New continuous production process for enantiopure (2R,5R)-hexanediol

Article abstract of DOI:10.1021/op020023t

A new continuous production process has been developed for optically active pure (2R,5R)-hexanediol. The process uses resting whole cells of Lactobacillus kefir DSM 20587 as a biocatalyst. The reduction of (2,5)-hexanedione to (2R,5R)-hexanediol was carried out in a 2-L continuously operated membrane reactor. Conversion of (2,5)-hexanedione was nearly quantitative and the selectivity between product and intermediate was 78% for the product. Enantioselectivity and diastereoselectivity were >99% for the whole period. The productivity of L. kefir could be increased by factor 30. (2R,5R)-Hexanediol was continuously produced over 5 days with a space-time yield of 64 g·L^-1·d^-1.

Full text of DOI:10.1021/op020023t

Organic Process Research & Development 2002, 6, 458−462
New Continuous Production Process for Enantiopure (2R,5R)-Hexanediol
Ju¨rgen Haberland,† Werner Hummel,‡ Thomas Daussmann,§ and Andreas Liese*,†
Institute of Biotechnology, Research Center Juelich GmbH, 52425 Juelich, Germany, Institute of Enzyme Technology,
Heinrich-Heine UniVersity of Duesseldorf, 52426 Juelich, Germany, and Juelich Fine Chemicals GmbH (JFC),
52428 Juelich, Germany
Abstract:
yield is only 30%.7 The second approach is the use of
oxazaborolidines in tetrahydrofurane as the solvent. At 75%
the described yields are moderate and only 45% ee was
reached.8,9 The third approach uses ruthenium-BINAP
complexes as catalysts for the reduction. The optical purities
seem to be moderate, but the yields are rather low 15-50%.10
Biotechnological approaches are not often found in the
literature, although they are used for production purposes.
The established industrial production process for (2R,5R)-
hexanediol is a multistep synthesis starting with an enantio-
selective acylation of the (R)-hydroxy function of the
racemic/meso (2,5)-hexanediol mixture catalyzed by a li-
pase.11,12 Subsequently, the nonacylated (S)-hydroxy function
of the meso-(R,S)-diol is inverted by chemical transformation
with methane sulfonyl chloride leading to the (R,R)-diol.
Reagents used in the production process are triethylamine,
methane sulfonyl chloride, dichloromethane, dimethyl for-
mamide, cesium acetate, methanol, and acidic resins (Am-
berlite IR 120). The maximum theoretical yield of this
process is 75%. However, no information has been published
on the real yields achieved in this process. Another microbial
approach using resting whole cells to produce (2R,5R)-
hexanediol was published by Ohta et al. in 1996.13 Pichia
farinosa is used as a biocatalyst to reduce (2,5)-hexanedione
to (2R,5R)-hexanediol. In batch experiments yields of about
83% are achieved. The enantiomeric excess is >99%, but
the diastereomeric excess is only >95%. The productivity
is very low in this process (12.5 mgproduct/gwetweight).
A new continuous production process has been developed for
optically active pure (2R,5R)-hexanediol. The process uses
resting whole cells of Lactobacillus kefir DSM 20587 as a
biocatalyst. The reduction of (2,5)-hexanedione to (2R,5R)-
hexanediol was carried out in a 2-L continuously operated
membrane reactor. Conversion of (2,5)-hexanedione was nearly
quantitative and the selectivity between product and intermedi-
ate was 78% for the product. Enantioselectivity and diastereo-
selectivity were >99% for the whole period. The productivity
of L. kefir could be increased by factor 30. (2R,5R)-Hexanediol
was continuously produced over 5 days with a space-time yield
of 64 g‚L-1‚d-1
.
Introduction
Enantio- and diastereomerically pure diols are important
and interesting building blocks for the synthesis of pharma-
ceuticals, agrochemicals, and fine chemicals.1 (2R,5R)-
Hexanediol is a versatile building block for the synthesis of
various chiral phosphine ligands, which are used in chiral
Wilkinson catalysts.2,3,4
Here we present the application of resting whole cells
from Lactobacillus kefir DSM 20587 as a biocatalyst5,6 for
the enantio- and diastereoselective reduction of (2,5)-
hexanedione 1 to (2R,5R)-hexanediol 3. The reaction is
shown in Figure 1. The respective enantio- and diastereo-
selectivities of the product were >99%.
In the literature there are several chemical routes published
leading to (2R,5R)-hexanediol. In principle only three dif-
ferent approaches are used. The first is the use of compounds
from the chiral pool to introduce chirality. D-Mannitol is a
possible starting compound. In a three-step reaction sequence
3 can be synthesized in high optical purities, but the overall
The production process introduced in this contribution
starts from a much cheaper substrate than that used industri-
ally, and in comparison to the whole-cell approach described
by Ohta et al. (see Continuous Reduction section) higher
productivities were achieved. Furthermore, the reduction with
L. kefir leads to quantitative optical purities. After extraction
with ethyl acetate and crystallization from isooctane the
product is isolated in its optically pure form. A further
* To whom correspondence should be sent. E-mail: a.liese@fz-juelich.de.
Telephone: +49-2461-616044. Fax: +49-2461-613870.
† Institute of Biotechnology, Research Center.
(7) Saravanan, P.; Raina, S.; Sambamurthy, T.; Singh, V. K. J. Org. Chem.
1997, 62, 2669-2670.
(8) Bach, J.; Berenguer, R.; Garcia, J.; Loscertales, T.; Manzanal, J.; Vilarrasa,
J. Tetrahedron Lett. 1997, 38(6), 1091-1094.
(9) Bach, J.; Berenguer, R.; Garcia, J.; Lopez, M.; Manzanal, J.; Vilarrasa, J.
Tetrahedron 1998, 54, 14947-14962.
‡ Institute of Enzyme Technology, Heinrich-Heine University of Duesseldorf.
§ Juelich Fine Chemicals GmbH.
(1) Liese, A.; Seelbach, K.; Buchholz, A.; Haberland, J. Processes. In Industrial
Biotransformations; Liese, A., Seelbach, K., Wandrey, C., Eds.; Wiley-
VCH: Weinheim, 2000; pp 93-392.
(2) Burk, M. J.; Feaster, J. E.; Harlow, R. L. Organometallics 1990, 9, 2653-
2655.
(10) Fan, Q.-H.; Yeung, C.-H.; Chan, A. S. C. Tetrahedron: Asymmetry 1997,
8(24), 4041-4045.
(3) Burk, M. J. J. Am. Chem. Soc. 1991, 113, 8518-8519.
(4) Burk, M. J.; Feaster, J. E.; Harlow, R. L. Tetrahedron: Asymmetry 1991,
2(7), 569-592.
(5) Hummel, W.; Liese, A.; Wandrey, C. Process for Reducing Keto-Group
Containing Compounds. Eur. Patent 00113127.51-2110, 2000.
(6) Haberland, J.; Kriegesmann, A.; Wolfram, E.; Hummel, W.; Liese, A. Appl.
Microbiol. Biotechnol. 2002, 58, 595-599.
(11) Taylor, S. J. C.; Holt, K. E.; Brown, R. C.; Keene, P. A.; Taylor, I. N.
Choice of Biocatalyst in the Development of Industrial Biotransformations.
In StereoselectiVe Biocatalysis; Patel, R. N., Ed.; Marcel Dekker: New York,
2000; pp 397-404.
(12) Nagai, H.; Morimoto, T.; Achiwa, K. Synlett 1994, 4, 289-290.
(13) Ikeda, H.; Sako, E.; Sugai, T.; Ohta, H. Tetrahedron 1996, 52(24), 8113-
8122.
458
•
Vol. 6, No. 4, 2002 / Organic Process Research & Development
10.1021/op020023t CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/08/2002

Figure 1. Microbial reduction of (2,5)-hexanedione to (2R,5R)-hexanediol with Lactobacillus kefir DSM 20587.
Figure 2. Flow scheme of the continuously operated stirred tank reactor with cell retention.
advantage is the realization of a continuous production
process.
Nitrogen gas was applied at 3 vvm to maintain anaerobic
conditions and to increase the gas flow of carbon dioxide
for off-gas analysis.
The metabolism of the cell is used to regenerate the
cofactor NADPH by providing glucose as cosubstrate. This
is another advantage of the whole cell approach because the
in vitro regeneration of NADP to NADPH is not easy to
realize. Glucose is metabolized to lactate, acetate, ethanol,
and carbon dioxide.
To our knowledge this is the first continuously operated
process using L. kefir as a biocatalyst for the synthesis of
optically pure diols.
Continuous Reductions
The reduction of (2,5)-hexanedione proceeds in a two-
step reaction. In the course of the reaction first the hydrox-
yketone 2 is formed and, depending on the reaction condi-
tions, accumulated. Subsequently, this intermediate is further
reduced to the diol.6 Since the first reduction step is faster
than the second one there is always an accumulation of the
intermediate. Therefore the selectivity between 2 and 3 is
an important parameter for product purity and can be defined
as follows:
Reactor Setup
Cells of L. kefir were cultivated, harvested, and stored at
temperatures -20 °C. Thus, the amount of cells used in each
experiment is easy to prepare (see Experimental Section).
The continuous reductions were carried out on a 2-L scale
under anaerobic conditions. For cell retention an ultrafiltra-
tion module with a membrane area of 1.7 m2 and a molecular
weight cutoff of 400 kD was used. A buffered glucose
solution was the main feed to the reactor. The substrate itself
was provided in a pure form at a very low flow rate. This
setup enabled easy variation of the substrate-to-glucose ratio.
Variations of the substrate concentration and also a change
to a different starting material can be realized. The reactor
setup is shown in Figure 2. The residence time in the reactor
was kept constant at 4 h for all experiments.
cproduct
selectivity )
(1)
cproduct+cintermediate
A biomass concentration of about 100 g‚L-1 wet cells
(approximately 21 g‚L-1 cell dry weight (cdw)) was applied
in the continuous reduction. The feed rates were 76 mmol‚h-1
for glucose and 56 mmol‚h-1 for (2,5)-hexanedione. The pH
was set to 6 and kept constant by feeding sodium hydroxide
solution controlled by a pH-stat. The residence time was set
to 4 h resulting in an overall flux of 500 mL‚h-1. The
resulting concentration time course for 1, 2, and 3 is shown
in Figure 3.
The pH was kept constant during biocatalysis at a value
of 6 by adding 4 N sodium hydroxide solution. This addition
was necessary, because of the large quantities of lactate and
acetate produced by the cells. The temperature of the
fermenter was 30 °C, the condenser temperature was 4 °C.
In the steady-state, a concentration of 90 mmol‚L-1 of
(2R,5R)-hexanediol 3 was established. The optical purity of
the product was very high (de, ee > 99%). As seen in Figure
1, the concentration for the intermediate 2 was 30 mmol‚L-1
and for 1 around 2 mmol‚L-1. A space-time yield of 63.8
Vol. 6, No. 4, 2002 / Organic Process Research & Development
•
459

Figure 3. Concentration time course for the continuous
production of (2R,5R)-hexanediol with L. kefir. Conditions: V
) 2 L, 30 °C, pH 6, τ ) 4 h, 0.05 mol‚L-1 phosphate buffer,
cglucose 152 mmol‚L-1, c(2,5)-hexanedione 112 mmol‚L-1, 21 gcdw L.
kefir.
Figure 5. Conversion and selectivity as function of time.
Conditions: see Figure 3.
Figure 6. Concentration time course for different steady states
during the reduction of (2,5)-hexanedione. Conditions: V ) 2
L, 30 °C, pH 6, τ ) 4 h, 0.05 mol‚L-1 phosphate buffer, cglucose
152 mmol‚L-1, c(2,5)-hexanedione 76 mmol‚L-1 (0-54 h), c(2,5)-hexanedione
120 mmol‚L-1 (55-95 h), 21 gcdw L. kefir.
Figure 4. Increase of product yield per gcdw used per experi-
ment.
g‚L-1‚d-1 could be achieved. The productivity of the cells
was increased drastically in comparison to previously used
production methods such as batch or fed-batch.6 Because
biomass is the cost-limiting factor the improvement of the
ratio of gproduct/gcdw is the key step towards a low-priced
enantiopure product. This ratio was increased by factor 30
(Figure 4).
The conversion is nearly quantitative over a range of 120
h. A selectivity of 78% for (2R,5R)-hexanediol was achieved.
In Figure 5 the conversion and the selectivity is plotted as
function of time. Selectivity is an important parameter for
downstream processing because crystallization of 3 is not
possible if the selectivity is lower than 60%.
By changing the feed rate of (2,5)-hexanedione during
continuous reduction different steady states of the reactor
are achieved. In Figure 6 the concentration time course of
1, 2, and 3 for two different steady states is shown. The feed
ratio of glucose to (2,5)-hexanedione was changed between
the steady states (0-54 h: glucose feed 75 mmol‚h-1, (2,5)-
hexanedione feed 38 mmol‚h-1; 55-95 h: glucose feed 75
mmol‚h-1, (2,5)-hexanedione feed 60 mmol‚h-1). All other
conditions were described in the Reactor Setup section.
In Figure 7 it can be seen that the reproducibility between
different continuous experiments is very high. The selectivity
for diol formation increases with increasing conversion.
Figure 7. Selectivity (eq 1) as function of conversion for
different continuous reduction processes.
Additionally, it is important to note that almost quantitative
conversion is needed to reach the high selectivity necessary
for easy downstream processing.
Feed-Specific Biomass Concentration
Beside the target molecule, (2R,5R)-hexanediol 3, also
the intermediate, (5R)-hydroxyhexane-2-one 2, is of great
industrial interest. One stereogenic center with an ee of
>99% and a second prochiral functionality that offers a broad
460
•
Vol. 6, No. 4, 2002 / Organic Process Research & Development

fermenter was stirred at 100 rpm for 2 days at a temperature
of 30 °C. After finishing this intermediate culture the whole
suspension was transferred into a 300 L fermenter (Chemap
AG, Volketswil, Switzerland) containing 190 L of MRS
medium. After a further 2 days of cultivation at 30 °C (300
rpm), OD660
) 4.5 was reached, and the cells were
nm
harvested with a cell separator (Typ SA 1, Westfalia
Separator AG, Oelde, Germany) and stored at -20 °C. 1.2
kg of wet cells was obtained.
Continuous Reduction Procedure. All biotransforma-
tions were carried out anaerobically in a 3-L Labfors
bioreactor with a 2-L working volume (H ) 38 cm, D )
11.6 cm, Infors, Basel, Switzerland). The reactor was
sterilized (121 °C for 20 min) prior to use, glucose solution
was sterilized by filtration in a presterilized vessel (autocla-
vable sterile capsule 0.45- and 0.2-mm pores, Sartorius AG,
Goettingen, Germany). The stirrer speed was 1200 rpm, the
fermenter temperature was set at 30 °C, and the condenser
temperature at 4 °C, controlled by a water bath and cryostat.
The applied nitrogen flow was 6 L‚min-1 (6 vvm). The pH
was kept at 6 ( 0.05 with 4 M NaOH via a pH-stat
(Metrohm, Herisau, Switzerland). The carbon dioxide con-
centration was measured in the off-gas (Fisher-Rosemount
carbon dioxide analyzer type Binos 100 range 0-2%).
LABVIEW 6.01 software (National Instruments, Munich,
Germany) was used for online data acquisition. For cell
retention an ultrafiltration module (KrosFlow, φ fibers 0.5
mm, A 1.7 m2, MWCO 400 kD, Membrapure, Bodenheim,
Germany) was integrated in an external loop (dead volume
of loop approximately 200 mL, flux 60 L‚h-1, permeate flux
500 mL‚h-1). The filtration module was sterilized before and
after operating the reactor by rinsing with 1 M NaOH
solution. Cells (220 g) were thawed and washed twice with
phosphate buffer (pH 6) and transferred to the fermenter.
Antifoam (0.2 mL)was added. Nitrogen flow was started,
and the cells were stirred for 15 min to remove all the carbon
dioxide from the suspension. After 15 min the filtration and
both feeds were started. Glucose was fed with 76 mmol‚h-1
and a flow of 460 mL‚h-1. (2,5)-Hexanedione was fed pure
with 56 mmol‚h-1 and a flow of 7 mL‚h-1.
Figure 8. Selectivity as function of feed-specific biomass
concentration (fsbc) for fed-batch (triangles) and continuous
mode (circles).
spectrum of possible reactions creates an interesting building
block.
The two parameters influencing the selectivity for the
synthesis of (2R,5R)-hexanediol 3 are the amount of biomass
and the feed rate of the substrate. The outcome of this is the
definition of the feed-specific biomass concentration (fsbc),
which correlates both parameters to the selectivity of the
following reaction.
biomass
feedrate
fsbc )
(2)
We have already described the use of the fsbc6 for
controlling selectivity in the fed-batch mode. This can be
seen in Figure 8 (triangles). Calculating the fsbc for the
continuous steady states, it can be shown that these data
points match the curve (Figure 8 (circles)). This means that
it is possible to control the selectivity by varying these two
parameters.
Therefore it should be possible to choose these two
parameters in such a way as to produce only the intermediate,
(5R)-hydroxyhexane-2-one 2, not only in a fed-batch mode
but also in a continuous mode.
Product Purification. From the gathered permeate solu-
tions the water was removed by reduced pressure (rotary
evaporator Rotavapor R111, Bu¨chi Laboratoriumstechnik
GmbH, Go¨ttingen, Germany). The concentrated aqueous
solution (500 mL) was extracted five times with ethyl acetate
(500 mL). Ethyl acetate was evaporated from the combined
organic phases. Subsequent addition of isooctane started the
product crystallization. The product can be recrystallized
from ethyl acetate if needed. Chemical and optical purity of
the final product was determined by the analytical methods
described in the Analysis section.
Conclusions
A new continuous process for the diastereomeric produc-
tion of (2R,5R)-hexanediol (ee >99%, de>99%) has been
developed. By changing from batch to a continuously
operated process the ratio of gproduct/gcdw was increased by
factor 30 to 15 g/g. A typical space-time yield for the
continuous process over 5 days is 64 g‚L -1‚d-1.
Experimental Section
Biomass Production. L. kefir was cultivated on MRS
medium: 10 g/L casein peptone, 5 g/L sodium acetate, 10
g/L meat extract, 2 g/L K2HPO4, 5 g/L yeast extract, 22 g/L
glucose‚H2O, 1 g/L Tween 80, 2 g/L diammonium hydrogen
citrate, 0.2 g/L MgSO4‚7H2O, 0.05 g/L MnSO4‚H2O at a
starting pH of 6.5 in 100-mL cultures. After 2 days of
cultivation at 30 °C on a reciprocating shaker set to 60 rpm
five 100-mL cultures were added to a 10-L glass fermenter
containing 9.5 L of the above-mentioned medium. The
Analysis
Optical densities were measured on a Shimadzu UV-160
spectrometer.
Quantification of (2,5)-hexanedione (1), 5-hydroxyhexane-
2-one (2) and (2,5)-hexanediol (3) was carried out on an
Agilent HP-5890A gas chromatograph with a Permabond
Carbowax 20M column (50 m × 0.32 mm i.d., Macherey-
Vol. 6, No. 4, 2002 / Organic Process Research & Development
•
461

Nagel, Dueren, Germany), at 210 °C, with a flame ionization
detector and helium as the carrier gas (1.5 bar). To minimize
the injection error (1,5)-pentanediol (4) was used as an
internal standard. Typical retention times were (1) 4.36 min,
(2) 4.89 min, (3) 5.82 min, (4) 8.18 min.
Cyclodex column (Chromatographie-Service, Langerwehe,
Germany) at 70 °C, with a flame ionization detector and
hydrogen as carrier gas (1 bar). Typical retention times were
(R,R)-(3) 21.45 min, (S,S)-(3) 22.03 min, meso-(3) 25.05 min.
To determine the enantio- and diastereoselectivity of all
diastereomers the analytes had to be derivatized with
trifluoroacetic acid anhydride prior to analysis. Each aqueous
sample (300 µL) was extracted twice with 300 µL CHCl3.
Trifluoroacetic acid (100 µL) was added to the organic phase
and incubated at 70 °C for 30 min. The sample was
evaporated to dryness and resolved in 500 µL CHCl3.
Determination of enantio- and diastereomeric excess was
performed on a HP-6890 gas chromatograph with a â-I/P-
Acknowledgment
This work was financially supported by BMBF (Project
No. 0312639B). We thank Professor Dr. C. Wandrey for his
continuing generous support.
Received for review February 8, 2002.
OP020023T
462
•
Vol. 6, No. 4, 2002 / Organic Process Research & Development