ANALYTICAL
BIOCHEMISTRY
207,236-240
(1992)
Resolution of Dihydroxyeicosanoates Dihydroxyeicosatrienoates by Chiral Phase Chromatography
and of
Jorge H. Capdevila,**T*’ Shouzou Wei,* Anil Kumar,* Jun Kobayashi,* James R. Snapper,* Darryl C. Zelclin,* Rama K. Bhatt,$ and John R. Falck$ Departments of *Medicine and TBiochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232; and $Department
of Molecular
Received
24, 1992
June
Genetics,
University
of Texas Southwestern
A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-uic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vie-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality. @ 1992 Academic Press, Inc.
The cytochrome P450 epoxygenase catalyzes the enantioselective NADPH-dependent oxidation of arachidonic acid to four regioisomeric ci.s-epoxyeicosatrienoic acids (EETs)’ (1). The EETs are enzymatically hydrated by cytosolic epoxide hydrolase to the corresponding Gc-dihydroxyeicosatrienoic acids (DHETs) (1,2). The DHETs have been linked to hormone-induced changes in rabbit renal hydrosmotic function (3) as well as inhibition of water flow and adenylate cyclase in the toad bladder (4). Importantly, Catella et uZ. ob-
r To whom correspondence should be addressed at Medical Center North S-3223, Vanderbilt University, Nashville, TN 37232. * Abbreviations used EET, ck-epoxyeicosatrienoic acid, EET-Me, EET methyl ester; EET-PFB, EET pentafluorobenzyl ester; DHET, ukdihydroxyeicosatrienoic acid, DHET-Me, DHET methyl ester; DHET-PFB, DHET pentafluorobenzyl ester; Ha-DHET, uklihydroxyeicosanoic acid Ha-DHET-PFB, He-DHET pentafluorobenzyl ester; TMS, trimethylsilyh GLC, gas-liquid chromatography; MS, mass spectrometry; pC1, positive ion chemical ionization; NICI, negative ion chemical ionization. 236
Medical
Center at Dallas, Dallas,
Texas 75235
served significant increases in the urinary excretion of DHETs during pregnancy-induced hypertension (5). While the absolute configurations of endogenous DHETs are unknown, recent studies in these laboratories have demonstrate& (a) enantioselective epoxidation of endogenous arachidonic acid pools in rat liver and kidney and in human kidney cortex (1) and, (b) enantioselective EET hydration by rabbit liver and lung cytosolic epoxide hydrolases (6). To facilitate the structural characterization of DHETs and the evaluation of their functional relevance and mechanism of formation, we report here an efficient chromatographic procedure for the direct stereochemical analysis of DHETs-Me and He-DHETs-PFB. In addition, these procedures provide convenient access to preparative quantities of all eight DHET and all eight HG-DHET antipodes. MATERIALS
AND
METHODS
The optical antipodes of 5,6-, 8,9-, 11,12-, and 14,15&-EETs and of 5,6- and 14,15-DHETs were prepared by total asymmetric synthesis according to published procedures (7-11). Racemic standards of all four regioisomeric EETs were prepared from [l-14C]arachidonic acid (0.01-2 mCi/mmol) as described (12). Methyl esters were prepared by reaction with an ethereal solution of CHzNz. Pentafluorobenzyl esters were prepared using pentafluorobenzyl bromide in N,N-diisopropylethylamine exactly as in (13). PFB and Me esters were purified by reversed-phase HPLC as in (13). Silylations were done utilizing bis(trimethylsily1) trifluoroacetamide in pyridine (14). Hydrogenations were performed using PtOz as catalyst (14). Chemical hydration of racemic or chiral EETs to DHETs was conducted as follows: samples of l-14C-labeled (lo-52 mCi/mmol) or unlabeled EETs (
BIOCHEMISTRY
207,236-240
(1992)
Resolution of Dihydroxyeicosanoates Dihydroxyeicosatrienoates by Chiral Phase Chromatography
and of
Jorge H. Capdevila,**T*’ Shouzou Wei,* Anil Kumar,* Jun Kobayashi,* James R. Snapper,* Darryl C. Zelclin,* Rama K. Bhatt,$ and John R. Falck$ Departments of *Medicine and TBiochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232; and $Department
of Molecular
Received
24, 1992
June
Genetics,
University
of Texas Southwestern
A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-uic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vie-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality. @ 1992 Academic Press, Inc.
The cytochrome P450 epoxygenase catalyzes the enantioselective NADPH-dependent oxidation of arachidonic acid to four regioisomeric ci.s-epoxyeicosatrienoic acids (EETs)’ (1). The EETs are enzymatically hydrated by cytosolic epoxide hydrolase to the corresponding Gc-dihydroxyeicosatrienoic acids (DHETs) (1,2). The DHETs have been linked to hormone-induced changes in rabbit renal hydrosmotic function (3) as well as inhibition of water flow and adenylate cyclase in the toad bladder (4). Importantly, Catella et uZ. ob-
r To whom correspondence should be addressed at Medical Center North S-3223, Vanderbilt University, Nashville, TN 37232. * Abbreviations used EET, ck-epoxyeicosatrienoic acid, EET-Me, EET methyl ester; EET-PFB, EET pentafluorobenzyl ester; DHET, ukdihydroxyeicosatrienoic acid, DHET-Me, DHET methyl ester; DHET-PFB, DHET pentafluorobenzyl ester; Ha-DHET, uklihydroxyeicosanoic acid Ha-DHET-PFB, He-DHET pentafluorobenzyl ester; TMS, trimethylsilyh GLC, gas-liquid chromatography; MS, mass spectrometry; pC1, positive ion chemical ionization; NICI, negative ion chemical ionization. 236
Medical
Center at Dallas, Dallas,
Texas 75235
served significant increases in the urinary excretion of DHETs during pregnancy-induced hypertension (5). While the absolute configurations of endogenous DHETs are unknown, recent studies in these laboratories have demonstrate& (a) enantioselective epoxidation of endogenous arachidonic acid pools in rat liver and kidney and in human kidney cortex (1) and, (b) enantioselective EET hydration by rabbit liver and lung cytosolic epoxide hydrolases (6). To facilitate the structural characterization of DHETs and the evaluation of their functional relevance and mechanism of formation, we report here an efficient chromatographic procedure for the direct stereochemical analysis of DHETs-Me and He-DHETs-PFB. In addition, these procedures provide convenient access to preparative quantities of all eight DHET and all eight HG-DHET antipodes. MATERIALS
AND
METHODS
The optical antipodes of 5,6-, 8,9-, 11,12-, and 14,15&-EETs and of 5,6- and 14,15-DHETs were prepared by total asymmetric synthesis according to published procedures (7-11). Racemic standards of all four regioisomeric EETs were prepared from [l-14C]arachidonic acid (0.01-2 mCi/mmol) as described (12). Methyl esters were prepared by reaction with an ethereal solution of CHzNz. Pentafluorobenzyl esters were prepared using pentafluorobenzyl bromide in N,N-diisopropylethylamine exactly as in (13). PFB and Me esters were purified by reversed-phase HPLC as in (13). Silylations were done utilizing bis(trimethylsily1) trifluoroacetamide in pyridine (14). Hydrogenations were performed using PtOz as catalyst (14). Chemical hydration of racemic or chiral EETs to DHETs was conducted as follows: samples of l-14C-labeled (lo-52 mCi/mmol) or unlabeled EETs (
