Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders

Abstract

Glucuronidated and/or methylated metabolites of the proanthocyanidin (PA) monomer (-)-epicatechin are detected in both blood and brain following feeding of rodents with a monomeric grape seed PA extract shown to reduce symptoms in a mouse model of Alzheimer's disease. To generate metabolites for future mechanistic studies, we investigated the ability of recombinant human glucuronosyl transferases of the UGT1A and UGT2B families to glucuronidate epicatechin or 3'-O-methyl epicatechin in vitro. Of twelve enzymes tested, UGT1A9 was the most efficient, producing epicatechin 3'-O-glucuronide as the major product. Incubation of UGT1A9 with 3'-O-methyl-epicatechin resulted in two major products, one of which was identified as 3'-O-methyl-epicatechin 5-O-glucuronide, a major metabolite found in blood plasma and brain tissue of the rodents following feeding with a grape seed extract. We also investigated in vitro methylation of epicatechin and epicatechin glucuronides by human catechol O-methyltransferase. Enzymatic production of 3'-O-methyl-epicatechin 5-O-glucuronide was optimized to 50% overall yield. These studies form a basis for generation of mg quantities of pure epicatechin (methyl) glucuronides of biological significance, and provide clarification of structure of previously identified epicatechin metabolites.

Figure 1

LC/MS comparisons of epicatechin metabolites found in the plasma of mice fed GSPE or generated from epicatechin in vitro by the activity of human UGT1A9. (A) the glucuronidated epicatechin products formed in vitro with UGT1A9. (B) the glucuronidated (epi)catechin metabolites found in the plasma of mice that had been fed monomer enriched GSPE. (C) the purified major product from the glucuronidation of 3′-O-methyl-epicatechin with UGT1A9. (D) the glucuronidated methyl-(epi)catechin compounds found in the plasma of rats that had been fed monomer enriched GSPE. Peak 5 is (−)-epicatechin-3′-O-glucuronide. Peak 4 is an epicatechin glucuronide and an exact match based on retention time and mass spectral data to peak 3 from the rat plasma. Peak 6 has been identified as 3′-O-methyl-epicatechin-5-O-glucuronide by microcoil NMR (Table 2) and is an exact match to peak 7 from the rat plasma based on retention time and mass spectral data. Mass spectral data for peaks 3,4,6, and 7 are shown in Supplemental Figure 1.

Figure 2

The structure of (−)-epicatechin with R groups at the 3′, 4′, or 5 positions which may be hydroxylated, O-methylated, or O-glucuronidated. 3′-OH, 4′-OH, 5-OH = epicatechin.

Figure 3

Glucuronidation of epicatechin with UGT1A9 and partial purification of the glucuronidated products. HPLC chromatograms were monitored at 280nm. (A) control reaction. (B) UGT1A9 reaction. (C) organic phase of the ethyl acetate extraction of (B). (D) aqueous phase of the ethyl acetate extraction of (B). Identified peaks are: 1, (−)-epicatechin; 2, (−)-epicatechin-3′-O-glucuronide; 3, (−)-epicatechin glucuronide.

Figure 4

Glucuronidation of 3′-O-methyl-epicatechin by UGT1A9. HPLC chromatograms were monitored at 280nm. (A) control reaction. (B) UGT1A9 reaction. Identified peaks are: 1, 3′-O-methyl-epicatechin; 2, 3′-O-methyl-epicatechin-5-O-glucuronide; peaks 3–5 are 3′-O-methyl-epicatechin glucuronides.

Figure 5

Modification of epicatechin by glucuronidation followed by methylation or methylation followed by glucuronidation. HPLC chromatograms were monitored at 280nm. (A) control glucuronidation reaction. (B) 22 h glucuronidation reaction with UGT1A9. (C) 22 h glucuronidation reaction followed by 6 h methylation reaction with COMT. (D) control COMT reaction. (E) 6 h COMT reaction. ( F) 6 h COMT reaction followed by 22 h glucuronidation reaction with UGT1A9. Identified peaks are: 1, (−)-epicatechin; 2, (−)-epicatechin-3′-O-glucuronide; 3, 3′-O-methyl-epicatechin; 4, 4′-O-methyl-epicatechin; 5, 3′-O-methyl-epicatechin-5-O-glucuronide; 6, 4′-O-methyl-epicatechin glucuronide.