Human UDP-glucuronosyltransferase UGT1A4 forms tertiary N-glucuronides predominately with the energetically less favored tautomer of substituted 1H-indazole (benzpyrazole)

Abstract

Abstract Background Human UDP-glucuronosyltransferase UGT1A4 is reported to convert a range of tertiary amine substrates to the respective quaternary glucuronides. In this study, we report on the in vitro glucuronidation of a substituted 1H-indazole (benzpyrazole), a transient receptor potential ankyrin 1 antagonist. Methods Depending on the mammalian liver used for production, two peaks with different UV spectra and slightly different MS/MS spectra were found in the LC-MS/MS analysis. An optimized HPLC method gave a baseline separation of the two glucuronides. Preparation and isolation allowed to assign their structure by 1H-, 13C-, and 15N-NMR. Results Both nitrogens of this structure were accessible to glucuronidation resulting in the respective tertiary glucuronides. As expected, the N-glucuronidation proceeded with marked species difference. Surprisingly, the glucuronide produced by human liver homogenate and by the majority of liver preparations from 11 animal species was attached at the energetically less favorable 2-position, indicating the regioselectivity of the respective enzyme. Among 13 recombinant human UDP-glucuronosyltransferases tested only the isoenzyme 1A4 showed significant formation of both glucuronides with the energetically less favorable N-glucuronide in 33-fold excess. An MS/MS fragmentation mechanism was proposed for a fragment loss of 134 Da that differentiated the spectra of the two glucuronides. Conclusions This investigation shows that assumptions on the structure of metabolites based on stability of tautomers may be wrong. A novel meachanism to form tertiary N-glucuronides was proposed.

Keywords

• Transient Receptor Potential Vanilloid
• Transient Receptor Potential
• Proton Affinity
• Axitinib
• Transient Receptor Potential Ankyrin