Chem. Res. Toxicol. 1989,2, 316-324
316
Studies of Intramolecular Rearrangements of Acyl-Linked Glucuronides Using Salicylic Acid, Flufenamic Acid, and (S)and ( R )-Benoxaprofen and Confirmation of Isomerization in Acyl-Linked A9-l 1-Carboxytetrahydrocannabinol Glucuronide Gretchen Bradow, Lou-sing Kan, and Catherine Fenselau*?t The Johns Hopkins University, Baltimore, Maryland 21205 Received October 31, 1988
NMR and HPLC have been used to investigate the rearrangements of l-0-acylglucuronides in vitro and the occurrence of rearranged isomers in urine. Glucuronides of flufenamic acid, ( S ) -and (R)-benoxaprofen, salicylic acid, and A9-11-carboxytetrahydrocannabinolwere synthesized, by use of immobilized enzymes, or purified from urine. Ester-linked isomers of these gluruconides were characterized, and isomers derived from flufenamic acid, (S)-benoxaprofen, and salicylic acid were purified for further study by NMR and HPLC. The positions of the new ester linkages could be identified by two-dimensional NMR. Shifts not only in the resonance of the proton adjacent to the esterfied hydroxyl group but also in the resonance of the anomeric proton on carbon 1of the glucuronic acid moiety could be correlated with the position of each isomeric ester bond. HPLC elution times also correlated with ester position in this small set of samples. The sequences of isomer formation were studied in situ by NMR and also a t p H 8 by HPLC. These studies indicate that, for the three cases examined, the C-2 ester is formed first, followed by formation of C-3 and C-4 esters. The purified isomeric esters were found not to re-form the high-energy l-0-acyl bond. All other rearrangement steps are reversible. In contrast to other glycosides and glycerol esters, no evidence could be found for rearrangements beyond nearest-neighbor hydroxyl groups in glucuronic acid. The sequence of formation and reversibility is consistent with an ortho ester intermediate, as has been proposed for rearrangements of other glycosides. Half-lives for the glucuronides studied fall in the middle of the range summarized by Rachmel, Hazelton, Yergey, and Liberato [ (1985) Drug Metab. Dispos. 13, 705-7101. The diastereomeric glucuronides of ( S ) -and (R)-benoxaprofen were found to undergo hydrolysis and rearrangement a t different rates, although the C-2 ester was the most stable in both diastereomeric series. The C-3 isomer of flufenamic acid was found to be the most stable. A single rearranged isomer of the l-0-acylglucuronide of salicylic acid was observed, which had relatively high stability and a different mechanism of formation. HPLC, TLC color tests, and also 'H NMR were applied to urine samples expected to contain l-0-acylglucuronides and their isomers formed from flufenamic acid, benoxaprofen, salicylic acid, and A 9 - l l carboxytetrahydrocannabinol, in order to evaluate both the suitability of the techniques and also the occurrence or not of rearranged isomers in urines from rabbits, rhesus monkeys, and humans.
Introduction A wide range of xenobiotic and endogenous substances can form glucuronides conjugated through a carboxylic acid functional group ( I ) . These acyl-linked glucuronides are chemically more reactive than other O-linked glucuronides ( 2 ) and have been shown to be more susceptible to hydrolysis ( l ) ,to transesterification by methanol ( 3 ) ,ammonia ( 4 ) , and ethanethiol (5), and to reaction with chemical nucleophiles such as NBP' (6). They have been observed to react with nucleophilic groups on biopolymers such as albumin (7-12). Reactive glucuronides are also reported ( 2 ) to undergo intramolecular rearrangement, in which the acyl-containing aglycon migrates from the glycosidic linkage at carbon 1to new ester linkages with hydroxyl groups on carbons 2 , 3 , and 4 of the glucuronic acid moiety. 'Present address: Chemistry and Biochemistry Department, University of Maryland Baltimore County, 5401 Wilkens Ave., Baltimore, MD 21228.
These ester-linked isomers have been found in human and animal urine ( 2 ) ,bile (13-15), and blood (7,16-18). Increased isomer levels have been reported to be associated with hepatobiliary and renal dysfunction (19, 20). Although the occurrence of suites of isomers resistant to 0-glucuronidase was originally recognized as an analytical complication, a growing number of studies indicate that glucuronide rearrangement influences both the elimination and the toxicity of drugs (2). The resistance of the isomers to 0-glucuronidase alters enterohepatic recirculation and excretion (14,18,21-23). It has been reported in two cases ( 4 9 )that glucuronide ester isomers form covalent bonds with serum albumin, in addition to acylation of albumin by the l-O-acylglucuronide itself. Most of the published studies of isomers and of the rearrangement process contain some structural characterization, including treatment with @-glucuronidase,reaction with specific spray reagents, and separation by Abbreviations: NBP, 4-(p-nitrobenzyl)pyridine; 2D COSY,two-dimensional homonuclear chemical shift correlation NMR spectroscopy.
0 1989 American Chemical Society
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 317
Acyl-Linked Glucuronide Rearrangement ,COOH
I
Flufenamic acid Benoxaprofen
ooH COOH
Salicylic acid
AH OH
A -1 l-carboxytetrahydrocannabinol
Figure 1. Structures of the compounds used in this study. The asterisk indicates an asymmetric center.
HPLC. I t is easy to distinguish the isomers with ester bonds from the glucuronide conjugated through a glycosidic bond. However, it has proven more difficult to distinguish one ester isomer from another. Mass spectrometry is potentially attractive for this purpose, with its excellent sensitivity and chromatographic compatibility; however, reports to date, using the electron impact, fast atom bombardment, and thermospray techniques, indicate that only the glycoside can be distinguished (15, 24-27). Extensive use of NMR to address similar structural questions in other areas of carbohydrate chemistry (28,29) led us to apply COSY techniques for proton analysis to identify rigorously the ester isomers and to follow their formation in situ. Our objectives included the use of the isomers identified by NMR to calibrate more sensitive HPLC assays and to certify a set of reactions with color reagents, and the evaluation of this ensemble of techniques for analysis of urine samples. Extensive studies were made of the sequence of formation of the various regioisomers, toward the objective that isomer structures might be assigned by their order of appearance, without the need to purify large amounts of material for two-dimensional NMR. We have studied the rearrangement of flufenamic acid glucuronide, chosen because its alkylating reactivity has been well characterized (6, 30)) the diastereomeric glucuronides of (S)- and (R)-benoxaprofen, and the acyllinked glucuronide of bifunctional salicylic acid. Urine was examined from a rabbit dosed with flufenamic acid, from rhesus monkeys receiving racemic benoxaprofen, from a volunteer who had taken aspirin, and from a participant in a supervised study of marijuana smoking (Figure 1).
Materials and Methods Materials. Flufenamic acid, salicylic acid, and deuterated solvents for NMR were obtained from Sigma Chemical Co. (St. Louis,MO). HPLC-grade acetonitrile was obtained from Burdick and Jackson (Muskegon, MI). Glass-distilled filtered water was used to prepare all solutions. Racemic benoxaprofen was provided by Wyeth Pharmaceutical Co. (Philadelphia,PA), and the resolved enantiomers were provided by Eli Lilly and Co. (Indianapolis, was obtained from the IN). AB-ll-Carboxytetrahydrocannabinol National Institute on Drug Abuse. All other chemicals were reagent grade. Enzymatic Synthesis of 1-0-Acylglucuronides. Small quantities of 1-0-acylglucuronideswere synthesized from salicylic acid, (S)-(+)-benoxaprofen, (R)-(-)-benoxaprofen, and A g - l l carboxytetrahydrocannabinolby a method that uses immobilized enzymes and has been previously described (31, 32). The benoxaprofen and salicylic acid incubations contained 15 mL of beads carrying i m m o b i l i i rabbit liver microsomal protein, 2 mM benoxaprofen or 4.3 mM salicylic acid, and 6.0 mM UDPGA in 10 mL of 0.1 M KH2P04,pH 6.0. They were incubated overnight at room temperature. The Ag-11-carboxytetrahydrocannabinol
incubation contained 28 mL of packed beads, 0.7 mM aglycon dissolved in 10 pL of methanol, 300 mg of human serum albumin (33),and 7.3 mM UDPGA in 40 mL of 0.01 M KHzP04,pH 6.4. The conjugates were purified on C-18 SepPak cartridges (Waters, Milford, MA) (34) and used as standards for TLC and HPLC. Flufenamic acid 1-0-acylglucuronidewas synthesized in incubations that contained 15 mL of packed beads, 3.6 mM flufenamic acid, and 5.7 mM UDPGA in 10 mL of 0.1 M KHzP04, pH 6.0. After 16 h at room temperature, the mixture was filtered through a coarse fritted funnel. The pH of the filtrate was adjusted to 2.0 with concentrated HC1, and the solution was maintained at room temperature for 10 min to allow precipitation of unreacted aglycon. The resulting suspension was filtered through a 0.45-pm filter (Millipore,Bedford, MA), and the aqueous filtrate was applied to a prewashed C-18 Sep-Pak cartridge. The cartridge was washed with 3 mL of HzO and 2 mL of diethyl ether; the glucuronide was eluted with 4 mL of acetonitrile. The glucuronide was further purified by semipreparative HPLC. Twenty incubations were processed, and the products were combined to provide a sample suitable for NMR analysis. Isolation of Benoxaprofen 1-0-Acylglucuronides from Monkey Urine. The diastereomeric glucuronides of benoxaprofen were isolated from monkey urine that had been collected from three rhesus monkeys receiving 37.5 mg/ (kpday) racemic benoxaprofen. The urine was collected in a mixture of ice and pH 4 citric acid solution. After each 24-h collection period, the urine was deep-frozen until use. The urine that was extracted had been collected on the sixth, seventh, and eighth days of drug administration. The urine was thawed immediately before extraction, filtered through cheesecloth, and applied to a column of 50 g of Amberlite XAD-2 (Mallinkrodt Co., St. Louis, MO), which had been prewashed with ethyl acetate, acetonitrile, and water. After application of the urine, the column was washed with 250 mL of water and the glucuronide was eluted with 300 mL of acetonitrile. The solvent was evaporated in vacuo, and the glucuronide was further purified by semipreparative HPLC. Isolation of Salicylic Acid 1-0-Acylglucuronide from Human Urine. Urine was collected during hours 4-40 from a healthy human subject who had received 9 g of aspirin over a 36-h period. The urine was stored at 4 "C until extraction. The pH of the urine was below 5 and so was not adjusted during collection. Immediately before extraction, urine was filtered and the pH was adjusted to 2.5 with concentrated HC1. The urine was then divided into 275-mL aliquots and applied to 300-mL capacity Tox-Elut columns of diatomaceous earth (Analytichem, Harbor City, CA). The glucuronide fraction was eluted with two 100-mL portions of ethyl acetate. All ethyl acetate fractions were combined, and the solvent was evaporated in vacuo. The acyl-linked glucuronide was further purified by semipreparative HPLC. Preparation of Rearranged Isomers. The regioisomers of flufenamic acid glucuronide were isolated after their formation in the time-dependent NMR experiment. The isomers of the glucuronides of isomeric benoxaprofen, salicylic acid, and Ag11-carboxytetrahydrocannabinolwere produced by dissolving the 1-0-acylglucuronide in 0.1 M KHzP04,pH 8.0, for 72 h at room temperature. Semipreparative HPLC. The 1-0-acylglucuronidesand their regioisomers were purified on a Whatman Magnum 9-ODSI11 C-18 column, 500 x 10 mm. The flow rate was maintained at 3 mL/min, and UV detection at 254 nm was used. Flufenamic acid glucuronide was purified by using an isocratic system of acetonitrile/l% aqueous acetic acid (55:45 v/v), Flufenamic acid glucuronide isomers were separated by using a gradient of acetonitrile/l% aqueous acetic acid, in which the amount of acetonitrile was increased from 30% to 60% in 15 min. The diastereomeric benoxaprofen glucuronides were isolated by using a gradient of acetonitrile/l% aqueous acetic acid, in which the percentage of acetonitrile was varied from 30% to 60% over 15 min. The diastereomeric glucuronides were separated from each other using a gradient of acetonitrile/0.05 M ammonium acetate, pH 6.0, in which the acetonitrile is taken from 25% to 35% in 20 min. The isomers of the glucuronide of (S)-benoxaprofenwere separated and purified on the Magnum 9 C-18 column in a gradient of acetonirile/0.05 M ammonium acetate, pH 6.0, in which the percentage of acetonitrile was varied from 25% to 35% in 20 min (32).
318 Chem. Res. Toxicol., Vol. 2, No. 5, 1989 Salicylic acid acyl-linked glucuronide was purified in two steps, both using an isocratic system of acetonitrile/0.05 M ammonium acetate, pH 6.0. The first step was performed by using 15% acetonitrile, and the second with 10% acetonitrile. The phenol-like glucuronide was separated from the acyl-linked glucuronide during the first step. The product of the rearrangement of salicylic acid glucuronide was purified by using an isocratic system of acetonitrile/0.05 M ammonium acetate, pH 6.0 (25:75 v/v). Analytical HPLC. Analytical HPLC was performed by using a Brownlee 5-pm/RP-18 column, 250 X 4.6 mm (Brownlee Co., Santa Clara, CA). The flow rate was 1.5 mL/min, and UV detection was at 254 nm. The isomers of the glucuronides of (R)-benoxaprofen and of AB-11-carboxytetrahydrocannabinol were not purified on a large scale but were characterized analytically. A gradient of acetonitrile and 0.05 M KH2P04,pH 4.5, was used, with acetonitrile increasing from 25% to 40% over 12 min, and subsequently to 60% through an additional 5 min. The flow rate was 1.5 mL/min, and UV detection was a t 254 nm. The retention time of the 1-0-acylglucuronide of (R)-benoxaprofenwas 9.0 min. The retention times of its isomers, in order of their formation, were 10.4,7.4, and 9.8 min. The retention time of the 1-0-acylglucuronide of A9-l l carboxytetrahyd"abino1 was 15.0 min, and its isomers eluted a t 14.2, 16.0, and 16.2 min. Kinetic studies of the isomerization of flufenamic acid and (S)-benoxaprofen glucuronides were made by using the same solvent system. The glucuronide of salicylic acid and its isomer were more polar and were analyzed with a gradient of acetonitrile and 0.05 M KHzPOI, pH 3.4, in which acetonitrile was increased from 5% to 15% in 10 min. TLC and Reactions with Color Reagents. Aluminumbacked silica GF-254 plates (EM Science, Cherry Hill, NJ) were developed in a system of benzene/butanol/methanol/water (1:2:1.25:1 v/v). This solvent system brought all the glucuronides studied to about the middle of the plate and did not separate glucuronides from isomers. The plates were visualized by UV light and naphthoresorcinol spray reagent (35),p-anisidine spray reagent (24), or NBP spray reagent (6). Characterization of 1-0-Acylglucuronides and Identification of Rearranged Isomers by NMR. AU NMR spectra were obtained by using a Bruker WM-300 spectrometer, equipped with an ASPECT 3000 data system. A 10-mm proton probe was used, and all spectra were acquired at 25 OC. The internal HOD signal was used as a lock reference and shift assignments were made relative to it. The samples were dried under vacuum in the presence of P206for 24 h, then dissolved in 500 pL of an acetonitrile-d3/Dz0 (3565 v/v) mixture, and analyzed in 5-mm tubes. The isomers of flufenamic acid glucuronide (- 100 pg each) were identified by a series of successive decoupling experiments, in which each proton on the glucuronic acid ring was decoupled separately. The isomers of (S)-benoxaprofen glucuronide and salicylic acid glucuronide (- 500 pg each) were characterized by 2D COSY NMR. These two NMR techniques provide the same type of information. The latter is more automated; however, it is less sensitive. 2D COSY information was obtained by a 90°-tl-450-FID(t2) pulse sequence. The second 45O pulse reduces the peak intensity on the diagonal lime (54). All sets of data consist of 1024 and 512 data points for tz and tl domains, respectively. These give a digital resolution of 4.7 Hz/point with a spectral window of 2400 Hz. A total of 256 t l values were taken during the sampling and then zero-filled to 512. Both time domains were multiplied by a sine bell function during the Fourier transform. A total of 32 scans were recorded for each tl value. Time-Dependent Study of the Rearrangements Monitored in Situ by NMR. A solution of about 2 mg of each 1-0-acylglucuronide in 500 pL of acetonitrile-d3/Dz0 (35:65 v/v) was prepared, and a reference spectrum was obtained. A total of 1 p L of a 0.4% solution of NaOD in D20 was added, giving a final concentration of NaOD of 0.2 mM. Spectra were obtained every 20 min for 12 h, beginning immediately after the addition of base. At each time point, data acquisition required about 3 min. The solvent used in these NMR experiments was chosen to optimize both the solubility and the reactivity of the samples. Preliminary experiments used deuterated dimethyl sulfoxide, in
Bradow et al. which the glucuronides and isomers are quite soluble; however, in situ experiments revealed no reactivity of flufenamic glucuronide, even after addition of strong base. The glucuronides and isomers are most reactive in aqueous solutions but are less soluble in these solutions. This factor is less important with the small samples needed for HPLC analysis but becomes prohibitive when more concentrated samples are needed, as for characterization by NMR. Addition of small amounts of acetonitrile to water enhances solubility without completely suppressing reactivity. The amount of base added to initiate the in situ experiments was chosen to provide approximately the same reactivity of flufenamic acid glucuronide as that observed in the aqueous solution a t pH 8. Time-DependentStudy of the Rearrangements Monitored by HPLC. A sample of each 1-0-acylglucuronide or one of the purified isomers (approximately 300 pg) was dissolved in 0.1 M KH2P04,pH 8.0, to give solutions that were about 2 mM. Aliquota were analyzed once an hour, beginning immediately upon dissolution. Each aliquot was withdrawn from the reaction mixture with a Hamilton syringe (Reno, NV) and injected immediately onto the HPLC column. Rearrangement was terminated by the lower pH of the HPLC solvent. Analytical HPLC was performed on a Brownlee 5-pm/RP-18 column, 250 X 4.6 mm (Brownlee Co.). A gradient of acetonitrile/O.O5 M KHzP04,pH 4.5 was employed, in which the percentage of acetonitrile was increased from 25% to 40% in 12 min and then further increased to 60% in 5 min. The flow rate was maintained at 1.5 mL/min, and UV detection a t 254 nm was used. This analytical HPLC protocol was also used to characterize the rearrangement products formed from the 1-0-acylglucuronide of Ag-11-carboxytetrahydrocannabinol and to examine urine extracts. Examination of Urine for 1-0-Acylglucuronides and Rearranged Isomers. The following four urine samples were analyzed: (1)Rabbit urine, pooled over 4 weeks during weekly ip administration of 30 mg of flufenamic acid in 2 mL of corn oil (37 OC). Urine pH was adjusted to below 4 by the presence of 2.2 M citric acid in the collecting flask. Before analysis, this urine (-800 mL) was extracted with 3 volumes of ether and was adjusted to pH 2. (2) Fresh human urine, collected 4 h after the administration of 1 g of aspirin. The p H of this sample was 7.0 and was not adjusted. The sample was used immediately. (3) Previously frozen Rhesus monkey urine donated by Wyeth Laboratories, collected on the fourth day of the administration of benoxaprofen, 37.5 mg/(kg.day). The p H of this sample was 3.3, having been collected in 2.2 M citric acid. (4) Previous frozen human urine, obtained from the Veteran's Administration Hospital in Oakland, CA, under the auspices of the National Institute on Drug Abuse, collected after the administration of marijuana during a long-term smoking study. The pH was 8.8 and had not bee adjusted on collection. All samples were filtered, and the pH was adjusted to 2.5 with concentrated HCl. Between 200 and 250 mL of each sample was applied to a 300-mL capacity Tox-Elut column, and the glucuronide fractions were eluted with two 100-mL portions of ether (flufenamic acid metabolites) or ethyl acetate. The solvent was evaporated in vacuo, and each fraction was analyzed by color reactions on TLC plates. Flufenamic acid metabolites were further purified on a 75-g column of XAD-2 resin. The loaded XAD-2 column was washed with 200 mL of HzO and 400 mL of H20/CH3CN, 1:l. The glucuronide and isomers were eluted with 200 mL of acetone. All urine fractions were analyzed by HPLC, using the analytical systems described above. HPLC fractions with the retention times of the standard glucuronides and isomers were collected and analyzed by color reagents on TLC plates. Each ethyl acetate or acetone fraction was then dried over Pz05,dissolved in 500 pL of acetonitrile-d3/Dz0,and analyzed by NMR. Mass Spectrometry and Infrared and Ultraviolet Spectroscopy. Fast atom bombardment mass spectrometry was carried out on a Kratos MS-50 instrument, equipped with an RF magnet and a DS-90 data system. Glycerol was used as a matrix, and both positive and negative ion spectra were recorded. Samples were prepared as KBr pellets for infrared characterization on a Perkin-Elmer 1420 recording ratio spectrophotometer. Samples were dissolved in water and analyzed at pH 7 and 12 on a Bausch
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 319
Acyl-Linked Glucuronide Rearrangement Table I. HPLC Retention Times and Carbohydrate Proton Signals in NMR Spectra of Isomers of the Glucuronide of Flufenamic Acid" NMR assignment, ppm retention isomer time, min H-1 H-2 H-3 H-4 H-5 I-0-acyl 9.0 6.091 4.001 3.937 3.910 4.179 2-0-acyl 9.6 5.790 5.245 4.471 4.406 4.077 3-0-acyl 7.6 5.716 4.495 5.061 4.877 4.160 4-0-acyl 10.4 5.607 4.295 4.715 5.541 5.183 glucuronic acid 4.914 4.006 3.785 3.559 4.369
A, ,0 0
a Retention times are obtained with the analytical protocol used to study rearrangement sequences (see Materials and Methods).
Table 11. HPLC Retention Times and Carbohydrate Proton Signals in the NMR Spectra of Isomers of the Glucuronide of (S)-Benoxaprofen" NMR assignment, ppm retention isomer time, min H-1 H-2 H-3 H-4 H-5 3.179 3.763 3.791 3.995 9.0 5.788 I-O-acyI* 2-0-acyl 10.6 5.624 5.041 4.145 3.891 4.020 5.562 4.502 5.254 4.150 4.203 3-0-acyl 7.4 4-0-acyl 10.0 5.477 3.868 3.940 5.278 4.024 Retention times are obtained with the analytical protocol used to study rearrangement sequences (see Materials and Methods). *The spectrum of the 1-0-acylglucuronide of (R)-benoxaprofen was shown to be congruent with that of (S)-benoxaprofen glucuronide.
Table 111. HPLC Retention Times and Carbohydrate Proton Signals in NMR Spectra of the Glucuronide of Salicylic Acid and Its Isomer" NMR assignment, ppm retention H-3 H-4 H-5 time, min H-1 H-2 isomer I-0-acyl 9.6 5.974 3.858 3.804 3.792 4.050 9.0 6.131 3.962 3.828 3.911 4.358 as in Figure 3 Retention times are obtained with the analytical protocol used to study the rearrangement sequences (see Materials and Methods). a
and Lomb Spectronic 21 W/visible spectrophotometer, from 255 to 320 nm.
Results and Discussion The 1-0-acylglucuronides of flufenamic acid, (S)-benoxaprofen, salicylic acid, (R)-benoxaprofen, and A 9 - l l carboxytetrahydrocannobinolwere all found to undergo rearrangement in aqueous solution buffered at pH 8.0 to form isomers not hydrolyzed by @-glucuronidase. The three ester-linked regioisomers formed from each of the first two metabolites (Tables I and 11) were purified by HPLC and identified by 2D NMR. Three isomers of each of the latter two were characterized by HPLC and color reactions on TLC plates. One stable rearranged product was detected and recovered from the solution of conjugated salicylic acid (Table 111),and a structure is proposed, based on mass spectrometry, 2D COSY NMR, and infrared and UV spectroscopy. Characterization of Glucuronides and Isomers by NMR. The patterns of signals obtained for the glucuronic acid protons are quite similar in NMR spectra of the 10-acylconjugates of flufenamic acid, (R)-benoxaprofen, (S)-benoxaprofen, and salicylic acid. The 2D COSY spectrum of salicylic acid 1-0-acylglucuronideis presented in Figure 2. For the purpose of clarity, only the H1 to H5 regions (3.5-6.2 ppm) of both 1D (bottom panel) and 2D (top panel) spectra are shown in the figure. It is evident that HI at 5.97 ppm couples to Hz (3.86 ppm) and H2
1
5 2 4
1 ' ' 1 ' 1 ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 6.0
5.5
5.0
9.5
4.0
Chemical S h i f t , ppm Figure 2. 1D (bottom panel) and 2D COSY (top panel) spectra of salicylic acid 1-0-acylglucuronide from human urine. Solid lines with arrows indicate the coupling network. Dotted lines connect the signals in the 2D contour plot to more informative 1D plots, and the identities of signals are indicated under the peak. Spectra A and B are slices of the 2D at 3.86 and 3.80 ppm, respectively.
couples to H3 (3.80 ppm). This relationship is reillustrated by spectrum A which is a slice of the 2D compilation at 3.86 ppm. Off-diagonal signals at 3.80 and 5.97 ppm in spectrum A correspond to H3and H1, respectively. H3 and H4 (3.79 ppm) resonate very closely. This is clear when the intensity of the multiplet at 3.8 ppm is examined. Finally, the coupling between H4 and H5 (4.05 ppm) is revealed by the 2D presentation and by spectrum B. The assignments of proton signals in other acylglucuronides and their isomers were accomplished in a similar manner. Chemical shifts for carbohydrate protons are summarized in Tables 1-111 for glucuronides of flufenamic acid, ( S ) benoxaprofen, and salicylic acid and their rearranged isomers. Spectra of the 1-0-acyl conjugates of (R)-and (S)-benoxaprofen were shown to be congruent by analysis of a mixture of the diasteromers. The protons on the aromatic aglycon moieties of these four conjugates were detected downfield of 7 ppm. Benoxaprofen, with a more complex chemical structure, also has signals due to the benzylic proton and methyl group protons, at 4.4 and 1.9 ppm, respectively. The spectra of the isomers of flufenamic acid and (S)-benoxaprofen glucuronides share two distinct trends. The first is the downfield shift of the signal for the proton at the position of the new ester linkage. This has considerable precedent in the carbohydrate literature (40-46). This shift is observed not only when position 2 , 3 , or 4 is substituted. The anomeric C-1 proton signal is shifted downfield in the 1-0-acyl conjugate, relative to when that position is free (as, for example, in free glucuronic acid). The second trend is the upfield shift of the C-1 proton signal as the position of acylation is further removed (40-43). In nonglucuronide carbohydrate systems (45,461, this upfield shift is also present, but the magnitude of the
320 Chem. Res. Toxicol., Vol. 2, No. 5, 1989 Table IV. C-1 Proton Signals of Anomers of the Isomers of Glucuronic Acid, Flufenamic Acid Glucuronide, and Ester Isomers a anomer @ anomer a anomer @ anomer 1-0-acyl ND" 6.091 4-0-acyl 5.789 5.607 2-0-acyl 5.990 5.790 glucuronic 5.525 4.914 3-0-acyl 5.758 5.716 acid a
ND, not detected. 0
Figure 3. Proposed structure of the stable isomer of salicylic acid glucuronide.
shift varies and the order of anomeric proton signals does not always correspond to the order of the position of acylation (40-43). Isomers of flufenamic acid glucuronide were generated and characterized directly in an in situ NMR experiment. These isomers were subsequently recharacterized after purification by HPLC with an acidic gradient elution. Each single HPLC peak was found by NMR to contain two stereoisomers, the a and ,6 C-1 anomers. The positions of the C-1 proton signal in these Stereoisomers, confirmed by decoupling experiments, are presented in Table IV. The signals of the a anomers are shifted slightly downfield relative to those of the ,6 isomers (37,38). We determined, for the set of samples studied here, that such artifactual anomerization could be eliminated if the samples were not exposed to pH conditions below 5.0 in their final purification. Only one product was obtained from the rearrangement of salicylic acid glucuronide, and its NMR spectrum is different from those of the other rearranged isomers (Table 111). As can be seen in Figure 2, the signals of the protons at carbons 2, 3, and 4 are shifted very little, in contrast to shifts in the spectra of the ester isomers of flufenamic acid and benoxaprofen. The anomeric proton signal is shifted slightly downfield rather than upfield, relative to that of the original glucuronide. By analogy with the previous interpretations this suggests that no ester bonds have been formed and that C-1 still carries an acetal group. The four aromatic aglycon protons are detected, with only small changes in shifts. Additional Studies of the Structure of the Stable Isomer of Salicylic Acid Glucuronide. A negative ion mass spectrum obtained by using fast atom bombardment contained a peak at ml2 313, corresponding to the (M H)- ions of a compound with the same molecular weight as the original 1-0-acylglucuronide. Anions of mass 137 were detected, comprising the salicylic acid and formed by loss of the glucuronic acid moiety. Because isomer formation by transacylation to ester isomers was excluded by the NMR studies, the possibility of phenolate attack (Figure 3) was examined. Ultraviolet spectra were measured at pH 5.0 and 12.0 to test for an unsubstituted, ionizable phenol group. Minimal differences between spectra obtained at the two pH values confirmed that the phenol is derivatized. In addition, phenolic OH stretches present in the infrared spectrum of salicylic acid at 3240 and 1660 cm-l were absent in the infrared spectrum of the isomer of the glucuronide. The infrared spectrum also indicated that the benzoic acid group was still esterified. A strong absorption at 1550
Bradow et al.
cm-' and a weaker one at 1410 cm-' were observed, characteristic of carboxylate anions and indicating that acid groups in the sample had been ionized. Any free acid groups would be dimerized in the KBr disk; however, neither the characteristic dimeric OH stretch (2700-2500 cm-') nor the carbonyl stretch (1710-1680 cm-' for aryl acid dimers) was detected. The carboxylate signals were assigned to the glucuronide moiety. Strong bands at 1750 and 1180 cm-' (carbonyl and CO stretches, respectively) demonstrate the presence of an aromatic acid ester and were consistent with a cyclic lactone (53). Although the stereochemistry at C-1 has not been specified experimentally, in the NMR spectra only one C-1 proton was detected, indicating that the rearrangement proceeded stereospecifically. Phenolate attack at the anomeric center, as illustrated in Figure 3, would lead to conventional opening of the carbohydrate ring and steric inversion. All the spectroscopic observations on the stable isomer of salicylic acid glucuronide are consistent with the structure proposed in Figure 3. No rearranged product such as that isolated at pH 8 in the present studies has been reported in studies of aspirin metabolism in vivo. A phenol-linked glucuronide was reported to be formed by rearrangement in vitro of diflunisal 1-0-acylglucuronide exposed to both acid and base (36). Analysis of the Rearrangement Sequence by NMR. Sample sizes of the order of 100 pg are required for the two-dimensional analyses used to assign structures to the various HPLC peaks. This requirement may be the reason that only a few laboratories (36,39,47) have used NMR to assign structures to the suite of chromatographic peaks that usually betrays the rearrangement. Some investigators have suggested (2) that the structures might be assigned according to the order in which they are formed in an in vitro system, and this is supported by NMR for one case (36). We undertook to test this suggestion by studying the appearance of characterized isomers both in isomerizations of the 1-0-acylglucuronides of flufenamic acid, (S)-benoxaprofen, and salicylic acid and in those of their purified ester isomers. Time-dependent NMR studies were carried out in situ in the NMR instrument as described under Materials and Methods. In all three samples studied the rearrangements proceeded more slowly in the mixed organic solvent required for this analysis. Smith and co-workers (16) have reported that glucuronides are stabilized toward isomerization in acetonitrile. The first time-dependent experiment was performed with flufenamic acid glucuronide. After the addition of base to begin the rearrangement reactions, 36 spectra were acquired over a 12-h period. Special attention was given to the region of the spectrum in which the various isomers could be distinguished by signals due to the protons on carbon 1 (Tables I-IV). Through time, the signal due to the C-1 proton in the original glucuronide decreases, and the signals due to the C-1 protons of the isomers emerge. The areas of the C-1 proton signals were measured in each of the spectra as an indication of the relative amounts of each isomer present at that time. We estimate the uncertainty of these measurements as &lo% of the peak area. The smallest peaks measured correspond to about 40 pg. These data are summarized graphically in Figure 4. This figure clearly indicates the sequential appearance of the isomers, with the 2-ester present after 20 min, and the 3and 4-esters appearing after 60 and 100 min, respectively. Hydrolysis to free glucuronic acid and flufenamic acid is a competing reaction and is measured in this experiment
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 321
Acyl-Linked Glucuronide Rearrangement
l 80 o o h
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Figure 4. Reiative amounts of isomers formed from the 1-0acylglucuronide of flufenamic acid monitored by NMR 1-0-acyl
Figure 5. Mixtures formed by rearrangement at pH 8.0 of (a) the 1-0-acylglucuronideof flufenamic acid and three purified ester isomers, (b) 2-ester, (c) 3-ester, and (d) 4-ester: 1-0-acyl ( 0 ) ; 2-ester (0);3-ester (A);4-ester (A);hydrolyzed aglycon (0).
( 0 ) ;2-ester (0);3-ester (A);4-ester (A);hydrolyzed glucuronic acid (0).
l80
by observing the anomeric proton signal of glucuronic acid. Under the conditions of this time-dependent experiment, formation of anomeric diastereomers was not observed. The lines in Figure 3 are drawn to aid interpretation and are not mathematically based. The glucuronide of (S)-benoxaprofen was analyzed under identical conditions. After 12 h under basic conditions, the original glucuronide still accounted for 98% of the mixture. A small amount of the 2-ester was present, and no other species could be detected. Salicylic acid glucuronide, like (S)-benoxaprofen glucuronide, rearranges very slowly under the conditions of the NMR experiment, and about 96% of the original conjugate remained at the end of the 12-h reaction period. In this case, however, the isomer detected is formed by phenolinitiated ring opening. The 2-ester could not be detected. Analysis of the Rearrangement Sequence by HPLC. Once isomeric structures had been rigorously assigned to the various HPLC peaks, by using NMR, then HPLC was applied to studies of the rearrangements of four 1-0acylglucuronides, carried out in aqueous solutions buffered at pH 8.0. The amounts of isomers, formed by rearrangement, and of aglycon, generated by hydrolysis, were determined by peak height measurements through 8 h and are shown in Figures 5-8. In addition to four glucuronides, the rearrangement and hydrolysis of three sets of purified isomers were analyzed. The lines in Figures 5-8 are not mathematically based but are drawn to aid interpretation. Coefficients of variation for relative proportions measured by HPLC are 2-12%, depending on the signal to noise ratio, and were determined from three rearrangement experiments with each purified isomer and glucuronide. Although the time resolution was poorer in the HPLC study (fewer data points were collected), the sequence of
6o
o
o
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h
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E 0
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T I M E (HOURS)
Figure 6. Mixtures formed by rearrangement at pH 8.0 of (a) the 1-0-acylglucuronideof (S)-(+)-benoxaprofenand three purified ester isomers, (b) %ester,(c) 3-ester, and (d) 4-ester. The symbols are as in Figure 4.
appearance of isomers of flufenamic acid glucuronide (Figure 5a) appears to proceed similarly to its isomerization in the NMR experiment. In the isomerization of (S)-benoxaprofen glucuronide, the order of appearance of the isomers in aqueous solution is clearer, with detection of the 2- and 3-ester isomers first, followed by the 4-ester.
Bradow et al.
322 Chem. Res. Toxicol., Vol. 2, No. 5, 1989
t
90
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.\
d
10
Table V. Half-Lives of Disappearance of the 1-0-Acylglucuronides and Rearranged Isomers in Buffered Aaueous Solution. DH 8.0, at Ambient Temperature isomer half-life, h isomer half-life, h
'b'
flufenamic acid 1-0-acyl 2-0-acyl 3-0-acyl 4-0-acyl (S)-benoxaprofen 1-0-acyl 2-0-acyl 3-0-acyl 4-0-acyl
L
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2
1
4
5
6
7
8
2
3
3
r
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6
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Figure 7. Mixtures formed by rearrangement at pH 8.0 of (a) the 1-0-acylglucuronideof salicylic acid and (b) its purified isomer: 1-0-acyl ( 0 ) :isomer (0).
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(R)-benoxaprofen 1- 0-acy1 salicyclic acid 1-0-acyl as in Figure 3
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Figure 8. Mixtures formed at pH 8.0 from the 1-0-acylglucuronide of (R)-(-)-benoxaprofen.Isomer structures are assigned based on order of appearance. The symbols are as in Figure 4.
The reactions of the purified isomers (Figures 5 and 6) also indicate a second general feature, that these rearrangements involve migration primarily between hydroxyl groups on adjacent carbon atoms. The 2-ester isomer of the (S)-benoxaprofen conjugate rearranged first to its 3-ester isomer, and subsequently the 4-ester appeared (Figure 6b). Purified 3-esters yielded both 2-ester and 4-ester isomers (Figures 5c and 6c). Both of the 4-ester isomers formed only the 3-esters (Figures 5d and 6d). The glucuronide pattern differs from that reported for acylated glucoses and glucosides, in which 1-0-acyl to 3-0-acyl migration was favored in the 0 anomers under various conditions (48-50). This discrepancy emphasizes the fact that, although rearrangement is found generally in carbohydrate chemistry, it may proceed differently in diverse substrates and various reaction conditions. The sequence and reversibility we observe are consistent with an ortho ester intermediate, proposed as well for rearrangements of glycerol and other glycoside esters. The third general feature of glucuronide isomerization that our experiments support is that each step is reversible, except the thermodynamically unfavorable rearrangement back to the original 1-0-acylglucuronide. Observations of other workers are consistent (15, 18, 19, 24, 26, 39, 511,
except for the study of diflunisal glucuronide isomers assayed with HPLC (36). Isomerization of the 1-0-acylglucuronide of (R)-benoxaprofen was also analyzed, as presented in Figure 8. Isomer structures are assigned here based on their order of appearance and the near coincidence of their HPLC retention times (Materials and Methods) with those for the ( S ) benoxaprofen series. Interestingly, rearrangement and hydrolysis of the conjugate of (R)-benoxaprofen did not proceed identically with that of the glucuronide of ( S ) benoxaprofen (Figure 6a). Although the sequence of appearance of isomers is qualitatively the same, the rate of hydrolysis is faster for (R)-benoxaprofenglucuronide. The overall half-lives for disappearance are similar, 7 and 8 h (Table V). Half-lives were calculated (Table V) for the disappearance of each glucuronide and purified ester isomer, reflecting both rearrangement and hydrolysis, based on the same sets of measurements used to generate Figures 5-8. Apparent first-order rate constants were calculated for disappearance, with the assumption that the rates all go to zero, and ignoring an apparent lag in the disappearance of flufenamic acid glucuronide. Linear regression analysis of the natural logarithms was used, following the procedures of Hasegawa (26) and Rachmel (27). Half-lives of the four 1-0-acylglucuronides studied here fall in the middle of the broad range of values, 3 X 10-'-3 X assembled by Rachmel et al. in their literature survey (27). Our studies of three sets of purified ester isomers indicate that these are all more stable than their corresponding 1-0-acyl conjugates. The 3-ester isomer of flufenamic acid glucuronide is significantly more stable than the 2- or 4-ester isomers. The range of the half-lives of the isomers of (S)-benoxaprofen is more narrow; however, within this set the 2-ester isomer is most stable. The isomer of salicylic acid glucuronide was found to be completely undegraded after 2 weeks. Examination of Urine for Acyl-Linked Glucuronides and Ester Isomers. The analysis of 1-0acylglucuronides and their isomers in physiologic fluids provides the most challenging application of the analytical methods developed here. Urines from a rabbit dosed with flufenamic acid (see Materials and Methods), a rhesus monkey that had received racemic benoxaprofen, a healthy subject who received 1 g of aspirin, and a volunteer in a marijuana smoking study were examined for the presence of 1-0-acylglucuronidesof flufenamic acid, benoxaprofen, salicylic acid, and A9-11-carboxytetrahydrocannabinol(52) and their rearranged isomers, respectively. The ethyl acetate extracts of these four urine samples were analyzed by NMR, by HPLC, and by color reagents on a TLC plate. Three TLC plates were developed for each sample; each plate was visualized by UV light and by one of three spray reagents. Naphthoresorcinol gives a positive reaction with uronic acids, p-anisidine with reducing sugars, and NBP
Acyl-Linked Glucuronide Rearrangement
with alkylating agents. The 1-0-acylglucuronidescan be distinguished from other glucuronides because they react with NBP as well as with naphthoresorcinol. The hemiacetal or reducing groups are free in the rearranged ester isomers, which react with all three reagents. The TLC system that was used separated isomers and 1-0-acylglucuronides from many other contaminants by moving them to the middle of the plate; however, this did not separate the glucuronides and isomers from each other. All four urine extracts provided UV-positive spots, coincident with reference conjugates, that reacted positively with all three color reagents. This was interpreted to suggest the presence of isomers of 1-0-acylglucuronides in the extracts. The extracts were also analyzed by HPLC, using the analytical conditions developed to provide separation of reference glucuronides and their isomers. In all four cases, peaks were detected at retention times coincident with those of the reference glucuronides and isomers (Tables 1-111 and Materials and Methods). These peaks were collected and subjected to the same TLC analysis as the crude extracts. TLC indicated that many of these HPLC fractions contained more than one compound. However, in all cases material was present that moved to the middle of the plate, coincident with standards. All of the putative ester isomers reacted with the three color reagents. The presumed 1-0-acylglucuronides reacted with naphthoresorcinol and NBP and not with p-anisidine. Extracts from the monkey and human urine were also analyzed directly by NMR. Signals corresponding to the C-1 protons of the 1-0-acylglucuronideand all three of the rearranged isomers of benoxaprofen were observed in the monkey urine extract. No signals in this distinctive region could be observed in spectra of the extracts containing salicylic acid or tetrahydrocannabinol metabolites. This is probably due to the fact that in both these cases the acyl-linked glucuronide is not present at high concentrations. In contrast, acyl conjugation with gluruconic acid is the major route of metabolism for benoxaprofen. Putative isomers of flufenamic acid glucuronide in the rabbit urine extract were purified by HPLC in sufficient quantities to characterize their C-1 protons by NMR. These spectra further supported the assignment of isomeric structures. Although the pH of the rabbit urine had been adjusted to between 3 and 4 during collection, relative heights of HPLC peaks indicated that the 1-0-acylglucuronide of flufenamic acid constituted less than 5% of the total of the four isomers. The HPLC analysis was repeated on an unextracted urine sample within 5 min of evacuation by the rabbit, and the same distribution of isomers was observed. This suggests that the enzymatically conjugated 1-0-acylglucuronide rearranges extensively before evacuation. This is not unexpected, because the pH of rabbit urine is approximately 8.5.
Acknowledgment. We thank Drs. Dale Whalen and John A. Joule, University of Maryland Baltimore County, and Dr. Hans Ruelius, Wyeth Pharmaceutical Co., for helpful discussions. This work was supported by US. Public Health Service Grants GM 21248 and T 2GM07626. The NMR studies were performed in the Biophysics NMR Facility Center, The Johns Hopkins University, established by U.S. Public Health Service Grant GM 27512, and mass spectra were measured in the Middle Atlantic Mass Spectrometry Center, an NSF Instrumentation Facility at The Johns Hopkins University School of Medicine.
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