Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides.
Two clenbuterol O-glucuronide diastereomers were synthesized by the Koenigs-Knorr reaction. Structures and glucuronidation sites of the glucuronides were characterized by tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The two diastereomers were used as standard compounds in studies of stereoselective glucuronidation of clenbuterol with liver microsomes from different species and with 15 human recombinant UDP-glucuronosyltransferases. In this study, chemical and enzymatic reactions produced only O-glucuronides of clenbuterol, although on the basis of the chemical structure of the aglycone, both O- and N-glucuronides of clenbuterol could be formed. Differences in the production of diastereomers of clenbuterol glucuronides were observed among liver microsomes from the various animals. Dog and bovine liver microsomes were significantly active, and also stereoselective, each producing only one but a different diastereomer. Liver microsomes from rabbit and rat were also rather actively glucuronidating clenbuterol, but human, pig, and moose liver microsomes produced only minor amounts of glucuronides. Human liver microsomes produced only one clenbuterol glucuronide diastereomer, and the same was true of the human UDP-glucuronosyltransferases that were active (formation of glucuronide: 1A9 > 1A10 >> 1A7). The marked differences in the stereoselective glucuronidation of clenbuterol show that UDP-glucuronosyltransferases in the livers of different animals do not have the same functions, activities, or distribution. This needs to be taken into account, particularly in toxicology testing.
Alonen
Anna
A
Faculty of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland.
Gartman
Minna
M
Aitio
Olli
O
Finel
Moshe
M
Yli-Kauhaluoma
Jari
J
Kostiainen
Risto
R
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002976:Clenbuterol / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D004285:Dogs; D020719:Glucuronides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D015394:Molecular Structure; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D053719:Tandem Mass Spectrometry
D020719:Glucuronides; D011994:Recombinant Proteins; D014453:Glucuronosyltransferase; D002976:Clenbuterol