Robust enzymatic resolution of 3-fluoromandelic acid with lipase PS supported on celite

Article abstract of DOI:10.1021/op300137a

The resolution of different mandelic acids using the lipase PS Amano SD enzyme is described. By supporting the lyophilized enzyme over Celite, both the activity and the stability of lipase PS in organic media were significantly improved, enabling the robust resolution scale-up of 3-fluoromandelic acid. The methodology was extended to produce a range of optically pure (R)-mandelic acids, avoiding tedious extractions or chromatography.

Full text of DOI:10.1021/op300137a

Article
pubs.acs.org/OPRD
Robust Enzymatic Resolution of 3-Fluoromandelic Acid with Lipase
PS Supported on Celite
́
Javier Mendiola,* Susana García-Cerrada,* Oscar de Frutos, and María Luz de la Puente
́
Centro de Investigacion, Lilly S.A., Avda. de la Industria, 30, Alcobendas-Madrid 28108, Spain
S
* Supporting Information
ABSTRACT: The resolution of different mandelic acids using the lipase PS “Amano” SD enzyme is described. By supporting
the lyophilized enzyme over Celite, both the activity and the stability of lipase PS in organic media were significantly improved,
enabling the robust resolution scale-up of 3-fluoromandelic acid. The methodology was extended to produce a range of optically
pure (R)-mandelic acids, avoiding tedious extractions or chromatography.
an internal kit of 24 hydrolytic enzymes.8 Both processes (see
1. INTRODUCTION
Schemes 2 and 3) were carried out following our standard
Mandelic acid and its derivatives are important building blocks
protocols.9
in bioactive compounds.1 They have been widely utilized as
chiral pool starting material and reagents for synthetic
purposes. Although an ample range of derivatives are
commercially available, optically pure materials are often
expensive, thus limiting their use in large- and medium-scale
processes.
Saponification (from 1 to 2) was run in an aqueous
phosphate buffer at 25 °C for 24 h. HPLC/MS monitoring
showed conversion close to 50% in 6 out of the 24 tests. Those
hit reactions were worked up and analyzed by chiral HPLC/MS
(Table 1). Only CAL-A (either lyophilized or immobilized)
showed optical purity exceeding of 98% for the ester derivative;
however, over-reaction (>50% of conversion) was mandatory
to ensure the desired optical purity (>98% ee).
For that reason, in the last 20 years, a range of different
approaches (Scheme 1) have been developed for the
preparation of mandelic acids in enantiomerically pure form:
(a) chemoselective synthesis (through the reduction of α-keto-
acid derivatives),2 (b) classical resolution (salt screening),3 (c)
chromatography (of racemic or diasteromeric derivatives),4 and
(d) biotransformation (via enzymatic resolution),5 deracemiza-
tion,6 and reduction with yeast.2b
3-(R)-Fluoromandelic acids were identified in our chemistry
group to cover a structure−activity relationship (SAR) study in
a project under development. After an extensive bibliographic
search, our group compiled a significant number of references
dealing with the enzymatic resolution of mandelic deriva-
tives.5−7 However, to the best of our knowledge, only two
papers6d,7 reported the direct resolution of such α-hydroxy
acids (in both cases achieved via lipase PS acetylation).7
On the basis of our findings, we envisaged the enzymatic
resolution of 3-fluoromandelic acid (2) via acetylation, using
lipase PS. Initially promising results were obtained at small scale
(50 mg, 50% conversion, >95% ee). However, attempts to scale
up the resolution to 1 g failed due to low conversion, under a
range of different temperatures or enzyme/substrate ratios.
In the present contribution the development of a robust
resolution of 3-fluoromandelic acid and the application of this
methodology to a further nine mandelic acids using lipase PS
supported over a ceramic support is described.
Acetylation screen (from 2 to 1) was performed in MTBE at
25 °C for 24 h using vinyl acetate as the source of acetate
(Table 2). All the reactions were monitored by HPLC/MS.
Among them, five showed a conversion close to 50%, and were
worked up for further chiral HPLC/MS analysis. Lipase PS
“Amano” SD and lipase AK “Amano” 20 showed very
promising results with optical purity ranging between 97%
and 98% ee for both the ester and the alcohol derivatives. While
CAL-A lyophilized enzyme showed adequate optical purity
(97% ee) for the ester due to a low conversion, the immobilized
CAL-A over-reacted and provided ee >98% for the hydroxyl
acid. Immobilized Thermomyces lanuginosus was nonselective.
As reported in the literature, conversion and optical purity
results confirmed lipase PS “Amano” SD as the enzyme of
choice for the enzymatic resolution of 3-fluoromandelic acid.
In order to improve the results of the resolution, more dilute
conditions (20 vol of MTBE) and addition of a higher excess of
vinyl acetate (5 equiv) were initially explored. Unexpectedly,
very low conversion (20%) was observed at both 1:1 and 2:1
enzyme/substrate ratio. Furthermore, conversion could not be
increased by modifying the temperature, the time of reaction,
or the solvent (dichloromethane, acetonitrile, toluene).
It is well-known that enzyme activity is compromised in
organic solvents or extreme conditions. However, it has been
reported that the immobilized enzymes are more stable and
active than the corresponding lyophilized forms. On the basis of
literature references about the activity of lipases over ceramic
2. RESULTS AND DISCUSSION
The conditions reported in the literature for the resolution of 3-
fluoromandelic acid failed during the scale-up studies. On the
basis of our previous experience on enzymatic transformations,
we designed two resolution screens (saponification and
acetylation) for the resolution of 3-fluoromandelic acid with
Received: May 24, 2012
Published: June 26, 2012
© 2012 American Chemical Society
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Scheme 1
Scheme 2
Scheme 3
at 20- and 200-g scale (reaction time was 96 h) to give
consistent results.
Table 1. Saponification screening for affinity enzyme/
substrate for 2 (24 h)
Additionally, an extensive solubility screen of the products
identified that the desired alcohol could be precipitated directly
from the reaction mixture by the addition of dichloromethane.
This finding gave an efficient and cost-effective process for the
preparation of desired pure 3-(R)-fluoromandelic acid. The
configuration of the product isolated was established as (R) by
comparison of the specific optical rotation value with the one
described in the literature.12
Furthermore, it was identified that 3-fluoromandelic acid
could be epimerized in strong basic media. This finding was
used to increase the yield of this simple and robust process.
Thus, the isolated undesired (S)-acetyl derivative was hydro-
lyzed with 15% aq NaOH/toluene at reflux to yield a racemic
mixture that could be resubjected to enzymatic resolution to
provide further 3-(R)-fluoromandelic acid (Scheme 4).
a
b
enzyme
acylase “Amano”
ES (1) (ee, %)
AL (2) (ee, %)
E
34 (ES1)
7 (ES2)
51 (AL2)
6 (AL1)
4
1
lipase AS “Amano” SD
D-aminoacylase “Amano”
CAL-A
81 (ES2)
>98 (ES1)
>98 (ES1)
15 (ES1)
77 (AL1)
81 (AL2)
75 (AL2)
19
49
31
3
CAL-A-IMB
protease from Aspergillus oyzae
49 (AL2)
a
b
Calculated by chiral HPLC, see Supporting Information. E value
calculated according to Rackels et al. (Enzyme Microb. Technol 1993,
15, 1051).
Table 2. Esterification screening for affinity enzyme/
substrate for 1 (24 h)
a
b
enzyme
ES (1) (ee, %)
AL (2) (ee, %)
E
lipase PS “Amano” SD
lipase AK “Amano” 20
CAL-A
>98 (ES2)
97 (ES2)
97 (ES2)
47 (ES2)
85 (ES2)
>98 (AL1)
>98 (AL1)
49 (AL1)
458
303
107
11
Scheme 4
CAL-A-IMB
>98 (AL1)
Thermomyces lanuginosus IMB
57 (AL1)
22
a
b
Calculated by chiral HPLC, see Supporting Information. E value
calculated according to Rackels et al. (Enzyme Microb. Technol 1993,
15, 1051).
supports,10 inexpensive Celite was selected as the immobilizing
support in our laboratories. A 1-to-1 w/w Celite/lipase PS
“Amano” SD mixture was suspended in 1 volume of water,
homogenized with mechanical stirring, and dried in an oven at
40 °C for 24 h, to provide the “Celite supported enzyme lipase
PS Amano SD” (KF11 analysis indicated <2% of water).
Acetylation was repeated at 300-mg scale using the freshly
supported immobilized enzyme. Under previous conditions (20
vol MTBE, 5 equiv vinyl acetate), 50% conversion in 72 h at 25
°C was obtained when a 2/1 lipase PS−Celite/substrate (1/1
enzyme:substrate) ratio was used. The procedure was repeated
To evaluate the potential recycling of the supported enzyme
as a further improvement to the protocol, immobilized lipase
PS “Amano” SD−Celite from the previous resolution was used
for a second cycle under identical reaction conditions. Analysis
of the results from this test showed that the activity of the
enzyme had not been impacted, from 20 g of racemic 3-
fluoromandelic acid, 11.9 g (60%) of 3-(R)-fluoromandelic
acid12 was isolated after two cycles (Scheme 4).
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Table 3. Resolution of different mandelic acids (3) using lipase PS “Amano” supported on Celite
d
d
entry
Ar−X
ee (AL) (25 °C) (%)
ee (ES) (25 °C) (%)
E
ee (AL) (40 °C) (%)
ee (ES) (40 °C) (%)
E
1
4-Br (3a)
3-Cl (3b)
4-CF3 (3c)
3-MeO (3d)
4-Me (3e)
4-MeO (3f)
2-F (3g)
88
79
86
84
88
91
45
97
98
b
98
97
98
93
98
98
99
96
98
b
290
159
276
73
99
97
99
97
96
99
60
a
98
96
98
83
97
97
99
a
525
207
525
45
2
3
4
5
290
316
310
207
458
−
259
347
367
−
−
−
6
7
8
H (3h)
9
4-F (3i)
a
a
10
11
4-OH (3j)
OCH2O (3k)
a
a
c
c
−
a
a
−
a
b
c
d
Resolution was not performed. There were solubility issues. Decomposition products were detected. E value calculated according to Rackels et
al. (Enzyme Microb. Technol 1993, 15, 1051).
In view of these results, Celite-supported lipase PS “Amano”
SD was utilized to determine the potential scope and generality
of this protocol for the resolution of other substituted mandelic
acids (Table 3, Scheme 5). As observed for 3-fluoromandelic
acid, the immobilized enzyme showed very high selectivity
towards one enantiomer.
acid could be increased via chemical racemization in basic
media, separation and subsequent acetylation resolution,
recycling the previous supported enzyme (two separate
steps). There is a reactivity trend for the enzymatic resolution
of substituted mandelic acids, reactivity was found as para >
meta > ortho.
4. EXPERIMENTAL SECTION
Scheme 5
Enzymes and reagents. This work has been conducted
with hydrolytic enzymes procured from Biocatalytics Co.
(USA) (Hydrolytic enzyme screening kit, ICR-2400), from
Amano Co. (Japan) and from Sigma (USA).
1
Analytical conditions. H NMR spectra were acquired on
a Bruker Avance DPX 300 MHz spectrometer.
HPLC(DAD)/MS was used for the determination of
reaction conversion and enantiomeric excess of both remaining
substrate and resulting product.
All analytical studies were performed on a Series 1100 Liquid
Chromatography/Mass Selective Detector LC/MSD (Agilent,
Waldbronn, Germany) driven by ChemStation software (Rev.
A10.02, Agilent Technologies).
Sample solution of reference materials and those products
yielded by enzymatic screen was prepared by dissolving 2 mg in
1 mL of a 9/1 hexane/ethanol mixture.
High Performance Liquid Chromatography coupled with
Diode Array and Mass Detection (HPLC/UV(DAD)/MS) was
used for the optical purity assessment of enzymatic hits (both
remaining substrate and resulting product) and isolated
products.
HPLC grade n-hexane and ethanol (EtOH) were purchased
from Merck (Darmstadt, Germany) while 2-propanol (iso-
propanol; IPA) was supplied by LabScan (Dublin, Ireland).
Spectrophotometric grade trifluoroacetic acid (TFA) was
provided by Sigma-Aldrich (Steinheim, Germany). Chromatog-
raphy was performed on Chiralpak AD [amylose tris (3,5-
dimethyl-phenyl carbamate)] and Chiralcel OJ [cellulose tris
(4-methyl benzoate)] columns from Daicel (Chiral Technol-
ogies Europe). The dimension of the analytical columns was
150 × 4.6 mm. with the enantioselective phase coated onto a 5
um silica-gel substrate. (Further information is compiled in the
Supporting Information).
Optical rotation was measured in a Perkin-Elmer Polarimeter
343 system. Specific rotation was calculated based on the
equation [α]λT = (100·α)/(l·c) where l is the cell path length in
decimeters (l = 1 in our unit); c is the concentration in g/100
mL (c = 1 for 10 mgml−1); T is the temperature (given in
The temperature and the reaction time were adjusted for
each substrate. First, a screen at 25 °C was performed (Table
3). Two samples failed (entries 10, 11) due to solubility issues
and cross reactivity and two samples (entries 8, 9) showed high
selectivity and accurate conversion for a success resolution
(>97% ee for hydroxyl acid, and >96% for the acetyl
derivative). A second screen at 40 °C showed excellent
selectivity and an accurate conversion (40−50%) for all
examples except for the o-fluoro derivative (entry 7, 60% ee
for hydroxyl acid, which performed with good ester selectivity
profile (99% ee) but poor conversion). This finding confirmed
the general trend that, in general, minor steric hindrance was
the major influence in the reactivity with supported enzyme,
and this was reflected in the need for further optimization for
the resolution of the o-fluoro-substituted mandelic acid.
Functionalization at ortho/meta/para required a reaction
temperature ∼40 °C, but reactivity was found as para > meta
> ortho on the basis of conversion data at 25 and 40 °C. Small
substituents such as F in the para/meta position or no
substitution provided good conversion at 25 °C.
3. CONCLUSIONS
A robust protocol for the enzymatic resolution of α-hydroxyl
mandelic acids has been designed, developed and validated. A
more stable form of the immobilized lipase PS “Amano” SD
enzyme was prepared for use in organic media by simply
supporting the enzyme on Celite. The stability and activity of
the new supported form was successfully utilized for the
resolution of a range of mandelic acids and the enzyme could
be recycled. The yield of the resolution of 3-fluoromandelic
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degrees Celsius) at which experiments have been run and λ is
the wavelength of light used for the observation in nanometers
(589 nm, the D line of a sodium lamp, in our protocol).
Concentration and solvent data is included.
7.27−7.19 (m, 2H), 7.15−7.09 (m, 1H), 6.14 (br, 1H), 5.07 (s,
1H).
ASSOCIATED CONTENT
* Supporting Information
■
General procedure for the enzymatic hydrolytic
screening. To 10 mg of each enzyme in a test tube, is
added 0.1 mmol of substrate dissolved/suspended in 1 mL of
phosphate buffer pH 7.5 0.1 M (a stock solution/suspension
can be used). The mixtures are incubated with agitation at 30
°C, and the progress of the reaction is monitored by any
preferred method over a 24 h period (HPLC and/or TLC), to
check the conversion of the ester to the alcohol (recom-
mended: 8 h, 16 h, 24 h). After 24 h, HCl 1N (1 mL) and
MTBE (2 mL) are added to each mixture. The organic phases
are collected and filtered through a 0.45 nylon filter. The solvent
is removed, and the conversion is monitored by LCMS.
S
Table 1 - retention time (in minutes) of the isomers of each
racemic alcohol (1, 3a−g) and its corresponding racemic acetyl
derivative (2, 4a−g); Table 1 - results from the optical purity
assessment in (1, 2, 3a−g, 4a−g). This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*mendiola_javier@lilly.com (J.M.); susana.garcia@lilly.com
(S.G.-C.)
■
Notes
General procedure for supporting the enzyme over
Celite. Lipase PS “Amano” SD (40 g) and Celite (40 g) in
water (40 mL) were well stirred inside a plastic container until
homogenization. This mixture was dried in the vacuum oven at
40 °C and 4 mbar until the quantity of water was below 2%.11
General procedure for the resolution of mandelic
acids. The enzyme supported over Celite (40 g) was
suspended in MTBE (400 mL) and then, racemic 3-
fluoromandelic acid (20 g, 0.118 mol) and vinyl acetate
(54.54 mL, 0.558 mol) were added. Reaction mixture was
stirred overnight at 23 °C for 96 h. The supported enzyme was
filtered and washed with TBME (93 mL) and the filtrate was
evaporated to dryness. The residue was stirred with DCM (10
mL) for 10 min. The white solid obtained was filtered and
washed with DCM (1.5 mL). Pure 3-(R)-fluoromandelic acid
was obtained (first batch). The fraction soluble in DCM was
concentrated and triturated again with DCM to obtain a second
batch of pure 3-(R)-fluoromandelic acid (Purity >99%, 100%
ee). Total amount: 7.02 g (70.2% corrected yield, 41.4 mmol).
Fraction soluble in DCM was concentrated to give a colourless
oil (76.6 mmol of mixture). It was dissolved in toluene (62 mL)
and 15% aq. NaOH (66 mL) was added. The mixture was
heated at 100 °C for 8 h. Then, reaction was cooled to 22 °C,
toluene was removed under vacuum, and 2 N aq. HCl was
added until pH: 3−4. The mixture was extracted with MTBE (2
× 100 mL), washed with brine (50 mL) and dried over
Na2SO4. It was filtered and solvent was removed under vacuum
to give a white solid (13.03 g, 76.6 mmol). For the second
resolution, the previous recovered supported enzyme mixture
was suspended in MTBE (400 mL) and then, racemic 3-
fluoromandelic acid (76.6 mmol) and vinyl acetate (37.44 mL,
0.383 mol) were added. Reaction mixture was stirred overnight
at 23 °C for 96 h. The supported enzyme was filtered and
washed with MTBE (50 mL) and the filtrate was evaporated to
dryness. The residue was stirred with DCM (6 mL) for 10 min.
The white solid obtained was filtered and washed with DCM (1
mL). Pure 3-(R)-fluoromandelic acid was obtained (first batch).
The soluble fraction in DCM was concentrated and triturated
again with DCM to obtain a second batch of pure 3-(R)-
fluoromandelic acid (Purity >99%, 100% ee). Total amount:
4.89 g (75.0% corrected yield, 28.7 mmol). Combining the
entire processes, after a first enzymatic resolution, subsequent
epimerization, and final second enzymatic resolution, 11.91 g
(60% overall yield, 79% corrected yield, 70.0 mmol) of 3-(R)-
fluororomandelic acid12 was isolated as a white solid. 1H NMR
(DMSO-d6, 300 MHz): 12.55 (br, 1H), 7.43−7.36 (m, 1H),
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. Lorraine Murtagh for her assessment and helpful
discussion, Dr. Carlos Jaramillo, Dr. Marta Cifuentes, and Dr.
■
́
Cristina Garcıa Paredes for their continuous support, and
Alcala Pilot Plant for confirming the robustness of the
́
enzymatic resolution at 200-g scale.
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