Ibuprofen as a Probe of Lipid Metabolism
The hypothesis that deracemization of racemic ibuprofen (IBU) occurs via the enolization of a Candida rugosa lipase (CRL)-ibuprofen ester (Scheme 1.6.2) was disproven by reacting racemic IBU under the conditions that were used to deracemize ibuprofen methyl ester (IME), namely pH 7.6. At pH 7.6, IME through IBU butyl esters were not formed in-situ and racemic IBU was not deracemized by reacting the corresponding primary alcohol with racemic IBU in the presence of CRL. At pH 7.6, hexyl through dodecyl IBU esters were formed in-situ by reacting the corresponding primary alcohol with racemic IBU in the presence of CRL. The longer chain alcohols reacted faster following the trend: hexanol, octanol, decanol, and dodecanol with values 9.0 x 10-4, 2.3 x 10-3, 3.9 x 10-3, and 6.4 x 10-3 hr-1, respectively. Initially it was believed that the formation of ester corresponded with an increase of (R)-IBU but after isolating (R)-IBU and IBU dodecyl ester, in a corresponding 48 and 45 % respective yield, it was determined that the monitoring of the reactions by chiral HPLC was affected by the difficulties in recovering the ester. The esters formed are therefore probably only of (S)-IBU. Molecular docking studies indicated that IBU through IBU propyl esters enter the hydrophobic pocket of CRL, isobutyl group first. However, longer chain IBU esters bind with the primary alkyl group in the hydrophobic pocket first. In addition, lower concentrations of the substrates, not using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, using the CRL supernatant versus the whole commercial enzyme, and reacting at pH 6.0 versus 7.6 all led to faster reactions of racemic IBU and dodecanol. For the reaction of racemic IBU and n-butanol catalyzed by CRL, formation of ester was favored at pH 6.0 and hydrolysis of the same ester was favored at pH 7.6. The hydrolysis of larger chain IBU esters was much slower. The observed slower hydrolysis is due to the hydrophobic anchoring of the primary alkyl group of long chain IBU esters in the hydrophobic pocket of CRL.
Rosales, Jose Antonio, "Ibuprofen as a Probe of Lipid Metabolism" (2019). ETD Collection for University of Texas, El Paso. AAI22585316.