Measurement of Internal Acyl Migration Reaction Kinetics Using

Andrew V. Stachulski, John R. Harding, John C. Lindon, James L. Maggs, B. Kevin Park, and Ian D. Wilson. Journal of Medicinal Chemistry 2006 49 (24), ...
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Anal. Chem. 1996, 68, 2564-2572

Measurement of Internal Acyl Migration Reaction Kinetics Using Directly Coupled HPLC-NMR: Application for the Positional Isomers of Synthetic (2-Fluorobenzoyl)-D-glucopyranuronic Acid Ulla G. Sidelmann,† Steen H. Hansen,† Claire Gavaghan,‡ Howard A. J. Carless,‡,§ John C. Lindon,‡ R. Duncan Farrant,‡ Ian D. Wilson,⊥ and Jeremy K. Nicholson*,‡

Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark, Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London WC1H 0PP, U.K., Department of Physical Sciences, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K., and Department of Safety of Medicines, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.

Ester glucuronides (1-O-acyl-β-D-glucopyranuronates) of many drugs may undergo internal acyl migration reactions, resulting in the formation of new positional isomers with both r- and β-anomers. We illustrate here a novel approach for the direct investigation of the acyl migration kinetics of ester glucuronides and show the application with respect to the isomers of synthetic (2-fluorobenzoyl)D-glucopyranuronic acid. Individual isomers were separated from an equilibrium mixture containing the β-1-Oacyl, r- and β-2-O-acyl, r- and β-3-O-acyl, and r- and β-4O-acyl isomers at pH 7.4 in 20 mM phosphate buffer. The interconverting isomers were separated using reversedphase HPLC and pumped directly into a dedicated online NMR flow probe in a 600 MHz NMR spectrometer. The flow was stopped with each isomer in the NMR flow probe, and sequential NMR spectra were collected at 25 °C, allowing direct measurement of the production of positional isomers from each selectively isolated glucuronide isomer. All of the positional isomers and anomers were characterized, and relative quantities determined, and a kinetic model describing the rearrangement reactions was constructed. The acyl migration reaction kinetics were simulated using a theoretical approach using nine first-order rate constants determined for the acyl migration reactions and six first-order rate constants describing the mutarotation each of the 2-, 3-, and 4-positional isomers. The rate constants (in h-1) for the rearrangement reactions of the 2-fluorobenzoyl glucuronide isomers were as follows: β-1-O-acyl, 0.29 ( 0.01; r-2-O-acyl, 0.11 ( 0.01; β-2-O-acyl, 0.07 ( 0.01; r-3-O-acyl, 0.10 ( 0.01; β-3-O-acyl, 0.09 ( 0.01; r-4-O-acyl, 0.09 ( 0.01; and β-4-O-acyl, 0.06 ( 0.01. The r- and β-anomerization rates were estimated on the basis of the kinetics model; the anomerization rates of the 4-O-acyl isomers were additionally determined experimentally using directly coupled HPLC-NMR. The fitted anomerization rates for the 4-O-acyl isomer were 0.80 (r f β) and 0.50 h-1 (β f r), whereas the experimentally estimated anomerization rates were 0.89 ( 0.1 and 0.52 ( 0.1 h-1, respectively. The dynamic stop-flow HPLCNMR approach allows unique kinetic information to be

obtained relating to glucuronide reactivity, and this approach will be useful in future structure-reactivity studies on drug ester glucuronides and their properties.

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S0003-2700(96)00014-5 CCC: $12.00

NMR spectroscopy is well established as a technique that gives detailed structural and dynamic information on molecules in solution. Recently it has been successful to directly couple highperformance liquid chromatography (HPLC) systems to NMR “detectors”, giving a hyphenated technique, HPLC-NMR, for combined separation and direct structure elucidation of compounds in complex mixtures,1-7 and this approach promises to be of value in numerous areas of pharmaceutical and bioanalytical chemistry. There are many instances where the compounds under study are structurally closely related and are present in complex equilibrium mixtures. Studies on the reactivity of such compounds often pose significant analytical problems, as is the case with D-glucopyranuronate esters of carboxylate-containing drugs, which readily undergo acyl migration reactions. We have used a novel “dynamic” HPLC-NMR approach to the study of such reactions in which NMR can be used noninvasively to monitor directly the spontaneous isomerization of separated drug glucuronides in aqueous solution. Most drugs containing carboxylate groups form β-1-O-acyl glucuronic acid conjugates in vivo via the enzyme UDP-glucuronosyl transferase. The nonsteroidal antiinflammatory drugs (NSAIDs) are compounds containing carboxylate groups, e.g., †

The Royal Danish School of Pharmacy. University of London. § Wellcome Research Laboratories. ⊥ Zeneca Pharmaceuticals. (1) Farrant, R. D.; Salman, S. R.; Lindon, J. C.; Cupid, B. C.; Nicholson, J. K. J. Pharm. Biomed. Anal. 1993, 11, 687-692. (2) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. J. Pharm. Biomed. Anal. 1992, 8, 601-605. (3) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. Anal. Chem. 1993, 65, 327-330. (4) Wilson, I. D.; Nicholson, J. K.; Spraul, M.; Hofmann, M.; Nicholson, J. K.; Lindon, J. C. J. Chromatogr. 1993, 617, 324-328. (5) Spraul, M.; Hofmann, M.; Wilson, I. D.; Lenz, E.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1993, 11, 1009-1016. (6) Seddon, M. J.; Spraul, M.; Wilson, I.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1994, 12, 419-424. (7) Spraul, M.; Hofmann, M.; Lindon, J. C.; Farrant, R. D.; Seddon, M. J.; Nicholson, J. K.; Wilson, I. D. NMR Biomed. 1994, 7, 295-303. ‡

© 1996 American Chemical Society

Scheme 1. Kinetic Model for the Acyl Migration Reactions of Drug Ester Glucuronides upon which the Mathematical Model is Based

ibuprofen, naproxen, diflunisal, and aspirin. Several glucuronideforming NSAIDs (zomepirac, benoxaprofen, indoprofen, and alclofenac) have been withdrawn from clinical use because of a high incidence of allergic reactions associated with drug therapy.8,9 These allergic reactions are thought to be mediated by covalent drug-protein adducts which act as haptens and cause immune responses.10-12 The propensity for rapid internal acyl migration in the glucuronide conjugates of these drugs appears to be related to the incidence of hypersensitivity reactions, and all the withdrawn compounds have highly reactive glucuronides with short half-lives of the β-1-O-acyl isomer in aqueous solution at pH 7.4.8

The acyl glucuronides are potentially reactive metabolites due to the susceptibility of the acyl group to nucleophilic reactions. (8) Spahn-Langguth, H.; Benet, L. Z. Drug Metabol. Rev. 1992, 24, 5-48. (9) Clive, D. M.; Stoff, J. F. N. Engl. J. Med. 1984, 310, 563-572. (10) Pohl, L. R.; Satoh, H.; Christ, D. D.; Kenna, J. G. Annu. Rev. Pharmacol. 1988, 28, 367-387. (11) Williams, A. M.; Worall, S.; Jersey, J.; Dickinson, R. G. Biomed. Pharmacol. 1992, 43, 745-755. (12) Smith, P. C.; McDonagh, A. F.; Benet, L. Z. J. Clin. Invest. 1986, 77, 934939.

They have been shown to undergo hydrolysis (regeneration of parent compound),8,13 intramolecular rearrangement (isomerization by acyl migration),14-16 and covalent adduct formation with both small nucleophiles (such as methanol) and proteins.15,17-19 The susceptibility of the ester linkage of the acyl glucuronides to nucleophilic reactions allows the acyl drug moiety to migrate from one hydroxyl group to an adjacent hydroxyl group on the glucuronic acid ring via an ortho ester intermediate.20 The drug moiety migrates from C-1 toward C-4, and both the R- and β-anomers of 2-, 3-, and 4-O-acyl positional isomers are formed (Scheme 1). The acyl migration reactions are reversible except for the migration from C-1 to C-2, as the 1-O-acyl glucuronide has not been observed to be re-formed, presumably because of a high energy barrier in re-formation of the anomeric C-O bond. The positional glucuronide isomers (excluding the β-1-O-acyl isomer) can also undergo anomerization, and, as can be seen from Scheme 1, there are nine reaction rate constants involved in the acyl migration reactions and six rate constants to describe the anomerization rates plus the hydrolysis rate constant from the β-1-O-acyl isomer. The model of acyl migration kinetics presented in Scheme 1 assumes (13) Smith, P. C.; Song, W. Q.; Rodriguez, R. J. Drug Metab. Dispos. 1992, 20, 962-965. (14) Bradow, G.; Kan, L. S.; Fenselau, C. Chem. Res. Toxicol. 1989, 2, 316-324. (15) King, A. R.; Dickinson, R. G. Biochem. Pharmacol. 1991, 42, 2289-2299. (16) Feneselau, C. In Conjugation-Deconjugation reactions in drug metabolism; toxicity; Kauffman, F. C., Ed.; Springer-Verlag: Berlin, 1994; pp 367-389. (17) Smith, P. C.; Benet, L. Z.; McDonagh, A. F. Drug Metab. Dispos. 1990, 18, 639-644. (18) Weiss, J. S.; Guatam, A.; Lauff, J. J.; Sundberg, M. W.; Jatlow, P.; Boyer, J. L.; Saligson, D. N. Engl. J. Med. 1983, 309, 147-150. (19) Ruelius, H. W.; Young, E. M.; Kirkman, S. K.; Schillings, R. T.; Sisenwine, S. F.; Janssen, F. W. Biochem. Pharmacol. 1985, 34, 451-452. (20) Fischer, E. Chem. Ber. 1920, 53, 1621-1633.

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that acyl migration takes place independently of the anomerization of the glucuronide positional isomers. The reactions are spontaneous in aqueous solution, and the migration rates are highly dependent upon the structure of the aglycon, pH, and temperature.8,15 The relationship between the chemical structure of the aglycon and acyl migration rates can be explained on the basis of steric and electronic factors.16 Bulky groups in the vicinity of the carbonyl group can obstruct the attack from an adjacent hydroxyl group on the carbonyl carbon, and inductive effects can make the carbonyl carbon more susceptible to nucleophilic attack. Acyl migration reactions are inhibited in acidic solution, but above pH 7, the migration rates increase markedly with increasing pH.8,15 The accepted mechanisms for covalent bonding between drug glucuronides and proteins are transacylation and glycation. Transacylation involves the direct nucleophilic displacement of the glucuronic acid moiety by a suitable nucleophilic group (normally SH, NH2, or OH) on a protein and is thought to be the mechanism of adduct formation for the 1-O-acyl glucuronide of carboxylate drugs.11,15,21 The second mechanism requires acyl migration and is similar to the nonenzymatic glycosylation of albumin.12,15,22 Covalent binding occurs after acyl migration between the reactive aldehyde of the open chain derived from the glucuronic acid ring and a nucleophilic amine group on the protein, resulting in the formation of an imine (a Schiff base), which can further undergo acid-catalyzed Amadori rearrangement17 to form a keto amine. The 4-O-acyl isomer of the various positional glucuronide isomers has been shown to be most reactive toward formation of covalent adducts to proteins,15,24,23 and the reactivity order for protein adduct formation is 4-O-acyl isomer > 3-O-acyl isomer > 2-O-acyl isomer > 1-O-acyl isomer.23 It follows that protein binding via transacylation plays only a small role for drug glucuronides that undergo fast intramolecular acyl migration. As acyl migration is closely associated with drugprotein adduct formation and possible consequent immunogenic reactions on the basis of the glycation mechanism, it is valuable to study the factors controlling the rates of intramolecular acyl migration reactions. We show that directly coupled 600 MHz HPLC-1H NMR in the “stop-flow” mode can be used to study the individual reaction kinetics of acyl migration and anomerization of acyl glucuronides separated by HPLC-NMR from the equilibrium mixture (Scheme 1). We have chosen the β-1-O-acyl 2-fluorobenzoyl glucuronide as a synthetic model to illustrate this general HPLC-NMR approach to the study of drug metabolite reactivity. We have previously developed HPLC separation methods for the isomeric acyl fluorobenzoyl glucuronides in potassium phosphate buffer (20 mM) at pH 7.4, 25 °C, resulting in efficient chromatographic resolution of the various positional isomers as well as their R- and β-anomeric forms.24 In the present study, the kinetics of the individual acyl migration rates were obtained from the equilibrium mixture using directly coupled dynamic stop-flow 600 MHz (21) Williams, A. M.; Dickinson, R. G. Biochem. Pharmacol. 1994, 47, 457467. (22) Higgins, P. J.; Bunn, H. F. J. Biol. Chem. 1981, 256, 5204-5208. (23) Dickinson, R. G.; King, A. R . Biochem. Pharmacol. 1991, 42, 2301-2306. (24) Sidelmann, U. G.; Gavaghan, C.; Carless, H. A. J.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. Anal. Chem. 1995, 67, 3401-3404.

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1HHPLC-NMR

in which individually separated isomers were held in the flow probe and the internal rearrangement reactions of each monitored noninvasively via sequential 1H NMR measurements. HPLC-NMR provides a unique method for simultaneously confirming the isomeric structure of each compound and estimating reaction kinetics under defined solvent conditions. This approach will be of value in the construction of structurereactivity relationships for drug and synthetic glucuronide conjugates and hence further the understanding of the toxicological properties of those compounds that are mediated via their acyl migration reactions.

EXPERIMENTAL SECTION Chemicals. All chemicals were of analytical chemical grade, purchased from Aldrich Chemical Co. Ltd. (Poole, U.K.). The 2-(fluorobenzoyl)-β-D-glucopyranuronic acid (I) was synthesized as previously described.25,26 Establishment of the Equilibrium Mixture of the 2-Fluorobenzoyl Glucuronide Isomers. A 1.5 mg/mL solution of I was incubated in potassium phosphate buffer (20 mM) at pH 7.4, 25 °C, for up to 24 h. The equilibration was monitored by HPLC, and when the relative amounts of the positional isomers had stabilized, the sample was stored at -20 °C prior to analysis. Analytical Chromatography. The HPLC system consisted of a Bruker LC22C pump (Rheinstetten, Germany) and a Bischoff Lambda 1000 variable-wavelength UV detector (operated at 200 nm). The outlet of the UV detector was connected to the HPLC-NMR flow probe via an inert poly(ethyl ether ketone) capillary (0.25 mm i.d.). A Mistral column oven (Spark Emmen, Holland) was used for controlling the column temperature at 25 °C. Data were collected using a Bruker Chromstar HPLC data system. Chromatography was performed on a Knauer column (120 mm × 4.6 mm i.d.) packed with Spherisorb ODS-2 (Phase Separations Ltd., Deeside, U.K.), 5 µm. The mobile phase was acetonitrile-0.2 M potassium phosphate (pH 7.4)-deuterium oxide (1:10:89 v/v/v), with a flow rate of 1 mL/min. Identification of the compounds corresponding to the HPLC peaks in the chromatograms was based on results obtained using directly coupled HPLC-NMR.24 NMR Spectroscopy. The HPLC-NMR data were acquired using a Bruker AMX-600 spectrometer equipped with a 1H flow probe (cell of 3 mm i.d., with a volume of 120 µL). 1H NMR spectra were obtained in the stop-flow mode21 at 600.14 MHz (dynamic stop-flow HPLC-NMR). Each HPLC UV absorption peak corresponding to an individual positional isomer was isolated in the NMR flow cell in the stop-flow mode, and the subsequent establishment of equilibrium was followed by NMR. This produced pseudo-two-dimensional plots with NMR frequency (chemical shift) on the horizontal axis and reaction time on the vertical axis. The anomerization rates were much faster (by a factor of ∼10) than the acyl migration rates, so anomeric equilibrium mixtures were quickly obtained. Therefore, only one UV peak(25) Schmidt, R. R.; Grundler, G. Synthesis 1981, 885-887. (26) Van Boeckel, C. C. A.; Delbressine, L. P. C.; Kaspersen, F. M. Recl. Trav. Chim. Pays-Bas 1985, 104, 259-265.

Figure 1. Pseudo-two-dimensional contour plot of the acyl migration reactions of the 4-O-acyl isomer of 2-fluorobenzoyl glucuronide, with δ 1H on the horizontal axis and reaction time on the vertical axis. The acyl migration reactions were followed by collection of 1D NMR spectra for 30 h. Contours for novel positional isomers are shown filled.

from the pair of anomers of each glucuronide isomer was studied and the acyl migration of both the R- and the β-anomers could be obtained simultaneously. The following procedure was used for NMR data acquisition: A one-dimensional version of the nuclear Overhauser effect spectroscopy (NOESY) pulse sequence was applied (Bruker, Rheinstetten, Germany) with double presaturation for suppression of the water and acetonitrile signals. Free induction decays (FIDs) were collected into 8K computer data points with a spectral width of 7246 Hz, 90° pulses were used with an acquisition time of 0.57 s, and the spectra were acquired by accumulation of 256 scans in 15 min time windows for investigation of acyl migration and 32 scans in 1 min time windows for investigation of anomerization rates. Prior to Fourier transformation, an exponential apodization function was applied to the FID corresponding to a line broadening of 1.1 Hz. Determination of Individual Acyl Migration Rates of Ester Glucuronides. Several key “reporter” 1H NMR resonances were identified for the different glucuronide positional isomers. These signals were well resolved from each other and subject to negligible chemical shift overlap at 600 MHz and, therefore, were suitable as quantitative markers of acyl migration/ mutarotation. The selected signals were followed in the stopflow HPLC-NMR spectra as a function of time in order to follow acyl migration reactions and calculate the individual acyl migration rates. For the R- and the β-anomers of the 4-Oacyl isomer, the doublets at δ 4.27 and 3.93 respectively were measured (5′ protons on the glucuronide ring).24 For the

R- and β-anomers of the 3-O-acyl isomer, the triplets at δ 5.38 and 5.20 were measured (3′ proton on the glucuronide ring).24 For the R- and the β-anomer of the 2-O-acyl isomer, the doublets at δ 4.10 and 3.75 were followed (5′ protons on the glucuronide ring).24 As the individual NMR spectra were accumulated over 15 min, the midpoint was used as the time data point. The sequential stop-flow HPLC-NMR data were displayed as a pseudo-two-dimensional contour plot of chemical shift versus the reaction time. All the spectra were baseline corrected, and representative spectra from the pseudo-two-dimensional plot were selected at different time points for integration. Integrals of the selected peaks were measured relative to those of the aromatic protons (assumed to be constant). The same integration intervals were used in all the spectra chosen. The individual acyl migration rates were determined on the assumption that the initial parts of the concentration decay curves, after anomeric equilibrium had occurred, followed firstorder kinetics. Hence, the logarithm of the integrals from the initial parts of the acyl migration curves were plotted against time, straight lines were obtained (with correlation coefficients > 0.9), and the slopes were used as an estimate of the rates. Both the decomposition and formation rates were estimated in the same way. These estimated acyl migration and formation rates were analyzed by a kinetics simulation program (Kinetics, courtesy of F. Neese, Department of Biology, University of Konstanz Am Giessberg, 7750 Konstanz, Germany). This program solves a series of differential equations (eqs 1-8), fitted to Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

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Figure 2. Measurement of anomerization rates using stop-flow HPLC-NMR. 1H NMR spectra selected at different time points from the respective pseudo-2D plots of the acyl migration reactions of the 2- (C), 3- (B), and 4-O-acyl isomers (A) of the 2-fluorobenzoyl glucuronides.

the model in Scheme 1, by a semiimplicit Euler method. Brackets denote concentration, A denotes the aglycon, and kh is the rate of hydrolysis. The relative anomerization rates were calculated from

[β1] ) -kβ1-2[β1]

(1)

[β2] ) kβ1-2[β1] + kβ3-2[β3] - kβ2-3[β2] - kβ-R2[β2 ] + kR-β2[R2] (2) [β3] ) -kβ3-2[β3] + kβ2-3[β2] + kβ4-3[β4] - kβ3-4[β3] kβ-R3[β3] + kR-β3[R3] (3) [β4] ) -kβ4-3[β4] + kβ3-4[β3] + kR-β4[R4] - kβ-R4[β4] (4) [R2] ) -kR-β2[R2] + kβ-R2[β2] + kR3-2[R3] - kR2-3[R2] (5) [R3] ) kR2-3[R2] - kR3-2[R3] + kR4-3[R4] - kR3-4[R3] kR-β3[R3] + kβ-R3[β3] (6)

brium state. The anomerization rates were then estimated by fitting the curves to the experimental data obtained from the NMR experiments. One set of anomerization rates (anomerization of the 4-O-acyl isomers) was estimated experimentally in order to evaluate the fitted anomerization rates. They were estimated by the same method as the acyl migration rates except that the sequential NMR spectra were collected at 1 min time intervals. The effect of 1% acetonitrile (necessary for efficient HPLC separation) on the acyl migration of the related 3-fluorobenzoyl glucuronides was evaluated independently by a parallel study of the acyl migration of the 1-O-acyl isomer (a) on-line in the NMR probe with 1% acetonitrile in 20 mM potassium phosphate buffer, pH 7.4, and (b) by taking aliquots at different time points from an incubation in 20 mM buffer, pH 7.4, without acetonitrile and analyzing them by HPLC. Both experiments were carried out at 25 °C. In experiment a, a rate of 0.23 h-1 was obtained, whereas in experiment b, a rate of 0.22 h-1 was obtained. From this preliminary experiment, it was deduced that 1% acetonitrile did not significantly affect the rate of acyl migration under the conditions studied.

[R4] ) kR3-4[R3] - kR4-3[R4] + kβ-R4[β4] - kR-β4[R4] (7) [Α] ) kh[β1]

(8)

the ratio of the R- and β-anomers in the anomeric equili-

RESULTS The first peak in the chromatographic UV trace (tR ) 1.99 min) corresponded to the R-anomer of the 4-O-acyl isomer, as assigned by directly coupled HPLC-NMR.24 This compound was isolated in the HPLC-NMR probe in the stop-flow mode, and its acyl Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

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Table 1. Individual Acyl Migration Half-Lives for Positional Isomers of (2-Fluorobenzoyl)-D-glucopyranuronates at 25 °C and pH 7.4a positional isomers 1-O-acyl 2-O-acyl 3-O-acyl 4-O-acyl

t0.5(R) (h)

t0.5(β) (h)

6.3 ( 1.1 6.9 ( 1.2 8.9 ( 1.1

3.7 ( 0.4 10.5 ( 1.0 8.2 ( 1.4 11.9 ( 1.4

a The rate constants (k) were estimated as the slope of the straight line obtained when the logarithms of the integrals (obtained from the NMR data) were plotted against time. t0.5 ) (ln 2)/k ( standard deviation. The standard deviation was estimated as the error on the slopes of the linear fitted curves.

migration reactions were studied over a time period of 30 h. The pseudo-two-dimensional plot of the spectral region δ 5.6-3.2, incorporating all the glucuronide ring proton signals, is shown in Figure 1. 1H NMR spectra selected at different time points in the pseudo-two-dimensional plot are displayed in Figure 2A. Formation of the positional isomers resulting from acyl migration can be clearly observed. The R-anomer and the β-anomer of the 3-O-acyl isomer were formed first, and then the R-anomer and the β-anomer of the 2-O-acyl isomer were formed, consistent with the hypothesis that acyl migration takes place via the neighboring hydroxyl group of the glucuronic acid ring only.13 A later UV peak (tR ) 5.80 min) corresponded to the R-anomer of the 3-Oacyl isomer.24 This compound was held in the flow probe in the stop-flow mode, and its rearrangement reactions were followed for 12 h. This experiment shows that the 2-O-acyl and 4-O-acyl positional isomers were formed simultaneously from the 3-O-acyl isomer, as acyl migration can occur in both directions. Selected NMR spectra from the pseudo-two-dimensional plot are shown in Figure 2B. The UV peak with a retention time of 9.91 min corresponded to the β-anomer of the 2-O-acyl isomer;24 this peak was held in the flow probe for 20.75 h, and the acyl migration reactions were followed by collection of sequential NMR spectra over this time. The R- and β-anomers of the 3-O-acyl isomer were formed first, followed by the R- and β-anomers of the 4-O-acyl isomers. Selected 1D spectra from the pseudo-twodimensional plot are shown in Figure 2C. There was no evidence for hydrolysis of the acyl-migrated glucuronides, i.e., no signals from the aglycon or glucuronic acid were observed from any of the 2-, 3-, or 4-positional glucuronide isomers over the time studied. The calculated acyl migration rates of the 2-, 3-, and 4-O-acyl 2-fluorobenzoyl glucuronides are presented as half-lives in Table 1, where they are compared with the acyl migration half-life of the β-1-O-acyl isomer obtained earlier by HPLC analysis.15 The R-anomers have shorter half-lives than the β-ano-mers, but the β-anomers are formed faster than the R-anomers (see Figure 3). It follows from the kinetic model shown in Scheme 1 that the anomerization rates are faster than the acyl migration rates and that the anomerization rate R f β is faster than the rate β f R, in line with the known greater proportion of the β-anomer at equilibrium. The acyl migration rate of the 1-O-acyl isomer was, however, faster (by a factor of 4) than the acyl migration rates of all the positional isomers, because the 1-O-acyl isomer is the only compound that undergoes significant hydrolysis and because the acyl migration reaction from the β-1-O-acyl isomer to the 2570 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

Table 2. Acyl Migration Rates (h-1) for Positional Isomers of 2-Fluorobenzoyl D-Glucopyranuronates Used To Simulate Acyl Migration Kineticsa rate constant

R-anomer

β-anomer

k1-2 k2-3 k3-2 k3-4 k4-3

0.11 ( 0.02 0.08 ( 0.01 0.10 ( 0.01 0.08 ( 0.01

0.22 ( 0.02 0.07 ( 0.02 0.08 ( 0.01 0.08 ( 0.01 0.06 ( 0.02

a The rate constants (k) were estimated as the slope of the straight line obtained when the logarithms of the integrals (obtained from the NMR data) were plotted against time. The standard deviations presented are the errors of the slopes to the linear-fitted curves.

β-2-O-acyl isomer is not reversible. The formation rates for the glucuronide positional isomers of the 2-, 3-, and 4-O-acyl isomers were calculated similarly (data not shown). The acyl migration and formation rates obtained (see Table 2) were used to simulate the acyl migration reactions in the kinetic simulation program, and from this the anomerization rates were estimated. The NMR integrals of the resonances of the positional isomers plotted as a function of time can be seen in Figure 3A-C. The simulated curves obtained from the calculated acyl migration rates and formation rates for the glucuronide isomers are shown in Figure 3D-F. The curves obtained correspond closely to those expected for the consecutive reactions described by the model. The curves obtained from the NMR data are affected by the signalto-noise ratio of the NMR acquisitions (reflected in the errors on the estimated half-lives in Table 1 and rates in Table 2), as only low concentrations of the glucuronide conjugates are present (as necessary for good chromatographic separation). However, as the simulated curves fit the experimental data obtained, it can be assumed that the kinetic model postulated in Scheme 1 is valid for this system. To evaluate the validity of the estimated anomerization rates, the anomerization rates for the 4-O-acyl isomer were estimated experimentally in a separate experiment. The integrals of the selected protons (the doublets at δ 4.27 and 3.93) in the pseudotwo-dimensional plot were plotted as a function of time, when the R-4-O-acyl isomer (see Figure 4A) and the β-4-O-acyl isomer (see Figure 4B) were isolated in the NMR flow cell. From the initial part of these curves, the anomerization rates could readily be calculated, as the anomerization rates are a factor of ∼10 faster than the acyl migration rates. The fitted anomerization rates for the 4-O-acyl isomer were 0.50 h-1 for the anomerization β f R and 0.80 h-1 for the anomerization R f β. The experimentally estimated anomerization rates were 0.52 ( 0.1 and 0.89 ( 0.1 h-1, respectively. It could therefore be concluded that the anomerization rates fitted via the kinetics simulation program were trustworthy estimates. DISCUSSION The use of directly coupled dynamic stop-flow HPLC-NMR is shown here for the first time to solve a complex kinetic problem in physical organic chemistry. The construction of a kinetic model of acyl migration of drug glucuronide acyl migration would be extremely difficult to solve by any other method, as it necessitates rapid purification of interconverting compounds and following isomeric rearrangements in real time. Using HPLC-NMR

Figure 3. 1H NMR integrals of selected resonances for each positional isomer obtained using stop-flow HPLC-NMR. The integrals in the NMR spectra were measured relative to those of the aromatic region of the spectra, which were assumed to be constant through the time periods followed. The integrals in the selected 1H NMR spectra from the pseudo-two-dimensional plots were plotted against time for (A) 4-, (B) 3-, and (C) 2-O-acyl isomers and are compared with the simulated acyl migration curves, (D) 4-, (E) 3-, and (F) 2-O-acyl isomers. Symbols: (9) R-4-O-acyl isomers, (0) β-4-O-acyl isomers, ([) R-3-O-acyl isomers, (]) β-3-O-acyl isomers, (2) R-2-O-acyl isomers, and (4) β-2-O-acyl isomers.

spectroscopy, it was possible to differentiate between the different glucuronide positional isomers as well as their R- and β-anomers.26 Since data collection by NMR is nondestructive, it was possible to study on-line the kinetics of acyl migration of all the positional isomers of I. Two conclusions can be drawn from the estimated acyl migration rates: (i) degradation of the R-anomers is consistently faster than that of the β-anomers for all positional isomers and (ii) The forward and reverse reaction rates (e.g., k2-3, k3-2, or k3-4, k4-3) are equal within the errors of determination. The acyl

migration rates derived in these studies will not necessarily be the same in absolute value to actual rates occurring in vivo, as, in addition to temperature differences, the presence of tissue and serum esterases and nonspecific protein binding would affect rates in vivo. Furthermore, the presence of 1% acetonitrile, 20 mM (27) Wertz, P. W.; Garver, J. C.; Anderson, L. J. Am. Chem. Soc. 1981, 103, 3916-3922. (28) Sidelmann, U.; Gavaghan, C.; Carless, H. A. J.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. Anal. Chem. 1995, 67, 4441-4445. (29) Sidelmann, U.; Nicholls, A. W.; Meadows, P.; Gilbert, J.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. J. Chromatogr. A 1995, 728, 377-385.

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conditions are the closest possible to biological conditions consistent with rapid chromatographic separation and acyl migration studies. Investigations on the kinetics of mutarotation of simple sugars in aqueous solution have earlier been presented involving very complex sample preparation, which adds inaccuracy on the time scale and is very time consuming.27 The kinetic problem presented here is much more complex, as there is not a common intermediate; its only limitations lies in the number of scans needed per NMR spectrum for identification of concentration changes. We have shown that a faster reaction, such as mutarotation, can be followed by accumulating 32 scans, giving 1 min time increments; however, for the slower reacting acyl migration reactions, 15 min time increments are adequate and necessary in order to follow the formation of positional isomers at the concentration level used here. In the more general view of studying acyl migration reactions by the dynamic HPLC-NMR approach, it will be possible to investigate congeneric series of synthetic glucuronides under solvent conditions similar to those used here. Such studies are currently being undertaken in our laboratories for synthetic 3- and 4-fluorobenzoyl28 and 2-, 3-, and 4-(trifluoromethyl)benzoyl glucuronides, as well as the 2-, 3-, and 4-fluorophenylacetic acid glucuronides.29 Hence, it will be possible to further the understanding of the structural physicochemical properties that determine drug glucuronide reactivity and their related adverse drug responses in man, and so to improve drug design criteria for carboxylate or procarboxylate group containing drugs.

Figure 4. 1H NMR integrals of selected resonances for each anomer of the 4-O-acyl isomer of the 2-fluorobenzoyl glucuronide obtained using stop-flow HPLC-NMR. The integrals in the NMR spectra were measured relative to those of the aromatic region of the spectra. The integrals in the selected 1H NMR spectra from the pseudo-twodimensional plots were plotted against time when (A) the R-4-O-acyl isomers and (B) the R-4-O-acyl isomers were isolated in the NMR flow cell. Symbols: (9) R-4-O-acyl isomers and (0) β-4-O-acyl isomers.

phosphate buffer, and D2O in the HPLC solvent may have some effects on the observed rate constants, although these solvent

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ACKNOWLEDGMENT We thank The Danish Research Academy and BBSRC for supporting this project. In addition, we are grateful to Dr. D. B. Davies for helpful discussions.

Received for review January 5, 1996. Accepted April 30, 1996.X AC960014G X

Abstract published in Advance ACS Abstracts, June 1, 1996.