Rapid internal acyl migration and protein binding of synthetic probenecid glucuronides

Chem Res Toxicol. 2002 Jun;15(6):765-72. doi: 10.1021/tx010166t.


Internal acyl migration reactions of drug 1-O-acyl-beta-D-glucopyranuronates (1beta-acyl glucuronides) are of interest because of their possible role in covalent binding to proteins and consequent adverse effects. The reactivity of the synthetic probenecid 1beta-acyl glucuronide (PRG), the principal metabolite of probenecid (PR) in humans, has been investigated in terms of acyl migration, hydrolysis, and covalent binding to proteins in phosphate buffer (pH 7.4) and human plasma at 37 degrees C. PRG primarily degraded by acyl migration according to apparent first-order kinetics and the 2-, 3-, and 4-acyl isomers sequentially appeared as both alpha- and beta-anomeric forms. In addition, small amounts of PRG and extremely labile 1alpha-acyl isomer existed in the equilibrated mixture favoring the 2alpha/beta-acyl isomer, that provided significant information regarding the mechanism of acyl migration. All of the positional isomers and anomers were characterized using preparative HPLC and NMR spectroscopy. Acyl migration was observed to predominate over hydrolysis in both media although the extent of hydrolysis in plasma was larger than that in the buffer. The overall degradation half-lives (h) in the buffer and plasma were 0.27 +/- 0.003 and 0.17 +/- 0.007, respectively. The covalent binding rapidly proceeded mainly via the Schiff's base mechanism and reached a plateau after 2 h of incubation. The maximal binding was 146 +/- 4.8 pmol/mg of protein, and ca. 10% of the initial concentration of PRG. These results indicated that PRG is most labile and susceptible to acyl migration of all the drug acyl glucuronides reported to date in the physiological conditions, and highly reactive to plasma proteins, that could provide a possible explanation for the immunologically based adverse effects of PR.

MeSH terms

  • Acylation
  • Blood Proteins / metabolism*
  • Buffers
  • Chromatography, High Pressure Liquid
  • Drug Stability
  • Glucuronides / blood*
  • Glucuronides / chemical synthesis
  • Glucuronides / chemistry
  • Glucuronides / metabolism
  • Half-Life
  • Humans
  • Hydrolysis
  • Kinetics
  • Nuclear Magnetic Resonance, Biomolecular
  • Probenecid / adverse effects
  • Probenecid / blood*
  • Probenecid / chemistry
  • Probenecid / immunology
  • Protein Binding
  • Serum Albumin / metabolism
  • Stereoisomerism


  • Blood Proteins
  • Buffers
  • Glucuronides
  • Serum Albumin
  • Probenecid