A thermodynamic study of the ketoreductase-catalyzed reduction of 2-alkanones in non-aqueous solvents

Article abstract of DOI:10.1016/j.jct.2004.08.002

Equilibrium constants K have been measured for the reactions (2-alkanone + 2-propanol = 2-alkanol + acetone), where 2-alkanone = 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, and 2-octanone and 2-alkanol = 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, and 2-octanol. The solvents used were n-hexane, toluene, methyl tert-butyl ether (MTBE), and supercritical carbon dioxide SCCO ₂ (pressure P - 10.0 MPa). The temperature range was T - (288.15 to 308.27) K. Chiral analysis of the reaction products showed that the enzyme used in this study was stereoselective for the 2-octanone reaction system, i.e. only (S)-(+)-2-octanol was formed. For the reactions involving butanone, pentanone, and hexanone, the products were racemic mixtures of the respective (S)-(+)-2-alkanol and the (R)-(-)-2-alkanol. Chiral analysis showed that for the 2-heptanone reaction system, the 2-alkanol product was a mixture of (S)-(+)-2-heptanol and (R)-(-)-2-heptanol, at the respective mole fractions of 0.95 and 0.05. The equilibrium constant for the reaction system involving 2-butanone carried out in n-hexane was measured at several temperatures. For this reaction, the values for the thermodynamic reaction quantities at T= 298. 15 K are: K= 0.838±0.013; the standard molar Gibbs free energy change Δ_r^gH_m^° = (0.44±0.040) kJ · mol^-1; the standard molar enthalpy change Δ_r^gH_m^° = -(1.2±1.7) kJ mol^-1, and the standard molar entropy change Δ_r^gH_m^° = -(5.5±5.7) J K^-1 mol^-1. Interestingly, inspection of the values of the equilibrium constants for these reactions carried out in n-hexane, toluene, MTBE, and SCCO₂ shows that these values are comparable and have little dependence on the solvent used to carry out the reaction. The values of the equilibrium constants decrease monotonically with increasing value of the number of carbons N_c and trend towards a limiting value of ≈0.30 for N_c > 8. Published by Elsevier Ltd.

Full text of DOI:10.1016/j.jct.2004.08.002

J. Chem. Thermodynamics 37 (2005) 89–96
www.elsevier.com/locate/jct
A thermodynamic study of the ketoreductase-catalyzed reduction
of 2-alkanones in non-aqueous solvents
a,
a
a
b
Yadu B. Tewari *, Michele M. Schantz , Karen W. Phinney , J. David Rozzell
a
Biotechnology and Analytical Chemistry Divisions, National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA
b
BioCatalytics, Inc., 129 N. Hill Ave., Pasadena, CA 91106, USA
Received 7 July 2004; revised 2 August 2004; accepted 2 August 2004
Available online 28 September 2004
Abstract
Equilibrium constants K have been measured for the reactions (2-alkanone + 2-propanol = 2-alkanol + acetone), where 2-alka-
none = 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, and 2-octanone and 2-alkanol = 2-butanol, 2-pentanol, 2-hexanol,
2-heptanol, and 2-octanol. The solvents used were n-hexane, toluene, methyl tert-butyl ether (MTBE), and supercritical carbon diox-
ide SCCO2 (pressure P = 10.0 MPa). The temperature range was T = (288.15 to 308.27) K. Chiral analysis of the reaction products
showed that the enzyme used in this study was stereoselective for the 2-octanone reaction system, i.e. only (S)-(+)-2-octanol was
formed. For the reactions involving butanone, pentanone, and hexanone, the products were racemic mixtures of the respective
(S)-(+)-2-alkanol and the (R)-(ꢀ)-2-alkanol. Chiral analysis showed that for the 2-heptanone reaction system, the 2-alkanol product
was a mixture of (S)-(+)-2-heptanol and (R)-(ꢀ)-2-heptanol, at the respective mole fractions of 0.95 and 0.05. The equilibrium con-
stant for the reaction system involving 2-butanone carried out in n-hexane was measured at several temperatures. For this reaction,
the values for the thermodynamic reaction quantities at T = 298.15 K are: K = 0.838 0.013; the standard molar Gibbs free energy
change DrGꢁ ¼ ð0:44 ꢂ 0:040Þ kJ ꢃ molꢀ1; the standard molar enthalpy change DrHꢁ ¼ ꢀð1:2 ꢂ 1:7Þ kJ ꢃ molꢀ1, and the standard
m
m
molar entropy change DrSꢁ ¼ ꢀð5:5 ꢂ 5:7Þ J ꢃ Kꢀ1 ꢃ molꢀ1. Interestingly, inspection of the values of the equilibrium constants for
m
these reactions carried out in n-hexane, toluene, MTBE, and SCCO2 shows that these values are comparable and have little depend-
ence on the solvent used to carry out the reaction. The values of the equilibrium constants decrease monotonically with increasing
value of the number of carbons NC and trend towards a limiting value of ꢄ0.30 for NC > 8.
Published by Elsevier Ltd.
Keywords: Acetone; 2-Alkanol; 2-Alkanone; EC 1.1.1.2; Equilibrium constant; Ketoreductase; 2-Propanol; Supercritical carbon dioxide; Thermo-
dynamics; Transhydroxylation
1. Introduction
lution of racemic mixtures. Recently, ketoreductase-cat-
alyzed reactions [8–12] have been used for the
production of chiral secondary alcohols from the corre-
sponding ketones. These chiral alcohols are useful inter-
mediates for the pharmaceutical, agrochemical, and
perfume industries. For several years, our laboratory
has been interested in the thermodynamics [13–18] of en-
zyme-catalyzed reactions in organic solvents. Recently,
our studies have been extended to include enzyme-
catalyzed reactions carried out in supercritical carbon
dioxide (SCCO2) [19]. Supercritical carbon dioxide is
an attractive solvent for biocatalysis [20–22] because of
Enzyme-catalyzed reactions in organic solvents have
proven to be useful for the stereoselective synthesis of
chiral intermediates for pharmaceuticals and agrochem-
icals [1–3]. Lipase-catalyzed esterification [4–6] and
transesterification [1,7] reactions in organic solvents
have been commonly used for the enantioselective reso-
*
Corresponding author.
E-mail addresses: yadu.tewari@nist.gov (Y.B. Tewari), miche-
le.schantz@nist.gov (M.M. Schantz), karen.phinney@nist.gov (K.W.
Phinney), davidrozzell@biocatalytics.com (J.D. Rozzell).
0021-9614/$ - see front matter. Published by Elsevier Ltd.
doi:10.1016/j.jct.2004.08.002

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Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
its easy removal from the reaction mixture, non-toxicity,
non-flammability, abundant availability, environmental
friendliness, and recyclability. The low viscosity and
high diffusivity of SCCO2 also help to provide favorable
mass transfer properties, which can enhance the cataly-
sis. The thermodynamic results obtained in these studies
are essential for the basic understanding of the energet-
ics of these reactions and also for the practical utiliza-
tion of these enzyme-catalyzed reactions in these
solvents.
2-octanoneðsolnÞ þ 2-propanolðsolnÞ ¼
2-octanolðsolnÞ þ acetoneðsolnÞ:
Here ‘‘soln’’ denotes any of the four organic solvents
ð5Þ
used in this study.
The activity of the ketoreductase depends on the
presence of a small catalytic amount of b-nicotina-
mide-adenine dinucleotide (reduced) {NADP(red)}.
The ketoreductase-catalyzed reaction proceeds in two
steps. In the first step, the 2-alkanone is reduced to the
corresponding 2-alkanol and NADP(ox). In the second
step, the NADP(ox) is reduced to NADP(red):
In the present investigation, we report results of equi-
librium measurements for the following ketoreductase-
catalyzed transhydroxylation reactions (see figure 1)
2NADPðredÞ þ 2-alkanone ¼
2-alkanol þ 2NADPðoxÞ;
ð6Þ
ð7Þ
2-butanoneðsolnÞ þ 2-propanolðsolnÞ ¼
2-butanolðsolnÞ þ acetoneðsolnÞ;
ð1Þ
ð2Þ
ð3Þ
ð4Þ
2NADPðoxÞ þ 2-propanol ¼
2NADPðredÞ þ acetone:
2-pentanoneðsolnÞ þ 2-propanolðsolnÞ ¼
2-pentanolðsolnÞ þ acetoneðsolnÞ;
Combination of equations (6) and (7) gives the over-
all reaction for the reduction of the 2-alkanone
2-hexanoneðsolnÞ þ 2-propanolðsolnÞ ¼
2-hexanolðsolnÞ þ acetoneðsolnÞ;
2-alkanone þ 2-propanol ¼ 2-alkanol þ acetone:
ð8Þ
The principal aims of this study were to determine
how the values of the equilibrium constants for the
above reactions vary as a function of the number of car-
bons in the respective alkanones and also to examine
2-heptanoneðsolnÞ þ 2-propanolðsolnÞ ¼
2-heptanolðsolnÞ þ acetoneðsolnÞ;
OH
OH
O
O
+
+
+
+
(1)
(2)
2-propanol
2-butanol
2-butanone
acetone
O
O
OH
OH
+
2-pentanol
2-pentanone
acetone
2-propanol
OH
OH
O
O
+
(3)
2-hexanone
2-propanol
2-hexanol
acetone
O
OH
OH
O
(4)
(5)
+
+
+
2-heptanol
acetone
+
2-heptanone
2-propanol
O
OH
OH
O
2-propanol
2-octanol
acetone
2-octanone
FIGURE 1. Structures of the substances in reactions (1) to (5).

Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
91
how the values of these equilibrium constants depend on
the solvent. The equilibrium constants for reaction (1)
were measured by using n-hexane, toluene, methyl tert-
butyl ether (MTBE), and supercritical carbon dioxide
(SCCO2) as the solvents. The temperature dependency
of the equilibrium constant for reaction (1) in n-hexane
was also measured. Reaction (2) was studied by using
hexane and SCCO2 as solvents. Reactions (3) and (4)
were studied by using n-hexane as the solvent. Finally,
reaction (5) was studied by using n-hexane, toluene,
and MTBE as the solvents. These solvents were selected
based upon their current and potential use in non-
aqueous enzymology [3].
In all cases, the chirality of the above reactions was
investigated. Chiral analysis of the 2-alkanols in reac-
tions (1) to (3) showed that the alkanols produced in
these reactions were racemic mixtures (equal mole
fractions) of both the (S)-(+)-2-alkanol and the (R)-
(ꢀ)-2-alkanol. However, reaction (4) had a much greater
degree of stereoselectivity and the product was primarily
the S-(+)-2-heptanol. For reaction (5), the only product
was (S)-(+)-2-octanol.
sure of the helium carrier gas was P = 283 kPa. The ini-
tial column temperature of T = 313 K was held for 3 min
and then raised to T = 513 K at a rate of 0.333 K Æ sꢀ1
and held at T = 513 K for 5 min.
Enantioselective separation of the (R)-(ꢀ)-2-alkan-
ols and (S)-(+)-2-alkanols was carried out with another
HP 5890 g.c. equipped with a FID and a fused silica c-
cyclodextrin trifluoroacetyl column (30 m long, 0.25
mm i.d., Chiraldex, Astec, Whippany, NJ, USA). For
all of the alkanols, the helium carrier gas flow was
held constant at 0.017 cm3 Æ sꢀ1 with a detector
make-up flow of 0.50 cm3 Æ sꢀ1. The injector and detec-
tor were held at T = 423 K. All injections were split
with a split flow rate of the alkanols, the g.c. oven
condition varied with the alkanol. The butanols were
not run on this column because the temperature
needed to separate the enantiomers was lower than
could be obtained with the apparatus. For the separa-
tion of the 2-pentanols, the oven was held at T = 305
K for 20 min and then raised to T = 313 K at a rate
of 0.0167 K Æ sꢀ1 and then held at T = 313 K for 2.5
min. For the separation of the 2-hexanols, the oven
was held at T = 308 K for 15 min and then raised to
T = 333 K at a rate of 0.0334 K Æ sꢀ1 and held at
T = 333 K for 2.5 min. For the separation of the 2-
heptanols, the oven was held at T = 308 K for 15
min and then raised to T = 333 K at a rate of
0.0167 K Æ sꢀ1 and held at T = 333 K for 5 min. For
the separation of the 2-octanols, the oven was held iso-
thermally at T = 323 K for 50 min.
Internal standards were used for the quantitative
determination of the concentrations of the substances
in reactions (1) to (5). The internal standards were:
(S)-(+)-2-octanol for reaction (1) carried out in n-hex-
ane, SCCO2, and MTBE; 1-hexanol for reaction (1)
carried out in toluene; 2-octanol for reactions (2)–(4)
carried out in n-hexane; 1-butanol for reaction (5) car-
ried out in n-hexane and in MTBE; and 1-hexanol for
reaction (5) carried out in toluene. By using the chro-
matographic conditions described above, the chroma-
tographic peaks of the compounds of interest and
their respective internal standards were well separated.
For the quantitative analysis of the reactants and
products in the equilibrated reaction mixtures in n-hex-
ane, toluene and MTBE, ꢄ4 cm3 of reaction mixture
and ꢄ0.05 cm3 of internal standard solution were grav-
imetrically added to a vial, and tightly capped. Approx-
imately 6 Æ 10ꢀ4 cm3 of this solution was then injected
into the g.c., and the reaction mixture was analyzed
for reactants and products. The concentrations c {ex-
pressed as mol (kg Æ sln)ꢀ1} of each of the substances in-
volved in the reactions were determined from their
respective chromatographic peak areas, the appropriate
response factor ratios, the concentration of the internal
standard solution, and the chromatographic peak area
of the internal standard.
2. Experimental
2.1. Materials
The substances used in this study, their Chemical Ab-
stract Service (CAS) registry numbers, empirical formu-
las, molar masses, sources, and purities as determined by
gas chromatography (g.c.) are given in table 1. 1 The en-
zyme used in this study was ketoreductase (EC 1.1.1.2)
from Biocatalytics, Inc., Pasadena, CA. This enzyme
was prepared from a recombinant bacterial source and
gene expressed in Escherichia coli; {NADP(red)} was
embedded in the ketoreductase preparation by the
vendor.
2.2. Chromatography and quantitative analysis
The quantitative analysis of the reactants and prod-
ucts was carried out by using a Hewlett-Packard (HP)
5890 g.c. (Agilent Technologies, Wilmington, DE,
USA), equipped with a flame ionization detector
(FID) and a fused silica Phenomenex ZB-FFAP capil-
lary column (30 m long, 0.53 mm id, 0.53 lm thick film
coating). The injector and detector temperatures were
T = 523 K and T = 543 K, respectively; the head pres-
1
Certain commercial equipment, instruments, or materials are
identified in this paper to specify the experimental procedures
adequately. Such identification is not intended to imply recommenda-
tion or endorsement by the National Institute of Standards and
Technology, nor is it intended to imply that the materials or equipment
identified are necessarily the best available for the purpose.

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Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
TABLE 1
Principal substances used in this study with their Chemical Abstracts Service (CAS) registry numbers, empirical formulas, and molecular masses Mr
Substance
CAS no.
Formula
C3H6O
Mr
x1
x2
Acetone
67-64-1
58.08
0.99
0.99
0.99
0.99
0.96
0.99
0.99
0.995
0.99
0.99
0.98
0.998
0.98
0.99
0.98
0.99
0.99
0.995
0.998
0.9999
(R)-(ꢀ)-2-Butanol
(S)-(+)-2-Butanol
2-Butanone
14898-79-4
4221-99-2
78-93-3
C4H10
C4H10
O
O
74.12
74.12
72.11
0.99
0.99
C4H8O
(R)-(ꢀ)-2-Heptanol
(S)-(+)-2-Heptanol
2-Heptanone
6033-24-5
6033-23-4
110-43-0
110-54-3
26549-24-6
52019-78-0
591-78-6
1634-04-4
5978-70-1
6169-06-8
111-13-7
31087-44-2
26184-62-3
107-87-9
67-63-0
C7H16
C7H16
C7H14
C6H14
C6H14
C6H14
C6H12
C5H12
O
116.20
116.20
114.19
86.18
0.96
0.99
O
O
n-Hexane
(R)-(ꢀ)-2-Hexanol
(S)-(+)-2-Hexanol
2-Hexanone
O
O
O
O
102.18
102.18
100.16
88.15
0.99
0.99
Methyl tert-butyl ether
(R)-(ꢀ)-2-Octanol
(S)-(+)-2-Octanol
2-Octanone
C8H18O2
C8H18O2
C8H16O2
130.23
130.23
128.22
88.15
0.98
0.99
(R)-(ꢀ)-2-Pentanol
(S)-(+)-2-Pentanol
2-Pentanone
C5H12
C5H12
C5H10
O
O
O
0.99
0.99
88.15
86.13
2-Propanol
Toluene
C3H8O
C7H8
60.10
92.14
108-88-3
All of these substances were obtained from Aldrich with the exception of toluene which was Reference Material NIMC-CRM-4003 from the
National Institute of Materials and Chemical Research of Japan. The total mole fraction purities x1 are those determined by the vendor by using g.c.
The chiral mole fraction purities x2 of the alcohols were determined in our laboratory (see Section 2.2).
2.3. Equilibrium studies
tograph (SFC, Agilent Technologies, Wilmington, DE,
USA) with a modified three-port valve was used to con-
nect the reaction vessel in the system.
The equilibrium measurements were carried out by
approaching equilibrium from both directions of reac-
tion. Solutions consisting of {2-alkanone + 2-propanol}
were used for the forward direction and solutions of
{(S)-(+)-2-alkanol + acetone} were used for the reverse
direction. The substrates were first dissolved in the sol-
vents n-hexane, MTBE, and toluene. The total volume
of the solutions used was ꢄ15 cm3. Then, 0.10 cm3 of
a solution containing the enzyme [0.010 g of ketoreduc-
tase dissolved in 0.10 cm3 of phosphate buffer {K2HPO4
(c = 0.10 mol Æ dm3), adjusted to pH 7.3 with H3PO4}]
was added to each reaction mixture. These reaction mix-
tures were then placed in a constant temperature bath
For the measurement of the equilibrium constant for
reaction (1) in SCCO2, ꢄ0.20 g of an equimolar solution
of (2-butanone + 2-propanol) was first prepared and
placed in the stainless steel reaction vessel. Then 0.10
cm3 of a solution containing the enzyme [0.010 g of keto-
reductase dissolved in 0.10 cm3 of phosphate buffer
{K2HPO4 (c = 0.10 mol Æ dm3), adjusted to pH 7.3 with
H3PO4}] was added to the reaction vessel. The reaction
vessel was then assembled and connected to the SFC sys-
tem. The reaction was allowed to proceed for five hours in
the SCCO2 media at T = 303.15 K and P = 10.0 MPa.
During the course of the reaction, the vessel was gently
shaken at approximately 15 min intervals. After comple-
tion of reaction, the vessel was depressurized by opening
the needle valve while bubbling CO2 (g) into a 20 cm3 vol-
umetric flask containing ꢄ7 cm3 of n-hexane in order to
collect the escaping reactants and products. Once the
pressure in the vessel reached p ꢄ 0.1 MPa, the vessel
was disconnected from the SFC system, and the cover
of the reaction vessel was removed. The n-hexane in the
volumetric flask was poured into the reaction vessel and
an additional quantity of n-hexane was added to the reac-
tion vessel to fill it completely. Then, ꢄ0.50 cm3 of this di-
luted reaction mixture, ꢄ0.10 cm3 of the internal standard
solution, and ꢄ10 cm3 of n-hexane were gravimetrically
added to a vial. Analysis for the concentrations of ace-
tone, 2-propanol, 2-butanone and 2-butanol was done
by using the first g.c. method (FID with ZB-FFAP col-
( 0.01 K) and shaken laterally at ꢄ30 shakes Æ minꢀ1
.
The solutions were periodically analyzed to determine
the extent of reaction. Reactions were considered to be
at equilibrium when the ratios of the g.c. peak area ra-
tios of products:reactants were essentially identical for
the forward and the reverse reaction mixtures. Addi-
tional enzyme solution was added to the reaction mix-
tures if it was found necessary to speed the approach
to equilibrium. Reaction (1) equilibrated in 4 h for all
three solvents. However, reactions (2) and (3) required,
respectively, 1 day and 3 days to reach equilibrium.
Reactions (4) and (5) required additional enzyme and
took ꢄ7 days to reach equilibrium.
For the equilibrium study in SCCO2, a stainless steel
reaction vessel (volume = 15 cm3) was used [19]. As de-
scribed previously [19], a HP supercritical fluid chroma-

Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
93
umn) described above. For the measurement of the equi-
K ¼cð2-alkanolÞ ꢃ cðacetoneÞ=fcð2-alkanoneÞꢃ
cð2-propanolÞg;
where c(2-alkanol) = c{(R)-(+)-2-alkanol} + c{(S)-(+)-
librium constants from the reverse direction, the reactions
were carried out in the same way except that the starting
mixtures were, respectively, equimolar mixtures of {(S)-
(+)-2-butanol + acetone} and of {(S)-(+)-2-pentanol +
acetone}.
ð16Þ
2-alkanol}. By using the relationships
cfðRÞ-ðþÞ-2-alkanolg ¼ cfðSÞ-ðþÞ-2-alkanolg;
cð2-alkanolÞ ¼2 ꢃ cfðRÞ-ðþÞ-2-alkanolg ¼
2 ꢃ cfðSÞ-ðþÞ-2-alkanolg;
ð17Þ
ð18Þ
3. Results and discussion
one obtains
3.1. Thermodynamic formalism
Kð8Þ ¼ 2 ꢃ Kð14Þ:
In the treatment of the results and in the discussion to
follow, we shall deal solely with equilibrium constants
for reactions (1) to (5) and that are defined by equations
(9) to (13) and where the total amount of 2-alkanol is
considered. The equilibrium constants that pertain to
reactions {see equation (14)} involving specific chiral
products can be calculated easily by using equation (18).
The respective equilibrium constants for reactions (1)
through (5) are:
K ¼cð2-butanolÞ ꢃ cðacetoneÞ=fcð2-butanoneÞꢃ
cð2-propanolÞg;
ð9Þ
ð10Þ
ð11Þ
ð12Þ
ð13Þ
K ¼cð2-pentanolÞ ꢃ cðacetoneÞ=fcð2-pentanoneÞꢃ
cð2-propanolÞg;
3.2. Results of equilibrium measurements
K ¼cð2-hexanolÞ ꢃ cðacetoneÞ=fcð2-hexanoneÞꢃ
cð2-propanolÞg;
The results of the equilibrium measurements for the
ketoreductase-catalyzed reduction of 2-butanone, 2-
pentanone, 2-hexanone, 2-heptanone, and 2-octanone to
the corresponding 2-alkanols in the several solvents used
in this study are given in table 2. Attempts were also
made to use SCCO2 as a solvent for reactions that use
2-hexanone, 2-heptanone, and 2-octanone. However,
the rates of reaction for these substances in this solvent
were not rapid enough to conveniently carry out meas-
urements of the desired equilibrium constants. It is
important to note that the values of the equilibrium con-
stants obtained from the forward and the reverse direc-
tions of reaction are either in agreement or very close to
agreement within their statistical uncertainties. Thus,
the reactions are judged to have reached equilibrium
in terms of the total amounts of the substances shown
in reactions (1) to (5). The values of the equilibrium con-
stants ÆKæ reported in the last column in table 2 are the
averages of the results obtained from the forward and
the reverse directions of reaction. The uncertainties gi-
ven in column 8 of table 2 are based on the random er-
rors in the measurements expressed as two estimated
standard deviations of the mean and do not include pos-
sible systematic errors in the measurements. It is judged
that reasonable estimates of the standard uncertainties
[23] due to possible systematic errors in the values of
the equilibrium constants for these reactions are:
0.03 Æ K in the g.c. measurements of the concentrations
of the reactants and products (this includes possible er-
rors in the values of the response factors) and 0.01 Æ K
due to possible sample impurities. These estimated
uncertainties have been combined in quadrature to-
gether with the statistical uncertainties, expressed as
one estimated standard deviation of the mean, to obtain
K ¼cð2-heptanolÞ ꢃ cðacetoneÞ=fcð2-heptanoneÞꢃ
cð2-propanolÞg;
K ¼cð2-octanolÞ ꢃ cðacetoneÞ=fcð2-octanoneÞꢃ
cð2-propanolÞg:
Here, c is the concentration of the indicated substance.
In organic solvents, the substrates involved in these
reactions are in a non-ionized form and their concentra-
tions are small. Hence, we have assumed that their activ-
ity coefficients are unity and that the calculated
equilibrium constants can be identified as thermody-
namic equilibrium constants defined in terms of activi-
ties of the reactants and the products.
It is also possible to consider reactions (1) to (5) in
terms of the formation of a specific chiral product. This
generic reaction, written in terms of the (S)-(+) isomer,
is
2-alkanone þ 2-propanol ¼
ðSÞ-ðþÞ-2-alkanol þ acetone:
The equilibrium constant for this reaction is
ð14Þ
K ¼cfðSÞ-ðþÞ-2-alkanolgꢃ
cðacetoneÞ=fcð2-alkanoneÞꢃ
cð2-propanolÞg:
ð15Þ
The equilibrium constant for the generic reaction {see
reaction (8)} involving the racemic mixture {equal
amounts of (R) and (S) isomers} is

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Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
TABLE 2
Results of equilibrium measurements of the ketoreductase-catalyzed reduction of 2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, and 2-
octanone to the corresponding 2-alkanols in several solvents {reactions (1) to (5)}
Solvent
Direction T/K
c(2-alkanone)
c(2-propanol)
c(2-alkanol)
c(acetone)
K
ÆKæ
mol Æ (kg Æ soln)ꢀ1 mol Æ (kg Æ soln)ꢀ1 mol Æ (kg Æ soln)ꢀ1 mol Æ (kg Æ soln)ꢀ1
2-Butanone(soln) + 2-propanol(soln) = 2-butanol(soln) + acetone(soln) (1)
n-Hexane Forward
Reverse
288.15 0.01117
288.15 0.01129
293.05 0.01106
293.05 0.01126
298.15 0.01097
298.15 0.01109
303.30 0.01107
303.30 0.01129
308.27 0.01095
308.27 0.00992
0.00499
0.00521
0.00635
0.00658
0.00604
0.00657
0.00690
0.00728
0.00821
0.00706
0.00557
0.00483
0.00630
0.00552
0.00671
0.00503
0.00658
0.00578
0.00933
0.00855
0.00844
0.01031
0.00940
0.01133
0.00837
0.01217
0.00986
0.01208
0.00777
0.00663
0.843 0.009 0.845 0.054
0.847 0.005
Forward
Reverse
0.843 0.003 0.844 0.055
0.844 0.005
Forward
Reverse
0.848 0.003 0.844 0.054
0.840 0.004
Forward
Reverse
0.849 0.004 0.849 0.054
0.850 0.003
Forward
Reverse
0.806 0.007 0.808 0.052
0.809 0.008
Toluene
SCCO2
MTBE
Forward
Reverse
298.15 0.01198
298.15 0.01391
0.01064
0.01009
0.01025
0.01039
0.01055
0.01157
0.848 0.011 0.853 0.056
0.857 0.014
Forward
Reverse
303.15 0.00570
303.15 0.01313
0.00355
0.00859
0.00681
0.01077
0.00242
0.00865
0.814 0.011 0.820 0.054
0.826 0.014
Forward
Reverse
298.15 0.01038
298.15 0.00798
0.01002
0.00653
0.00935
0.00793
0.00909
0.00533
0.817 0.007 0.814 0.052
0.811 0.006
2-Pentanone(soln) + 2-propanol(soln) = 2-pentanol(soln) + acetone(soln) (2)
0.00483
0.00629
n-Hexane Forward
Reverse
298.15 0.00572
298.15 0.00677
0.00584
0.00772
0.00433
0.00523
0.626 0.003 0.627 0.040
0.629 0.002
SCCO2
Forward
Reverse
303.15 0.02318
303.15 0.02848
0.01394
0.01622
0.02534
0.04951
0.00796
0.00601
0.624 0.008 0.634 0.041
0.644 0.010
2-Hexanone(soln) + 2-propanol(soln) = 2-hexanol(soln) + acetone(soln) (3)
0.00674
0.00650
n-Hexane Forward
Reverse
298.15 0.00917
298.15 0.00900
0.00333
0.00315
0.00846
0.00856
0.456 0.003 0.459 0.029
0.461 0.005
2-Heptanone(soln) + 2-propanol(soln) = 2-heptanol(soln) + acetone(soln) (4)
0.00271
0.00279
n-Hexane Forward
Reverse
298.15 0.00872
298.15 0.00849
0.00532
0.00561
0.00576
0.00580
0.336 0.005 0.338 0.022
0.340 0.002
2-Octanone(soln) + 2-propanol(soln) = 2-octanol(soln) + acetone(soln) (5)
0.00695
0.00405
n-Hexane Forward
Reverse
298.15 0.00558
298.15 0.00855
0.00343
0.00110
0.00351
0.00992
0.310 0.005 0.313 0.020
0.315 0.001
Toluene
MTBE
Forward
Reverse
298.15 0.00442
298.15 0.00451
0.00533
0.00545
0.00141
0.00129
0.00520
0.00592
0.311 0.007 0.311 0.021
0.311 0.007
Forward
Reverse
298.15 0.00434
298.15 0.00439
0.01172
0.01139
0.00326
0.00322
0.00488
0.00488
0.313 0.003 0.313 0.020
0.314 0.002
Abbreviations are: MTBE, methyl tert-butyl ether; and SCCO2, supercritical carbon dioxide (P = 10.0 MPa). The concentrations of the substrates
involved in the reaction {2-alkanone(soln) + 2-propanol(soln) = 2-alkanol(soln) + acetone(soln)} are given in columns 4 to 7. The 2-butanol,
2-pentanol, and 2-hexanol are racemic mixtures {equal amounts of the respective (R) and (S) isomers}. The 2-heptanol is a mixture of (S)-
(+)-2-heptanol and (R)-(ꢀ)-2-heptanol, at the respective mole fractions of 0.95 and 0.05. The 2-octanol consists solely of (S)-(+)-2-octanol. Each
concentration is the average of five measurements. The values of the equilibrium constants K (column 8) are calculated from the concentrations given
in columns 4 to 7 and by using equations (9) to (13). The quantity ÆKæ is the average of the equilibrium constants which were measured from the
forward and the reverse directions of reaction. The uncertainties in the values of K (column 8) are based on two estimated standard deviations of the
mean. The uncertainties in the values of ÆKæ (column 9) are combined expanded uncertainties (see Section 3.2).
combined standard uncertainties [23]. These combined
uncertainties are then multiplied by two to obtain the
expanded uncertainties given in the average values of
K (see column 9 in table 2).
Chiral analysis of the reaction products showed that
the enzyme used in this study was stereoselective for the
2-octanone system – only (S)-(+)-2-octanol was formed.
For the reactions involving butanone, pentanone, and
hexanone, the products were racemic mixtures of the
respective (S)-(+)-2-alkanol and the (R)-(ꢀ)-2-alkanol.
Chiral analysis showed that for the 2-heptanone system
reaction (4), the 2-alkanol product was a mixture of (S)-
(+)-2-heptanol and (R)-(ꢀ)-2-heptanol, at the respective
mole fractions of 0.95 and 0.05.

Y.B. Tewari et al. / J. Chem. Thermodynamics 37 (2005) 89–96
95
A comparison of the values of the equilibrium con-
stants for reaction (1) in n-hexane, toluene, MTBE,
and SCCO2 shows that these values have a small
dependence (<0.036 Æ K) on the solvent used to carry
out the reaction. Similarly, the values of the equilibrium
constants for reaction (2) in n-hexane and SCCO2 and
for reaction (5) in n-hexane, toluene, and MTBE do
not vary with the solvent. This result has potential impli-
cations for process design. Specifically, since the theoret-
ical product yields for these reactions do not vary
significantly with the solvent, one can select the solvent
based on kinetic, economic, environmental, or separa-
tion considerations.
We have calculated [24] values of the standard molar
Gibbs free energy change DrGꢁm, the standard molar
enthalpy change DrHꢁm, and the standard molar entropy
change DrSꢁm from the temperature dependency of the
equilibrium constant for reaction (1) carried out in n-
hexane. In performing this calculation, it was assumed
that that the standard molar heat capacity change
DrCꢁm, for this reaction was zero. The calculated values
for these thermodynamic quantities at T = 298.15 K
2-heptanone produced 0.95 mole fraction of (S)-(+)-2-
heptanol and 0.05 mole fraction of (R)-(ꢀ)-2-heptanol
while shorter chain length 2-alkanones produced
racemic mixtures of both (S)-(+)-2-alkanol and (R)-
(ꢀ)-2-alkanol. Clearly, these findings have implications
for understanding the interaction of the 2-octanone with
the active site of the ketoreductase enzyme and for the
mechanism of these reactions. In any case, if one wishes
to produce optically active alkanols, there are additional
enzymes that might be utilized. Specifically, Kruse et al.
[8] used alcohol dehydrogenase coupled with formate
dehydrogenase for the production of chiral (S)-(+)-
alcohols from the corresponding ketones. Also, Nanduri
et al. [10] have recently purified a NAD(red) dependent
ketoreductase from Gluconobactor Oxydans with a high
enantioselectivity. They [10] used this enzyme, coupled
with formate dehydrogenase, for the stereoselective pro-
duction of (S)-(+)-2-pentanol from 2-pentanone. Hav-
ing the thermodynamic results in place for the
reactions studied herein and for additional representa-
tive reactions can play an important role in the engineer-
ing of a process used to make the desired products in
bulk and with a high product yield. The results obtained
herein are believed to be the first thermodynamic results
obtained on these reactions.
are:
K = 0.838 0.013;
DrGꢁ ¼ ð0:44 ꢂ 0:040Þ kJ ꢃ
molꢀ1; DrHꢁ ¼ ꢀð1:2 ꢂ 1:7Þ kJ ꢃmmolꢀ1; and DrSꢁ ¼ ꢀ
m
m
ð5:5 ꢂ 5:7Þ J ꢃ Kꢀ1 ꢃ molꢀ1. These uncertainties are equal
to two estimated standard deviations of the mean.
Shown in figure 2 is a plot of the equilibrium con-
stants for reactions (1) to (5) carried out in n-hexane
as a function of the number of carbons NC in the 2-
alkanone. The values of the equilibrium constants de-
crease monotonically with increasing value of NC and
trend towards a limiting value of ꢄ0.30 for NC > 8.
The ketoreductase used in this study has been re-
ported to be stereoselective [12] for the production of
(S)-(+)-2-octanol from 2-octanone. This result was con-
firmed in the chiral analysis performed in this study.
However, in our study we also found that reduction of
Acknowledgment
We thank Dr. Robert N. Goldberg for helpful discus-
sions and suggestions.
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FIGURE 2. The equilibrium constants K for reactions (1) to (5)
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was n-hexane and the temperature was 298.15 K.

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JCT 04-146