Measurement of Internal Acyl Migration Reaction Kinetics Using Directly Coupled HPLC−NMR:  Application for the Positional Isomers of Synthetic (2-Fluorobenzoyl)-d-glucopyranuronic Acid

Abstract

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.

Stereospecific pH‐dependent degradation kinetics of R‐ and S‐naproxen‐β‐l‐O‐acyl‐glucuronide

“…56 In contrast, it was found by dynamic stoppedflow HPLC- 1 H NMR experiments that the a-and b-anomers of the 4-O-acyl isomer of (2-fluorobenzoyl)-D-glucopyranuronic acid were more stable than the corresponding a-and b-anomers of the 2-and 3-O-acyl isomers. 41 Diflunisal b-1-Oacyl glucuronide hydrolyzes faster than any of the acylmigrated isomers, 56 similar to the findings in the present study, where k b1-D is higher than k X-D for both R-and S-naproxen glucuronide isomers at the pH values studied. The a-anomers of the 2-, 3-and 4-O-acyl isomers of (2-fluorobenzoyl)-D-glucopyranuronic acid were found to be less stable than the corresponding b-anomers.…”

“…The a-anomers of the 2-, 3-and 4-O-acyl isomers of (2-fluorobenzoyl)-D-glucopyranuronic acid were found to be less stable than the corresponding b-anomers. 41 Acylmigration rates were faster between the a-anomers than between the b-anomers, and also the equilibrium between the individual a/b-anomers favored the b-anomer, reflecting the higher reactivity of the a-anomers.…”

“…Stopped-flow HPLC-1 H NMR has been used in several cases to identify the acyl-migrated isomers of b-1-O-acyl glucuronides unambiguously 9,12 and dynamic NMR has been used to quantify the individual acyl-migrated isomers in mixtures. 10,13,40,41 Another approach is to use HPLC with UV detection and the method of sequential appearance. The general acyl migration scheme depicted in Figure 2 shows the sequential migration of the aglycone from the biosynthetic b-1-O-acyl glucuronide via the 2-O-acyl isomer to the 3-O-acyl isomer and finally the 4-O-acyl isomer.…”

Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates

“…As indicated by the almost identical chromatographic profiles obtained following preparation of multiple batches of the isomer mixture, the relative amounts of each positional isomer was similar. This result was consistent with earlier publications 21, 22. The final mixture was used for column screening and further chromatographic method development.…”

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

“…By combining the strength of nuclear magnetic resonance (NMR) spectroscopy with the ability to separate complex mixtures by HPLC, it is possible to determine the structures of compounds of interest rapidly and efficiently. While most HPLC/NMR applications have been reported in the pharmaceutical industry on studies of combinatorial chemistry,5 on studies of chemical impurities,6 drug formulations and composition7 and drug metabolism,8–10 results have also been reported for natural products,11–14 chemical reactivity15 and polymer research 16. In addition, the complementarity of the structural information derived from NMR spectroscopy and MS is enhanced if these data are collected in the same separation run.…”