Computational methods for the study of enzymic reaction mechanisms III: a perturbation plus QM/MM approach for calculating relative free energies of protonation

We describe a coupling parameter, that is, perturbation, approach to effectively create and annihilate atoms in the quantum mechanical Hamiltonian within the closed shell restricted Hartree-Fock formalism. This perturbed quantum mechanical atom (PQA) method is combined with molecular mechanics (MM) methods (PQA/MM) within a molecular dynamics simulation, to model the protein environment (MM region) effects that also make a contribution to the overall free energy change. Using the semiempirical PM3 method to model the QM region, the application of this PQA/MM method is illustrated by calculation of the relative protonation free energy of the conserved OD2 (Asp27) and the N5 (dihydrofolate) proton acceptor sites in the active site of Escherichia coli dihydrofolate reductase (DHFR) with the bound nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. For a number of choices for the QM region, the relative protonation free energy was calculated as the sum of contributions from the QM region and the interaction between the QM and MM regions via the thermodynamic integration (TI) method. The results demonstrate the importance of including the whole substrate molecule in the QM region, and the overall protein (MM) environment in determining the relative stabilities of protonation sites in the enzyme active site. The PQA/MM free energies obtained by TI were also compared with those estimated by a less computationally demanding nonperturbative method based on the linear response approximation (LRA). For some choices of QM region, the total free energies calculated using the LRA method were in very close agreement with the PQA/MM values. However, the QM and QM/MM component free energies were found to differ significantly between the two methods.     

Heterobimetallic reductive cross-coupling of benzonitrile with carbon dioxide, pyridine, and benzophenone

Described herein are heterobimetallic radical cross-coupling reactions between the benzonitrile adduct of the molybdenum(III) complex Mo(N[t-Bu]Ar)3 (Ar = 3,5-C6H3Me2) and titanium(III) complexes with carbon dioxide, pyridine, and benzophenone. The titanium(III) system employed was either Ti(N[t-Bu]Ar)3 (Ar = 3,5-C6H3Me2) or Ti(N[t-Bu]Ph)3. Crystal structure studies are described for the Mo/PhCN/CO2/Ti coupled system and for an analogue of the Mo/PhCN/Ph2CO/Ti coupled system in which PhCN is replaced with 2,6-Me2C6H3CN. In the case of the couplings involving pyridine and benzophenone, C-C bond formation takes place with dearomatization, with the new C-C bond being formed between the nitrile carbon of PhCN and the para carbon of pyridine or one of the benzophenone phenyl groups. Of the radical metal complex/substrate adducts invoked in this work, that between titanium(III) and CO2 is the only one not directly observable. In all cases, the selective cross-coupling reactions are interpreted as arising by heterodimerization of titanium(III) substrate complexes (substrate = CO2, py, or Ph2CO) with the persistent molybdenum-PhCN radical adduct. All of the heterobimetallic coupling products are diamagnetic, and the metal ions Ti and Mo in them both are assigned to the formal 4+ oxidation state.     

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

Summary R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand–ligand and ligand–protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.     

Phosphaalkynes from acid chlorides via P for O(Cl) metathesis: a recyclable niobium phosphide (P(3-)) reagent that effects C-P triple-bond formation

Reported herein is a new, metathetical P for O(Cl) exchange mediated by an anionic niobium phosphide complex that furnished phosphaalkynes (RCP) from acid chlorides (RC(O)Cl) under mild conditions. The niobaziridine hydride complex, Nb(H)(tBu(H)C=NAr)(N[Np]Ar)2 (1, Np = neopentyl, Ar = 3,5-Me2C6H3), has been shown previously to react with elemental phosphorus (P4), affording the mu-diphosphide complex, (mu2:eta2,eta2-P2)[Nb(N[Np]Ar)3]2, (2), which can be subsequently reduced by sodium amalgam to the anonic, terminal phosphide complex, [Na][PNb(N[Np]Ar)3] (3). It is now shown that treatment of 3 with either pivaloyl (t-BuC(O)Cl) or 1-adamantoyl (1-AdC(O)Cl) chloride provides the thermally unstable niobacyles, (t-BuC(O)P)Nb(N[Np]Ar)3 (4-t-Bu) and (1-AdC(O)P)Nb(N[Np]Ar)3 (4-1-Ad), which are intermediates along the pathway to ejection of the known phosphaalkynes t-BuCP (5-t-Bu) and 1-AdCP(5-1-Ad). Phosphaalkyne ejection from 4-t-Bu and 4-1-Ad proceeds with formation of the niobium(V) oxo complex ONb(N[Np]Ar)3 (6) as a stable byproduct. Preliminary kinetic measurements for fragmentation of 4-t-Bu to 5-t-Bu and 6 in C6D6 solution are consistent with a first-order process, yielding the thermodynamic parameters DeltaH = 24.9 +/- 1.4 kcal mol-1 and DeltaS = 2.4 +/- 4.3 cal mol-1 K-1 over the temperature range 308-338 K. Separation of volatile 5-t-Bu from 6 after thermolysis has been readily achieved by vacuum transfer in yields of 90%. Pure 6 is recovered after vacuum transfer and can be treated with 1.0 equiv of triflic anhydride (Tf2O, Tf = O2SCF3) to afford the bistriflate complex, Nb(OTf)2(N[Np]Ar)3 (7), in high yield. Complex 7 provides direct access to 1 upon reduction with magnesium anthracene, thus completing a cycle of element activation, small-molecule generation via metathetical P-atom transfer, and deoxygenative recycling of the final niobium(V) oxo product.     

Recognition of base-pairing by DNA polymerases during nucleotide incorporation: the properties of the mutagenic nucleotide dPTP alphaS

The highly mutagenic nucleoside dP (6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) is a bicyclic analogue of N4-methoxy-2'-deoxycytidine. It exists as a mixture of its imino and amino tautomers in solution with a ratio of about 10:1 based on its tautomeric constant. The bicyclic nature of the heterocycle P restrains the amino substituent in an anti conformation and permits effective Watson-Crick base-pairing using either tautomer. The specificity of incorporation of dP by the 3'-5'-exonuclease-free Klenow fragment of DNA polymerase I (exo-free Klenow) has been studied using the 5'-(1-thio)triphosphate dPTP alphaS in combination with phosphorothioate-specific sequencing of the DNA products. The method provides a convenient qualitative assay for studying nucleotide incorporation and reveals for the first time a potential role for the minor tautomeric forms of the natural DNA bases in base misinsertion (substitution mutagenesis) during replication.