Systematic investigation of the kinetic resolution of 3-hydroxy fatty acid esters using Candida antarctica lipase B (CALB) and the influence of competing oligomerization on the enantiomeric ratios

Article abstract of DOI:10.1080/00397911.2010.535132

The kinetic resolution of 3-hydroxy fatty acid esters C8:0 to C16:0 with Candida antarctica lipase B shows common plots of the enantiomeric excesses of the product and substrate, respectively, versus the conversion and an enantiomeric ratio E of 27 calculated from ee(p). Differences in E, either calculated from the products or the substrates, could be explained by competing oligomerization as a second substrate-consuming process. This reaction is slow compared to acylation, and the remaining enantiomer was oligomerized. Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397911.2010.535132

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Systematic Investigation of the Kinetic
Resolution of 3-Hydroxy Fatty Acid
Esters Using Candida antarctica Lipase B
(CALB) and the Influence of Competing
Oligomerization on the Enantiomeric
Ratios
Markus Braner a , Stefan Zielonka a , Sylvia Auras a & Stefan H.
Hüttenhain a
a Hochschule Darmstadt, Fachbereich Chemie und Biotechnologie ,
Darmstadt , Germany
Accepted author version posted online: 06 Sep 2011.Published
online: 16 Dec 2011.
To cite this article: Markus Braner , Stefan Zielonka , Sylvia Auras & Stefan H. Hüttenhain (2012)
Systematic Investigation of the Kinetic Resolution of 3-Hydroxy Fatty Acid Esters Using Candida
antarctica Lipase B (CALB) and the Influence of Competing Oligomerization on the Enantiomeric
Ratios, Synthetic Communications: An International Journal for Rapid Communication of Synthetic
Organic Chemistry, 42:7, 1019-1025, DOI: 10.1080/00397911.2010.535132
To link to this article: http://dx.doi.org/10.1080/00397911.2010.535132
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Synthetic Communications1, 42: 1019–1025, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2010.535132
SYSTEMATIC INVESTIGATION OF THE KINETIC
RESOLUTION OF 3-HYDROXY FATTY ACID ESTERS
USING CANDIDA ANTARCTICA LIPASE B (CALB) AND
THE INFLUENCE OF COMPETING OLIGOMERIZATION
ON THE ENANTIOMERIC RATIOS
Markus Braner, Stefan Zielonka, Sylvia Auras, and
Stefan H. Hu¨ttenhain
Hochschule Darmstadt, Fachbereich Chemie und Biotechnologie,
Darmstadt, Germany
GRAPHICAL ABSTRACT
Abstract The kinetic resolution of 3-hydroxy fatty acid esters C8:0 to C16:0 with Candida
antarctica lipase B shows common plots of the enantiomeric excesses of the product and
substrate, respectively, versus the conversion and an enantiomeric ratio E of 27 calculated
from ee(p). Differences in E, either calculated from the products or the substrates, could be
explained by competing oligomerization as a second substrate-consuming process. This
reaction is slow compared to acylation, and the remaining enantiomer was oligomerized.
Keywords CALB; 3-hydroxy fatty acid ester; kinetic resolution
Received October 21, 2010.
Address correspondence to Stefan H. Huttenhain, Hochschule Darmstadt, Fachbereich Chemie
¨
und Biotechnologie, Hochschulstrasse 2, 64289 Darmstadt, Germany. E-mail: stefan.huettenhain@h-da.de
1019

1020
M. BRANER ET AL.
INTRODUCTION
3-Hydroxy fatty acid esters are important building blocks for a wide range of
target molecules, and research for convenient and highly selective methods for their
synthesis is still ongoing.[1–5] As the 3-oxo species are readily available,[6,7] one stan-
dard procedure to get the hydroxy esters is by reducing them. The gold standard is
doubtlessly the Noyjori’s Ru–Binap-catalyzed hydrogenation, which always leads to
greater than 95% conversion and enantiomeric excess (ee), as recently reported by
Bruckner. Thus, depending on the catalyst, nearly quantitative yields of each desired
¨
enantiomer can be obtained.[8,9] However, one alternative to this somewhat expens-
ive and sensitive catalyst is the enzymatic resolution of the racemic mixture from the
conventional hydrogenation of the respective oxo-ester.[10,11] Though this method
will produce, in principle, only 50% of the theoretical yield of the racemic starting
mixture, it is effective when both separated enantiomers are needed. In our search
for easily obtained, homochiral, liposome-building, amphiphilic molecules, we used
Candida antarctica lipase B (CALB, Novozym 435) for the resolution of the
homolog-even 3-hydroxy fatty acid esters C8:0 to C16:0, according to Eq. (1):
Absolute configuration of the products was assumed as described.[10,11]
Surprisingly, there are few reports regarding the kinetic resolution of
3-hydroxy fatty acid esters. Bornscheuer et al. investigated the influence of diverse
factors, such as solvent polarity and water content, on the transesterification of
1a=b-, 2a=b-, and 3-hydroxy hexanoic acid methyl ester using Pseudomonas cepia
lipase (PCL). They obtained moderate to good enantiomeric ratios E (5–17), depend-
ing on the solvent, and they reported the kinetic resolution of these three molecules
using PCL in supercritical CO2.[12,13] The same lipase was used by Ikunaka for the
resolution of 3-hydroxy tetradecanoic acid and its methyl ester to gain homochiral
compounds for the synthesis of important pharmaceutical molecules.[14] Other than
those mentioned, only short-chain homologs seem to be investigated (e.g., Garcia
used CALB to separate the enantiomers of 4-chlorobutyrate and valeric acid ester
by aminolysis of the esters).[15] To our knowledge, there has been no systematic
investigation of the kinetic resolution of long-chain fatty acid esters.
Using vinyl acetate as solvent and acylating reagent, we found significant simi-
larity in the reaction rates of all hydroxy esters. There is, within experimental error,
only one common plot for all data from 1a=b to 4a=b, with up to a 50% conversion
rate. Only the most hydrophobic 5a=b ester had a significantly slower reaction

3-HYDROXY FATTY ACID ESTERS
1021
Figure 1. Resolution of 3-hydroxy fatty acid esters C8:0–C16:0 by CALB and vinyl acetate. Plots of
2
2
~
conversion versus time for C8–C14 (^) y ¼ ꢀ0.0048x þ 0.9903x þ 2.205; R ¼ 0.9775 and C16 (
)
y ¼ ꢀ0.0009x2 þ 0.3958x þ 0.2193; R2 ¼ 0.9978.
(Fig. 1). As CALB is known to hydrolyze long-chain triglycerides very slowly, we
were not astonished by the slower reaction but by the homogeneity of the other data
and the sudden drop from one homolog to the next.
Amazingly, when plotting conversion rate versus the ee of the remaining
substrates, ee(p) or the respective products ee(s), all five compounds were represented
by common curves (Figs. 2a and b).
Calculation of the average enantiomeric ratio at a 30% conversion from the
regression curves of the product using the equations of Chen and Shi showed
E ¼ 27. The calculation of E is according to the equations in Refs. 16 (see also Refs.
10 and 11). This is different from the results reported by Adam et al. for the resol-
ution of 2-hydroxy esters, which differ significantly in ee (21% to 98%, respectively)
with the enantiomeric ratios ranging from 5.8 to 55, respectively.[16] Moreover,
E ¼ 27 is much greater than that found by Bornscheuer et al., who used PCL for
the resolution of 1a=b-, 2a=b-, and 3-hydroxy hexanoic acid methyl ester in his
systematic investigation of the influence of solvents on this reaction.[12]
Incomprehensibly, however, E differs significantly when calculated from either
the ee of the subtrate [ee(s)] or the ee of the product [ee(p)]; because of the oligomer-
ization of the bifunctional molecules, the enantiomeric ratio amounts to only 13
when calculated from ee(s). The summation of the relative percentages for the (R)
forms of the compounds 1a=b and 1c=d showed an excess of 4.46% for a 40%
conversion and 12.3% at about an 80% conversion, respectively. This was not limited
to compound 1 but was found less marked for all others as well. From Fig. 3, one
can see that the common graph for the esters 1a=b to 5a=b has up to 6% excess of
the (R) forms, and the curve for the hydroxy octanoic ester is clearly separated
and significantly steeper.
Acidic racemization of the alcohol group could be discounted; a filtered charge
of hydroxy ester 5, with a defined ratio of (R)- and (S)-alcohol and acetate (5a ¼
20.02%; 5b ¼ 42.53%, 5c ¼ 1.78%, 5d ¼ 35.67%), was stirred for several days in the

1022
M. BRANER ET AL.
Figure 2. Common graphs of enantiomeric excess (ee) versus conversion for the resolution of 3-hydroxy
&
~
fatty acid esters C8:0–C16:0 by CALB and vinyl acetate. c8 (^), c10 ( ), c12 ( ), c14 ( ), and c16 (ꢀ).
*
(a) y ¼ 0.0046x2 þ 1.161x ꢀ 3.8909; R2 ¼ 0.9711; (b) y ¼ꢀ0.004x2 ꢀ 0.131x þ 97.264; R2 ¼ 0.9205.
Figure 3. Plots of conversion versus excess of the (R)-form of the 3-hydroxy esters C8 (^) y ¼
2
2
2
2
~
ꢀ0.0014x þ 0.0458x þ 0.3237; R ¼ 0.9963 and C10–C16 ( ) y ¼ꢀ0.0009x þ 0.0264 þ 0.4608; R ¼ 0.9484.

3-HYDROXY FATTY ACID ESTERS
1023
respective vinyl acetate without a significant change in the ratios, which is clear
evidence for the involvement of the enzyme in the process.
A closer look at the absolute amounts [gas chromatographic (GC) area counts]
of the substrate isomers in the reaction solution revealed that over time, the sum of
the area counts of both (R) and (S) forms decreased strongly, but the (S)-form
reduction was stronger. We determined that there must be another process competing
with the acylation that consumed the hydroxy esters. As no free hydroxy acid could be
detected, an enzymatic hydrolysis of the substrates could be ruled out.
In a separate experiment, pure 1a=b was incubated with CALB in hexane.
Continuous GC=MS analysis over 8 days showed a 14% decline in the amount of
the hydroxy ester, but no decomposition product was formed. A workup of the reac-
tion by short-way distillation easily separated the remaining hydroxy ester from the
residue. It showed an ee of 5% for the (S) form. An alkaline methanolysis of the resi-
due resulted in educt 1a/b, with an ee of 6% for the (R) alcohol. From this, one has to
conclude that beside the desired acylation reaction, oligomerization of the 3-hydroxy
esters also occurred. Because of the preferred acylation of the (R) form, the concen-
tration of the (S) form rose in the solution, and as the oligomerization seems to be
nonselective (Eꢁ1), its concentration decreased more rapidly than that of the (R)
form. As confirmed by Fig. 3, this process should increase with decreasing concen-
tration of the (R) form.
Lipases, especially CALB, are known to catalyze the synthesis of polyesters
from methyl diesters and diols and the lactonization of x-hydroxy fatty acid
esters.[18,19] This is clearly the case for the reported experiments in which one ester
acts as the acyl donor while the hydroxyl group of another molecule plays the role
of the substrate to form an oligomer or polyester. This is contrary to the results
reported by Adlercreutz for the reaction of lactic acid and its esters.[20] From the
absence of dimer formations, they concluded that the hydroxy group would not play
the role of a nucleophile, and they did not find any CALB-catalyzed esterifications of
lactic acid and dodecanoic acid. As Adam et al. have shown, a-hydroxy esters
undergo enzyme-catalyzed acylation, and lactic acid is probably a special exception.
In most other cases, it is likely that an oligomerization side reaction will occur with
other bifunctional molecules as well.
EXPERIMENTAL
Unless otherwise stated, all materials used were of analytical grade and
bought from Merck, Darmstadt. CALB Batch No. LC2 00009 was provided by
Novo Nordisk.
Preparation of 3-Hydroxy Fatty Acid Methyl Ester
The 3-hydroxy fatty acid methyl esters were synthesized from the respective
keto esters by sodium borohydride reduction in methanol.[21] In the cases of homo-
logs C14:0 and C16:0, tetrahydrofuran (THF)=methanol (1:1) was used as the sol-
vent to increase solubility. After quantitative conversion, the products were
purified by short-way distillation. Yields were not determined, but the purity was
measured using gas chromatography.

1024
M. BRANER ET AL.
Keto esters were prepared according to Clay et al.’s method b.[22]
Kinetic Resolution Experiments
Five ml of the enantiomeric mixture a=b and 10 ml of vinyl acetate were added to
30 mg of CALB in 1 ml of the respective solvent. GC analysis of the reaction mixture
was performed at regular time intervals. The details of the GC are as follows: HP
6890 GC, Autosampler 7683, split=splitless injector, mass selective detector (MSD)
5973, Cyclodex-B (30 m, i.d. 0.25-mm, 0.25-mm film) from Agilent, He 1 ml=min.
All separations were carried out isothermally. The temperature and retention times
for each mixture are as follows: 1 (140 ꢂC), [a: 9.6 min, b: 9.9 min, c: 11.8 min, d:
12.1 min]; 2 (150 ꢂC), [a: 15.9 min, b: 16.4 min, c: 19.8 min, d: 20.2 min]; 3 (160 ꢂC),
[a: 25.7 min, b: 26.4 min, c: 31.3 min, d: 31.9 min]; 4 (160 ꢂC), [a: 63.3 min, b:
65.3 min, c: 76.1 min, d: 77.6 min]; and 5 (160 ꢂC), [a: 159.7 min, b: 164.4 min, c:
189.2 min, d: 192.8 min].
The conversion of compound 5 in vinyl acetate was stopped by filtering off
the enzyme after 4 days (5a ¼ 20.02%; 5b ¼ 42.53%, 5c ¼ 1.78%, 5d ¼ 35.67%). The
enzyme-free solution was stirred for an additional 4 days with regular GC inspec-
tions and showed no racemization or any other change in enantiomer distribution.
OLIGOMERIZATION
For the oligomer identification, 0.5 mmol of 1a=b (87 mg, 92 ml, respectively)
were added to 550 mg of CALB in 3 ml of hexane. The mixture was stirred at room
temperature, and 50-ml analytic samples were taken regularly, diluted with 500 ml of
hexane, and measured by GC. After 8 days, the solvent was evaporated and
short-way distillation was done with a Buchi BKR-51 (1.7 ꢃ 10ꢀ1 mbar=100 ꢂC) to
¨
give a clean fraction of compound 1 (59 mg).
Two ml of 10% KOH in methanol were added to the distillation residue, and
the mixture was stirred at 60 ꢂC for 15 min to transform the nonvolatile oligomer
mixture to the methyl ester. The methanol phase was extracted with 1 ml of isooc-
tane, which was directly analyzed and showed 1 as the main compound.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of ZFE, Hochschule
Darmstadt.
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