Enzymatic kinetic resolution of tropic acid

Article abstract of DOI:10.1016/j.tetasy.2005.10.032

A new strategy has been developed for the CAL-B catalysed kinetic resolution of tropic acid by which both enantiomers of tropic acid can be obtained in good enantiomeric excess. (R)-Tropic acid was synthesised with 90% ee and (S)-tropic acid butyl ester in 99% ee by the hydrolysis of tropic acid butyl ester. The other enantiomers were available through the enzymatically catalysed reaction of tropic acid lactone with butanol to give (S)-tropic acid lactone and (R)-tropic acid ester in >98% ee.

Full text of DOI:10.1016/j.tetasy.2005.10.032

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3892–3896
Enzymatic kinetic resolution of tropic acid
Dirk Klomp, Joop A. Peters and Ulf Hanefeld*
Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
Received 24 September 2005; accepted 14 October 2005
Abstract—A new strategy has been developed for the CAL-B catalysed kinetic resolution of tropic acid by which both enantiomers
of tropic acid can be obtained in good enantiomeric excess. (R)-Tropic acid was synthesised with 90% ee and (S)-tropic acid butyl
ester in 99% ee by the hydrolysis of tropic acid butyl ester. The other enantiomers were available through the enzymatically catalysed
reaction of tropic acid lactone with butanol to give (S)-tropic acid lactone and (R)-tropic acid ester in >98% ee.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
has been studied intensively, but any literature on the
enzymatic kinetic resolutions of 2-aryl-3-hydroxycom-
pounds such as tropic acid, is scarce. Only recently,
was an enzymatic kinetic resolution of this hydroxy acid
utilising lipase PS (Pseudomonas cepacia lipase) to target
the primary alcohol reported.20 The ethyl ester of tropic
acid was resolved, to give (S)-acetoxy tropic acid ethyl
ester in 39% yield and 87% ee and (R)-tropic acid ethyl
ester in 46% yield and 94% ee. Until now, only a few
reports on the resolution or enantioselective synthesis
of tropic acid have been published. So far, the most con-
venient method for the preparation of enantiopure (S)-
tropic acid is the hydrolysis of hyoscyamine and similar
compounds isolated from plants. Racemic tropic acid
can be resolved in various ways, for example, by
employing quinine21 or other co-crystallisation agents,22
preparative LC23 or electrophoreses.24 A report on an
elaborate synthesis of (R)-tropic acid appeared a few
years ago.25
(S)-Tropic acid (S)-1 is an important building block for
biologically active alkaloids such as hyoscyamine 2 and
hyoscine 3 (Fig. 1), which are well-established pharma-
ceuticals. The synthesis of the enantiomers of tropic acid
1 is a big challenge, since this compound racemises
under alkaline conditions. Analogously, hyoscyamine
and hyoscine racemise easily into their racemates atro-
pine and scopolamine. The enantiomers of tropic acid
can not only be applied in alkaloid synthesis but are also
building blocks in polyester synthesis. Poly-3-hydroxy-
acids are of great interest for bioerodable and biode-
gradable polymers,1 while the enantiomeric ratio of the
monomers has a dramatic effect on the crystallinity
and solubility of the resulting polymer.2,3
The enzymatic kinetic resolution of 2-arylpropionic
acids, mainly ibuprofen,4–12 and related structures,13–19
N
N
OH
O
HO
OH
OH
O
O
O
O
O
(S)-tropic acid 1
hyoscyamine 2
hyoscine 3
Figure 1. Tropic acid and some tropic acid containing alkaloids.
*
Corresponding author. Tel.: +31 15 278 9304; fax: +31 15 278 4289; e-mail: U.Hanefeld@tnw.tudelft.nl
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.10.032

D. Klomp et al. / Tetrahedron: Asymmetry 16 (2005) 3892–3896
3893
Table 1. Enzymes screened for the hydrolysis of 5 and their activity
Herein, we report a successful strategy for the enzymatic
kinetic resolution of tropic acid. In contrast to the enzy-
matic resolution described in the literature, this can be
achieved through functionalisation of the carboxylate
rather than of the primary alcohol group.
Enzyme
Reaction
time (h)a
Initial reaction
rate (mmol/h)b
Acylase
a-Chymotrypsin
—
—
—
—
18
—
—
—
96
—
—
—
—
0.071
—
—
—
0.018
Achromobacter sp. lipase
Candida rugosa lipase
Candida antarctica lipase B
Humicola lanuginosa lipase
Pig liver esterase
2. Results and discussion
2.1. Screening of enzymes for the hydrolysis of esters of
tropic acid
Rhizomucor miehei lipase
Subtilisin
— Indicates no activity.
A hydrolysis reaction was chosen as an enzyme screening
experiment, since in this way the reaction could be fol-
lowed in real time by monitoring the hydroxide con-
sumption. Initially, tropic acid lactone 4 was studied as
a test substrate. This b-lactone was synthesised via a
Mitsunobu reaction in 75% yield (Scheme 1).26 The
major advantages of this compound are that both the
hydroxyl and carboxylic acid groups are protected and
that the ester is activated as a result of the ring tension
in the four-membered lactone. The dual protection also
blocks polyester formation. The hydrolysis reactions
were carried out at 25 ꢁC in a 10 mM phosphate buffer
at pH 7.
a Time needed for separation of the enantiomers.
b Initial reaction rate for 200 U enzyme.
the other enzymes tested showed unexpectedly low activ-
ity towards this substrate. CRL was reported to be suc-
cessful in the resolution of 2-aryl propionic acids,9 but
apparently the additional hydroxyl group of tropic
acid renders this enzyme inactive towards ester 5.
The enzyme a-CT, which promoted the hydrolysis of
2-benzyl-3-hydroxypropanoic acid methyl ester, alth-
ough not enantioselectively,32 does not show any reac-
tivity with tropic acid as the substrate. The lactone
(3R,4S)-3-benzyl-4-(bromoethyl)oxetan-2-one, a similar
compound to 4, was reported to bind to and act as an
inhibitor for this enzyme.33 Inhibition of the enzyme
with 4 or 5 in an analogous pathway (an SN2 reaction
forming an ether) is unlikely to take place, however, still
no reaction was observed. Subtilisin showed some activ-
ity towards the hydrolysis of the tropic acid butyl ester,
but the reaction took over 4 days to complete.
OH
PPh3
DEAD
O
OH
THF
O
-78 oC to -10 oC
O
4
1
75%
Scheme 1. Synthesis of tropic acid lactone 4.
Attempts at the hydrolysis of tropine acetate with CAL-
B or subtilisin were unsuccessful, hence a coupling with
tropic acid to synthesise hyoscyamine by these enzymes
seemed to be unfeasible. The two parts are probably too
bulky to be able to fit in the active site, preventing an
enzymatic coupling. Based on the results described,
CAL-B was selected as the enzyme for the enzymatic
kinetic resolution of tropic acid.
The blank reaction, however, showed that 4 is too reac-
tive in the buffer. The starting material was converted to
racemic 1 within 24 h and therefore tropic acid butyl
ester 5 was chosen as an alternative test substrate. The
blank reaction now showed <2% conversion in 24 h.
The hydrolysis (Scheme 2) was performed with the com-
mercially readily available enzymes that were earlier
used successfully in the kinetic resolution of phenylprop-
ionic acids.
2.2. Enzymatic kinetic resolution in toluene
From the enzymes tested (see Table 1), CAL-B showed
the most promising results. After 18 h, (R)-tropic acid
(R)-1 and (S)-tropic acid butyl ester (S)-5 were obtained
in 90% and >99% ee, respectively. CAL-B is not gener-
ally known to resolve racemic acids, although a few
examples are known in which a transesterification with
amines27,28 or alcohols29,30 takes place. A successful
application of the resolution of a-methylene b-lactones
using this enzyme has also been reported.31 Some of
With an enzyme in hand that was proven to be very sta-
ble and selective in organic solvents, our attention next
focussed on the esterification reactions in toluene.
Therefore, b-lactone 4, which is activated due to the ring
strain, was selected as the starting compound.
The enzymatic resolution of 4 with butanol, employing
CAL-B as the catalyst in toluene, gave a very fast and
clean reaction towards butyl ester (R)-5 (Scheme 3).
OH
OH
OH
OH
enzyme
O
O
phosphate buffer
+
O
O
O
pH7
25 oC
5
(R)-1
(S)-5
Scheme 2. Hydrolysis of tropic acid butyl ester 5.

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D. Klomp et al. / Tetrahedron: Asymmetry 16 (2005) 3892–3896
OH
O
O
CaL-B
O
OH
+
O
O
O
toluene
80 oC
4
(R)-5
(S)-4
Scheme 3. Enzymatic resolution of tropic acid lactone 4.
The resolution of lactone 4 towards (R)-5 and (S)-4 pro-
ceeds within 70 min with an initial reaction rate of
2.6 mmol/h/200U (Scheme 3, Fig. 2). Both compounds
were obtained in an enantiomeric excess of >98% in
almost quantitative yields. With longer primary alcohols
such as octanol, the results and reaction times were sim-
ilar. The inversion of the stereoselectivity of the enzyme
by the long-chain alcohols which was shown for CRL
in the esterification of 3-hydroxybutyric acid,34 was
not observed for CAL-B. Remarkably, CAL-B does
not attack the primary alcohol that is released upon ring
opening of the lactone, no dimers or oligomers were
formed during the experiment. It is also striking that
in the resolution of the similar 2-phenylbutyric acid
vinyl ester with butanol, the product is also the (R)-butyl
ester.29 This means that the relative stereochemistry is
opposite from that observed with lactone 4. Apparently,
substitution of the hydroxyl group in tropic acid by a
methyl group causes the enzyme to change its preference
for the different groups in the molecule.
and (R)-tropic acid lactone were also obtained in high
yields in 90% and >98% ee, respectively. As described
earlier, the primary alcohol group of tropic acid is not
a substrate for CAL-B20 and therefore, no polyesters
were formed.
CAL-B is an outstanding enzyme for the resolution of
this b-hydroxy acid and it can be expected that it is also
a good enzyme for the resolution of 3-hydroxy-2-aryl-
propionic acids in general.
4. Experimental
4.1. General remarks
All experiments were performed in dried glassware
under a nitrogen atmosphere unless stated otherwise.
All chemicals were purchased from Aldrich or Acros.
Anhydrous solvents and solids were used as received.
Enzymes were purchased from Sigma, immobilised
Candida antarctica lipase B (CAL-B) as Chirazyme
L2,c-f.C2, lyo was a gift from Roche Diagnostics. Enan-
tiomeric excesses were determined and reactions were
followed by gas chromatography using a Shimadzu
GC-17A gas chromatograph, equipped with a 25 m ·
0.32 mm chiral column ChrompackTM Chirasil-Dex CB,
split injector (1/97) at 220 ꢁC, a Flame Ionisation Detec-
tor at 220 ꢁC and He as carrier gas. Retention times
(min) at 120 ꢁC isotherm: (R)-3-phenyl-oxetan-2-one 4
(9.9), (S)-3-phenyl-oxetan-2-one 4 (10.7), (S)-3-hydr-
oxy-2-phenyl-propionic acid butyl ester 5 (56.3), (R)-3-
hydroxy-2-phenyl-propionic acid butyl ester 5 (58.4),
(R)-3-hydroxy-2-phenyl-propionic acid methyl ester
(63.9), (S)-3-hydroxy-2-phenyl-propionic acid methyl
ester (70.0). NMR spectra were recorded on a Varian
Unity Inova-300 spectrometer at 25 ꢁC. For column
chromatography, Fluka silica gel 60 was used and
Merck aluminium sheets with silica gel 60 F254 were
used for TLC. Elution was carried out with mixtures
of petroleum ether 40–65 ꢁC (PE) and ethyl acetate
(EtOAc).
50
40
(R)-4
30
20
10
0
(S)-4
(R)-5
(S)-5
0
20
40
60
time/min
Figure 2. Reaction progress of the resolution of tropic acid lactone 4.
Since an enzymatic coupling of tropic acid and tropine
was not possible, a chemical coupling of (S)-4 and
endo-tropine was attempted. Due to the alkaline proper-
ties of the tertiary amine in tropine, the lactone was eas-
ily opened in this way. However, the stereogenic centre
in tropic acid is also readily racemised under these con-
ditions. The classical syntheses of atropine by refluxing
the two components in hydrochloric acid35,36 or by
treating tropic acid subsequently with acetyl chloride,
thionyl chloride and tropine hydrochloride21 do how-
ever offer an alternative route, starting from enantiopure
(S)-tropic acid and will give enantiopure hyoscyamine.
4.2. 3-Hydroxy-2-phenyl-propionic acid butyl ester 5
Tropic acid (3.3 g, 20 mmol) and butanol (1.8 mL,
20 mmol) were dissolved in toluene (50 mL). The mix-
ture was refluxed in a Dean–Stark set-up for 6 h and
the volatiles evaporated. After column chromatography
(PE/EtOAc 17:3), 3.8 g (17 mmol, 85%) of the ester 5
was obtained as a liquid. H NMR (300 MHz, CDCl3)
d 0.87 (t, 3H, J = 7.20 Hz, CH3), 1.25 (sextet, 2H,
CH2–CH3), 1.56 (quintet, 2H, J = 7.20 Hz, CH2–CH2–
3. Conclusion
The enzymatic resolution of tropic acid with CAL-B was
very successful using the strategies described. Both (R)-
and (S)-tropic acid butyl esters can be obtained in >98%
ee and high yields. The complementary (S)-tropic acid
1

D. Klomp et al. / Tetrahedron: Asymmetry 16 (2005) 3892–3896
3895
CH3), 2.38 (br s, 1H, OH) 3.83 (t, 2H, J = 5.70 Hz,
CH2–CH2–CH2–CH3), 4.04–4.18 (m, 3H, CH–CH2–
OH), 7.22–7.40 (m, 5H, ArH).
CAL-B (50.0 mg, 160 U) added. After 70 min, the reac-
tion was stopped by filtering off the enzyme. The filtrate
was concentrated. The products were separated by col-
umn chromatography (PE/EtOAc 17:3) giving 204 mg
25
4.3. 3-Phenyl-oxetan-2-one 4
0.94 mmol, 47%, ee >98%, ½aꢁD ¼ þ40:0 (c 3.4, CHCl3)
of (R)-3-hydroxy-2-phenyl-propionic acid butyl ester
DEAD (diethyl azodicarboxylate) (1.89 mL, 12 mmol)
was added dropwise to a stirred solution of triphenyl-
phosphine (3.18 g, 12 mmol) in THF (40 mL) at
ꢀ78 ꢁC. After about 30 min, the suspension became
white and then a solution of tropic acid (2.00 g,
12 mmol) in THF (40 mL) was added dropwise. The
resulting mixture was stirred and warmed in about 2 h
to ꢀ10 ꢁC, at which temperature the solution was homo-
geneous. After concentration at room temperature and
purification by column chromatography (PE/EtOAc
17:3), the product was isolated as a clear liquid in
1.30 g yield (9.00 mmol, 75%). 1H NMR (300 MHz,
CDCl3) d = 4.34 (dd, 1H, J = 4.76, 5.12 Hz, CH2),
4.64 (dd, 1H, J = 5.12, 6.78 Hz, CH2), 4.92 (dd, 1H,
J = 4.76, 6.78 Hz, CH), 7.26–7.42 (m, 5H, Ar-H), 13C
NMR (75 MHz, CDCl3) d = 56.91 (CH), 66.38 (CH2),
127.15 (2 · Ar-C), 128.31 (Ar-C), 129.19 (2 · Ar-C),
132.63 (Ar-C), 169.54 (CO).
(R)-5 as a colourless liquid and 139 mg 0.92 mmol,
25
46%, ee >98%, ½aꢁD ¼ þ24:0 (c 4.0, CHCl3), mp:
45.6 ꢁC of (S)-3-phenyl-oxetan-2-one (S)-4 as a white
crystalline solid. Hydrolysis of (S)-4 yielded (S)-1 with
25
15
½aꢁD ¼ ꢀ63:3 (c 0.3, acetone); Lit.21 ½aꢁD ¼ ꢀ83:3 (c
1.8, acetone).
Acknowledgements
We thank Prof. Maschmeyer for enabling this research.
U.H. thanks the Royal Netherlands Academy of Arts
and Sciences (KNAW) for a fellowship. The authors
gratefully acknowledge Diagnostics Penzberg (Dr. W.
Tischer) for the generous gift of CAL-B.
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