Both plant and mammalian cells express glucuronosyltransferases that catalyze glucuronidation of polyphenols such as flavonoids and other small molecules. Oral administration of select polyphenolic compounds leads to the accumulation of the corresponding glucuronidated metabolites at μM and sub-μM concentrations in the brain, associated with amelioration of a range of neurological symptoms. Determining the mechanisms whereby botanical extracts impact cognitive wellbeing and psychological resiliency will require investigation of the modes of action of the brain-targeted metabolites. Unfortunately, many of these compounds are not commercially available. This article describes the latest approaches for the analysis and synthesis of glucuronidated flavonoids. Synthetic schemes include both standard organic synthesis, semi-synthesis, enzymatic synthesis and use of synthetic biology utilizing heterologous enzymes in microbial platform organisms.

Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3′-O-methyl-quercetin-3-O-β-d-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4′-O-methyl-quercetin-7-O-β-d-glucuronide (4), 3-O-methyl-quercetin-3′-O-β-d-glucuronide (5), and 4′-O-methyl-quercetin-3′-O-β-d-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4′-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.

The first step towards eukaryotic genome duplication is loading of the replicative helicase, the Mcm2-7 complex, onto chromatin. This so-called "licensing" step is executed by the Pre- Replication Complex (Pre-RC) whose assembly on chromatin is nucleated by the DNA-binding activity of the Origin Recognition Complex (ORC). It is thought that metazoan ORC, like the yeast complex, is recruited directly to chromatin by its ATP-dependent DNA binding and encirclement activity. However, we have previously shown that this DNA binding mode is dispensable for chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. We show here that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this activity is regulated by IDR phosphorylation. In vitro studies show that the IDR alone binds DNA and this bestows the ORC holocomplex with a high-affinity ATP-independent DNA binding mode. Interestingly, we find that Orc1 IDRs have diverged so markedly across metazoans that they are unrecognizable as orthologs and yet we find that these compositionally homologous sequences retain DNA and chromatin binding activity down to basal metazoans. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity and we propose that this necessitates IDR-dependent chromatin tethering which poises ORC to opportunistically encircle nucleosome free regions as they become available. This work reveals a novel step in metazoan replication licensing and expands our understanding of disordered protein homology and evolution by stretching the relationship between primary structure and function.