One-Pot Phosphate-Mediated Synthesis of Novel 1,3,5-Trisubstituted Pyridinium Salts: A New Family of S. aureus Inhibitors.

Polysubstituted pyridinium salts are valuable pharmacophores found in many biologically active molecules. Their synthesis typically involves the use of multistep procedures or harsh reaction conditions. Here, we report water-based phosphate mediated reaction conditions that promote the condensation of arylacetaldehydes with amines to give 1,3,5-pyridinium salts. The reaction, carried out at pH 6, provides conditions suitable for the use of less stable aldehydes and amines in this Chichibabin pyridine condensation. The evaluation of selected 1,3,5-trisubstituted pyridinium salts highlighted that they can inhibit the growth of S. aureus in the low µg/mL range. The synthetic accessibility of these compounds and preliminary growth inhibition data may pave the way towards the discovery of new anti-bacterials based on the 1,3,5-trisubstituted pyridinium scaffold.

Tetrahydroisoquinolines affect the whole-cell phenotype of Mycobacterium tuberculosis by inhibiting the ATP-dependent MurE ligase.

OBJECTIVES: (S)-Leucoxine, isolated from the Colombian Lauraceae tree Rhodostemonodaphne crenaticupula Madriñan, was found to inhibit the growth of Mycobacterium tuberculosis H37Rv. A biomimetic approach for the chemical synthesis of a wide array of 1-substituted tetrahydroisoquinolines was undertaken with the aim of elucidating a common pharmacophore for these compounds with novel mode(s) of anti-TB action. METHODS: Biomimetic Pictet-Spengler or Bischler-Napieralski synthetic routes were employed followed by an evaluation of the biological activity of the synthesized compounds. RESULTS: In this work, the synthesized tetrahydroisoquinolines were found to inhibit the growth of M. tuberculosis H37Rv and affect its whole-cell phenotype as well as the activity of the ATP-dependent MurE ligase, a key enzyme involved in the early stage of cell wall peptidoglycan biosynthesis. CONCLUSIONS: As the correlation between the MIC and the half-inhibitory enzymatic concentration was not particularly strong, there is a credible possibility that these compounds have pleiotropic mechanism(s) of action in M. tuberculosis.

The C-glycosylation of flavonoids in cereals.

Flavonoids normally accumulate in plants as O-glycosylated derivatives, but several species, including major cereal crops, predominantly synthesize flavone-C-glycosides, which are stable to hydrolysis and are biologically active both in planta and as dietary components. An enzyme (OsCGT) catalyzing the UDP-glucose-dependent C-glucosylation of 2-hydroxyflavanone precursors of flavonoids has been identified and cloned from rice (Oryza sativa ssp. indica), with a similar protein characterized in wheat (Triticum aestivum L.). OsCGT is a 49-kDa family 1 glycosyltransferase related to known O-glucosyltransferases. The recombinant enzyme C-glucosylated 2-hydroxyflavanones but had negligible O-glucosyltransferase activity with flavonoid acceptors. Enzyme chemistry studies suggested that OsCGT preferentially C-glucosylated the dibenzoylmethane tautomers formed in equilibrium with 2-hydroxyflavanones. The resulting 2-hydroxyflavanone-C-glucosides were unstable and spontaneously dehydrated in vitro to yield a mixture of 6C- and 8C-glucosyl derivatives of the respective flavones. In contrast, in planta, only the respective 6C-glucosides accumulated. Consistent with this selectivity in glycosylation product, a dehydratase activity that preferentially converted 2-hydroxyflavanone-C-glucosides to the corresponding flavone-6C-glucosides was identified in both rice and wheat. Our results demonstrate that cereal crops synthesize C-glucosylated flavones through the concerted action of a CGT and dehydratase acting on activated 2-hydroxyflavanones, as an alternative means of generating flavonoid metabolites.

Characterization and engineering of the bifunctional N- and O-glucosyltransferase involved in xenobiotic metabolism in plants.

The glucosylation of pollutant and pesticide metabolites in plants controls their bioactivity and the formation of subsequent chemical residues. The model plant Arabidopsis thaliana contains >100 glycosyltransferases (GTs) dedicated to small-molecule conjugation and, whereas 44 of these enzymes catalyze the O-glucosylation of chlorinated phenols, only one, UGT72B1, shows appreciable N-glucosylating activity toward chloroanilines. UGT72B1 is a bifunctional O-glucosyltransferase (OGT) and N-glucosyltransferase (NGT). To investigate this unique dual activity, the structure of the protein was solved, at resolutions up to 1.45 A, in various forms including the Michaelis complex with intact donor analog and trichlorophenol acceptor. The catalytic mechanism and basis for O/N specificity was probed by mutagenesis and domain shuffling with an orthologous enzyme from Brassica napus (BnUGT), which possesses only OGT activity. Mutation of BnUGT at just two positions (D312N and F315Y) installed high levels of NGT activity. Molecular modeling revealed the connectivity of these residues to H19 on UGT72B1, with its mutagenesis exclusively defining NGT activity in the Arabidopsis enzyme. These results shed light on the conjugation of nonnatural substrates by plant GTs, highlighting the catalytic plasticity of this enzyme class and the ability to engineer unusual and desirable transfer to nitrogen-based acceptors.

'Dopamine-first' mechanism enables the rational engineering of the norcoclaurine synthase aldehyde activity profile.

Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the Pictet-Spengler condensation of dopamine and an aldehyde, forming a substituted (S)-tetrahydroisoquinoline, a pharmaceutically important moiety. This unique activity has led to NCS being used for both in vitro biocatalysis and in vivo recombinant metabolism. Future engineering of NCS activity to enable the synthesis of diverse tetrahydroisoquinolines is dependent on an understanding of the NCS mechanism and kinetics. We assess two proposed mechanisms for NCS activity: (a) one based on the holo X-ray crystal structure and (b) the 'dopamine-first' mechanism based on computational docking. Thalictrum flavum NCS variant activities support the dopamine-first mechanism. Suppression of the non-enzymatic background reaction reveals novel kinetic parameters for NCS, showing it to act with low catalytic efficiency. This kinetic behaviour can account for the ineffectiveness of recombinant NCS in in vivo systems, and also suggests NCS may have an in planta role as a metabolic gatekeeper. The amino acid substitution L76A, situated in the proposed aldehyde binding site, results in the alteration of the enzyme's aldehyde activity profile. This both verifies the dopamine-first mechanism and demonstrates the potential for the rational engineering of NCS activity.

Phosphate mediated biomimetic synthesis of tetrahydroisoquinoline alkaloids.

A one-pot synthesis of tetrahydroisoquinoline alkaloids in a phosphate buffer has been achieved, and a reaction mechanism proposed. The utilisation of mild reaction conditions readily afforded a range of isoquinolines, including norcoclaurine.

Role of a carboxylesterase in herbicide bioactivation in Arabidopsis thaliana.

Arabidopsis thaliana contains multiple carboxyesterases (AtCXEs) with activities toward xenobiotics, including herbicide esters that are activated to their phytotoxic acids upon hydrolysis. On the basis of their susceptibility to inhibition by organophosphates, these AtCXEs are all serine hydrolases. Using a trifunctional probe bearing a fluorophosphonate together with biotin and rhodamine to facilitate detection and recovery, four dominant serine hydrolases were identified in the proteome of Arabidopsis. Using a combination of protein purification, capture with the trifunctional probe and proteomics, one of these hydrolases, AtCXE12, was shown to be the major carboxyesterase responsible for hydrolyzing the pro-herbicide methyl-2,4-dichlorophenoxyacetate (2,4-D-methyl) to the phytotoxic acid 2,4-dichlorophenoxyacetic acid. Recombinant expression of the other identified hydrolases showed that AtCXE12 was unique in hydrolyzing 2,4-D-methyl. To determine the importance of AtCXE12 in herbicide metabolism and efficacy, the respective tDNA knock-out (atcxe12) plants were characterized and shown to lack expression of AtCXE12 and have greatly reduced levels of 2,4-D-methyl-hydrolyzing activity. Young atcxe12 seedlings were less sensitive than wild type plants to 2,4-D-methyl, confirming a role for the enzyme in herbicide bioactivation in Arabidopsis.