Mechanism of enzymatic Birch reduction: stereochemical course and exchange reactions of benzoyl-CoA reductase

Dearomatizing benzoyl-coenzyme A reductases (BCR) from facultatively anaerobic bacteria are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of benzoyl-CoA (BCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA). A Birch reduction mechanism involving alternate electron transfer and protonation steps has been proposed for BCR. In this work we reacted BCoA in H2O and D2O, and d5-BCoA in H2O with BCR and the second enzyme of the pathway, dienoyl-CoA hydratase (DCH). The 1,4 hydration product formed from the dienoyl-CoA, 6-hydroxycyclohex-1-ene-1-carbonyl-CoA, was analyzed by several NMR techniques. The results obtained indicate that BCR stereoselectively forms the trans-dienoyl-CoA product, and DCH stereoselectively catalyzes a trans-1,4 water addition. Moreover, unexpected proton exchanges at C-2 and C-6 were observed. They indicate that a free radical intermediate with an unusual low pKa is formed during BCR catalysis. This finding provides evidence for the proposed Birch reduction mechanism of BCR and is in agreement with the established radical mechanism of homologous alpha-hydroxyacyl-CoA dehydratases.     

Analysis of chemical shift changes reveals the binding modes of isoindolinone inhibitors of the MDM2-p53 interaction

In this study we present a method for defining the binding modes of a set of structurally related isoindolinone inhibitors of the MDM2-p53 interaction. This approach derives the location and orientation of isoindolinone binding, based on an analysis of the patterns of magnitude and direction of chemical shift perturbations for a series of inhibitors of the MDM2-p53 interaction. The MDM2-p53 complex is an attractive target for therapeutic intervention in cancer cells with intact tumor suppressor p53, as it offers the possibility of releasing p53 by blocking the MDM2-p53 binding site with a small molecule antagonist to promote apoptosis. Isoindolinones are a novel class of MDM2-antagonists of moderate affinity, which still require the development of more potent candidates for clinical applications. As the applicability of conventional structural methods to this system is limited by a number of fundamental factors, the exploitation of the information contained in chemical shift perturbations has offered a useful route to obtaining structural information to guide the development of more potent compounds. For a set of 12 structurally related isoindolinones, the data suggests 4 different orientations of binding, caused by subtle changes in the chemical structure of the inhibitors.     

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)‐benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing ²H and ³H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA‐thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.     

Quinolinone and pyridopyrimidinone inhibitors of DNA-dependent protein kinase

8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).     

Experimental Study of Hydrogen Bonding Potentially Stabilizing the 5′‐Deoxyadenosyl Radical from Coenzyme B12

Coenzyme B₁₂ can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co? C bond homolysis of coenzyme B₁₂ to cob(II)alamin and the 5′‐deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19? H to the 3′‐O of the ribose moiety of the incipient 5′‐deoxyadenosyl radical, which is stabilized by 30 kJ mol^?1 (B. Durbeej et al., Chem. Eur. J. 2009 , 15, 8578–8585). The diastereoisomers (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin were used as models for coenzyme B₁₂. A downfield shift of the NMR signal for the C19? H proton was observed for the (R)‐isomer (δ=4.45 versus 4.01 ppm for the (S)‐isomer) and can be ascribed to an intramolecular hydrogen bond between the C19? H and the oxygen of CHOH. Crystal structures of (R)‐ and (S)‐2,3‐dihydroxypropylcobalamin showed C19? H???O distances of 3.214(7) Å (R‐isomer) and 3.281(11) Å (S‐isomer), which suggest weak hydrogen‐bond interactions (?ΔG<6 kJ mol^?1) between the CHOH of the dihydroxypropyl ligand and the C19? H. Exchange of the C19? H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3–11. No exchange was found for any of the cobalt oxidation states. 3′,5′‐Dideoxyadenosylcobalamin, but not the 2′,5′‐isomer, was found to act as a coenzyme for glutamate mutase, with a 15‐fold lower k_cat/K_M than 5′‐deoxyadenosylcobalamin. This indicates that stabilization of the 5′‐deoxyadenosyl radical by a hydrogen bond that involves the C19? H and the 3′‐OH group of the cofactor is, at most, 7 kJ mol^?1 (?ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2′‐OH and 3′‐OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19? H and the 3′‐O of the ribose moiety of the 5′‐deoxyadenosyl group is concluded not to exceed 6 kJ mol^?1 (?ΔG).     

Revisiting the reaction of hydroxyl radicals with vicinal diols in water

The carbonyl products of the reactions of hydroxyl radicals with three vicinal diols (ethane-1,2-diol, propane-1,2-diol and butane-2,3-diol) have been identified and quantified. Hydroxyl radicals were produced by γ-radiolysis of N(2)O-saturated aqueous solutions. The reactions result in the formation of alkoxyl radicals (~15%) followed by β-fragmentation, and α-hydroxyl alkyl radicals that undergo H(2)O elimination. The latter process is part of a radical chain reaction at higher diol concentrations.