Axial-selective H/D exchange of glycine-derived 1H-benzo[e][1,4]diazepin-2(3H)-ones: kinetic and computational studies of enantiomerization.

Glycine-derived 1H-benzo[e][1,4]diazepin-2(3H)-ones (BZDs) 5d-g featuring C9- and N1- substitution exhibit enantiomerization barriers too high to be measured by (1)H NMR coalescence experiments. To address this problem, we found that room-temperature H/D exchange of these compounds is remarkably selective, affording only the axial-d(1) isotopomers. (1)H NMR spectroscopy was then employed to measure the rate of conformational inversion of these d(1)-compounds at elevated temperatures. These studies reveal the highest enantiomerization barriers (up to 28 kcal/mol) ever determined for a BZD. Density functional theory calculations match the experimental enantiomerization barriers within 1.2 kcal/mol.

Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase.

UPPS (undecaprenyl pyrophosphate synthase) catalyses consecutive condensation reactions of FPP (farnesyl pyrophosphate) with eight isopentenyl pyrophosphates to generate C55 UPP, which serves as a lipid carrier for bacterial peptidoglycan biosynthesis. We reported the co-crystal structure of Escherichia coli UPPS in complex with FPP. Its phosphate head-group is bound to positively charged arginine residues and the hydrocarbon moiety interacts with hydrophobic amino acids including L85, L88 and F89, located on the alpha3 helix of UPPS. We now show that the monophosphate analogue of FPP binds UPPS with an eight times lower affinity (K(d)=4.4 microM) compared with the pyrophosphate analogue, a result of a larger dissociation rate constant (k(off)=192 s(-1)). Farnesol (1 mM) lacking the pyrophosphate does not inhibit the UPPS reaction. GGPP (geranylgeranyl pyrophosphate) containing a larger C20 hydrocarbon tail is an equally good substrate (K(m)=0.3 microM and kcat=2.1 s(-1)) compared with FPP. The shorter C10 GPP (geranyl pyrophosphate) displays a 90-fold larger K(m) value (36.0+/-0.1 microM) but similar kcat value (1.7+/-0.1 s(-1)) compared with FPP. Replacement of L85, L88 or F89 with Ala increases FPP and GGPP K(m) values by the same amount, indicating that these amino acids are important for substrate binding, but do not determine substrate specificity. With GGPP as a substrate, UPPS still catalyses eight isopentenyl pyrophosphate condensation reactions to synthesize C60 product. Computer modelling suggests that the upper portion of the active-site tunnel, where cis double bonds of the product reside, may be critical for determining the final product chain length.

Mode of action of 4-hydroxyphenylpyruvate dioxygenase inhibition by triketone-type inhibitors.

A series of 2-(2-nitrobenzoyl)cyclohexane-1,3-dione analogues (1-9) were designed, synthesized, and evaluated for inhibition of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), a key enzyme involved in the catabolism of tyrosine which catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate. The correlations between the results of enzyme inhibition, ferric chloride tests, and the conformational analysis suggested that the tight binding between triketone-type inhibitors and 4-HPPD is likely due to chelation of the enzyme-bound ferric iron with the enol tautomer of 1,3-diketone moiety of the triketones. The presence of a 2-carbonyl group in the triketone is an essential structural feature for potent 4-HPPD inhibition. Modification of the 3-carbonyl group of triketone moiety to other functionality will reduce the overall planarity and thus prevent keto-enol tautomerization, resulting in a decrease or lack of inhibition activity.

SAR studies of 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one as inhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver. The inhibition results indicated that well-positioned dicarbonyl groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC(50) of 15 nM, the rest of the synthesized analogues were less potent inhibitors than the parent compound.