Evidence for substrate binding-induced zwitterion formation in the catalytic Cys-His dyad of the SARS-CoV main protease.

The coronavirus main protease (M(pro)) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M(pro) and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M(pro) active site that lead to the Cys(-)/His(+) zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate-enzyme and inhibitor-enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M(pro) employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured pKa values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M(pro) activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M(pro) toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors.

QM/MM investigations of organic chemistry oriented questions.

About 35 years after its first suggestion, QM/MM became the standard theoretical approach to investigate enzymatic structures and processes. The success is due to the ability of QM/MM to provide an accurate atomistic picture of enzymes and related processes. This picture can even be turned into a movie if nuclei-dynamics is taken into account to describe enzymatic processes. In the field of organic chemistry, QM/MM methods are used to a much lesser extent although almost all relevant processes happen in condensed matter or are influenced by complicated interactions between substrate and catalyst. There is less importance for theoretical organic chemistry since the influence of nonpolar solvents is rather weak and the effect of polar solvents can often be accurately described by continuum approaches. Catalytic processes (homogeneous and heterogeneous) can often be reduced to truncated model systems, which are so small that pure quantum-mechanical approaches can be employed. However, since QM/MM becomes more and more efficient due to the success in software and hardware developments, it is more and more used in theoretical organic chemistry to study effects which result from the molecular nature of the environment. It is shown by many examples discussed in this review that the influence can be tremendous, even for nonpolar reactions. The importance of environmental effects in theoretical spectroscopy was already known. Due to its benefits, QM/MM can be expected to experience ongoing growth for the next decade.In the present chapter we give an overview of QM/MM developments and their importance in theoretical organic chemistry, and review applications which give impressions of the possibilities and the importance of the relevant effects. Since there is already a bunch of excellent reviews dealing with QM/MM, we will discuss fundamental ingredients and developments of QM/MM very briefly with a focus on very recent progress. For the applications we follow a similar strategy.

New Route of Tire Rubber Devulcanization Using Silanes.

The disposal of tires at the end of their lifespan results in societal and environmental issues. To tackle this, recycling and reuse are effective solutions. Among various recycling methods, devulcanization is considered to be a very sustainable option, as it involves the controlled breakdown of crosslinks while maintaining the polymer backbones. The objective of this study is to develop a sustainable devulcanization process for passenger car tire rubber using silanes. In this study, a thermo-mechanical-chemical devulcanization process was conducted to screen six potential devulcanization aids (DAs). Silanes were chosen as they are widely used in tire rubber as coupling agents for silica. The efficiency of the devulcanization was studied by the degree of network breakdown, miscibility of the devulcanized material, and mechanical properties of the de- and revulcanized material. Compared to the parent compound, a 55-60% network breakdown was achieved for the devulcanizate along with 50-55% of tensile strength recovery. In addition to superior devulcanization efficiency, this DA offers a sustainable alternative to the conventional ones, such as di-phenyl-di-sulphide, due to its compliance with safety regulations. The devulcanizate can be utilized in high-performance applications, such as tires and seals, while 100% devulcanizate can be employed in low-strength technical rubber products.

A flexible route to chiral 2-endo-substituted 9-oxabispidines and their application in the enantioselective oxidation of secondary alcohols.

A new and flexible route to enantiomerically pure bi- and tricyclic 9-oxabispidines has been developed with use of (1R,5S)-7-methyl-2-oxo-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylic acid tert-butyl ester as the common late-stage intermediate. The 9-oxabispidines synthesized were evaluated as the chiral ligands in the Pd(II)-catalyzed oxidative kinetic resolution of secondary alcohols giving good to excellent selectivity factors of up to 19.

Origin of the reactivity differences of substituted aziridines: CN vs CC bond breakages.

Aziridines are broadly used as starting materials for various chemical syntheses, and the underlying reactions (CN vs CC bond breaking accompanied by an attack of a nucleophile or a dipolarophile) are strongly influenced by the substitution pattern. The present study investigates reaction courses of possible ring-opening reactions accompanied by the attack of a nucleophile for different substitution patterns of the aziridine. Information is obtained through the computation of the underlying potential energy surfaces and reaction paths. The results provide insight into the mechanisms of different ring-opening reactions and explain how the kinetics and thermodynamics of the reaction are influenced by substituents. This allows predicting substitution patterns that steer the reaction course to either CN or CC bond cleavage.

Tautomeric equilibria of 3-formylacetylacetone: low-temperature NMR spectroscopy and ab initio calculations.

Keto-enol tautomerization of 3-formylacetylacetone has been studied by NMR spectroscopy, ab initio, and DFT calculations in the gas phase and continuum solvation. By employing very low temperatures in a freonic solvent, tautomeric and conformational equilibria in the slow exchange regime were analyzed in detail. The beta-tricarbonyl compound always adopts a structure with an enolized keto group irrespective of an increasing dielectric constant of the solvent when lowering the temperature of the Freon mixture. This experimentally observed tautomeric distribution of 3-formylacetylacetone is correctly reproduced by continuum solvated DFT calculations.

Benchmark Study for the Cysteine-Histidine Proton Transfer Reaction in a Protein Environment: Gas Phase, COSMO, QM/MM Approaches.

Proton transfer reactions are of crucial interest for the investigation of proteins. We have investigated the accuracy of commonly used quantum chemical methods for the description of proton transfer reactions in different environments (gas phase, COSMO, QM/MM) using the proton transfer between the catalytic dyad residues cysteine 145 and histidine 41 of SARS coronavirus main protease as a case study. The test includes thermodynamic, kinetic, and structural properties. The study comprises computationally demanding ab initio approaches (HF, CC2, MP2, SCS-CC2, SCS-MP2, CCSD(T)), popular density functional theories (BLYP, B3LYP, M06-2X), and semiempirical methods (MNDO/d, AM1, RM1, PM3, PM6). The approximated coupled cluster approach LCCSD(T) is taken as a reference method. We find that the robustness of the tested methods with respect to the environment correlates well with the level of theory. As an example HF, CC2, MP2, and their SCS variants show similar errors for gas phase, COSMO, or QM/MM computations. In contrast for semiempirical methods, the errors strongly diversify if one goes from gas phase to COSMO or QM/MM. Particular problems are observed for the recent semiempirical methods PM6 and RM1, which show the best performance for gas phase calculations but possess larger errors in conjunction with COSMO. Finally, a combination of SCS-MP2 and B3LYP or M06-2X allows reliable estimates about remaining errors.

Mechanistic study of the reaction of thiol-containing enzymes with alpha,beta-unsaturated carbonyl substrates by computation and chemoassays.

We investigated the reactions between substituted alpha,beta-unsaturated carbonyl compounds (Michael systems) and thiols by computations as well as chemoassays. The results give insight into variations in the underlying mechanisms as a function of the substitution pattern. This is of interest for the mechanisms of inhibition of the SARS coronavirus main protease (SARS-CoV M(pro)) by etacrynic acid derivatives as well as for the excess toxicity of substituted alpha,beta-unsaturated carbonyl compounds. This study compares possible reaction courses including 1,4-addition followed by a ketonization step, and underscores the importance of a base-catalyzed step for the reactivity of thiol groups in enzymes. Phenyl and methyl substituents at the Michael system decrease the reactivity of the electrophilic compound, but chlorophenyl substituents partly recover the reactivity. Computations also indicate that electron-pushing substituents lead to a change in the reaction mechanism. The conformation of the Michael system is also found to significantly influence reactivity: the s-cis conformation leads to higher reactivity than the s-trans conformation. The computed data explain the trends in measured inhibition potencies of substituted alpha,beta-unsaturated carbonyl compounds and of reaction rates in chemical assays. They also indicate that the reversibility of inhibition does not stand in contrast to the formation of a new covalent bond between inhibitor and protease.