic procedure developed
for such alcohols was also extended to the preparative resolution
and desymmetrization of corresponding rac- and meso- biphenyl
dimers 5–8. The high enantioselectivity of lipase AK allowed the
complete separation of single diastereoisomers of mixtures, pro-
viding the single products in enantiopure form.
Kinetic data of the reactions performed on single diastereoiso-
meric species evidenced that desymmetrization of meso-6
proceeded with comparable reaction rate with respect to racemic
resolution of ( )-1, whereas in the case of dimer ( )-5 the res-
olution process was slower. This experiment also indicated that
the lipase maintained the same stereodifferentiation ability, dis-
criminating independent enantiomeric molecules as in ( )-1 or
stereogenic opposite centers on a same molecule as meso-6 in com-
parable rate.
(
)-5 was built considering the sum of both monoacetate and diac-
Acknowledgment
etate, that contributes for two transformed R-centers, the overall
speed of esterification appeared about halved. This finding can be
explained considering that the exposure of one R-center of R,R-diol
to the active site of the enzyme makes the second one on the same
molecule not available for acylation in same time unit.
The research reported in this paper was financed by RAS project
“Molecular diversity in the chemical synthesis of biologically active
compounds with social impact” (L.R. 7/2007 Regione Autonoma
Sardegna) which is acknowledged.

C. Sanfilippo et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 107–113
113
Appendix A. Supplementary data
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