Measurement of internal acyl migration reaction kinetics using directly coupled HPLC-NMR: Application for the positional isomers of synthetic (2-fluorobenzoyl)-D-glucopyranuronic acid

Ester glucuronides (1-O-acyl-beta-D-glucopyranuronates) of many drugs may undergo internal acyl migration reactions, resulting in the formation of new positional isomers with both alpha- and beta-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 beta-1-O-aryl, alpha- and beta-2-O-acyl, alpha- and beta-3-O-acyl, and alpha- and beta-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 online NMR now probe ina 600 MHz NMR spectrometer. The now was stopped with each isomer in the NMR now probe, and sequential NMR spectra were collected at 25 degrees 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: beta-1-O-acyl, 0.29 +/- 0.01; alpha-2-O-acyl, 0.11 +/- 0.01; beta-2-O-acyl, 0.07 +/- 0.01; alpha-3-O-acyl, 0.10 +/- 0.01; beta-3-O-acyl, 0.09 +/- 0.01; alpha-4-O-acyl, 0.09 +/- 0.01; and beta-4-O-acyl, 0.06 +/- 0.01. The alpha- and beta-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 (alpha --> beta) and 0.50 h(-1) (beta --> alpha), 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.