Electrostatic Control of Chemistry in Terpene Cyclases

Electrostatic interactions play a major role in stabilizing transition states in enzymes. A crucial question is how general this electrostatic stabilization principle is. To address this point, we study a key common C–C bond formation step in a family of enzymes that is responsible for the biosynthesis of 60% of all natural products. In these terpene cyclases, we have previously shown that the enzymes gain <i>chemical control</i> by raising the energy of initial carbocation intermediates along the reaction coordinate to bypass the formation of unwanted side products. Here we employ hybrid quantum mechanics–molecular mechanics free energy simulations to show that this energy tuning is achieved by modulation of electrostatic interactions. The tempering of electrostatic interactions allows enzymatically directed chemical control that <i>slows down</i> the reaction temporarily by introducing thermodynamic and activation barriers. We show that this electrostatic control in terpene cyclases is achieved by a unique binary active site architecture with a highly charged region flanked by a hydrophobic region. In the charged region, negatively and positively charged moieties are arranged in approximately a layered manner relative to the carbocation binding pocket, with alternating negative and positive layers. We suggest that this active site architecture can be utilized for rational design

Chemical Control in the Battle against Fidelity in Promiscuous Natural Product Biosynthesis: The Case of Trichodiene Synthase

Terpene cyclases catalyze the highly stereospecific molding of polyisoprenes into terpenes, which are precursors to most known natural compounds. The isoprenoids are formed via intricate chemical cascades employing rich, yet highly erratic, carbocation chemistry. It is currently not well understood how these biocatalysts achieve chemical control. Here, we illustrate the catalytic control exerted by trichodiene synthase, and in particular, we discover two features that could be general catalytic tools adopted by other terpenoid cyclases. First, to avoid formation of byproducts, the enzyme raises the energy of bisabolyl carbocation, which is a general mechanistic branching point in many sesquiterpene cyclases, resulting in an essentially concerted cyclization cascade. Second, we identify a sulfur–carbocation dative bonding interaction that anchors the bisabolyl cation in a reactive conformation, avoiding tumbling and premature deprotonation. Specifically, Met73 acts as a chameleon, shifting from an initial sulfur−π interaction in the Michaelis complex to a sulfur–carbocation complex during catalysis

Multinuclear Magnetic Resonance Spectroscopy and Density Function Theory Calculations for the Identification of the Equilibrium Species in THF Solutions of Organometallic Complexes Suitable As Electrolyte Solutions for Rechargeable Mg Batteries

We present a multinuclear nuclear magnetic resonance (NMR) and density functional theory (DFT) study of electrolyte solutions based on organometallic complexes with aromatic ligands. These solutions constitute a unique electrolyte family possessing a wide electrochemical window, making them suitable for rechargeable magnesium batteries. In our previous study we identified equilibrium species in the solutions based on a combination of Raman spectroscopy and single-crystal XRD analyses, and herein we extend our studies to include multinuclear NMR analyses. These solutions are comprised of the metathesis reaction products of MgCl_2–<i>x</i>Ph_<i>x</i> and AlCl_3–<i>y</i>Ph_<i>y</i> in various proportions, in THF. In principle, these reactions involve the exchange of ligands between the magnesium and the aluminum based compounds, forming ionic species and neutral molecules, such as Mg₂Cl₃⁺·6THF, MgCl₂·4THF and AlCl_4–<i>y</i>Ph_<i>y</i>^– (<i>y</i> = 0–4). The identification of the solution phase species from the spectroscopic results is supported by spectral analyses of specially synthesized reference compounds and DFT quantum-mechanical calculations. The current approach reveals new aspects about the NMR shift of the organometallic complexes and, in particular, facilitates differentiation between ionic and neutral species

Analysis of the Spectroscopic Aspects of Cationic Dye Basic Orange 21

Spectroscopic properties of cationic dye basic orange 21 (BO21) in solutions, in solids, and within leukocytes were examined. Results obtained with solutions indicate that influence of variables such as pH, viscosity, salt composition, and various proteins on the absorption spectrum of BO21 is negligible. However, in the presence of heparin, a blue shift (484–465 nm) is observed, which is attributed to the aggregation of BO21 on the polyanion. Applying density functional theory demonstrates that in aqueous solutions (a) the formation of BO21 oligomers is thermodynamically favorable, they are oriented in an antiparallel dipolar arrangement, and their binding energies are lower than those of parallel dipolar arrangements, (b) association between BO21 aggregates and heparin is highly favorable, and (c) the blue shift is due to the mixing of π → π* transitions caused by BO21 molecule stacking. However, when embedded in basophils, the absorption spectra of intracellular BO21 is extremely red-shifted, with two peaks (at 505 and 550 nm) found to be attributed to BO21 and the BO21–heparin complex, respectively, which are intracellularly hosted in nonaqueous environments. Initial evidence of the ability to differentiate between leukocyte types by BO21 is presented

Identification of Highly Promising Antioxidants/Neuroprotectants Based on Nucleoside 5′-Phosphorothioate Scaffold. Synthesis, Activity, and Mechanisms of Action

With a view to identify novel and biocompatible neuroprotectants, we designed nucleoside 5′-thiophosphate analogues, <b>6</b>–<b>11</b>. We identified 2-SMe-ADP­(α-S), <b>7A</b>, as a most promising neuroprotectant. <b>7A</b> reduced ROS production in PC12 cells under oxidizing conditions, IC₅₀ of 0.08 vs 21 μM for ADP. Furthermore, <b>7A</b> rescued primary neurons subjected to oxidation, EC₅₀ of 0.04 vs 19 μM for ADP. <b>7A</b> is a most potent P2Y₁-R agonist, EC₅₀ of 0.0026 μM. Activity of <b>7A</b> in cells involved P2Y_1/12-R as indicated by blocking P2Y₁₂-R or P2Y₁-R. Compound <b>7A</b> inhibited Fenton reaction better than EDTA, IC₅₀ of 37 vs 54 μM, due to radical scavenging, IC₅₀ of 12.5 vs 30 μM for ADP, and Fe­(II)-chelation, IC₅₀ of 80 vs >200 μM for ADP (ferrozine assay). In addition, <b>7A</b> was stable in human blood serum, <i>t</i>_1/2 of 15 vs 1.5 h for ADP, and resisted hydrolysis by NPP1/3, 2-fold vs ADP. Hence, we propose <b>7A</b> as a highly promising neuroprotectant

Structural and Kinetic Studies of Formate Dehydrogenase from <i>Candida boidinii</i>

The structure of formate dehydrogenase from <i>Candida boidinii</i> (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO₂ fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD⁺) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10^–7 (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented

Odd–Even Effect in Molecular Electronic Transport via an Aromatic Ring

A distinct odd–even effect on the electrical properties, induced by monolayers of alkyl-phenyl molecules directly bound to Si(111), is reported. Monomers of H₂CCH–(CH₂)_<i>n</i>–phenyl, with <i>n</i> = 2–5, were adsorbed onto Si–H and formed high-quality monolayers with a binding density of 50–60% Si(111) surface atoms. Molecular dynamics simulations suggest that the binding proximity is close enough to allow efficient π–π interactions and therefore distinctly different packing and ring orientations for monomers with odd or even numbers of methylenes in their alkyl spacers. The odd−even alternation in molecular tilt was experimentally confirmed by contact angle, ellipsometry, FT-IR, and XPS with a close quantitative match to the simulation results. The orientations of both the ring plane and the long axis of the alkyl spacer are more perpendicular to the substrate plane for molecules with an even number of methylenes than for those with an odd number of methylenes. Interestingly, those with an even number conduct better than the effectively thinner monolayers of the molecules with the odd number of methylenes. We attribute this to a change in the orientation of the electron density on the aromatic rings with respect to the shortest tunneling path, which increases the barrier for electron transport through the odd monolayers. The high sensitivity of molecular charge transport to the orientation of an aromatic moiety might be relevant to better control over the electronic properties of interfaces in organic electronics

Unique Behavior of Dimethoxyethane (DME)/Mg(N(SO₂CF₃)₂)₂ Solutions

Mg­(N­(SO₂CF₃)₂)₂ (MgTFSI₂) solutions with dimethoxyethane (DME) exhibit a peculiar behavior. Over a certain range of salt content, they form two immiscible phases of specific electrolyte concentrations. This behavior is unique, as both immiscible phases comprise the same constituents. Thus, this miscibility gap constitutes an exceptionally intriguing and interesting case for the study of such phenomena. We studied these systems from solutions structure perspective. The study included a wide variety of analytical tools including single-crystal X-ray diffraction, multinuclei NMR, and Raman spectroscopy coupled with density functional theory calculations. We rigorously determined the structure of the MgTFSI₂/DME solutions and developed a plausible theory to explain the two-phase formation phenomenon. We also determined the exchange energy of the “caging” DME molecules solvating the central magnesium ion. Additionally, by measuring the ions’ diffusion coefficients, we suggest that the caged Mg²⁺ and TFSI^– move as free ions in the solution. Knowledge of the arrangement of the solvent/cation/anion structures in these solutions enables us to explain their properties. We believe that this study is important in a wide context of solutions chemistry and nonaqueous electrochemistry. Also, MgTFSI₂/DME solutions are investigated as promising electrolyte solutions for rechargeable magnesium batteries. This study may serve as an important basis for developing further MgTFSI₂/ether based solutions for such an interesting use

Unique Behavior of Dimethoxyethane (DME)/Mg(N(SO₂CF₃)₂)₂ Solutions

Mg­(N­(SO₂CF₃)₂)₂ (MgTFSI₂) solutions with dimethoxyethane (DME) exhibit a peculiar behavior. Over a certain range of salt content, they form two immiscible phases of specific electrolyte concentrations. This behavior is unique, as both immiscible phases comprise the same constituents. Thus, this miscibility gap constitutes an exceptionally intriguing and interesting case for the study of such phenomena. We studied these systems from solutions structure perspective. The study included a wide variety of analytical tools including single-crystal X-ray diffraction, multinuclei NMR, and Raman spectroscopy coupled with density functional theory calculations. We rigorously determined the structure of the MgTFSI₂/DME solutions and developed a plausible theory to explain the two-phase formation phenomenon. We also determined the exchange energy of the “caging” DME molecules solvating the central magnesium ion. Additionally, by measuring the ions’ diffusion coefficients, we suggest that the caged Mg²⁺ and TFSI^– move as free ions in the solution. Knowledge of the arrangement of the solvent/cation/anion structures in these solutions enables us to explain their properties. We believe that this study is important in a wide context of solutions chemistry and nonaqueous electrochemistry. Also, MgTFSI₂/DME solutions are investigated as promising electrolyte solutions for rechargeable magnesium batteries. This study may serve as an important basis for developing further MgTFSI₂/ether based solutions for such an interesting use