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lipase-catalyzed enantioselective esterification of ibuprofen in organic solvents under controlled water activity author links open overlay panelamélieducretmichaeltranirobertlortie

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It has been well established that enantiomers of non-steroidal anti-inflammatory drugs of the aryl propionic class exhibit diverse pharmacological and toxicological properties. The therapeutic action of racemic mixtures of these drugs is mainly due to the (S)-isomers.



The S-enantiomer of flurbiprofen (2-fluoro-a-methyl-[1,1'-biphenyl]-4-acetic acid) possesses most of its anti-inflammatory action, but the presence of the R-enantiomer is reported to greatly enhance its gastrointestinal toxicit. A later study reported that R-flurbiprofen was less potent as an analgesic than S-flurbiprofen, but it caused little toxicity in comparison to its antipode. This makes the resolution of the enantiomers of this drug particularly desirable. The lipase-catalyzed enantioselective esterification of racemic flurbiprofen has been reported .


Reaction conditions. The reaction mixture consisted of 300 mg enzyme, 500 mg of each of the appropriate salt hydrates, and 10 ml of a stock solution consisting of 0.6 mM racemic flurbiprofen and 50 mM caffeine dissolved in the solvent under study. The reaction mixture was allowed to equilibrate at the desired water activity by shaking in a constant temperature shaker bath (Blue M Magniwhirl) at 30°C for 24 hours. The mild temperature and solvent medium conditions were unlikely to compromise the stability of the enzymes over the duration of the reaction.

Analytical methodology. The LC system consisted of an Altex Model 110A pump, a Spectroflow 757 UV detector, a Dionex 4270 integrator and a Rheodyne 7125 injector. Enantiospecific HPLC analysis of flurbiprofen enantiomers in the samples was accomplished using a chiral a1-acid glycoprotein column by modifying a literature meth. The mobile phase consisted of 4% 2-propanol and 4 mM N,N-dimethyloctylamine in 20 mM potassium phosphate buffer adjusted to pH 6.5. The flow rate was 1.1 ml/min and the wavelength of detection was 234 nm. Samples were prepared by evaporation of solvent under vacuum and reconstitution of residue with 100 mL of mobile phase..

Reaction parameters. The initial rate is the absolute value of the slope of the initial linear portion of the hyperbolic curve obtained by plotting substrate concentration as a function of time, and is reported in units of nmol/(g of enzyme.hr). The initial rate of the faster reacting enantiomer is presented in the Results and Discussion section. The enantiomeric excess of the substrate, ees, at a given time was calculated using:

[B] - [A]

ees =

[A] + [B]

where [A] and [B] are the molar concentrations of the faster and slower reacting enantiomer respectively. The extent of conversion at any given time, c, was calculated using:

[A] - [B]

c = 1-

[A]o + [B]o

where the subscript o denotes initial concentrations.

Results and Discussion

Solvent dependence. It has been established that increasing hydrophobicity exponentially increases the rate of lipase-catalyzed reactions . The effectiveness of enzyme biocatalysis in organic solvents improves, as a rule, as the log P of the solvent increases, but only up to a limit. One of the goals of this study was to find the limiting solvent hydrophobicity above which the initial rate for the lipase-catalyzed esterification of flurbiprofen with n-butanol cannot be further enhanced. Preliminary studies showed that the reaction proceeded slowly in toluene (no measurable substrate loss in 96 hours) and faster in isooctane than in n-heptane with Candida rugosa lipase. If the higher reaction rate is due to the increased hydrophobicity of the solvent, then it should increase further with n-nonane.

Before speculating on what distinguishes isooctane from the other two solvents, it is worth noting if this phenomenon is also observed with the other lipases used in this study. With the other two enzymes, no significant differences in reaction rates were observed in the three solvents, as shown in  for Mucor javanicus lipase and in  for porcine pancreatic lipase..

Enantioselectivity. The solvent dependence of the enantioselectivity of the enzymes parallels the manner in which their catalytic efficiency depends on solvent hydrophobicity. For Candida rugosa lipase, the reaction with the S-enantiomer as substrate proceeds much faster than that of the R-enantiomer for all three solvents, but enantioselectivity is clearly greatest in isooctane .

The results seem to indicate that a limiting hydrophobicity of the solvent medium exists beyond which increasing hydrophobicity will result in neither improved reaction rate nor improved enantioselectivity. Using solvents more hydrophobic than the ones used in this study might pose practical problems since they tend to be quite viscous.







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