Lipase-catalyzed separation of geometrical isomers: Geraniol-nerol

Article abstract of DOI:10.1246/cl.2007.1110

The substrate/lipase ratio as well as pH of the buffer medium played important roles in the resolution of geometrical isomeric mixture of geraniol-nerol. Based on the results, an immobilized lipase from Pseudomonas fluorescens (PFL) was found effective in selective transesterifications whereas Pseudomonas sp. Lipase (PSL) was found to be useful in hydrolyzing the esters. Copyright

Full text of DOI:10.1246/cl.2007.1110

1
110
Chemistry Letters Vol.36, No.9 (2007)
Lipase-catalyzed Separation of Geometrical Isomers: Geraniol–Nerol
Ã
Pankaj Gupta, Subhash C. Taneja, Bhahwal A. Shah, Vijay K. Sethi, and Ghulam N. Qazi
Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India
(Received June 4, 2007; CL-070594; E-mail: sc taneja@yahoo.co.in)
The substrate/lipase ratio as well as pH of the buffer medi-
um played important roles in the resolution of geometrical iso-
meric mixture of geraniol–nerol. Based on the results, an immo-
bilized lipase from Pseudomonas fluorescens (PFL) was found
effective in selective transesterifications whereas Pseudomonas
sp. Lipase (PSL) was found to be useful in hydrolyzing the
esters.
R O/Py
2
OH
OR
R = Ac, E = 64%,
Z = 36%
R = Pr, E = 65.3%
Z = 34.7%
E = 63%
Z = 37%
Transesterification
Hydrolysis
Geraniol and nerol (trans and cis isomers of 3,7-dimethyl-
,6-octadiene-1-ol) are acyclic monoterpene primary alcohols
OAc
OH
2
1
and the important constituents of a number of aromatic plants.
Besides, their use in food and flavour industry these alcohols are
R = Ac, E > 95%,
Z < 05%
R = Pr, E > 99%
E > 88%
Z < 12%
2
used for the synthesis of Vitamins A and E. Geraniol also called
rhodinol, is the primary constituent of the oil of rose and palmar-
osa, and is associated with a sweet rose-like odor, for which it is
commonly used in perfumes. High content of geraniol is mainly
responsible for the characteristic odor of palmarosa oil, which
has its use as antiseptic, mosquito repellent, and pain relieving
Scheme 1. Lipase-catalyzed transesterification and hydrolysis.
experimental conditions of pH, temperature, and substrate:lipase
ratios.
In the course of biocatalytic approach, we were able to
achieve the separation of two geometrical isomers with moderate
to high selectivity. The various microorganisms used in the pres-
ent study include Trichosporon beigilli (DSMZ 11829), Arthro-
bacter sp. (MTCC no. 5125), Aspergillus niger (Lipase AS,
Amano), Mucor miehi (MM), Pseudomonas sp. (PS), Candida
crusie (CC), Candida rugosa (CR), etc.
3
agent. Different biological activities have been attributed to
geraniol and its geometrical isomer nerol. The trans isomer
4
has modest in vitro and in vivo immunosuppressive activity
and also inhibits the growth and polyamine biosynthesis in
5
,6
human colon cancer cells.
Geraniol reportedly inhibited
7
MCF-7 proliferation in human breast cancer cells. It also inhib-
ited HMG-CoA (3-hydroxy-3-methylglutaryl Coenzyme A) re-
ductase activity as the increase in HMG-CoA reductase activity
is characteristic of a tumor type. Geraniol is also capable of po-
tentiating the anti tumor effect of 5-florouracil.9
Table 1. Enzyme-catalyzed hydrolysis of (E/Z) geranyl/neryl
Ã
acylates using PSL
8
Time Convn
Substrate
Solvent (1:5)
ACN:Buffer
Acetone:Buffer 87.6:12.4 71
E:Z
/h
/%
The Z isomer nerol is a starting material for the synthesis
of nerol oxide,10 an important perfumery material. The small
Geranyl
acetate
95.6:4.4
71
42
50
ꢀ
difference in boiling points (nerol 227–228 C and geraniol
ꢀ
229–230 C) makes their separation more cumbersome even
(
E/Z)
DMSO:Buffer
Methanol:Buffer 90.2:9.8
85:15
71
71
71
49
60
35
45.7
36
38
49.5
45.7
18
if an efficient fractional distillation method may be used for their
separation. Though, biocatalytic methods including hydrolases
have extensively been used for the resolution of racemic
mixtures, however, their application in the separation of geomet-
Geranyl
propionate
(
ACN:Buffer
ACN:Buffer
ACN:Buffer
>99
82.3:17.7 71
96:4
a
E/Z)
120
71
120
71
41
41
1
1
rical isomers has been uncommon as well as less effective. In
the present study, we envisaged to exploit the biocatalytic route
for the separation of the cis and trans isomers. An earlier attempt
to resolve a mixture of geraniol and nerol, was achieved via
selective acylation with acid anhydrides in the presence of
Acetone:Buffer >99
Acetone:Buffer 93:7
Methanol:Buffer 90.1:9.9
Geranyl
butyrate (E/Z) ACN:Buffer
ACN:Buffer
>99
80:20
a
21.8
1
2
Porcine pancreatic lipase (PPL) as a catalyst. However, they
were able to achieve only a partial separation resulting in enrich-
ment level of 90:10 of E:Z isomers. In this communication,
results of our attempts towards the development of a more
efficient method of kinetic resolution of geometrical isomers
are presented (Scheme 1).
For the kinetic resolution studies, a panel of lipases belong-
ing to institute’s repository as well as procured from commercial
sources, were used for the selective hydrolysis of various
acylates of natural geraniol–nerol mixture (63:37) under varying
Ã
ꢀ
All the reactions were performed at 20–22 C, Quantity of
lipase (w/w) = 1:49. a, 1:19. Product was analyzed by in-
jecting a 0.5-mL aliquot in a split less mode into a gas–liquid
chromatograph (GLC) equipped with a flame ionization de-
tector. A BP-10 fused silica capillary column heated isother-
ꢀ
mally at 170 C was used to separate and identify the prod-
ucts. Injector and detector temperatures were set at 250 and
2
flow rate of 5 mL/m. Product yield and selectivity were cal-
culated using peak area integration by an on-line computer.
ꢀ
60 C respectively. Helium was the carrier gas with a total
Copyright ꢀ 2007 The Chemical Society of Japan

Chemistry Letters Vol.36, No.9 (2007)
1111
Table 2. Enzyme-catalyzed transesterification reactions of ger-
aniol/nerol using PFL
isomers geraniol and nerol was successfully achieved by bioca-
talytic approach. In this study, we observed that during hydrol-
ysis, selectivity depended largely on the biocatalyst–substrate
ratio and pH, and the best results were obtained using geranyl
propionate as a substrate, however, in transesterification
reactions it also depended on the nature of the acylating agent
while the effect of the organic solvent was not so significant.
Temp
ꢀ
Time
/h
Convn
/%
f
S.No
Solvent
ACN
ACN
Hexane
Hexane
DIE
E:Z
/
C
a
1
88:12
88:12
85:15
30–32
18–20
30–32
20
20
20
20
20
20
24
24
3.5
3.5
3.5
72
72
44
36
48
42
42
57
51
41.5
73
68
7
a
2
a
3
a
4
87.:13 18–20
30–32
30–32
The authors are thankful to the CSIR, New Delhi for
supporting the project.
a
5
83:17
85:15
a
6
Acetone
a
Ionic liquidg 89.:11 18–20
7
References and Notes
1
a
Ionic liquidh 88:12
8
18–20
30–32
30–32
30–32
30–32
30–32
b
Sigma-Aldrich Fine Chemicals, Flavors and Fragrances,
International Edition, 2001–2002.
b
9
Hexane
Hexane
Hexane
ACN
69:31
67:33
70:30
93:7
1
1
1
1
0c
d
2
3
4
C. Mercier, P. Chabardes, Pure Appl. Chem. 1994, 66, 1509.
V. S. Dubey, R. Luthra, Phytochemistry 2001, 57, 675.
P. Ji, M. S. Si, Y. Podnos, D. K. Imagawa, Transplant. Proc.
2002, 34, 1418.
1
e
2
20
31
e
3
DME
90:10
a
5
S. Carnesecchi, Y. Schneider, J. Ceraline, B. Duranton,
F. Gosse, N. Seiler, F. Raul, J. Pharmacal. Exp. Ther.
2001, 298, 197.
Acylating agents Vinyl acetate. Isobutyric anhydride.
Butyric anhydride. Propionic anhydride. Isopropenyl ace-
tate. ACN, acetonitrile; DIE, diisopropyl ether; DME, dime-
thoxyethane. 1-Butyl-3-methylimidazolium tetrafluorobo-
rate. 1-Butyl-3-methylimidazolium hexafluorophosphate.
c
d
e
f
g
6
7
8
9
S. Carnesecchi, K. Langley, F. Exinger, F. Gosse, F. Raul,
J. Pharmacal. Exp. Ther. 2002, 300, 625.
R. E. Duncan, D. Lau, A. El-Sohemy, Biochem. Pharmacol.
2004, 68, 1739.
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Zeisela, F. Gosse, M.-F. Pouponb, F. Raula, Cancer Lett.
2004, 215, 53.
h
Improved selectivity was observed for the propionate deriv-
ative followed by acetate derivative in enzymatic hydrolysis
of acyl esters.13 It has also been observed that pH played a key
role during the course of the reaction, as little variation during
hydrolysis effected the purity of the resolved products. There-
fore, all the reactions were performed strictly in a narrow
range of pH 7–7.1, that was maintained by adding 0.5 M NaOH
solution. Table 1 summarizes the results obtained from the
hydrolysis of acylates. The reactions were performed up to
grams scale level. The enzyme to substrate ratio played a very
important role in resolution, as at lower concentration of lipase
e.g. lipase:substrate (1:49), higher resolution of the geometrical
isomers was observed. Increasing the concentration of lipase was
detrimental to the selectivity. Much lower concentration i.e.
10 a) P. Gupta, V. K. Sethi, S. C. Taneja, B. A. Shah, S. S.
Andotra, S. S. Chimni, G. N. Qazi, Helv. Chim. Acta 2007,
90, 196. b) V. K. Sethi, S. C. Taneja, S. S. Andotra, P. Gupta,
G. N. Qazi, USP 7,166,728 dt 23-01-2007.
11 a) R. D. Schmid, R. Verger, Angew. Chem., Int. Ed. 1998,
37, 1608. b) K. Takabe, N. Mase, T. Hisano, H. Yoda, Tetra-
hedron Lett. 2003, 44, 3267.
12 J.-D. Fourneron, M. Chiche, G. Pieroni, Tetrahedron Lett.
1990, 31, 4875.
<
2% decreased the rate of hydrolysis. The reason for the
fall in selectivity at higher concentrations of biocatalyst may
be attributed to the presence of other lipases in an organism in
minor amounts which also played their roles during hydrolysis.
Besides, transesterification reactions of the isomeric mixture
were also studied using various immobilized enzymes, however,
Pseudomonas fluorescens lipase (PFL) displayed better resolu-
tion than other lipases, therefore detailed studies were undertak-
en with PFL.14 Table 2 depicts the results of transesterfication
reactions with PFL in the presence of various acyl transfer
agents. Notably, vinyl acetate proved to be the best acyl transfer
reagent that gave a product ratio (E:Z) of 89:11 in 24 h in an ion-
ic liquid, as almost similar results were obtained in acetonitrile.
Increasing the size of the acyl transfer agent did not improve
the resolution efficacy, though rate of esterification increased
considerably in some cases e.g. butyric/isobutyric anhydride.
The progress of all the enzymatic reactions was monitored by
GC as well as TLC.
13 Lipase-catalyzed hydrolysis: In a typical experiment, a
mixture of the substrate (1 g), organic solvent (5 mL), the
crude powder of enzyme PS lipase (20 mg) in 25 mL of
ꢀ
phosphate buffer (pH 7.0, 0.1 M) was stirred at 20–22 C.
pH of the reaction was maintained with 0.5 M NaOH
solution. The course of the reaction was monitered by
GLC. After a certain degree of conversion, the reaction
was terminated and the contents extracted with hexane and
organic phase washed with water, dried over sodium sulfate
and concentrated in vacuo to get the product.
14 Lipase-catalyzed transesterification: Ester synthesis was
performed in screw-capped test tubes in which 1 mmol of
terpene alcohol and 1.2 mmol vinyl ester or alkanoic anhy-
dride was added to 6-mL solvent followed by 0.5 times
(w/w of reactant) of lipase. Samples were kept in a
shaker at 320 rpm at room temp. After the incubation period,
samples were removed and diluted with hexane and filtered
to remove the enzyme that could be recycled again.
In conclusion, the resolution of a mixture of geometrical
Published on the web (Advance View) August 4, 2007; doi:10.1246/cl.2007.1110