Abstract
Selectivity of catechol O-methyltransferase has been examined for the three ring-fluorinated norepinephrines to elucidate the role of acidity of the phenolic groups in their methylation. Substitution of fluorine at the 5-position of norepinephrine reverses the selectivity of catechol O-methyltransferase so that p-O-methylation predominates. The 5-fluoro substituent also causes the pKa of the p-hydroxyl group to decrease substantially. In contrast, 2- and 6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group can play an important role in its ability to be methylated by catechol O-methyltransferase. Percentages of p-O-methylation of norepinephrine and its fluorinated derivatives increase with pH. This relative increase in p-O-methylation appears to accompany ionization of a group with pKa of 8.6, 7.7, 7.9, and 8.4 for norepinephrine and its 2-, 5-, and 6-fluoro derivative, respectively. These pKa values are the same as or similar to the pKa values of a phenolic hydroxyl group of these substrates. 3,4-Dihydroxybenzyl alcohol and its 5-fluoro derivative are O-methylated by catechol O-methyltransferase to form p- and m-O-methyl products in approximately 1:1 and 4:1 ratios, respectively, at all pH values. Based on the above results, a catechol-binding site model for catechol O-methyltransferase is proposed in which the two phenolic hydroxyl groups of catechol substrates are postulated to be approximately equally spaced from the methyl group of the cosubstrate S-adenosylmethionine.
Highlights
From the $Laboratoryof Molecular Pharmacology, Divisionof Biochemistry and Biophysics, Centerfor Drugs and Biologics, Food and Drug Administration, Bethesda, Maryland20205 and the §Laboratory of Chemistry and Uuboratoryof Bioorganic Chemistry
2- and6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group. These results suggest that acidity of a phenolic group canplay an important role in its ability to be methylated by catechol 0-methyltransferase
Nucleophilicity of the phenolic hydroxyl groups of catechol substrates are pos- nolic hydroxyl groups was accorded a secondary role to the tulated to be approximately spaced from the methyl group of the cosubstrateS-adenosylmethionine
Summary
2- and6-fluoronorepinephrines are methylated predominantly at the m-hydroxyl group These results suggest that acidity of a phenolic group canplay an important role in its ability to be methylated by catechol 0-methyltransferase. Hol and its 5-fluor0 derivative are 0-methylated by polar ionic groups on the side chain cause the substrate to catechol 0-methyltransferaseto form p- andm-0- bind predominantly in the orientation that brings only the methyl products in approximately 1:1 and 4: 1 ratios, m-hydroxyl group in the vicinity of the methyl group being respectively, at all pH values. 1,3, and 4 and references therein) leading to their metabolic inactivation or detoxication in most inevidence that methylation occurred predominantly atthe phenolic hydroxylic group which ionized more readily.These studies, provided strong evidence that nucleophilicity of the phenolic hydroxyl group played a more important role than polarity of the groups on the side chain in determining the site of methylation. Thesestudies were designed to elucidate the role of stereoelectronic factors in the regioselectivity of catechol 0-methyltransferase
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