A thermodynamic study of ketoreductase-catalyzed reactions 3. Reduction of 1-phenyl-1-alkanones in non-aqueous solvents

Article abstract of DOI:10.1016/j.jct.2005.12.014

The equilibrium constants K for the reactions (1-phenyl-1-alkanone + 2-propanol = 1-phenyl-1-alkanol + acetone) in the solvents n-pentane and n-hexane have been determined by using gas chromatography at the temperature 298.15 K. The 1-phenyl-1-alkaones included in this study were: 1-phenyl-1-ethanone, 1-phenyl-1-propanone, 1-phenyl-1-butanone, 1-phenyl-1-pentanone, 1-phenyl-1-hexanone, and 1-phenyl-1-heptanone. The equilibrium constants for the reaction involving 1-phenyl-1-ethanone were measured in the solvent n-hexane as a function of temperature (288 K to 308 K). The calculated thermodynamic quantities for the 1-phenyl-1-ethanone reaction at T = 298.15 K are: K = 0.2177 ± 0.0018; the standard molar Gibbs free energy change, Δ_r G_m^{{ring operator}} = (3.78 ± 0.02) kJ · mol^{- 1}, the standard molar enthalpy change, Δ_r H_m^{{ring operator}} = (4.53 ± 0.87) kJ · mol^{- 1}, and the standard molar entropy change, Δ_r S_m^{{ring operator}} = (2.5 ± 2.9) J · K^{- 1} · mol^{- 1}. The equilibrium constants of 1-phenyl-1-alkanone with an odd number of carbons in alkyl side chain are higher than the equilibrium constants of the preceding 1-phenyl-1-alkanone having an even number of carbons in the side chain and follow a zig-zag pattern with increasing carbon number in the alkyl side chain.

Full text of DOI:10.1016/j.jct.2005.12.014

J. Chem. Thermodynamics 38 (2006) 1165–1171
www.elsevier.com/locate/jct
A thermodynamic study of ketoreductase-catalyzed reactions
3. Reduction of 1-phenyl-1-alkanones in non-aqueous solvents
a,
b
c
a
Yadu B. Tewari *, Nand Kishore , J. David Rozzell , David J. Vanderah ,
d
Michele M. Schantz
a
Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
b
Chemistry Department, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India
c
BioCatalytics, Inc., 129 N. Hill Avenue, Pasadena, CA 91106, USA
Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA
d
Received 18 November 2005; received in revised form 14 December 2005; accepted 15 December 2005
Available online 3 March 2006
Abstract
The equilibrium constants K for the reactions (1-phenyl-1-alkanone + 2-propanol = 1-phenyl-1-alkanol + acetone) in the solvents
n-pentane and n-hexane have been determined by using gas chromatography at the temperature 298.15 K. The 1-phenyl-1-alkaones
included in this study were: 1-phenyl-1-ethanone, 1-phenyl-1-propanone, 1-phenyl-1-butanone, 1-phenyl-1-pentanone, 1-phenyl-1-hexa-
none, and 1-phenyl-1-heptanone. The equilibrium constants for the reaction involving 1-phenyl-1-ethanone were measured in the solvent
n-hexane as a function of temperature (288 K to 308 K). The calculated thermodynamic quantities for the 1-phenyl-1-ethanone reaction
at T = 298.15 K are: K = 0.2177 0.0018; the standard molar Gibbs free energy change, DrGꢀ ¼ ð3:78 ꢁ 0:02Þ kJ ꢂ molꢃ1, the standard
m
molar enthalpy change, DrHꢀ ¼ ð4:53 ꢁ 0:87Þ kJ ꢂ molꢃ1, and the standard molar entropy change, DrSꢀ ¼ ð2:5 ꢁ 2:9Þ J ꢂ Kꢃ1 ꢂ molꢃ1
.
m
m
The equilibrium constants of 1-phenyl-1-alkanone with an odd number of carbons in alkyl side chain are higher than the equilibrium
constants of the preceding 1-phenyl-1-alkanone having an even number of carbons in the side chain and follow a zig-zag pattern with
increasing carbon number in the alkyl side chain.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Acetone; Equilibrium constant; n-Hexane; Ketoreductase; 2-Propanol; 1-Phenyl-1-alkanol; 1-Phenyl-1-alkanone; Thermodynamics
1. Introduction
perfume industries [3–5]. Lipase-catalyzed reactions in
organic solvents have been used for the stereoselective res-
olution of racemic mixtures [4,6–8]. Ketoreductase cata-
lyzed reduction of carbonyl groups [9–16] have been used
for the synthesis of chiral secondary alcohols which are
useful intermediates for pharmaceuticals, agrochemicals,
and liquid crystals.
There have been several studies from our laboratory
dealing with the thermodynamics of enzyme-catalyzed
reactions in organic solvents [17–22]. The thermodynamic
results obtained in these studies are essential for the basic
understanding of the energetics of these reactions and
also for the practical utilization of enzyme-catalyzed
reduction reactions of 1-phenyl-1-alkanone in non-aque-
ous solvents.
In recent years, the use of biocatalysis for organic syn-
thesis [1,2] in non-aqueous media has become an attractive
alternative to traditional chemical synthetic methods. The
use of enzyme-catalyzed reactions has made it possible to
obtain desired stereoselective products which are useful
intermediates in the pharmaceutical, agrochemical, and
*
Corresponding author. Tel.: +1 301 975 2583; fax: +1 301 330 3447.
E-mail addresses: yadu.tewari@nist.gov (Y.B. Tewari), nandk
@ether.chem.iitb.ernet.in (N. Kishore), davidrozzell@biocatalytics.com
(J.D. Rozzell), david.vanderah@nist.gov (D.J. Vanderah), michele.
schantz@nist.gov (M.M. Schantz).
0021-9614/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jct.2005.12.014

1166
Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
O
OH
H
O
OH
R
R
+
=
+
2-propanol
1-phenyl-1-alkanone
1-phenyl-1-alkanol
acetone
FIGURE 1. The structures of the substances involved in the reduction of 1-phenyl-1-alkanones {reactions (1) to (6)}, where R = CH3, C2H5, C3H7, C4H9,
C5H11, or C6H13
.
In the present investigation, we report results of equilib-
rium measurements for the following ketoreductase-
catalyzed reduction of 1-phenyl-1-alkanone reactions
(see figure 1).
produced in these reduction reactions were racemic mix-
tures (equal mole fractions) of both the (R)-(+)-1-phenyl-
1-alkanol and the (S)-(ꢃ)-1-phenyl-1-alkanol.
2. Experimental
1-phenyl-1-ethanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-ethanolðsolnÞ þ acetoneðsolnÞ;
1-phenyl-1-propanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-propanolðsolnÞ þ acetoneðsolnÞ;
1-phenyl-1-butanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-butanolðsolnÞ þ acetoneðsolnÞ;
1-phenyl-1-pentanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-pentanolðsolnÞ þ acetoneðsolnÞ;
1-phenyl-1-hexanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-hexanolðsolnÞ þ acetoneðsolnÞ;
1-phenyl-1-heptanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-heptanolðsolnÞ þ acetoneðsolnÞ.
ð1Þ
ð2Þ
ð3Þ
ð4Þ
ð5Þ
ð6Þ
2.1. Materials
The substances used in this study, their Chemical
Abstract Service (CAS) registry numbers, empirical formu-
lae, molar masses, sources, and purities as determined by
gas chromatography (g.c.) are given in table 1. The mass
fraction of water listed in table 1 for the compounds
involved in these reactions and the internal standard,
1-hexanol were determined by Karl Fischer titration [23].
The enzyme 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 E. coli and required NAD(red)
catalytic activities.
Here ‘‘soln’’ denotes the organic solvent, n-pentane or
n-hexane, have been used in this study.
2.2. Chromatography and quantitative analysis
A small amount of b-nicotinamide-adenine dinucleotide
(reduced), {NAD(red)} is required for catalytic activity of
this ketoreductase. As described in our previous papers
[21,22], the ketoreductase catalyzed reactions proceed in
two steps. In the first step, the 1-phenyl-1-alkanone is
reduced to the corresponding 1-phenyl-1-alkanol and
NAD(ox). Then in the second step, the NAD(red) is regen-
erated by reduction of NAD(ox) in presence of 2-propanol:
The quantitative analysis of the reactants and products
was carried out by using a Hewlett-Packard (HP) 5890
g.c. (Agilent Technologies, Wilmington, DE, USA),1
equipped with a flame ionization detector (FID) and a
fused silica Phenomenex ZB-FFAP capillary column
(30 m long, 0.53 mm i.d., 0.53 lm thick film coating). The
injector and detector temperatures were 523 K and
543 K, respectively; the head pressure of the helium carrier
gas was P = 283 kPa. The initial column temperature of
313 K was held for 3 min and then raised to T = 513 K
at a rate of 0.333 K Æ sꢃ1 and held at T = 513 K for
5 min. Under these conditions, the chromatographic peaks
of the compounds of interest and the internal standard
were well separated.
1-phenyl-1-alkanoneðsolnÞ þ 2NADðredÞ ¼
1-phenyl-1-alkanolðsolnÞ þ 2NADðoxÞ
2NADðoxÞ þ 2-propanolðsolnÞ ¼
2NADðredÞ þ acetoneðsolnÞ.
ð7Þ
ð8Þ
Thus, the combination of equations (7) and (8) leads to an
overall reaction for the reduction of the 1-phenyl-1-
alkanone:
Enantioselective separation of the (R)-(+)-1-phenyl-1-
alkanol and (S)-(ꢃ)-1-phenyl-1-alkanol was carried out
with another HP 5890 g.c. equipped with an FID and a
fused silica c-cyclodextrintrifluoroacetyl capillary column
1-phenyl-1-alkanoneðsolnÞ þ 2-propanolðsolnÞ ¼
1-phenyl-1-alkanolðsolnÞ þ acetoneðsolnÞ.
ð9Þ
The principal aim of this study was to determine the
equilibrium constants for the above 1-phenyl-1-alkanone
reactions as a function of the number of carbons in the
alkyl chain. The chirality of these reactions has also been
investigated. The chiral analysis of the 1-phenyl-1-alkanols
in reactions (1) to (6) showed that the 1-phenyl-1-alkanols
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 recommendation 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.

Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
1167
TABLE 1
Principal substances used in this study with their Chemical Abstracts Service (CAS) registry numbers, empirical formulas, relative molecular masses Mr,
Supplier (A, Aldrich; B, BioCatalytics; R, Roche; S, synthesized; and T, TCI America), mole fraction purity x as stated by the vendor, mass fraction
moisture contents w determined by Karl Fischer analysis, x(chiral) is the chiral mole fraction purity of alcohols as stated by vendors, r designates racemic
mixture, and the methods used to determine the mole fraction purity and chiral purity
Substance
CAS no.
Formula
Mr
Supplier
x
w
x (chiral) Method
Acetone
n-Hexane
1-Hexanol
Ketoreductase (EC 1.1.1.2)
NAD(red)a
67-64-1
110-54-3
111-27-3
C3H6O
C6H14
58.08
86.18
102.18
A
A
A
B
R
A
A
A
A
A
A
A
S
0.99
0.99
0.99
0.0103
g.c.
g.c.
g.c.
C6H14
O
0.0011
24292-60-2 C21H26N7Na2P3O17 787.4
109-66-0
22144-60-1
22135-49-5 C10H14
495-40-9
1517-69-7
1445-91-6
98-86-2
0.97
0.99
0.97
0.97
0.99
0.99
0.99
0.99
0.99
0.98
0.99
0.98
0.99
Enzymatic assay
g.c.
FT-n.m.r., g.c.
FT-n.m.r., g.c.
FT-n.m.r., FT-IR, g.c.
g.c.
g.c.
FT-n.m.r., FT-IR, g.c.
g.c., n.m.r.
g.c.
g.c., n.m.r.
g.c.
g.c.
n-Pentane
C5H12
10H14
72.15
150.22
150.22
148.21
122.17
122.17
120.15
192.29
190.28
178.26
176.25
164.24
162.23
136.19
136.19
134.18
60.10
(R)-(+)-1-Phenyl-1-butanol
(S)-(ꢃ)-1-Phenyl-1-butanol
1-Phenyl-1-butanone (butyrophenone)
(R)-(+)-1-Phenyl ethanol
(S)-(ꢃ)-1-Phenyl ethanol
1-Phenyl-1-ethanone-1 (acetophenone)
1-Phenyl-1-heptanol
1-Phenyl-1-heptanone (n-heptanophenone) 1671-75-6
1-Phenyl-1-hexanol
1-Phenyl-1-hexanone (n-hexanophenone)
1-Phenyl-1-pentanol
1-Phenyl-1-pentanone (valerophenone)
(R)-(+)-1-Phenyl-1-propanol
(S)-(ꢃ)-1-Phenyl-1-propanol
1-Phenyl-1-propanone (propiophenone)
2-Propanol
C
O
O
O
0.0018 0.98
0.0060 0.99
0.0036
0.0066 0.99
0.0047 0.98
0.0026
0.0009
0.0019
0.0008
0.0010
0.0025
C10H12
C8H10
C8H10
O
O
C8H8O
C
C
13H20
13H18
O
O
O
O
O
O
r
r
r
T
S
C12H18
C12H16
C11H16
C11H14
942-92-7
583-03-9
1009-14-9
1565-74-8
613-87-6
93-55-0
T
T
T
A
A
A
A
0.995 0.0016
FT-n.m.r., g.c.
g.c.
g.c.
FT-n.m.r., FT-IR
FT-n.m.r., FT-IR
C9H12
C9H12
C9H10
O
O
O
0.99
0.99
0.99
0.0029 0.99
0.0015 0.99
0.0024
67-63-0
C3H8O
0.995 0.0006
Some common names of the substances are given in parentheses.
a
NAD(red) is the reduced form of b-nicotinamide-adenine dinucleotide phosphate, disodium salt.
(30 m long, 0.25 mm i.d., Chiraldex, Astec, Whippany, NJ,
USA). For analysis of (R) and (S) isomers of 1-phenyl-1-
alkanols, the helium carrier gas flow was held constant at
perature of the mixture was maintained at T ꢄ 293 K.
This reaction mixture was gently stirred overnight under
reduced pressure at room temperature (ꢄ293 K). The
methanol was removed from the reaction mixture and
ꢄ50 cm3 of water was added to the residue. The product
was then extracted twice into 50 cm3 diethyl ether. The
ether containing the 1-phenyl-1-alkanol was dried over
MgSO4 and removed in vacuo.
0.017 cm3 Æ sꢃ1 with
a
detector make-up flow of
0.50 cm3 Æ sꢃ1. The injector and detector were held at
T = 423 K. All injections were split with a split flow rate
of 0.50 cm3 Æ sꢃ1. For the separation of (R) and (S) isomers
of 1-phenyl ethanol through 1-phenyl-1-butanol in reac-
tions (1) to (3), the oven was held at T = 303 K for
15 min followed by a temperature ramp of 0.167 K Æ sꢃ1
to 423 K, where the oven was held for 10 min. For the sep-
aration of the (R) and (S) isomers of 1-phenyl-1-pentanol
in reaction (4), the oven was held isothermally at
T = 393 K for 40 min. For the separation of the (R) and
(S) isomers of 1-phenyl-1-hexanol in reaction (5) and the
(R) and (S) isomers of 1-phenyl-1-heptanol in reaction
(6), the oven was held at T = 373 K for 20 min followed
by a temperature ramp of 0.067 K Æ sꢃ1 to T = 473 K,
where the oven was held for 13 min.
The product yield (mole fraction) of crude 1-phenyl-1-
alkanol product was >0.90. The 1-phenyl-1-alkanol prod-
uct was further purified by vacuum distillation at
T ꢄ 293 K and at P = 120 Pa for 1-phenyl-1-hexanol, and
at T ꢄ 377 K and at P = 66.7 Pa for 1-phenyl-1-heptanol.
The purity of these 1-phenyl-1-alkanols was then checked
1
by using H n.m.r. and gas chromatography. The chirality
check on an enantioselective c-cyclodextrintrifluoroacetyl
capillary g.c. column showed that the 1-phenyl-1-hexanol
was a racemic mixture of (R)-(+)-1-phenyl-1-hexanol and
(S)-(ꢃ)- 1-phenyl-1-hexanol, and the 1-phenyl-1-heptanol
was also a racemic mixture of (R)-(+)-1-phenyl-1-heptanol
and the (S)-(ꢃ)-1-phenyl-1-heptanol.
2.3. Synthesis of 1-phenyl-1-hexanol and 1-phenyl-1-
heptanol
2.4. Response factor ratios
First, 0.05 mol of n-hexanophenone or n-heptanophe-
none was dissolved in 100 cm3 of methanol maintained at
T ꢄ 288 K. Another solution containing n = 0.018 mol of
and then diluted with 9 cm3 of water was also prepared
[24]. The NaBH4 solution was slowly added to n-hexan-
ophenone or n-heptanophenone solution so that the tem-
As described previously [22], a standard solution of
acetone, 1-phenyl-1-ethanone, 1-phenyl-1-ethanol, 2-pro-
panol, and 1-hexanol was prepared gravimetrically in
n-hexane. Using this solution the response factors (ratio
of concentration to peak area) with reference to the internal
standard, 1-hexanol, were determined for acetone, 1-phenyl-
NaBH4 dissolved in 1 cm3 of NaOH (c = 2.0 mol Æ dmꢃ3
)

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Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
1-ethanone, 1-phenyl-1- ethanol, and 2-propanol. For reac-
tions (2) to (6) standard solutions of 1-phenyl-1-alkanone,
its 1-phenyl-1-alkanol, and 1-hexanol were prepared in
n-hexane and their response factor ratios with respect to
1-hexanol were determined. Similarly, for the reactions in
n-pentane as solvent, a solution of acetone, 2-propanol,
and 1-hexanol was gravimetrically prepared in n-pentane,
and the response factor ratios of acetone and 2-propanol
with respect to 1-hexanol [22] were determined in n-pentane.
K ¼ cð1-phenyl ethanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcðacetophenoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg;
K ¼ cð1-phenyl-1-propanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcðpropiophenoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg; ð11Þ
K ¼ cð1-phenyl-1-butanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcðbutyrophenoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg; ð12Þ
K ¼ cð1-phenyl-1-pentanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcð1-phenyl-1-pentanoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg;
ð13Þ
ð10Þ
2.5. Equilibrium studies
K ¼ cð1-phenyl-1-hexanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcð1-phenyl-1-hexanoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg;
ð14Þ
K ¼ cð1-phenyl-1-heptanolðsolnÞÞ ꢂ cðacetoneðsolnÞÞ=
fcð1-phenyl-1-heptanoneðsolnÞÞ ꢂ cð2-propanolðsolnÞÞg.
ð15Þ
Here, c is the concentration of the indicated substance in
the organic solvent expressed as mol (kg Æ sln)ꢃ1. Since
these reactions are symmetrical with respect to reactant
and products, the equilibrium constants of these reactions
are dimensionless quantities and are independent of the
units chosen to express the concentrations. In organic sol-
vents, the substrates involved in these reactions are in a
non-ionized form, and their concentrations are small.
Hence, we have assumed that their activity coefficients
are unity and that the calculated equilibrium constants
can be identified as thermodynamic equilibrium constants
defined in terms of activities of the reactants and the
products.
The equilibrium measurements were carried out by
approaching equilibrium from both directions of reaction.
For the forward direction, the starting solution consisted of
{1-phenyl-1-alkanone + 2-propanol}, and for the reverse
direction, the starting solutions were {1-phenyl-1-alka-
nol + acetone}. The substrates were first dissolved in
ꢄ15 cm3 of solvent, n-hexane or n-pentane and ꢄ10 cm3
of these mixtures were used to carryout reactions. Then,
ꢄ0.10 cm3 of a solution containing the enzyme [0.010 g of
ketoreductase dissolved in 0.10 cm3 of phosphate buffer
{K2HPO4 (c = 0.10 mol Æ dmꢃ3), adjusted to pH 7.3 with
H3PO4} containing NAD(red) (c = 1.0 Æ 10ꢃ3mol Æ dmꢃ3)]
was added to each reaction mixture. These reaction mix-
tures were then placed in a constant temperature bath
( 0.01 K) and shaken laterally at ꢄ30 shakes Æ minꢃ1
.
The solutions were periodically analyzed by g.c. to deter-
mine the extent of reaction. A reaction was considered to
be at equilibrium when the ratios of the g.c. peak of prod-
ucts/reactants were essentially identical for the forward and
the reverse reaction mixtures. All the reactions that were
studied required the addition of freshly prepared enzyme
solution in order to achieve equilibrium within 7–15 days.
One to three such enzyme additions (the same amount of
enzyme as was used initially) were required.
The method of analysis of the reactants and products in
the equilibrated reaction mixtures in n-hexane and n-pen-
tane was similar to the method described previously
[21,22]. For quantitative analysis ꢄ 4 cm3 of reaction mix-
ture and ꢄ0.10 cm3 of internal standard solution (1-hexa-
nol) were gravimetrically added to a vial, and tightly
capped. Approximately, 6 Æ 10ꢃ4 cm3 of this solution was
then injected into the g.c., and the reaction mixture was ana-
lyzed for reactants and products. The concentrations
c{expressed as mol (kg Æ soln)ꢃ1} of each of the substances
involved in the reactions were determined from their respec-
tive chromatographic peak areas, the response factor ratios,
the concentration of the internal standard solution, and the
chromatographic peak area of the internal standard.
If we consider reactions (1) to (6) in terms of the forma-
tion of a specific chiral product, the generic reaction for the
(R)-(+)-1-phenyl-1-alkanol isomer, is
1-phenyl-1-alkanoneðsolnÞ þ 2-propanolðsolnÞ ¼
ðRÞ-ðþÞ-1-phenyl-1-alkanolðsolnÞ þ acetoneðsolnÞ. ð16Þ
The equilibrium constant for reaction (16) is
K ¼ cfðRÞ-ðþÞ-1-phenyl-1-alkanolðsolnÞgꢂ
cðacetoneðsolnÞÞ=fcð1-phenyl-1-alkanoneðsolnÞÞꢂ
cð2-propanolðsolnÞÞg.
ð17Þ
The equilibrium constant for the generalized reaction (9)
involving the racemic mixture {equal amounts of the (R)
and (S) isomers of 1-phenyl-1-alkanol} is
K ¼ cð1-phenyl-1-alkanolðsolnÞÞꢂ
cðacetoneðsolnÞÞ=fcð1-phenyl-1-alkanoneðsolnÞÞꢂ
cð2-propanolðsolnÞÞg.
ð18Þ
ð19Þ
3. Results and discussion
Here,
3.1. Thermodynamic formalism
cð1-phenyl-1-alkanolðsolnÞÞ ¼
cfðRÞ-ðþÞ-1-phenyl-1-alkanolðsolnÞgþ
cfðSÞ-ðꢃÞ-1-phenyl-1-alkanolðsolnÞg.
The respective equilibrium constants for reactions (1) to
(6) are

Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
1169
But the g.c. analysis of the reaction mixture has shown that
This leads to
the products are a racemic mixture {equal amounts of (R)
and (S) isomers}. Thus,
Kð18Þ ¼ 2 ꢂ Kð17Þ.
ð21Þ
Henceforth, we will be dealing with equilibrium constants
for reactions (1) to (6) as defined by equations 10 to 15,
where the total amount of the 1-phenyl-1-alkanol is consid-
ered. The equilibrium constants for specific chiral products
can be calculated by using equation (21).
cð1-phenyl-1-alkanolÞ ¼
2 ꢂ cfðRÞ-ðþÞ-1-phenyl-1-alkanolg ¼
2 ꢂ cfðSÞ-ðꢃÞ-1-phenyl-1-alkanolg;
ð20Þ
TABLE 2
Results of equilibrium measurements of the ketoreductase-catalyzed reduction of 1-phenyl-1-alkanone to the corresponding 1-phenyl-1-alkanol carried
out in n-pentane and n-hexane {reactions (1) to (6)}
Solvent
Direction T/K
c(phenylalkanone) c(2-propanol)
mol Æ (kg soln)ꢃ1 mol Æ (kg soln)ꢃ1 mol Æ (kg soln)ꢃ1 mol Æ (kg soln)ꢃ1
1-Phenyl-1-ethanone(soln) + 2-propanol(soln) = 1-phenyl-1-ethanol(soln) + acetone(soln) (1)
c(phenylalkanol) c(acetone)
K
ÆKæ
K
n-Hexane
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
288.36 0.06074
0.00730
0.00669
0.00701
0.00615
0.00900
0.00767
0.00866
0.00863
0.01032
0.00935
0.00618
0.00491
0.00422
0.00387
0.00525
0.00492
0.00490
0.00522
0.00542
0.00473
0.01456
0.01700
0.02069
0.02111
0.02275
0.01696
0.02467
0.02296
0.02601
0.02853
0.2029 0.0025 0.2033 0.0048
0.2036 0.0024
0.2125 0.0016 0.2132 0.0054
0.2138 0.0009
0.2151 0.0018 0.2171 0.0050
0.2190 0.0008
0.2233 0.0020 0.2227 0.0052
0.2221 0.0019
288.36 0.06127
292.83 0.05860
292.83 0.06214
297.93 0.06170
297.93 0.04967
303.04 0.06252
303.04 0.06253
308.05 0.05895
308.05 0.06232
0.2317 0.0012 0.2317 0.0056
0.2316 0.0019
n-Pentane Forward
298.15 0.01278
298.15 0.01168
0.01337
0.00668
0.00296
0.00185
0.01207
0.00880
0.2091 0.0022 0.2089 0.0053
0.2087 0.0019
Reverse
1-Phenyl-1-propanone (soln) + 2-propanol(soln) = 1-phenyl-1-propanol(soln) + acetone(soln) (2)
0.00919
0.01895
n-Hexane
Forward
Reverse
298.15 0.00869
298.15 0.01073
0.00159
0.00335
0.000509
0.000648
0.3385 0.0046 0.3401 0.0086
0.3416 0.0068
n-Pentane Forward
298.15 0.01754
298.15 0.01710
0.00231
0.00315
0.000694
0.001262
0.02115
0.01549
0.3623 0.0028 0.3626 0.0094
0.3629 0.0045
Reverse
1-Phenyl-1-butanone(soln) + 2-propanol(soln) = 1-phenyl-1-butanol(soln) + acetone(soln) (3)
0.00823
0.01706
n-Hexane
Forward
Reverse
298.15 0.00949
298.15 0.01032
0.00164
0.00318
0.000485
0.000492
0.2565 0.0063 0.2562 0.0067
0.2558 0.0076
n-Pentane Forward
298.15 0.01262
298.15 0.01221
0.00248
0.00141
0.000400
0.000327
0.02059
0.01402
0.2632 0.0012 0.2648 0.0062
0.2663 0.0087
Reverse
1-Phenyl-1-pentanone(soln) + 2-propanol(soln) = 1-phenyl-1-pentanol(soln) + acetone(soln) (4)
0.02165
0.02305
n-Hexane
Forward
Reverse
298.15 0.01114
298.15 0.01105
0.00462
0.00474
0.00113
0.00110
0.4753 0.0024 0.4797 0.0119
0.4841 0.0048
n-Pentane Forward
298.15 0.01355
298.15 0.02034
0.00214
0.002844
0.000776
0.00145
0.01810
0.01953
0.4844 0.0064 0.4870 0.0123
0.4895 0.0048
Reverse
1-Phenyl-1-hexanone(soln) + 2-propanol(soln) = 1-phenyl-1-hexanol(soln) + acetone(soln) (5)
0.01514
0.02581
n-Hexane
Forward
Reverse
298.15 0.02735
298.15 0.03293
0.02695
0.03191
0.01499
0.01325
0.3079 0.0064 0.3167 0.061
0.3255 0.0037
n-Pentane Forward
298.15 0.02830
298.15 0.02932
0.02421
0.03151
0.01423
0.01538
0.01411
0.01815
0.2931 0.0048 0.2976 0.0049
0.3021 0.0031
Reverse
1-Phenyl-1-heptanone(soln) + 2-propanol(soln) = 1-phenyl-1-heptanol(soln) + acetone(soln) (6)
0.01695
0.03180
n-Hexane
Forward
Reverse
298.15 0.01655
298.15 0.02832
0.03293
0.02845
0.01826
0.01448
0.5679 0.0053 0.5697 0.0167
0.5715 0.0056
n-Pentane Forward
298.15 0.01866
298.15 0.01732
0.01129
0.01730
0.01132
0.01540
0.01035
0.01123
0.5561 0.0074 0.5667 0.0169
0.5772 0.0071
Reverse
The 1-phenyl-1-alkanones are: 1-phenyl-1-ethanone, 1-phenyl-1-propanone, 1-phenyl-1-butanone, 1-phenyl-1-pentanone, 1-phenyl-1-hexanone and
1-phenyl-1-heptanone. The concentrations of the substrates involved in the reaction {1-phenyl-1-alkanone (soln) + 2-propanol(soln) = 1-phenyl-1-alkanol
(soln) + acetone(soln)} are given in columns 4 to 7; each reported concentration is an average of five measurements. The values of the equilibrium
constants K listed in column 8 are calculated from the concentrations given in columns 4 to 7 and by using equations (10) to (15). The quantity ÆKæ is the
average of the equilibrium constants which were obtained from the forward and the reverse directions of a reaction. The uncertainties in the values of K
(column 8) are based on two estimated standard deviations of the mean. The uncertainties given in column 9 are the combined expanded uncertainties in
the values of ÆKæ.

1170
Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
3.2. Results of equilibrium measurements
Clark and Glew equation [26] with the assumption that
the standard molar heat capacity change DrCꢀp;m for this
reaction is zero. The calculated values for the thermody-
namic quantities at T = 298.15 K are: K = 0.2177
0.0018; DrGmꢀ ¼ ð3:78 ꢁ 0:02Þ kJ ꢂ molꢃ1; DrHꢀm ¼ ð4:53ꢁ
The equilibrium constants for the ketoreductase-cata-
lyzed reduction of 1-phenyl-1-alkanone to the corre-
sponding 1-phenyl-1-alkanol are given in table 2. This
table includes equilibrium constants for reactions (1) to
(6) in n-hexane and n-pentane at 298.15 K and also the
equilibrium constants for reaction (1) in n-hexane as
function of temperature. The equilibrium constant K
listed in column 8 was calculated from the ‘‘c’’ of reac-
tants and products, reported in columns 4 to 7 and is
an average of five measurements. The uncertainties given
in this column are based on the random errors in the
measurements expressed as two estimated standard devi-
ations of the mean and do not include possible systematic
errors in the measurements. The equilibrium constants
ÆKæ reported in the last column are the averages of the
equilibrium constants obtained from the forward and
the reverse directions. It is important to note that the val-
ues of the equilibrium constants obtained from the for-
ward and the reverse directions of a reaction are
generally in agreement within their statistical uncertain-
ties. It is judged that reasonable estimates of the standard
uncertainties [25] due to possible systematic errors in the
values of the equilibrium constants for these reactions
are: 0.05 Æ K in the g.c. measurements of the concentra-
tions of the reactants and products (this includes possible
errors in the values of the response factors) and 0.01 Æ K
due to possible sample impurities. These estimated uncer-
tainties have been combined in quadrature together with
the statistical uncertainties, expressed as one estimated
standard deviation of the mean, to obtain combined stan-
dard uncertainties [25]. These combined expanded uncer-
tainties are reported in the last column with the average
values of the equilibrium constants ÆKæ (see column 9 in
table 2).
0:87Þ kJ ꢂ molꢃ1
,
and DrSꢀ ¼ ð2:5 ꢁ 2:9ÞJ ꢂ molꢃ1 ꢂ Kꢃ1
.
m
These uncertainties are equal to two estimated standard
deviations of the mean.
The chiral g.c. analysis of the reaction mixture has
shown that in every reaction the products were a racemic
mixture {equimolar mixture of (R)-(+)-1-phenyl-1-alkanol
and (S)-(ꢃ)-1-phenyl-1-alkanol isomers}. Thus, the ketore-
ductase used in our study for reduction of 1-phenyl-1-alka-
nones was not stereoselective. However, Zhu et al. [16]
have recently used NADP(red) dependent ketoreductase
which were produced via genomic mining and protein engi-
neering. In their study they have coupled the ketoreductase
with D-glucose dehydrogenase and D-glucose for regenera-
tion of NADP(red) and have investigated the stereoselec-
tive reduction of a series of arylketones.
The present study of 1-phenyl-1-alkanone is similar
to our previous studies of 2-alkanone reactions [21].
Comparing the two studies, the phenyl group in the 1-phenyl-
1-alkanone reaction replaces the methyl group of the
2-alkanone reactions. In 2-alkanone reactions [21], the val-
ues of the equilibrium constants decrease monotonically
with increasing value of the number of carbons and
approaches a limiting value of ꢄ0.30 for the number of car-
bons >8. In figure 2, the equilibrium constants for reactions
(1) to (6) carried out in n-hexane at T = 298.15 K is plotted
as a function of the number of carbons NC in the alkanone
side chain in the 1-phenyl-1-alkanones. In 1-phenyl-1-alka-
none reactions, the presence of the phenyl group has a pro-
nounced effect on the values of the equilibrium constants.
The values of the equilibrium constants of 1-phenyl-1-alka-
none with an odd number of carbons in alkyl side chain are
higher than the values of the equilibrium constants of the
preceding 1-phenyl-1-alkanone having an even number of
carbons in the side chain. Here, the values of the equilib-
rium constants of 1-phenyl-1-alkanone systems follow a
zig-zag pattern with increasing carbon number in the alkyl
For reaction (1) we have calculated the standard molar
Gibbs free energy change DrGꢀm, the standard molar
enthalpy change DrHꢀm, and the standard molar entropy
change DrSꢀm from the temperature dependency of the equi-
librium constant. In this calculation, we have used the
0.7
0.6
0.5
0.4
K
0.3
0.2
0.1
0
0
1
2
3
4
5
6
7
8
NC
FIGURE 2. The equilibrium constant K at T = 298.15 K for the reduction of 1-phenyl-1-alkanones {reactions (1) to (6)} in n-hexane plotted against the
number of carbons NC in the alkanone side chain.

Y.B. Tewari et al. / J. Chem. Thermodynamics 38 (2006) 1165–1171
1171
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side chain. Presently, we are unable to offer an explanations
for this behavior of 1-phenyl-1-alkanone reduction.
Acknowledgements
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JCT 05-289