Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction

To investigate fundamental features of enzyme catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within enzymes. We have modelled an important model enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the enzyme are 7.4-11.0 kcal mol(-1)(MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7-16.1 kcal mol(-1)(B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of Delta H(++)= 12.7 +/- 0.4 kcal mol(-1). The results provide unequivocal evidence of transition state (TS) stabilization by the enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol(-1)(MP2/6-31+G(d)/PCM) and 24.6 kcal mol(-1)(B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-fold rate acceleration by the enzyme. The substrate is found to be significantly distorted in the enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the enzyme, involving reaction from a 'near-attack conformer' of the substrate, indicates that adoption of this conformation is not in itself a major contribution to catalysis. Transition state stabilization (by electrostatic interactions, including hydrogen bonds) is found to be central to catalysis by the enzyme. Several hydrogen bonds are observed to shorten at the TS. The active site is clearly complementary to the transition state for the reaction, stabilizing it more than the substrate, so reducing the barrier to reaction.     

Open tubular supercritical fluid chromatography of triterpene acids from Dysoxylum pettigrewianum (Meliaceae)

Open tubular supercritical fluid chromatography (SFC) with carben dioxide as the mobile phase has been investigated for the separation of four triterpene acids, two of which were structural isomers. These compounds, isolated from Dysoxylum pettigrewianum (Meliaceae), were chromatographed on three stationary phases with different selectivities. 30%-biphenylmethylpolysiloxane and polyethylene glycol (Carbowax 20M) phases exhibited poor chromatographic selectivity for the compounds. The separation was improved by employing the shape selectivity of a liquid crystalline biphenylcarboxylate ester polysiloxane and utilizing simultaneous temperature and pressure programming. The elution order was explained on the basis of the molecular structures of the triterpene acids, and their interaction with the liquid crystal phase.     

A concise total synthesis of salinosporamide A

A concise and straightforward 14-step total synthesis of (+/-)-salinosporamide A, based on a diastereoselective acid-catalysed intramolecular cyclisation of to the pyrrolidinone , and a regioselective reduction of the malonate derivative 8b to the aldehyde 9, is described.     

Triterpenoid acids and lactones from the leaves of Fadogia tetraquetra var. tetraquetra (Rubiaceae)

Four triterpenoids isolated from the leaves of Fadogia tetraquetra var. tetraquetra, 3beta-hydroxy-11alpha, 12alpha-epoxyoleanan-28,13beta-olide (1), 3beta-hydroxyurs-11-en-28,13beta-olide (2), oleanolic acid (3), and ursolic acid (4), were evaluated for their antiviral and antibacterial properties. Compound 4 showed potent activity against the Semliki Forest virus with an IC50 of 14.7 microM, but was also found to be significantly cytotoxic (68% reduction in cell viability after 24 hours exposure at 50 microM) towards baby hamster kidney (BHK21) host cells. A viability assay on the mammalian human hepatocellular carcinoma (Huh-7) cell line showed no significant effects on intracellular ATP content after 48 hours exposure to compounds 1-4 at this concentration. Compound 4 also inhibited Staphylococcus aureus (MIC 12.5 microM), but was inactive against Enterobacter aerogenes, Escherichia coli, and Pseudomonas aeruginosa. Compounds 1-3 were inactive against all tested bacterial strains at 50 microM concentration.     

An expert system for the choice of factors for a ruggedness test in liquid chromatography

Method validation is an important and costly step in the development of liquid chromatographic methods, especially for pharmaceutical samples. A ruggedness test is valuable for finding the analytical conditions which give the best performance. By varying the factors that influence method performance, it is possible to design a more rugged method before a study of reproducibility is started. The choice of the factors to test is important. An expert system is described for the selection of factors in ruggedness tests. The system proved satisfactory for 10 of the 11 cases of pharmaceutical formulations tested.     

On well‐posedness for some thermo‐piezoelectric coupling models

There is an increasing reliance on mathematical modelling to assist in the design of piezoelectric ultrasonic transducers since this provides a cost‐effective and quick way to arrive at a first prototype. Given a desired operating envelope for the sensor, the inverse problem of obtaining the associated design parameters within the model can be considered. It is therefore of practical interest to examine the well‐posedness of such models. There is a need to extend the use of such sensors into high‐temperature environments, and so this paper shows, for a broad class of models, the well‐posedness of the magneto‐electro‐thermo‐elastic problem. Because of its widespread use in the literature, we also show the well‐posedness of the quasi‐electrostatic case. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantum Mechanics/Molecular Mechanics Simulations Show Saccharide Distortion is Required for Reaction in Hen Egg-White Lysozyme

Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on lysozyme show significant distortion of the bound saccharide is required to facilitate the catalytic reaction.     

Electronic structure benchmark calculations of CO2 fixing elementary chemical steps in RuBisCO using the projector-based embedding approach

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is the main enzyme involved in atmospheric carbon dioxide (CO₂) fixation in the biosphere. This enzyme catalyzes a set of five chemical steps that take place in the same active-site within magnesium (II) coordination sphere. Here, a set of electronic structure benchmark calculations have been carried out on a reaction path proposed by Gready et al. by means of the projector-based embedding approach. Activation and reaction energies for all main steps catalyzed by RuBisCO have been calculated at the MP2, SCS-MP2, CCSD, and CCSD(T)/aug-cc-pVDZ and cc-pVDZ levels of theory. The treatment of the magnesium cation with post-HF methods is explored to determine the nature of its involvement in the mechanism. With the high-level ab initio values as a reference, we tested the performance of a set of density functional theory (DFT) exchange-correlation (xc) functionals in reproducing the reaction energetics of RuBisCO carboxylase activity on a set of model fragments. Different DFT xc-functionals show large variation in activation and reaction energies. Activation and reaction energies computed at the B3LYP level are close to the reference SCS-MP2 results for carboxylation, hydration and protonation reactions. However, for the carbon–carbon bond dissociation reaction, B3LYP and other functionals give results that differ significantly from the ab initio reference values. The results show the applicability of the projector-based embedding approach to metalloenzymes. This technique removes the uncertainty associated with the selection of different DFT xc-functionals and so can overcome some of inherent limitations of DFT calculations, complementing, and potentially adding to modeling of enzyme reaction mechanisms with DFT methods.     

Macrocyclic Olefin Metathesis at High Concentrations by Using a Phase‐Separation Strategy

Macrocyclic olefin metathesis has seen advances in the areas of stereochemistry, chemoselectivity, and catalyst stability, but strategies aimed at controlling dilution effects in macrocyclizations are rare. Herein, a protocol to promote macrocyclic olefin metathesis, one of the most common synthetic tools used to prepare macrocycles, at relatively high concentrations (up to 60 mM ) is described by exploitation of a phase‐separation strategy. A variety of macrocyclic skeletons could be prepared having either different alkyl, aryl, or amino acids spacers.     

Thieno[3,4‐b]pyrazines and Its Extended Analogs: Important Buildings Blocks for Conjugated Materials

This review describes the synthesis and characterization of thieno[3,4‐b]pyrazines and its extended fused‐ring analogs as important building blocks for the production of low‐band gap conjugated materials.