BIOTECHNOLOGY LETTERS Volume 17 No.5 (May 1995) pp.525-530 Received as revised 5th April

INFLUENCE OF CHIRAL CARVONES ON SELECTIVITY

OF PURE LIPASE-B FROM Candida antarctica.

Miguel ARROYO and Jos~ Vicente SINISTERRA"

Department of Organic & Pharmaceutical Chemistry. Faculty of Pharmacy. Universidad Complutense. 28040 Madrid. SPAIN.

S-mmary. In the esterification reaction in non-aqueous media lipase-B from Candida antarctica is stereoselective towards the R-isomer of ketoprofen in an achiral solvent such as isobutyl methyl ketone and in S(+) carvone. On the contrary, S(+) ketroprofen is esterified quicker in R(-) carvone. In addition, the esterification yield changes depending on the stereochemistry of the carvone used as solvent. The formation of disteromeric complexes (chiral solvent + chiral substrates) may explain this finding.

INTRODUCTION

Lipases are one of the most adventageous enzymes employed in

organic synthesis because no cofactor regeneration is required. In

addition, lipases are highly stereospecific biocatalysts suitable

for preparative resolution, e.g. for racemic 2-substituted

propionic acids by esterification reaction in non-aqueous media

(Kirchner et al., 1985; B6dnar et al., 1990; Mustranta, 1992;

Arroyo and Sinisterra, 1994). So far, it is not possible to predict

the effect of the solvent in the catalytic process (Faber et al.,

1993). In fact, different and sometimes opposite correlations have

been found between enzyme activity and/or stereoselectivity and the

physicochemical properties of the solvent (Parida and Dordick,

1993; Kitaguchi et al., 1989). Recently, some authors suggested

that solvent can specifically interact with the enzyme (Secundo et

al., 1992) and/or with the substrate (Ottolina et al., 1994),

giving some diastereomeric complexes whose properties depend not

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only on the physicochemical characteristics of the solvent but also

on their geometry. This last work was carried out using R(-) and

S(+) carvone as chiral solvents, some racemic esters or alcohols as

substrates and several hydrolases as biocatalysts (adsorbed or in

the native form). As a consequence, many variables were introduced

and clear conclusions were not achieved.

In order to analyze the simplest model of the interaction

between chiral substrates and solvents, the esterification of pure

R(-) or S(+) 2-(3-benzoyl-phenyl) propionic acid (ketoprofen, !)

with l-propanol in R(-) and S(+) carvone has been carrried out. So

far, the same reaction was performed in an achiral ketone :

isobutyl methyl ketone (IBMK). Pure lipase-B from Candida

antarctica was used as biocatalyst to avoid the problems of the

presence of lipase A which has different enzymatic specificity

(Heldt-Hansen et al., 1985).

O H3C H O H3C H

,Y ~ "COOCH2CH2CH3 i . . . . l )' './'4

R(- ) - 1 ] Upa=, B from C.antarafca, R( - ) -2_ .- .OH ]

" organic solvent ,

O H CH 3 O H CH 3

s(+) -1 S(*) -2

MATERIALS AND METHODS

Materials. Pure lipase B from Candida antarctica (SP525) was kindly supplied by Novo Nordisk Bioindustrial S.A. (Madrid, Spain). R(-) and S(+) ketoprofen were gifts from Laboratorios Menarini S.A. (Badalona, Spa~n). R(-) and S(+) carvone were suplied by Fluka (Buchs, Switzerland) and isobutyl methyl ketone by Merck (Germany).

General Procedure for Esterification. The reaction mixture was composed of organic solvent (5 ml), ketoprofen (66 mM) and l-propanol (66 mM). The reaction was started by adding the enzyme podwer (7 mg of SP525 per ml of solvent) to the solution. The reactions were performed at 24°C by stirring (500 rpm) in 25 ml flasks. Per~odically, 100 HI of the solution were withdrawn from the reaction and added to 1.4 ml of the same solvent to analyze the ester conversion by capillary gas chromatography (CC).

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Determination of the degree of conversion. GC was performed in a Shimadzu GC-14A gas chromatograph equipped with a FID detector, a split inyector (1:2) and a SPBTM-I sulfur column 15 m. x 0.32 mm. (Supelco Inc. Bellafonte, P.A. USA). Inyector temperature was 300°C and the detector temperature was 350°C; oven temperature was maintained at 165"C. Carrier gas was nitrogen (carrier flow: 30 ml/min. An external standard method was employed to quantify the remaining acid and the formed ester.

RESULTS & DISCUSSION

The esterification of R(-) and S(+) ketoprofen was

carried out in isobutyl methyl ketone employed as the achiral

solvent (Figure 1).

Ester yield (%) 8 0 - -

6 0 - -

40 -J

2 0 ~

ISOEUTYL METHYL KETONE

7

• / & i

J • R(-) ketoprofen

" [ a S(÷) ketoprofen ! /

/

I , -J

f / I

• _.__=

f

0 100 200 300 400 500 Time (hours}

Figure i. Esterification of R(-) (|)and S(+) ketoprofen (A) with n-propanol in isobutyl methyl ketone (IHMK). Conditions: 66 mM acid and alcohol in 5 ml of IBMK, 24°C of temperature, concentration of enzyme: 7 mg SP525/ml, no water added to the solution, stirring speed: 300 rpm.

We can observe that R(-)-! is esterified at a higher reaction

rate than S(+)-! acid. The same results has been described with the

same lipase immobilized on a polymeric support (Arroyo and

sinisterra, 1994). The same process was carried out using R(-) or

S(÷) carvone as chirai solvent. The results are shown in Figure 2.

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Ester yield (%) 30 - -

2 o -

< l

i 0 " " i i

0 50 I00

S4÷) CARV~NE / . . . . ~

/

ll(.) ilei©profen $(~, ) klilop rlifen I

I I I ~50 200 250

Time ~ours)

Ester yield (%) 25 - -

J ] R(-) CARVONE

20- - / 15-- / 0!

/

0 I I I I I 0 SO 100 I~ 200 250

r l n e (hou~)

a b

Figure 2. Esterification of S(+) (m) and R(-) ketoprofen (i) with n-propanol ~n S(+J carvone (Fig 2a) or R(-) carvone (Fig 2b). Conditions: 66 mM acid and alcohol in 5 ml of solvent; temperature: 24°C; concentration of enzyme: 7 mg/ml; no addition of water; stirring speed: 300 rpm.

The results of Figure 2 indicate that the chiral solvent plays

an important role in the selectivity of the enzyme. S(+) carvone

reduces the lipase stereoselectivity (Fig.2a) in comparison with

the achiral solvent (Fig.l) whereas R(-) carvone changes its

selectivity in the esterification process.

S(+) carvone : R(-)-! ~ S(+)-!

R(-) carvone : R(-)-! < S(+)-!

Isobutyl methyl ketone : R(-)-! > S(+)-!

Therefore we can conclude that the chiral solvent gives a kind

of diastereomeric solvent-acid complex, where the interaction

forces depend on the geometry of the carvone. A possible

explanation could be that the conformation of each carvone favours

or disfavours the solvation of the acid group with their carbonyl

group which has different steric hindrance in both enantiotopic

faces ( the most stable chair of each solvent has been calculated

by MM2 program ). If we admit that the interaction ketone-acid

takes place through the polar groups (COOH and O=C) in the least

528

steric hindered face,

interactions:

we can postulate these schematic

R(-) ~m,ono . R(-) * ~ , o m f e n m , , ~ o x R(.) c ~ v o , o . S(+) keto~ofen o~,~Wx $(÷) om,or, e . $(÷) o r R(.) *etopmlm ~ml~exes

H=C-'~_.. " .. ~-o H , c ~ ,:..::'~c', ;-o CH3 ~ / s ---"

HaC"'\ H ' " , / ~ ~___~..~0 (H) HIC'~"- ' '~0 / (CH3}I-i N O~H

_3 _4 _s

The interaction between S(+) carvone and R or S acids (5)

would have similar energetic value due to the localization of the

acid molecule near the flat zone of the enone group. Nevertheless

the interaction of a(-) carvone with R(-) acid (!) or S(+) acid (~)

would show different energetic level due to the sterical reasons

(higher in ! than in !) related to the interaction of s-methyl

group of the acid with the ring of the ketone (see scheme).

As a consequence R(-)-! and S(+)-! would be solvated at the

same extend in S(+) carvone and the complex substrate-solvent would

be broken at the same rate. Therefore, the selectivity would be the

same than in the case of the achiral ketone, isooctane or

cyclohexane (Arroyo and Sinisterra, 1994) where this selectivity is

controlled by the B-lipase from C.antarctica : R(-)-! > S(+)-!

(result obtained in Fig 2a). However, in the case R(-) carvone, the

complex with R(-) acid, !, would be more stable than the complex

with the S(+) enantiomer, A- As a consequence, the molecules of

S(+) ketoprofen would be liberated quicker than the R(-) ones. This

hypothesis could explain why S(+)-! is esterified at higher

reaction rate in R(-) carvone than a(-)-! (Figure 2b).

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These results support the hypothesis of Ottolina et al. about

the control of the reaction by the formation of diastereomeric

complexes and explains their results obtained in R or in S carvone.

In order to confirm the hypothesis, the esterification of other

pure and racemic 2-arylpropionic acids is in progress.

REFERENCES

Arroyo, M. and Sinisterra, J.V. (1994) J. Org.Chem. 59, 4410-7. B6dnar, J.; Gubicza, L. and Szab6, L.P. (1990) J. Mol. Catal. 61, 353-361. Bossetti, A.; Bianchi, D.; Cesti, P. and Golini, P. (1994) Biocatalysis 9, 71-77. Faber, K.; Ottolina, G. and Riva, S. Biocatalysis 8, 91-132. Heldt-Hansen, H.P.; Ishii, M.; Patkar, S.A.; Hansen, T.T. and Eigtved, P. (1989) Biocatalysis in Agricultural Biotechnology, ACS Symp. Ser. 389 (Whitaker, J.R. and Sonnet, P.E. eds.) 157-172. Kirchner, G.; Scollar, M.P. and Klibanov, A.M. (1985) J. Am. Chem. Soc. iii, 3094-5. Mustranta, A. (1992) Appl. Microbiol. Biotechnol. 38, 61-66. Ottolina, G.; Bovara, R.; Riva, S. and Carrea, G. (1994) Biotechnol. Lett. 16, 923-8. Parida, S. and Dordick, J.S. (1993) J. Org. Chem. 58, 3238-3244. Secundo, F.; Riva, S. and Carrea, G. (1992) Tetrahedron Assym. 3, 267-280.

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