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J. Chem. Thermodynamics 40 (2008) 661–670

A thermodynamic study of ketoreductase-catalyzed reactions5. Reduction of substituted ketones in n-hexane

Yadu B. Tewari a,*, David J. Vanderah a, Michele M. Schantz a, Robert N. Goldberg a,J. David Rozzell b, Joel F. Liebman c, Raymond Wai-Man Hui c, Yitzy Nissenbaum c,

Ahmad Reza Parniani c

a Biochemical Science and Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USAb Codexis, Inc., 129 N. Hill Avenue, Pasadena, CA 91106, USA

c Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA

Received 1 October 2007; received in revised form 31 October 2007; accepted 31 October 2007Available online 7 November 2007

Abstract

The equilibrium constants K for the ketoreductase-catalyzed reduction reactions of 1-benzyl-3-pyrrolidinone, ethyl 2-oxo-4-phenylbu-tyrate, ethyl 4-chloroacetoacetate, 1-benzyl-4-piperidone, and 1-benzyl-3-piperidone were measured in n-hexane at T = 298.15 K byusing gas chromatography. The equilibrium constants for the reaction involving 1-benzyl-4-piperidone were also measured as a functionof temperature (288.15 to 308.05) K. The calculated thermodynamic quantities for the reaction (1-benzyl-4-piperidone + 2-propanol =1-benzyl-4-hydroxypiperidine + acetone) reaction carried out in n-hexane at T = 298.15 K are: K = (26.2 ± 1.7); DrG

�m ¼

�ð8:10� 0:16Þ kJ �mol�1; DrH �m ¼ �ð3:44� 0:42Þ kJ �mol�1; and DrS�m ¼ ð15:6� 1:4Þ J �K�1 �mol�1. The chirality of the hydroxyl

products of the reactions (1)–(3) and (5)has also been investigated. The results showed that the stereoselectivity of the hydroxyl productsformed can be controlled by the selection of the solvent and enzyme used in these reactions. The thermochemical results for these reac-tions are compared with the results for reactions that have analogous structural features as well as with the results of quantum chemicalcalculations.Published by Elsevier Ltd.

Keywords: 1-Benzyl-4-piperidone; 1-Benzyl-3-piperidone; 1-Benzyl-3-pyrrolidinone; Equilibrium constant; Enthalpy; Ethyl 4-chloroacetoacetate; Ethyl 2-oxo-4-phenylbutyrate; Ketoreductase

1. Introduction

For industrially important syntheses, biocatalysis innon-aqueous media [1–3] has become an attractive alterna-tive to traditional synthetic methods. Lipase-catalyzedreactions in organic solvents have been used for the stereo-

0021-9614/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.jct.2007.10.011

* Corresponding author. Tel.: +1 301 975 2583; fax: +1 301 330 3447.E-mail addresses: yadu.tewari@nist.gov (Y.B. Tewari), david.vanderah

@nist.gov (D.J. Vanderah), michele.schantz@nist.gov (M.M. Schantz),robert.goldberg@nist.gov (R.N. Goldberg), david.rozzell@codexis.com(J.D. Rozzell), jliebman@umbc.edu (J.F. Liebman).

selective resolution of racemic mixtures [4–8] and esterifica-tion reactions [5,9,10]. In recent years [11–14] emphasis hasbeen on ketoreductase-catalyzed reduction of ketones toobtain the desired chiral alcohols that are useful intermedi-ates in the multi-step chiral synthesis of pharmaceuticalproducts [1,15–17]. Our interest [18–21] has been to carryout the thermodynamic investigations of these reactions.The thermodynamic data obtained in these studies are use-ful for a basic understanding of these reactions and for pro-cess optimization that can lead to improved product yields.

In this study, we report the results of equilibrium mea-surements for the following ketoreductase-catalyzed reduc-tion reactions (see figure 1) in n-hexane:

1-benzyl-3-pyrrolidinone 2-propanol (±)-1-benzyl-3-pyrrolidinol acetone

ONO

OH

NOH

+ +=

ethyl 2-oxo-4-phenylbutyrate (±)-ethyl 2-hydroxy-4-phenylbutyrate

O

O

O

O

O

OH

2-propanol

OH+ =

acetone

O

+

(±)-ethyl 4-chloro-3-hydroxybutyrateethyl 4-chloroacetoacetate

Cl

O

O

2-propanol

OH+ =

acetone

O

+

O

Cl

OH

O

O

(1)

(2)

(3)

2-propanol acetone

O

OH+ +=

2-propanol

OH+ =

acetone

O

+

1-benzyl-4-piperidone

N

O

1-benzyl-4-hydroxypiperidine

N

HO

1-benzyl-3-piperidone

N

O

(±)-1-benzyl-3-hydroxypiperidine

N

HO

(4)

(5)

FIGURE 1. The structures of the substances involved in reactions (1) to (5).

662 Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670

1-benzyl-3-pyrrolidinoneðsolnÞ þ 2-propanolðsolnÞ ¼ð�Þ-1-benzyl-3-pyrrolidinolðsolnÞ þ acetoneðsolnÞ; ð1Þ

ethyl 2-oxo-4-phenylbutyrateðsolnÞ þ 2-propanolðsolnÞ ¼ð�Þ-ethyl 2-hydroxy-4-phenylbutyrateðsolnÞþacetoneðsolnÞ; ð2Þ

ethyl 4-chloroacetoacetateðsolnÞ þ 2-propanolðsolnÞ ¼ð�Þ-ethyl 4-chloro-3-hydroxybutyrateðsolnÞþacetoneðsolnÞ; ð3Þ

1-benzyl-4-piperidoneðsolnÞ þ 2-propanolðsolnÞ ¼1-benzyl-4-hydroxypiperidineðsolnÞ þ acetoneðsolnÞ;

ð4Þ

1-benzyl-3-piperidoneðsolnÞ þ 2-propanolðsolnÞ ¼ð�Þ-1-benzyl-3-hydroxypiperidineðsolnÞ þ acetoneðsolnÞ:

ð5Þ

Here ‘‘soln’’ denotes the n-hexane. The activity of the keto-reductase depends on the presence of a small amount ofcofactor, b-nicotinamide adenine dinucleotide phosphate(reduced form), abbreviated NADP(red). Since theNADP(red) is insoluble in n-hexane, it is highly likely thatthe reaction requires a small amount of water which isadded to the reaction mixture (see Section 2.6) and thatthe reaction proceeds in this aqueous phase. Thus, the stepsin which the overall reaction proceeds are

Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670 663

1-benzyl-4-piperidoneðsolnÞ ¼ 1-benzyl-4-piperidoneðaqÞ;ð6Þ

2-propanolðsolnÞ ¼ 2-propanolðaqÞ; ð7Þ1-benzyl-4-piperidoneðaqÞ þ 2 NADPðredÞðaqÞ ¼1-benzyl-4-hydroxypiperidineðaqÞ þ 2 NADPðoxÞðaqÞ;

ð8Þ2 NADPðoxÞðaqÞ þ 2-propanolðaqÞ ¼

2 NADPðredÞðaqÞ þ acetoneðaqÞ; ð9Þ1-benzyl-4-hydroxypiperidineðaqÞ ¼ð�Þ-1-benzyl-4-hydroxypiperidineðsolnÞ; ð10Þ

2-propanolðaqÞ ¼ 2-propanolðsolnÞ: ð11Þ

Ketoreductase KRED-A1J and KRED-A1L catalyze reac-tion (8) and KRED-104 catalyzes reaction (9). The overallreaction (4) is obtained as the sum of reactions (6)–(11).Note that NADP(red) is regenerated by reaction (9). Sincethe concentration measurements were made in the n-hexanephase, the measured equilibrium constant pertains to reac-tion (4). The above equations, while they pertain specifi-cally to reaction (4), are also applicable to the otherreactions that are the subject of this study. Also, it shouldbe recognized that the aqueous phase contains a smallamount of water (the solubility of n-hexane in water is1.43 Æ 10�4 mol Æ dm�3 [22]) and the n-hexane phase con-tains a small amount of water (a value for this solubilitydoes not appear to have been reported in the literature).Nevertheless, the recognition of this fact does not invali-date the Hess’s law cycle given above. The critical pointis that if the sampling of the reaction mixture is done fromthe n-hexane phase (albeit with a small amount of water init), the equilibrium constant that is measured pertains tothe reaction in the n-hexane phase. Clearly, the value of thisequilibrium constant is independent of both how the bioca-talysis occurs and the fact that there is an aqueous phase inproximity to the n-hexane phase.

The principal aim of this study was to determine theequilibrium constants for reactions (1) to (5) in n-hexane.The temperature dependency of the equilibrium constantfor reaction (4) has also been studied. With the exceptionof reaction (4), there is chirality associated with one ofthe products in each reaction. For this reason, we have alsoinvestigated the chirality of the hydroxyl products in reac-tions (1)–(3) and (5).

1 Certain commercial equipment, instruments, computer programs, ormaterials are identified in this paper to specify the experimental proce-dures adequately. Such identification is not intended to imply recommen-dation or endorsement by the National Institute of Standards andTechnology, nor is it intended to imply that the materials or equipmentidentified are necessarily the best available for the purpose.

2. Experimental

2.1. Materials

The chemicals used in this study, their ChemicalAbstract Service (CAS) registry numbers, empirical formu-lae, molar masses, sources, and purities as determined byvarious methods are given in table 1. The enzymes usedin this study were ketoreductase (EC 1.1.1.2) from Biocat-alytics, Inc., Pasadena, CA. These recombinant ketoreduc-

tases were prepared via genome mining and proteinengineering [13].

2.2. Chromatography and quantitative analysis

The quantitative analysis of the reactants and productswas carried out by using a Hewlett-Packard (HP) 5890GC (Agilent Technologies, Wilmington, DE, USA),1

equipped with a flame ionization detector (FID) and afused silica Phenomenex ZB-WAX capillary column(30 m long, 0.53 mm i.d., 0.53 lm thick film coating). Thechromatographic conditions used for the analysis of reac-tants and products are as follows: for reaction (1), T(injec-tor) = 493 K, T(detector) = 513 K, and the initial columntemperature of T = 313 K was held for 4 min and thenraised to T = 513 K at a rate of 0.417 K Æ s�1 and held atT = 513 K for 8 min; for reaction (2), T(injector) = 493 K,T(detector) = 513 K, and the initial column temperature ofT = 313 K was held for 4 min and then raised to T = 513 Kat a rate of 0.333 K Æ s�1 and held at T = 513 K for 8 min;for reaction (3), T(injector) = 373 K, T(detector) = 513 K,and the initial column temperature of T = 313 K was heldfor 4 min and then raised to T = 423 K at a rate of0.25 K Æ s�1 and held at T =423 K for 10 min; for reactions(4) and (5), T(injector) = 493 K, T(detector) = 513 K, andthe initial column temperature of T = 313 K was held for4 min and then raised to T = 513 K at a rate of 0.25 K Æ s�1

and held at T = 513 K for 8 min. In all cases, the head pres-sure of the helium carrier gas was P = 283 kPa.

2.3. Synthesis of 1-benzyl-3-hydroxypiperidine

The substance (±)-1-benzyl-3-hydroxypiperidine wasprepared by the reduction of 1-benzyl-3-piperidone usingestablished procedures [23]. A solution containing 0.014 mol(0.53 g) of NaBH4 in 9.0 cm3 of water containing 1.0 cm3

of NaOH (concentration c = 2.0 mol Æ dm�3) was addedslowly to 0.0266 mol (6.00 g) of 1-benzyl-3-piperidonehydrochloride in 50 cm3 of methanol so that the temperaturewas maintained at T � 278 K. This solution was stirred for�18 h at room temperature. The reaction solution wasconcentrated under reduced pressure and �50 cm3 of waterwas added to the residue. The aqueous layer was thenextracted twice into 50 cm3 of diethyl ether. The ether layerswere combined and dried over MgSO4. The ether was thenremoved to yield 4.31 g (0.72 mass fraction yield) of 1-benzyl-3-hydroxypiperidine. The 1-benzyl-3-hydroxypiperi-dine product was further purified by vacuum distillation to>0.99 mole fraction purity as determined by 1H n.m.r. andby g.c. The substance (±)-ethyl 2-hydroxy-4-phenylbutyratewas also prepared and purified in a similar way.

TABLE 1Principal substances used in this study with their Chemical Abstracts Service (CAS) registry numbers, empirical formulas, relative molar masses Mr,Supplier (A = Aldrich, B = BioCatalytics, F = Fluka, 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, and the methods used by vendors to determine the mole fraction purity andenantiomeric purity

Substance CAS no. Formula Mr Supplier x w Methoda

Acetone 67-64-1 C3H6O 58.08 A 0.99 0.0103 g.c.; i.r.; u.v.(R)-(�)-1-Benzyl-3-hydroxypiperidine 91599-81-4 C12H17NO 191.27 A 0.97 0.0075 g.c.; i.r.; o.r.(±)-1-Benzyl-3-hydroxypiperidine 14813-01-5 C12H17NO 191.27 S 0.98 0.0131 g.c.; n.m.r.1-Benzyl-4-hydroxypiperidine 4727-72-4 C12H17NO 191.27 A 0.97 0.0122 i.r.; n.m.r.; g.c.1-Benzyl-4-piperidone 3612-20-0 C12H15NO 189.25 A 0.99 0.0103 g.c.; i.r.1-Benzyl-3-piperidone hydrochloride 50606-58-1 C12H16NOCl 225.72 A 0.95 0.0795 i.r.; n.m.r.; t.l.c.(S)-(�)-1-Benzyl-3-pyrrolidinol 101385-90-4 C11H15NO 177.24 A 0.99 0.0101 i.r.; o.r.; g.c.(R)-(+)-1-Benzyl-3-pyrrolidinol 101930-07-8 C11H15NO 177.24 A 0.98 0.0106 i.r.; o.r.; g.c.1-Benzyl-3-pyrrolidinone 775-16-6 C11H13NO 175.23 A 0.98 0.0125 i.r.; g.c.Ethyl-4-chloroacetoacetate 638-07-3 C6H9ClO3 164.59 T 0.95 0.0012 i.r.; g.c.Ethyl (R)-(+)-4-chloro-3-hydroxybutyrate 90866-33-4 C6H11ClO3 166.60 T 0.98 0.0125 g.c.; o.r.; r.i.Ethyl (S)-(�)- 4-chloro-3-hydroxybutyrate 86728-85-0 C6H11ClO3 166.60 T 0.96 0.0095 g.c.; o.r.; r.i.Ethyl (R)-(�)-2-hydroxy-4-phenylbutyrate 90315-82-5 C12H16O3 208.25 F 0.99 0.0113 g.c.; n.m.r.; r.i.(±)-Ethyl 2-hydroxy-4-phenylbutyrate 7226-83-7 C12H16O3 208.25 S 0.99 0.0078 g.c.; n.m.r.Ethyl 2-oxo-4-phenylbutyrate 64920-29-2 C12H14O3 206.24 A 0.97 0.0141 g.c.; n.m.r.n-Hexane 110-54-3 C6H14 86.18 A 0.99 g.c.1-Hexanol 111-27-3 C6H14O 102.18 A 0.99 0.0011 g.c.Ketoreductase (EC 1.1.1.2)b 9028-12-0 Bb-Nicotinamide adenine dinucleotide phosphate, reduced

form, tetra sodium salt2646-71-1 C21H26N7Na4P3O17 833.35 R 0.97 Enzymatic

assay2-Propanol 67-63-0 C3H8O 60.10 A 0.995 0.001 g.c.

a The methods used by the vendors for the characterization of the substances are: gas chromatography (g.c.), infra red (i.r.), nuclear magnetic resonance(n.m.r.), optical rotation (o.r.), refractive index (r.i.), thin layer chromatography (t.l.c.), and ultra violet (u.v.).

b Three different kinds of ketoreductases were used in this study (see Section 1). They were designated by the vendor as KRED-A1J, KRED-A1L, andKRED-104.

664 Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670

2.4. Chiral separations

Separation of the chiral products for reactions (1) to (3)was carried out by using a Hewlett-Packard (HP) 5890 GC,equipped with a flame ionization detector (FID) and anAstec b-cyclodextrin dimethyl capillary column (30 m long,0.25 mm i.d.). The injector and detector temperatures wereset at 473 K. The carrier gas was helium (flow rate =0.020 cm3 Æ s�1) with a detector make-up flow of 0.50cm3 Æ s�1. All injections were split at a ratio of 30:1. Theinjection volume was 0.0010 cm3. For separation of ethyl(R)-(�)-2-hydroxy-4-phenylbutyrate and ethyl (S)-(+)-2-hydroxy-4-phenylbutyrate, the oven was held at T =353 K for 5 min followed by a temperature ramp of0.167 K Æ s�1 to T = 423 K and then held at T = 423 Kfor 10 min. For separation of ethyl (S)-(�)-4-chloro-3-hydroxybutyrate and ethyl (R)-(+)-4-chloro-3-hydroxybu-tyrate the oven was held isothermally at T = 363 K for38 min. Again for separation of (R)-(+)-1-benzyl-3-pyrro-lidinol and (S)-(�)-1-benzyl-3-pyrrolidinol the oven washeld at T = 353 K for 5 min followed by a temperatureramp of 0.167 K s�1 to T = 423 K and held at T = 423 Kfor 10 min. We attempted the use of several chiral capillarycolumns {b-cyclodextrin dimethyl (30 m long, 0.25 mm i.d.),c-cyclodextrin trifluoroacetyl (30 m long, 0.25 mm i.d.),b-cyclodextrin hydroxypropyl (30 m long, 0.25 mm i.d.), andc-cyclodextrin propionyl (20 m long, 0.25 mm i.d.)} for theseparation of (R)-(�)-1-benzyl-3-hydroxypiperidine and

(S)-(+)-1-benzyl-3-hydroxypiperidine. However, none ofthese columns provided a satisfactory separation. There-fore, a Perkin Elmer Model 341 polarimeter was used todetermine the chirality of 1-benzyl-3-hydroxypiperidine inreaction (5).

2.5. Response factor ratios

For reaction (4), a standard solution of 1-benzyl-4-pip-eridone, 1-benzyl-4-hydroxypiperidine, 1-hexanol, 2-pro-panol, and acetone was gravimetrically prepared inn-hexane. Using this solution, the response factors (ratioof concentration to peak area) with reference to the inter-nal standard 1-hexanol were determined for acetone,2-propanol, 1-benzyl-4-piperidone, and 1-benzyl-4-hydrox-ypiperidine. For reactions (1) and (2), standard solutions of1-benzyl-3-pyrrolidinone, (S)-(�)-1-benzyl-3-pyrrolidinol,and 1-hexanol, ethyl-2-oxo-4-phenylbutyrate, ethyl-2-hydroxy-4-phenylbutyrate, and 1-hexanol; respectively,were used to determine response factor ratios of theketones and their hydroxyl products with respect to 1-hex-anol. Due to the use of a different injector temperature inreaction (3), a standard solution of ethyl-4-chloroacetoace-tate, ethyl 3-hydroxy-4-chlorobutyrate, 1-hexanol, 2-pro-panol, and acetone was prepared in n-hexane todetermine their response factor ratios with respect to 1-hex-anol. For reaction (5), 1-benzyl-3-piperidone hydrochloridewas first dissolved in 1 cm3 of sodium hydroxide

Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670 665

(c = 2.0 mol Æ dm�3); 1-benzyl-3-hydroxypiperidine, 1-hex-anol, and n-hexane were then quantitatively added. Then-hexane phase was used for determination of the responsefactor ratios of 1-benzyl-3-piperidone and 1-benzyl-3-hydroxypiperidine with respect to 1-hexanol.

2.6. Equilibrium studies

The equilibrium measurements were carried out byapproaching equilibrium from both directions of the reac-tion. For reaction (1), the forward direction solutionconsisted of (1-benzyl-3-pyrrolidinone + 2-propanol) inn-hexane, and the reverse direction solution was {an equi-molar mixture of (R)-(+)- and (S)-(�)-1-benzyl-3-pyrro-lidinol + acetone} in n-hexane. The total volume of thesolutions was �10 cm3. Then, 0.20 cm3 of a solutioncontaining the enzyme [�0.010 g of KRED-104, �0.010 gof KRED-A1J, and �0.010 g of KRED-A1L in 0.20 cm3

of phosphate buffer [{K2HPO4 (c = 0.10 mol Æ dm3), adjustedto pH 7.3 with H3PO4} containing NADP(red) (c = 0.0011mol Æ dm�3)] was added to each reaction mixture. Due tothe low solubility of n-hexane in water [22], this aqueousenzyme solution exists as a separate phase from the mainn-hexane phase. While it is not possible to see the pointof contact of these two optically clear phases, which haverespective volumes of 0.20 cm3 and 10 cm3, there was noevidence of any emulsion formation.

These reaction mixtures were then placed in a constanttemperature water bath (±0.01 K) and shaken laterally at�30 shakes Æ min�1. The reaction mixtures were periodi-cally analyzed by g.c. to determine the extent of reaction.Since the aim was to study the chemical equilibrium in then-hexane phase, extreme care was taken to carefully with-draw with a syringe only a small portion of the n-hexanephase (the upper one) in the reaction vessel. A reactionwas considered to be at equilibrium when the ratios of theg.c. peak areas of products/reactants were essentially iden-tical for the forward and the reverse reaction mixtures. Anadditional amount of freshly prepared enzyme solution(�0.10 cm3) was added as needed to the reaction mixturesto speed up the equilibration process. The same procedurewas used for reactions (2) to (4). In performing reactionsfrom the reverse reaction direction, the racemic forms ofthe hydroxyl products were used in all cases with the excep-tion of reaction (4), where the product, 1-benzyl-4-hydrox-ypiperidine, does not possess a chiral center. For reaction(5), the reactant was in the form of a hydrochloride. There-fore, 1-benzyl-3-piperidone hydrochloride was dissolved in1.0 cm3 of sodium hydroxide (c = 2.0 mol Æ dm�3) and thenn-hexane was gravimetrically added to the vial. The n-hex-ane phase with 1-benzyl-3-piperidone was then added to avial containing 2-propanol to prepare the forward mixtureof reaction (5). The remainder of the procedure for thestudy of reaction (5) was identical to that used to studythe other four reactions. All reactions required additionalenzyme solution for equilibration. The time needed to reachequilibrium for these reactions varied from 5 d to 20 d.

For quantitative analysis of the reactants and productsin the equilibrated reaction mixtures, �4 cm3 of reactionmixture and �0.10 cm3 of internal standard solution (1-hexanol) were gravimetrically added to a vial, and tightlycapped. This solution was then injected into the g.c., andthe reaction mixture was analyzed for reactants and prod-ucts. The concentrations c {expressed as mol (kg Æ soln)�1}of each of the substances involved in the reactions weredetermined from their respective chromatographic peakareas, their appropriate response factor ratios, the concen-tration of the internal standard solution, and the chro-matographic peak area of the internal standard.

3. Results and discussion

3.1. Chirality of the reactions

Experiments were performed to examine the chirality ofthe hydoxyl products formed in reactions (1)–(3), and (5).Since the product of reaction (4), 1-benzyl-4-hydrpoxypi-peridine, does not possess an asymmetric carbon, thereare no chirality issues associated with this reaction. Theresults of these chirality experiments are given in table 2.In examining these results, it is seen that, in all cases, theracemic mixture of the hydroxyl products is obtained whenone uses the enzyme combination {KRED-104 + a 50:50mixture of KRED-A1J and KRED-A1L}. In a few cases,2-propanol was used as the solvent. The reason for thisselection is that, for industrial applications, the use of thissolvent serves, via Le Chaltelier’s principle, to increase theamount of the hydroxyl product that is formed. Interest-ingly, the chirality of the products obtained with reactions(2) and (3) carried out in 2-propanol and with the enzymecombination (KRED-104 + KRED-A1L) had (R)-stereo-chemistry as distinct from the (S)-stereochemistry of theproducts obtained when n-hexane was used as the solvent.Thus, the results given in table 2 demonstrate that the chi-rality of the products can be determined by the exclusiveuse of either KRED-A1L or KRED-A1J and by the sol-vent that is used. Clearly, an explanation of these resultsrequires an understanding of the mechanism(s) of thesereactions. In this regard, a critical first step would be adetermination of the structure of the enzyme.

3.2. Thermodynamic formalism

The respective equilibrium constants for reactions (1) to(5) are

K¼fcðð�Þ-1-benzyl-3-pyrrolidinolðsolnÞÞ�cðacetoneðsolnÞÞg=fcð1-benzyl-3-pyrrolidinoneðsolnÞÞ�cð2-propanolðsolnÞÞg; ð12Þ

K¼fcðð�Þ-ethyl 2-hydroxy-4-phenylbutyrateðsolnÞÞ�cðacetoneðsolnÞÞg=fcðethyl 2-oxo-4-phenylbutyrateðsolnÞÞ�cð2-propanolðsolnÞÞg; ð13Þ

TABLE 2Chirality of the products of reactions (1)–(3) and (5) at T = 298.15 Ka

Starting materials Solvent Enzyme combination Chirality of the product

Reaction (1)

1-Benzyl-3-pyrrolidinone + 2-propanol n-Hexane KRED-104 + KRED-A1L (S)-(�)-1-benzyl-3-pyrrolidinol (x = 0.99)1-Benzyl-3-pyrrolidinone + 2-propanol n-Hexane KRED-104 + KRED-A1J (R)-(+)-1-benzyl-3-pyrrolidinol (x = 0.99)1-Benzyl-3-pyrrolidinone + 2-propanol n-Hexane KRED-104 + (50:50 mixture of

KRED-A1J and KRED-A1L)Racemic mixture of (S)-(�)- and (R)-(+)-1-benzyl-3-pyrrolidinol

Racemic mixture of (S)-(�)- and (R)-(+)-1-benzyl-3-pyrrolidinol + acetone

n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

(R)-(+)-1-benzyl-3-pyrrolidinol (x = 0.66)b

Reaction (2)

Ethyl-2-oxo-4-phenyl butyrate + 2-propanol 2-Propanol KRED-104 + KRED-A1L Ethyl (R)-(�)-2-hydroxy-4-phenylbutyrate(x = 0.94)

Ethyl-2-oxo-4-phenyl butyrate + 2-propanol 2-Propanol KRED-104 + KRED-A1J Ethyl (S)-(+)-2-hydroxy-4-phenylbutyrate(x = 0.97)

Ethyl-2-oxo-4-phenyl butyrate + 2-propanol n-Hexane KRED-104 + KRED-A1L Ethyl (S)-(+)-2-hydroxy-4-phenylbutyrate(x = 0.90)

Racemic mixture of ethyl (S)-(+)- and ethyl (R)-(�)-2-hydroxy-4-phenylbutyrate + acetone

n-Hexane KRED-104 + KRED-A1L Ethyl (S)-(+)-2-hydroxy-4-phenylbutyrate(x = 0.87)

Ethyl-2-oxo-4-phenyl butyrate + 2-propanol n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of ethyl (S)-(+)- and ethyl(R)-(�)-2-hydroxy-4-phenylbutyrate

Racemic mixture of ethyl (S)-(+)- and ethyl (R)-(�)-2-hydroxy-4-phenylbutyrate + 2-Propanol

n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of ethyl (S)-(+)- and ethyl(R)-(�)-2-hydroxy-4-phenylbutyrate

Reaction (3)

Ethyl 4-chloroacetoacetate + 2-propanol n-Hexane KRED-104 + KRED-A1L Ethyl (S)-(�)-4-chloro-3-hydroxybutyrate(x > 0.99)

Ethyl 4-chloroacetoacetate + 2-propanol 2-Propanol KRED-104 + KRED-A1L Ethyl (R)-(+)-4-chloro-3-hydroxybutyrate(x > 0.99)

Ethyl 4-chloroacetoacetate + 2-propanol n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of ethyl (S)-(�)- and ethyl(R)-(+)-4-chloro-3-hydroxybutyrate

Racemic mixture of ethyl (S)-(�)- and ethyl (R)-(+)-4-chloro-3-hydroxybutyrate + acetone

n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of ethyl (S)-(�)- and ethyl(R)-(+)-4-chloro-3-hydroxybutyrate

Reaction (5)

1-Benzyl-3-piperidone + 2-Propanol n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of (R)-(�)- and (S)-(+)-1-benzyl-3-hydroxypiperidine

Racemic mixture of (R)-(�)- and (S)-(+)-1-benzyl-3-hydroxypiperidine + acetone

n-Hexane KRED-104 + (50:50 mixture ofKRED-A1J and KRED-A1L)

Racemic mixture of (R)-(�)- and (S)-(+)-1-benzyl-3-hydroxypiperidine

a The quantity x is the mole fraction of the specified substance. The product of reaction (4), 1-benzyl-4-hydrpoxypiperidine, does not possess anasymmetric carbon.

b This reaction mixture was not given sufficient time to reach equilibrium.

666 Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670

K¼fcðð�Þ-ethyl 4-chloro-3-hydroxy-4-chlorobutyrateðsolnÞÞ�cðacetoneðsolnÞÞg=fcðethyl 4-chloroacetoacetateðsolnÞÞ�cð2-propanolðsolnÞÞg; ð14Þ

K¼fcð1-benzyl-4-hydroxypiperidineðsolnÞÞcðacetoneðsolnÞÞg=fcð1-benzyl-4-piperidoneðsolnÞÞ�cð2�propanolðsolnÞÞg; ð15Þ

K¼fc�ð�Þð1-benzyl-3-hydroxypiperidineðsolnÞÞ�cðacetoneðsolnÞÞg=fcð1-benzyl-3-piperidoneðsolnÞÞ�cð2-propanolðsolnÞÞg: ð16Þ

In the above equations, c is the concentration {mol(kg Æ soln)�1} of the indicated substance. Since these aresymmetrical reactions with respect to reactants and prod-ucts, the equilibrium constants are dimensionless quantitiesand are independent of the units chosen to express the con-centrations. In organic solvents, the substrates in thesereactions are in a non-ionized form and their concentra-

tions are low. Hence, we have assumed that their activitycoefficients are unity and that the measured equilibriumconstants can be identified as thermodynamic equilibriumconstants defined in terms of activities of the reactantsand the products.

3.3. Results of equilibrium measurements

The results of the equilibrium measurements for thereactions (1) to (5) are given in table 3. The equilibriumconstants K (column 7) are calculated from the concentra-tions of reactants and products (reported in columns 3 to6). The uncertainties given in this column are equal totwo estimated standard deviations of the mean based onlyon the random errors in the measurements and do notinclude possible systematic errors. The equilibrium con-stants ÆKæ reported in column 8 are the averages of theequilibrium constants obtained from the forward and thereverse directions. It is important to note that the values

TABLE 3Results of equilibrium measurements on reactions (1) to (5) carried out in n-hexanea

Direction T c(ketone) c(2-propanol) c(hydroxyl product) c(acetone) K ÆKæ

K mol Æ (kg soln)�1 mol Æ (kg soln)�1 mol Æ (kg soln)�1 mol Æ (kg soln)�1

1-Benzyl-3-pyrrolidinone(soln) + 2-propanol(soln) = (±)-1-benzyl-3-pyrrolidinol(soln) + acetone(soln) (1)

Forward 298.15 0.00705 0.00615 0.01395 0.01464 4.71 ± 0.27 4.91 ± 0.45Reverse 298.15 0.00787 0.00578 0.01298 0.01792 5.11 ± 0.39

Ethyl 2-oxo-4-phenylbutyrate(soln) + 2-propanol(soln) = (±)-ethyl 2-hydroxy-4-phenylbutyrate(soln) + acetone(soln) (2)

Forward 298.15 0.00308 0.00767 0.03003 0.02970 37.75 ± 0.85 37.8 ± 2.7Reverse 298.15 0.00420 0.00430 0.02734 0.02501 37.86 ± 1.8

Ethyl 4-chloroacetoacetate(soln) + 2-propanol(soln) = (±)-ethyl 3-hydroxy-4-chlorobutyrate(soln) + acetone(soln) (3)

Forward 298.15 0.0000537 0.01324 0.03518 0.02693 1333 ± 70 (1.43 ± 0.13) Æ 103

Reverse 298.15 0.000873 0.000758 0.02528 0.04004 1530 ± 101

1-Benzyl-4-piperidone(soln) + 2-propanol(soln) = 1-benzyl-4-hydroxypiperidine(soln) + acetone(soln) (4)

Forward 288.12 0.00295 0.00327 0.01705 0.01544 27.29 ± 0.22 27.5 ± 1.8Reverse 288.12 0.00337 0.00329 0.01153 0.02662 27.68 ± 0.16Forward 293.21 0.00321 0.00320 0.01750 0.01554 26.47 ± 0.50 27.0 ± 1.8Reverse 293.21 0.00353 0.00362 0.01286 0.02744 27.61 ± 0.31Forward 298.15 0.000631 0.01302 0.01333 0.01544 25.05 ± 0.51 26.1 ± 1.8Reverse 298.15 0.00636 0.00222 0.01071 0.03591 27.24 ± 0.68Forward 303.09 0.00266 0.00381 0.02025 0.01275 25.48 ± 0.27 25.7 ± 1.7Reverse 303.09 0.00704 0.00141 0.00962 0.02670 25.88 ± 0.45Forward 308.04 0.00426 0.00387 0.02216 0.01865 25.07 ± 0.18 25.1 ± 1.6Reverse 308.04 0.00637 0.00150 0.00944 0.02547 25.16 ± 0.30

1-Benzyl-3-piperidone(soln) + 2-propanol(soln) = (±)-1-benzyl-3-hydroxypiperidine(soln) + acetone(soln) (5)

Forward 298.15 0.000397 0.01062 0.00881 0.01230 25.70 ± 0.59 26.6 ± 1.8Reverse 298.15 0.00594 0.00612 0.02867 0.03478 27.43 ± 0.58

a The concentrations of the substrates involved in these reactions are given in columns 3 to 6; each reported concentration is the average of fivemeasurements. The enzyme combination used to carry out this reaction was: �0.010 g of KRED-104, �0.010 g of KRED-A1J, and �0.010 g of KRED-A1L in 0.20 cm3 of phosphate buffer [{K2HPO4 (c = 0.10 mol Æ dm3), adjusted to pH 7.3 with H3PO4} containing NADP(red) (c = 0.0011 mol Æ dm�3)].The values of the equilibrium constants K listed in column 7 are calculated from the concentrations given in columns 3 to 6 by using Eqs. (8) to (12). Thequantity ÆKæ is the average of the equilibrium constants which were obtained from the forward and the reverse directions of reaction. The uncertainties inthe values of K (column 7) are based on two estimated standard deviations of the mean. The uncertainties given in column 8 are the expanded uncertaintiesin the values of ÆKæ (see Section 3.3).

Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670 667

of the equilibrium constants obtained from the forwardand the reverse directions of a reaction are either in agree-ment or in near agreement within their statistical uncertain-ties. Also, the respective racemic products were obtained inour studies of reactions (1) to (4). If this was not the case,different values of the equilibrium constants would havebeen obtained and it would have been necessary to applyadjustments for the entropy of mixing of the (R) and (S)forms of the respective products [24] in order to obtain val-ues of the equilibrium constants for the racemic mixtures.It is judged that reasonable estimates of the standarduncertainties [25] due to possible systematic errors in thevalues of the equilibrium constants for these reactionsare: ±0.03 Æ K in the g.c. measurements of the concentra-tions of the reactants and products (this includes possibleerrors in the values of the response factors) and±0.01 Æ K due to possible sample impurities. Also, in afew cases where the difference jK(forward) � K(reverse)jwas larger than the statistical uncertainty, it was necessaryto add an additional component of uncertainty for this dif-ference. These estimated uncertainties were combined inquadrature together with the statistical uncertainties,expressed as one estimated standard deviation of the mean,

to obtain combined standard uncertainties uc [25]. Thereported uncertainties in the values of K (see column 8 intable 3) are expanded uncertainties equal to 2 Æ uc.

The standard molar Gibbs free energy change DrG�m, the

standard molar enthalpy change DrH �m, and the standardmolar entropy change DrS

�m for reaction (4) in n-hexane

were calculated from the temperature dependency of theequilibrium constant using the Clark and Glew equation[26]. In this calculation, it was assumed that the standardmolar heat capacity change DrC

�p;m for the reaction is zero.

The calculated thermodynamic quantities at T = 298.15 Kare: K = (26.2 ± 0.10); DrG

�m ¼ �ð8:10� 0:01Þ kJ �mol�1;

DrH �m ¼ �ð3:44� 0:42Þ kJ �mol�1; and DrS�m ¼ ð15:6�

1:4Þ J �K�1 �mol�1 for reaction (4) in n-hexane. The uncer-tainties in these values are equal to two estimated standarddeviations of the mean. Consideration of the possible sys-tematic errors in the equilibrium measurements (see above)leads to the following values with expanded uncertain-ties: K = (26.2 ± 1.7); DrG

�m ¼ �ð8:10� 0:16Þ kJ �mol�1;

DrH �m ¼ �ð3:44� 0:42Þ kJ �mol�1; and DrS�m ¼ ð15:6�

1:4Þ J �K�1 �mol�1 for reaction (4) in n-hexane at T =298.15 K. Here it was assumed that any systematic errorsin the values of K will be the same at all temperatures

668 Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670

and that the value of DrH �m, which is calculated from thetemperature dependence of K, is not affected by these pos-sible systematic errors. The values of DrG

�m=ðkJ �mol�1Þ

at T = 298.15 K for reactions (1)–(3) and (5) are, respec-tively, �(3.94 ± 0.23), �(9.00 ± 0.18), �(18.01 ± 0.22), and�(8.13 ± 0.17). The uncertainties in these values of DrG

�m

are the based on the expanded uncertainties in the valuesof the equilibrium constants.

3.4. Thermochemical analogies

Because the results reported in this study appear to bethe first in the literature, direct comparisons with previousresults is not possible. Additionally, there do not appear tobe any thermochemical cycles that can be used to calculatethe measured quantities. Nevertheless, it is possible toexamine analogous reactions for which thermochemicaldata have been reported. These analogies required the useof the reactants and products in phases other than as sol-utes in hexane as well as the use of substances that arechemically similar to those studied herein. The first consid-ered analogy involves reactions (1) and (5), which have astructural relation similar to the previously measured(Tewari et al. [19]) redox reactions for cyclopentanoneand cyclohexanone. Therefore, the ratio of the equilibriumconstants for reactions (1) and (5), (K1/K5), is expected tobe approximately equal to the ratio of the equilibrium con-stants (K17/K18) for the following reactions:

cyclopentanoneðsolnÞ þ 2-propanolðsolnÞ ¼cyclopentanolðsolnÞ þ acetoneðsolnÞ; ð17Þ

cyclohexanoneðsolnÞ þ 2-propanolðsolnÞ ¼cyclohexanolðsolnÞ þ acetoneðsolnÞ: ð18Þ

This expectation is based on the assumptions that mediumeffects are negligible and that the effect of the benzylatednitrogen replacing a b-methylene group in the two ring sys-tems will be the same. Using the values of the equilibriumconstants for reactions (17) and (18) from Tewari et al.[19], the aforementioned ratio (K17/K18) is 0.02, while the ra-tio of equilibrium constants (K1/K5) for reactions (1) and (5)from the current study is 0.18. As discussed earlier, [19]cyclohexanol has virtually no steric strain, whereas the ben-zylated nitrogen replacing a b-methylene group precludestetrahedral symmetry for 1-benzyl-3-hydroxypiperidine inreaction (5). Also, based upon steric effects, one expects aninter-ring repulsion and a consequent, thermochemicaldestabilization (an increase in the values of the standard mo-lar enthalpies of formation DfH �m) in both the alcohols andketones. Additionally, both heterocycles are a-aminoke-tones, which have also been found (e.g., see the combustioncalorimetry results of Welle et al. [27]) to exhibit thermo-chemical destabilization. Thus, the approximate agreementin the ratios of the equilibrium constants is reasonable.

We now consider the results for reaction (2). There arefew thermochemical results for compounds with carbonylgroups adjacent to each other and fewer yet with a carbonyl

and alcohol group adjacent to each other. As such, there arefew substances with which one can make comparisons of thethermochemistry of reaction (2). Since substances contain-ing carbonyl groups are known to be more polarized thansubstances containing carbon–oxygen single bonds as inethers, the alcohol bearing carbon will be less positivelycharged than that in the ketone. Evidence for this comesfrom the fact that the values of the standard molar enthal-pies of vaporization DvapH �m for ketones are greater thanthe values of DvapH �m for the corresponding ethers that con-tain the same number of carbons [28]. Thus, one expects acoulombic repulsion between these carbons and also thatthe hydroxybutyrate part of (±)-ethyl 2-hydroxy-4-phen-ylbutyrate will be less destabilized than the correspondinga-ketobutyrate part of ethyl 2-oxo-4-phenylbutyrate.Accordingly, the value of the equilibrium constant for reac-tion (2) is expected to be increased by this effect. Veryroughly paralleling reaction (2) is the following reaction

ðC6H5Þ2CHOHðsÞ þ C6H5COCOC6H5ðsÞ ¼ðC6H5Þ2COðsÞ þ C6H5COCHOHC6H5ðsÞ: ð19Þ

In the absence of DfH �m data for these species as gases orany other sets of so related ketones, alcohols, or keto-alco-hols, we have relied for our calculations on data for the so-lid phase. Use of Pedley’s [29] tabulated values of standardmolar enthalpies of formation DfH �m for the substances inreaction (19) gives DrH �m ¼ �25 kJ �mol�1 for this reac-tion. Assuming that DrS

�m ¼ 0, one obtains an equilibrium

constant K � 2 Æ 104 for this reaction. Clearly several majorassumptions were made in this calculation, and this valueis, perhaps, too large. Nevertheless, the direction of reac-tion is clearly indicated and it is consistent with the resultobtained in this study.

We now consider the experimental results for reaction(3). The hydroxyl carbon in (±)-ethyl 4-chloro-3-hydroxy-butyrate is expected to be less positively charged than thesame carbon in ethyl 4-chloroacetoacetate. Thus, the cou-lombic repulsion between this carbon and the terminalchlorine-bearing carbon in (±)-ethyl 4-chloro-3-hydroxy-butyrate will be less than in ethyl 4-chloroacetoacetate.This would tend to increase the value of the equilibriumconstant for reaction (3). Going beyond this qualitativestatement requires thermochemical data for (±)-ethyl 4-chloro-3-hydroxybutyrate and for ethyl 4-chloroacetoace-tate. Since this data is not available, the following reaction,analogous to reaction (3), is considered

ClCH2CHOðgÞ þ CH3CH2OHðgÞ ¼ClCH2CH2OHðgÞ þ CH3CHOðgÞ: ð20Þ

From the literature, we have a computationally derivedvalue of Df H �m for chloroacetaldehyde(g) [30], an experi-mentally derived (combustion calorimetry) value of DfH �mfor 2-chloroethanol(g), [31], and values of Df H �m for bothethanol(l) and acetaldehyde(l) [29]. Ignoring an adjustmentfor the standard molar enthalpies of vaporization of chlo-roacetaldehyde(g), we calculate DrH �m � �22 kJ �mol�1 for

Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670 669

reaction (20). If we also ignore the entropic contributionand all chemical effects from the carbonyl and alcoholgroups, we obtain an equilibrium constant K � 7 Æ 103 forreaction (20). Considering the several approximationsmade in this calculation, this value of K for an analogousreaction can be considered to be in accord with the exper-imental equilibrium constant K = (1.43 ± 0.13) Æ 103 ob-tained herein for reaction (3).

We now consider the results for the 3- and 4-hydroxypi-peridines. The subtraction of reaction (4) from reaction (5)gives

1-benzyl-4-hydroxypiperidineðsolnÞþ1-benzyl-3-piperidoneðsolnÞ ¼ð�Þ-1-benzyl-3-hydroxypiperidineðsolnÞþ

1-benzyl-4-piperidoneðsolnÞ: ð21Þ

The equilibrium constant for the above reaction is

K ¼ fcfð�Þ-1-benzyl-3-hydroxypiperidineðsolnÞg�cf1-benzyl-4-piperidoneðsolnÞg=½fcð1-benzyl-3-piperidoneðsolnÞÞg�fcð1-benzyl-4-hydroxypiperidineðsolnÞÞg�: ð22Þ

Based on the equilibrium constants of reactions (4) and (5),the equilibrium constant for reaction (21) is equal to1.015 ± 0.092 and DrG

�m ¼ �ð0:04� 0:23Þ kJ �mol�1.

The standard molar enthalpies of formation of a varietyof substituted piperidines have recently been reportedby Ribeiro da Silva and Cabral [32–34]. These authorsreport [32] Df H �m¼�ð289:2�1:7Þ kJ �mol�1 and Df H �m¼�ð298:3�1:7Þ kJ �mol�1, respectively, for 1-methyl-3-hydroxypiperidine(l) and 1-methyl-4-hydroxypiperidine(l).The corresponding values for the gas phase species areDf H �m¼�ð225:5�2:0Þ kJ �mol�1 and DfH �m¼�ð226:8�1:8ÞkJ �mol�1. We are uncertain whether the pure liquid or puregas phase species should be compared with the solutionphase results in the current study, or how to directlyemploy these literature results with the values of the equi-librium constants obtained herein. Nevertheless, we pro-ceed by assuming that the equilibrium constants forreactions such as reaction (21) are essentially independentof the choice of alkyl substitution (cf. the results obtained

TABLE 4Calculated (6-31G*//6-31G*) thermally corrected standard molar enthalpies H �m(G�m ¼ H �m � TS�m) of the compounds involved in reaction (19) at the temperat

Compound H�m=ðkJ �mol�1Þ3-Hydroxypiperidineb �852908.14-Hydroxypiperidinec �852902.03-Piperidinoned �849931.84-Piperidinoned �849939.1

a The values of H �m are relative to the totally ionized atomic species correctedthe gas phase.

b The most stable conformer is N-equatorial, OH-axial.c The most stable conformer is N-equatorial, OH-equatorial.d The most stable conformer is N-equatorial.

by Tewari et al. [21]), and we therefore consider the analo-gous reaction

4-hydroxypiperidineþ 3-piperidone ¼3-hydroxypiperidineþ 4-piperidone: ð23Þ

Also of direct interest is the question of whether or not theisomerization reactions

3-hydroxypiperidine ¼ 4-hydroxypiperidine; ð24Þ3-piperidone ¼ 4-piperidone; ð25Þ

individually have negligible standard molar Gibbs free en-ergy changes, i.e., that 3- and 4-substitution of piperidineby hydroxyl and keto have essentially identical thermo-chemical effects. To attempt to answer this question, quan-tum chemical calculations were performed for each of thefour molecules in reactions (24) and (25). These ab initio

(Hartree–Fock) calculations of geometry optimized struc-tures and associated total energies, thermal corrections,and total absolute entropy were performed with the SPAR-TAN suite of computer programs [35] with the 6-31G*

basis set. The results of these calculations are summarizedin table 4.

Using these computational results, the substance 3-hydroxypiperidine is found to have a calculated standardmolar enthalpy of formation Df H �m (from total energy +thermal correction) that is 6.1 kJ Æ mol�1 more negative than4-hydroxypiperidine and a standard molar Gibbs freeenergy of formation DfG

�m that is 4.9 kJ Æ mol�1 more nega-

tive. These results are consistent with the hydrogen bondsuggested spectroscopically for the 3-isomer [36] and theresultant stabilization and the seeming absence of such sta-bilization for the 4-isomer [37]. However, it is not consistentwith the calorimetrically determined standard molar enthal-pies of formation [32]. Conversely, the standard molarenthalpy of formation and the standard molar Gibbs freeenergy of formation of 4-piperidinone is calculated to be7.3 kJ Æ mol�1 more stable than the 3-isomer. This is compat-ible with the earlier enunciated destabilization [27] of a-ami-noketones, acyclic counterparts of 3-piperidinone that sharetheir N–C–C(O)-framework. However, no such destabiliza-tion of b-aminoketones such as 4-piperidinone has beenindicated from calorimetric measurements [33]. Both the

, standard molar entropies S�m, and standard molar Gibbs free energies G�mure T = 298.15 K and the pressure p = 0.1 MPaa

S�m=ðJ �K�1 �mol�1Þ G�m=ðkJ �mol�1Þ322.9 �853004.4327.1 �852999.5324.7 �850028.6324.5 �850035.9

for thermal energies and unscaled zero-point energies. All substances are in

670 Y.B. Tewari et al. / J. Chem. Thermodynamics 40 (2008) 661–670

quantum chemical calculations and the results of combus-tion calorimetry suggest that DrG

�m for reaction (21) will be

��10 kJ Æ mol�1 as opposed to the experimental resultDrG

�m ¼ �ð0:04� 0:23Þ kJ �mol�1. At the present time, we

are unable to explain this possible discrepancy between theexperimental results reported herein and the computationaland analogous literature results for this reaction. One possi-ble explanation could lie with the solvent effects which wereignored in this discussion.

Acknowledgements

The authors thank Dr. Kejun Cheng, National Instituteof Drug Abuse, National Institutes of Health, for perform-ing the polarimetric measurements.

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JCT 07-309