Enantioselective synthesis of 3-hydroxytetradecanoic acid and its methyl ester enantiomers as new antioxidants and enzyme inhibitors

Article abstract of DOI:10.1007/s00706-012-0917-z

Optically pure R and S enantiomers of 3-hydroxytetradecanoic acid and its methyl esters were synthesised by porcine pancreas lipase catalysed hydrolysis of racemic methyl 3-hydroxytetradecanoate in aqueous medium, with the aim of determining their antioxidant, antielastase and antiurease activities. The effects of the weight ratio of substrate/lipase and the reaction time were investigated. Optimum reaction conditions were determined. The resolution reaction with porcine pancreas lipase afforded (R)-3-hydroxytetradecanoic acid, which is a component of bacterially important lipid A, with greater than 99 % ee in excellent enantiomeric ratio (>900) after 7 h incubation with a substrate/lipase weight ratio of 3:1 and 43 % conversion of the substrate. Methyl (S)-3-hydroxytetradecanoate, which is the unreacted enantiomer of the racemic substrate, could be recovered with 98 % ee after 7 h resolution with a substrate/lipase weight ratio of 1:1 and 60 % conversion. (R)-3- Hydroxytetradecanoic acid was converted to its ester and the S methyl ester to its acid. This biocatalytic enantioselective resolution in aqueous medium presents an environmentally friendly and green chemistry method for the synthesis of R and S enantiomers of 3-hydroxytetradecanoic acid and its methyl esters.

Full text of DOI:10.1007/s00706-012-0917-z

Monatsh Chem (2013) 144:1087–1091
DOI 10.1007/s00706-012-0917-z
ORIGINAL PAPER
Enantioselective synthesis of 3-hydroxytetradecanoic acid
and its methyl ester enantiomers as new antioxidants
and enzyme inhibitors
Hatice Baspinar K u¨ c u¨ k
Ay s¸ e Yusufo g˘ lu
•
Received: 7 August 2012 / Accepted: 28 December 2012 / Published online: 5 March 2013
Ó Springer-Verlag Wien 2013
Abstract Optically pure R and S enantiomers of 3-hy-
droxytetradecanoic acid and its methyl esters were
synthesised by porcine pancreas lipase catalysed hydrolysis
of racemic methyl 3-hydroxytetradecanoate in aqueous
medium, with the aim of determining their antioxidant,
antielastase and antiurease activities. The effects of the
weight ratio of substrate/lipase and the reaction time were
investigated. Optimum reaction conditions were deter-
mined. The resolution reaction with porcine pancreas lipase
afforded (R)-3-hydroxytetradecanoic acid, which is a
component of bacterially important lipid A, with greater
than 99 % ee in excellent enantiomeric ratio ([900) after
Introduction
Bacterial lipopolysaccharides (LPS) are major constituents
of the outer membranes of cell walls in Gram-negative
bacteria, and these molecules play an important role in the
development of septic shock in humans [1, 2]. Lipid A
bound to a polysaccharide portion is a glycolipid compo-
nent of LPS. The lipid A is responsible for most of the
endotoxic activities of LPS. Optically active (R)-3-hy-
droxytetradecanoic acid ((R)-1a) is the most common fatty
acid constituent of lipid A. Fukase et al. [3] reported that
the lipid A analogue with the S configuration was more
strongly bioactive than the natural R type. The methyl
esters of (R)- and (S)-3-hydroxytetradecanoic acids ((S)-1b
and (R)-1b) are also valuable compounds in the synthesis
of biologically active natural compounds [4, 5]. Therefore,
many synthetic methods have been described for the syn-
thesis of (R)-1a, (S)-1a, (S)-1b and (R)-1b, including
synthesis from (S)-3-hydroxy-c-butyrolactone [6] and (S)-
epichlorohydrin [7] and asymmetric reduction by fermen-
tation with baker’s yeast [8] and use of modified nickel
catalysts [9–11]. In our previous study [12], (S)-1a with
57 % ee was obtained by asymmetric reduction with chi-
7
h incubation with a substrate/lipase weight ratio of 3:1
and 43 % conversion of the substrate. Methyl (S)-3-hy-
droxytetradecanoate, which is the unreacted enantiomer of
the racemic substrate, could be recovered with 98 % ee
after 7 h resolution with a substrate/lipase weight ratio of
1
:1 and 60 % conversion. (R)-3-Hydroxytetradecanoic acid
was converted to its ester and the S methyl ester to its acid.
This biocatalytic enantioselective resolution in aqueous
medium presents an environmentally friendly and green
chemistry method for the synthesis of R and S enantiomers
of 3-hydroxytetradecanoic acid and its methyl esters.
rally modified NaBH . One of the effective ways to
4
Keywords Enzymes Á Enantiomeric resolution Á
Lipid A Á Enzymatic hydrolysis
produce these enantiomerically pure compounds consists of
the enzymatic kinetic resolution of racemic mixtures.
Bornscheur et al. [5] reported that the kinetic resolution of
racemic 3-hydroxyesters by lipase-catalysed transesterifi-
cation was achieved in different organic solvents. Optically
pure 3-hydroxyalkanoates were obtained by enzymatic
hydrolysis and enzymatic transesterification [4]. On the
other hand, lipase-catalysed resolution of 3-hydroxytetra-
decanoic acid methyl ester in tetrahydrofuran using vinyl
acetate gave the enantiomerically pure R acid, R ester and
S acid [13].
H. B. K u¨ c u¨ k Á A. Yusufo g˘ lu (&)
Department of Chemistry, University of Istanbul,
Istanbul, Turkey
e-mail: ayseserg@istanbul.edu.tr
123

1
088
H. B. K u¨ c u¨ k, A. Yusufo g˘ lu
This present study has two aims. One aim is to investigate
the resolution of racemic methyl 3-hydroxytetradecanoate
(R,S)-1b) by porcine pancreas lipase (PPL) in the water
Results and discussion
(
Preparation of racemic methyl
3-hydroxytetradecanoate ((R,S)-1b)
phase (Scheme 1), because in the literature survey, no
report was found describing the use of PPL in aqueous
buffer solution for the synthesis of 3-hydroxytetradecanoic
acid and its methyl ester enantiomers. The effects of
reaction parameters such as concentration of substrate and
enzyme, reaction time, conversion and enantioselectivity
were considered. The optimised reaction conditions,
including the most effective weight ratio of the substrate to
enzyme and the reaction time for the lipase-catalysed
hydrolysis of (R,S)-1b in aqueous medium, are reported.
The other aim of this study is to analyse the antioxidant,
elastase and urease activities of the optically pure R and
S enantiomers of 3-hydroxytetradecanoic acid and its methyl
ester via synthesised in this work. According to the literature,
no other study has examined the antioxidant, elastase and
urease activities of b-hydroxy fatty acid isomers except our
published study which reports the excellent antielastase,
antiurease and antioxidant activities of racemic 3–13
monohydroxyeicosanoic acid isomers [14]. In recent years
elastase, urease and antioxidant activities have gained great
importance. Elastase inhibition is a protective tool against
skin diseases and aging. Urease inhibition is of medical,
agricultural and environmental significance. New antioxi-
dants for foods, cosmetics and medicines are increasing too.
No report exists on the antioxidant and enzyme inhibition
activities of chirally pure b-hydroxytetradecanoic acid and
its methyl ester isomers.
The starting material, racemic methyl 3-hydroxytetrade-
canoate, was synthesised using the acetoacetic ester and
1
methods [15] and characterised by FT-IR, H NMR
NaBH
4
and elemental analyses. The spectroscopic data were
identical to those of (S)-1b and (R)-(1b).
Choice of lipase species
Lipase is a useful catalyst for fatty acid derivatives, espe-
cially in enantioselective reactions [16]. In kinetic
resolution of racemic compounds, the enzyme distin-
guishes between the two enantiomers of a racemic mixture,
so that one enantiomer is readily transferred to the product
faster than the other. PPL is an economic, commercially
available and non-microbial enzyme that has high enanti-
oselectivity. Many lipases react selectively with the
R enantiomer [17–19]. The enzyme PPL chosen in this
study hydrolysed selectively the R enantiomer making the
formation of (R)-1a possible. The unreacted (S)-1b was
recovered after purification by column chromatography
with good to excellent optical purities. (R)-1a isolated
(Table 1, entry 3) was esterified easily to (R)-1b by treat-
ment with BF /MeOH. (S)-1a was obtained via chemical
3
hydrolysis of the unreacted (S)-1b (Table 1, entry 1)
(Scheme 1). The conditions in entry 3 of Table 2 were the
best conditions to synthesize (R)-1a with greater than 99 %
ee. The conditions in entry 1 of Table 2 can be used for the
synthesis of enantiomerically pure (S)-1b (Scheme 1).
Scheme 1
OH O
C11H23
OCH3
R,S)-1b
(
Effects of the substrate/lipase weight ratio and reaction
time on the enantioselective enzymatic hydrolysis
Porcine pancreas
Buffer solution, pH 7.6
H2O
(R,S)-1b was exposed to kinetic resolution using PPL
(300 units/mg) as biocatalyst with various reaction times. pH
(
R,S)-1b/PPL=3:1
(R,S)-1b/PPL=1:1
(Table 2, entry 1)
was kept constant at 7.6 using citric acid/Na HPO buffer
2 4
(
Table 2, entry 3)
solutions (0.02 M). The optimum temperature was set to
6 °C. Experiments were performed as described in Table 2
entries 1–8) to determine the optimum weight ratio of the
3
(
OH
O
OH O
substrate to lipase and the reaction time. The resolution
reaction with PPL was stopped via dichloromethane after the
indicated reaction times shown Table 2. In entry 3, the reac-
tion time was selected as 7 h. In order to optimize the reaction
time, 7 h was halved to 3.5 h in entries 6 and 8 and then
doubled to 14 h in entries 5 and 7. The lipase amount was held
constant and the substrate/PPL weight ratio was changed.
Table 1 shows the results of the resolution entries,
conversion (X) and enantiomeric ratio (E) in the course of
hydrolysis of (R,S)-1b by PPL.
C11H23
OH
C11H23
OCH3
(R)-1a >99% ee
(S)-1b 98% ee
Esterification
BF3/MeOH
Hydrolysis
KOH/EtOH
OH
O
OH
O
C11H23
OH
C11H23
OCH3
(R)-1b >99% ee
(S)-1a 98% ee
1
23

Synthesis of 3-hydroxytetradecanoic acid
1089
Table 1 Enantioselective resolution of (R,S)-1b with PPL
Entry
(R)-1a produced
(S)-1b recovered
2
D
0
-1 -1
g
3
b
p
20
D
-1 -1
g
3
cm
b
s
[
(
a]
/ ° dm
c = 1, CHCl
cm
ee
/%
Conversion
(X)/% [20]
E [20]
[a]
/ ° dm
ee
/%
a
c
3
)
3
(c = 1, CHCl )
1
2
3
4
5
6
7
8
-10
63
75
60
54
43
45
45
53
54
55
19
20
?18.1
?16.3
?14
98
88
76
73
75
89
89
75
-12
-16
[99
89
[900
38
-14.2
-14.5
-12.5
-12.2
-14.7
?13.5
?13.8
?16.5
?16.4
?13.9
91
48
78
24
76
22
92
54
E enantiomeric ratio
a
b
c
25
25
a]
Enantiomeric excess values (ee, %) were determined by chiral HPLC
3
For methyl (R)-3-hydroxytetradecanoate [a] = -18.5 (c = 1.05, CHCl ) [8]
D 3 D 3
[ = -16.2 (c = 1, CHCl ) [8], [a] = -15.1 (c = 1.06, CHCl ) [13]
D
greater than 99 % ee in excellent enantioselectivity
(E [ 900). (S)-(1b) was obtained with 98 % ee.
Table 2 PPL-catalysed hydrolysis of (R,S)-1b with varying weight
ratios of substrate/PPL and reaction times
a
The described process in this study is enantioselective,
economical and convenient requiring only a facile sepa-
ration by column chromatography. The desired four
enantiomers (R)-1a, (S)-1a, (S)-1b and (R)-1b could be
obtained without the need for additional crystallisations.
This enzymatic route reduces the number of operations to
two simple procedures: hydrolysis and separation. The use
of PPL for this enzymatic resolution in an aqueous envi-
ronment proved to be an excellent methodology for the
preparation of these enantiopure bioactive products which
will be analysed for their antioxidant, elastase and urease
activities.
Entry Substrate/mg/
suspension/cm
PPL/mg/sus- Substrate/PPL Reaction
3
3
pension/cm
weight ratio
time/h
b
1,000 :56
b
1
2
3
4
5
6
7
8
a
1,000:56
1:1
2:1
3:1
6:1
3:1
3:1
6:1
6:1
7
1,000:28
500:28
7
1,000:18.67
1,000:9.33
1,000:18.67
1,000:18.67
1,000:9.33
1,000:9.33
333:18.67
166.6:9.33
333:18.67
333:18.67
166.6:9.33
166.6:9.33
7
7
14
3.5
14
3.5
Methyl (R,S)-3-hydroxytetradecanoate (1,000 mg, 3.88 mmol) was
used in these experiments (pH 7.6, temperature 36 °C)
b
3
cm of lipase suspension, prepared from 1 g of lipase and 15 cm
3
1
of water, hydrolysed 50 mg trimyristine [21]
Experimental
The best resolution for (R)-1a was entry 3. (R)-1a was
synthesised with greater than 99 % ee in excellent enanti-
oselectivity (E [ 900) after 7 h incubation with a
substrate/PPL weight ratio 3:1 and 43 % conversion of
PPL (porcine pancreas lipase (type II crude)) was supplied
from Sigma Chemical Co. (St. Louis, USA). All reagents
and solvents were of analytical grade and used as provided
without further purification. The reactions were monitored
using TLC plates (Merck 60 F-254). Column chromatog-
(R,S)-1b (entry 3). (S)-1b could be obtained in optically
pure ratio only according to entry 1.
1
raphy was performed on silica gel 60 (70–230 mesh). H
1
and C NMR spectroscopy were carried out in CDCl and
3
3
recorded on a Varian 400 MHz spectrometer using TMS as
an internal standard. Mass spectroscopy (ESI) was per-
formed on a Thermo Finnigan spectrometer. A Mattson
1000 series spectrometer was used for IR spectroscopy.
Optical rotations were measured on an Optical Activity
AA-55 digital polarimeter and the results were reported
from the averages of at least five measurements. Enantio-
meric excesses (ee) of products were determined with a
Conclusion
We have carried out an efficient enantioselective strategy
to prepare pharmacologically valuable enantiomers of
3
-hydroxytetradecanoic acid and its methyl esters via PPL-
catalysed kinetic resolution of the corresponding racemic
R,S)-1b in aqueous medium. (R)-1a was synthesised with
(
123

1
090
H. B. K u¨ c u¨ k, A. Yusufo g˘ lu
Shimatsu\DGU-20A5 HPLC apparatus fitted with a Chi-
ralcel OD Chiral column.
corresponding methyl ester (R)-1b, and analysed on the
Chiralcel OD column.
Methyl (R)-3-hydroxytetradecanoate ((R)-1b, C H O )
1
5 30 3
3
Chiral analysis
(R)-1a (100 mg, 0.407 mmol) was dissolved in 3 cm
3
methanol. Then 3 cm BF /MeOH reagent was added, and
3
The enantiospecifity of the enzymatic reaction was deter-
mined after purification of the products (R)-1a and (S)-1b
by column chromatography. The enantiomeric excesses of
the products (R)-1a and (S)-1b were determined by chiral
HPLC using a Chiralcel OD column. (R)-1a and (S)-1a did
not give any signals on the Chiralcel OD column. On the
other hand, their corresponding methyl esters (R)-1b and
the solutions were refluxed at 100 °C for 2 min. After the
reaction was completed, excess alcohol was removed under
3
vacuum at room temperature. Then 30 cm petroleum ether
3
and 20 cm water were added, and the solution was shaken
vigorously for 5 min before the layers were allowed to
separate. The bottom layer was dried over anhydrous
Na SO and the solvent was removed in vacuo to give
2
4
(
S)-1b could be detected on the Chiralcel OD column. In
(R)-1b as white crystals in 100 % yield (105 mg) and
2
0
-1 -1
g
3
order to determine the ee values by chiral HPLC the
racemic substrate (R,S)-1b was separated on Chiralcel OD
column into its enantiomers (R)-1b and (S)-1b. (R)-1b
elutes before (S)-1b. (R)-1a was transformed to its corre-
sponding methyl ester (R)-1b for measurement of its ee.
Conversion (X) and enantiomeric ratio (enantioselectivity,
E) were calculated according to Chen et al.’s equation [20].
The absolute configurations of the acid and ester were
assigned by comparison of their optical rotations with the
literature data. The results are summarised in Table 1.
greater than 99 % ee. [a]D = -18.4/ ° dm
cm
(c = 1, CHCl ). HPLC analysis: Chiralcel OD chiral
column, mobile phase n-hexane/2-propanol 98:2, flow rate
3
3
0.8 cm /min, wavelength 220 nm; t : 8.05 min for the (R)-
R
isomer, not observed for the (S)-isomer. The spectroscopic
data were identical to those of (S)-1b.
Methyl (S)-3-hydroxytetradecanoate ((S)-1b, C H O )
1
5 30 3
This compound was obtained as white crystals in 98 % ee
(
Table 1, entry 1). M.p.: 30–31 °C (Ref. [8] 28–30 °C);
20 -1 -1 3
[
a] = ?18.1/ ° dm
g
cm (c = 1, CHCl ). HPLC
3
D
analysis: Chiralcel OD chiral column, mobile phase
3
n-hexane/2-propanol 98:2, flow rate 0.8 cm /min, wave-
General procedure for the enantioselective synthesis
of 3-hydroxytetradecanoic acid and its methyl ester
length 220 nm; t : 8.05 min for the (R)-isomer, 10.70 min
R
for the (S)-isomer.
Enzymatic hydrolyses were carried out using the conditions
described in Table 2 varying the substrate/PPL weight ratios
and reaction times (entries 1–8). Different suspension
amounts of the racemic ester (R,S)-1b (1,000 mg,
(
S)-3-Hydroxytetradecanoic acid ((S)-1a, C H O )
14 28 3
3
S)-1b (100 mg, 0.387 mmol), 0.09 g KOH in 1 cm water
(
3
and 1 cm methanol were placed in a round-bottomed flask
which was mounted over a magnetic stirrer and refluxed for
3
.88 mmol) were incubated with PPL (Sigma L-3126) in
6
0 min. Unreacted ester, if any, was removed by ether
water at pH 7.6 (citric acid/Na HPO buffer solutions
2
4
extraction and the carboxylic acid was recovered by the
methods given below. The aqueous portion was acidified to
(
0.02 M)) and 36 °C. The enzyme was settled, unless the
reaction mixture was stirred or shaken. Thus, the resulting
suspension was shaken in an ultrasonic bath. The progress of
the reaction was monitored by TLC (developing solvent
CHCl /CH OH/CH COOH = 100:10:1). After the time
pH 2 with 6 N hydrochloric acid and extracted with ether
3
3 9 5 cm ). The combined ether extracts were dried over
(
Na SO and concentrated on a rotary evaporator. The acid
4
2
3
3
3
(S)-1a was obtained after drying under vacuum as a white
powder in 80 % yield (76 mg) and 98 % ee (Table 1, entry 1).
shown in Table 2, the reaction was stopped with the addition
of CH Cl . The enzyme was removed by vacuum filtration
2
2
20
-1 -1
3
[a]D = ? 16.1/ ° dm
analysed on Chiralcel OD column as its methyl ester (S)-1b.
g
cm (c = 1, CHCl ). It was
3
over Celite and washed with CH Cl . The organic phase was
2
2
separated and washed with distilled water. After drying over
anhydrous Na SO , the solvent was removed in vacuo to give
The spectroscopic data were identical to those of (R)-1a.
2
4
a mixture of (R)-1a and (S)-1b that was separated via column
chromatography (flash SiO , developing solvent CHCl /
Acknowledgments This work was supported by Scientific Research
Projects Coordination Unit of Istanbul University, project number
T-1195/0112001.
2
3
CH OH/CH COOH = 100:10:1).
3
3
(
This compound was obtained as a white powder in greater
R)-3-Hydroxytetradecanoic acid ((R)-1a, C H O )
14 28 3
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g
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