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Directly coupled HPLC-lH NMR was used in the "stopfiaw"mode to separate and rapidlyidenwan equilibrated mixture of ester glucuronide isomers formed spontaneously by intramolecular rearrangement reactions ( i t e d acyl migration and mutarotation) of 2-fluorobenzoic acid B-1-glucuronide (1-0-(2-fluorobenzoyl)-~-glucopyranuronic acid). The equilibrated mixture of isomers was obtained by incubation of the synthetic 2-fluorobenzoic acid glucuronide in buffer solution (PH 7.4) at 25 "C for 24 h. The B-anomer of the 1-0-acyl glucuronide, and the 2-, 3-, and 4-positional glucuronide isomers (all three as both a-and B-anomers) present in the equilibrium mixture, were all characterized after separation in an isocratic chromatographic system containing phosphate buffer at pH 7.4 and 1% acetonitrile in the mobile phase. The HPLC-NMR investigations also elucidated the mutarotation of the positional glucuronide isomers as well as showing the bene& of the HPLC-NMR technique as a primary analytical tool. This HPLC-NMR method will be of particular value in studies on the acyl migration reactions of nonsteroidal ant3da"atory drug glucuronides which may be related to their toxicological properties. Abstract published in Advance ACS Abstracts, August 15, 1995.

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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 α- 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-O-acyl, α- and β-2-O-acyl, α- and β-3-O-acyl, and α- and β-4-O-acyl isomers at pH 7.4 in 20 mM phosphate buffer. The interconverting isomers were separated using reversed-phase HPLC and pumped directly into a dedicated on-line 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; α-2-O-acyl, 0.11 ± 0.01; β-2-O-acyl, 0.07 ± 0.01; α-3-O-acyl, 0.10 ± 0.01; β-3-O-acyl, 0.09 ± 0.01; α-4-O-acyl, 0.09 ± 0.01; and β-4-O-acyl, 0.06 ± 0.01. The α- 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 (α → β) and 0.50 h(-)(1) (β → α), whereas the experimentally estimated anomerization rates were 0.89 ± 0.1 and 0.52 ± 0.1 h(-)(1), respectively. The dynamic stop-flow HPLC-NMR 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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