Interactions In Transition State Research Articles

Transition State Stabilizing Effects of Oxygen and Sulfur Chalcogen Bond Interactions.

Non-covalent chalcogen bond (ChB) interactions have found utility in many fields, including catalysis, organic semiconductors, and crystal engineering. In this study, the transition stabilizing effects of ChB interactions of oxygen and sulfur were experimentally measured using a series of molecular rotors. The rotors were designed to form ChB interactions in their bond rotation transition states. This enabled the kinetic influences to be assessed by monitoring changes in the rotational barriers. Despite forming weaker ChB interactions, the smaller chalcogens were able to stabilize transition states and had measurable kinetic effects on the rotational barriers. Sulfur stabilized the bond rotation transition state by as much as -7.2 kcal/mol without electron-withdrawing groups. The key was to design a system where the sulfur -hole was aligned with the lone pairs of the chalcogen bond acceptor. Oxygen rotors also could form transition state stabilizing ChB interactions but required electron-withdrawing groups. For both oxygen and sulfur ChB interactions, a strong correlation was observed between transition state stabilizing abilities and electrostatic potential (ESP) of the chalcogen, providing a useful predictive parameter for the rational design of future ChB systems.

Competing C and N as Reactive Centers for Microsolvated Ambident Nucleophiles CN-(H2O)n=0-3: A Theoretical Study of E2/SN2 Reactions with CH3CH2X (X = Cl, Br, I).

As an ambident nucleophile, CN- has both C and N atoms that can act as the reactive center to facilitate substitution reactions. We investigate in detail the potential energy profiles of CN-(H2O)0-3 with CH3CH2X (X = Cl, Br, I) to explore the influence of solvent molecules on competition between the different nucleophilic atoms C and N involving the SN2 and E2 pathways. The energy barrier sequence for the transition states follows C@inv-SN2 < N@inv-SN2 < C@anti-E2 < N@anti-E2. When two different atoms act as nucleophilic atoms, the SN2 reaction is always preferred over the E2 reaction, and this preference increases with microsolvation. For the ambident nucleophiles CN-(H2O)0-3, C as the reactive center always has stronger nucleophilicity and basicity than N acting as the reactive center. Regarding the leaving group, the height of the energy barrier is positively correlated with the acidity of the CH3CH2X substrate for the E2 pathway and with X-heterolysis for the SN2 pathway. Furthermore, we found that in the gas phase, the energy barrier for different leaving group systems decreases gradually in the order Cl > Br > I, while in the SMD solution, the energy barrier and product energy increase slightly in the system from X = Cl to Br; this change may be due to the significantly weakened transition-state interaction for the X = Br system. Our activation strain, quantitative molecular orbital, and charge analyses reveal the physical mechanisms underlying the various computed trends. In addition, we also demonstrate the two points recently proposed by Vermeeren, P. . Chem. Eur. J. 2020, 26, 15538-15548.

From covalent transition states in chemistry to noncovalent in biology: from β- to Φ-value analysis of protein folding.

Solving the mechanism of a chemical reaction requires determining the structures of all the ground states on the pathway and the elusive transition states linking them. 2024 is the centenary of Brønsted's landmark paper that introduced the β-value and structure-activity studies as the only experimental means to infer the structures of transition states. It involves making systematic small changes in the covalent structure of the reactants and analysing changes in activation and equilibrium-free energies. Protein engineering was introduced for an analogous procedure, Φ-value analysis, to analyse the noncovalent interactions in proteins central to biological chemistry. The methodology was developed first by analysing noncovalent interactions in transition states in enzyme catalysis. The mature procedure was then applied to study transition states in the pathway of protein folding - 'part (b) of the protein folding problem'. This review describes the development of Φ-value analysis of transition states and compares and contrasts the interpretation of β- and Φ-values and their limitations. Φ-analysis afforded the first description of transition states in protein folding at the level of individual residues. It revealed the nucleation-condensation folding mechanism of protein domains with the transition state as an expanded, distorted native structure, containing little fully formed secondary structure but many weak tertiary interactions. A spectrum of transition states with various degrees of structural polarisation was then uncovered that spanned from nucleation-condensation to the framework mechanism of fully formed secondary structure. Φ-analysis revealed how movement of the expanded transition state on an energy landscape accommodates the transition from framework to nucleation-condensation mechanisms with a malleability of structure as a unifying feature of folding mechanisms. Such movement follows the rubric of analysis of classical covalent chemical mechanisms that began with Brønsted. Φ-values are used to benchmark computer simulation, and Φ and simulation combine to describe folding pathways at atomic resolution.

Interatomic interactions in molecules, associates, crystals and transition states in terms of electronic force density fields

Interatomic interactions in molecules, associates, crystals and transition states in terms of electronic force density fields

Electrostatic Interactions in Asymmetric Organocatalysis.

ConspectusElectrostatic interactions are ubiquitous in catalytic systems and can be decisive in determining the reactivity and stereoselectivity. However, difficulties quantifying the role of electrostatic interactions in transition state (TS) structures have long stymied our ability to fully harness the power of these interactions. Fortunately, advances in affordable computing power, together with new quantum chemistry methods, have increasingly enabled a detailed atomic-level view. Empowered by this more nuanced perspective, synthetic practitioners are now adopting these techniques with growing enthusiasm.In this Account, we narrate our recent results rooted in state-of-the-art quantum chemical computations, describing pivotal roles for electrostatic interactions in the organization of TS structures to direct the reactivity and selectivity in the realm of asymmetric organocatalysis. To provide readers with a fundamental foundation in electrostatics, we first introduce a few guiding principles, beginning with a brief discussion of how electrostatic interactions can be harnessed to tune the strength of noncovalent interactions. We then describe computational approaches to capture these effects followed by examples in which electrostatic effects impact structure and reactivity. We then cover some of our recent computational investigations in three specific branches of asymmetric organocatalysis, beginning with chiral phosphoric acid (CPA) catalysis. We disclose how CPA-catalyzed asymmetric ring openings of meso-epoxides are driven by stabilization of a transient partial positive charge in the SN2-like TS by the chiral electrostatic environment of the catalyst. We also report on substrate-dependent electrostatic effects from our study of CPA-catalyzed intramolecular oxetane desymmetrizations. For nonchelating oxetane substrates, electrostatic interactions with the catalyst confer stereoselectivity, whereas oxetanes with chelating groups adopt a different binding mode that leads to electrostatic effects that erode selectivity. In another example, computations revealed a pivotal role of CH···O and NH···O hydrogen bonding in the CPA-catalyzed asymmetric synthesis of 2,3-dihydroquinazolinones. These interactions control selectivity during the enantiodetermining intramolecular amine addition step, and their strength is modulated by electrostatic effects, allowing us to rationalize the effect of introducing o-substituents. Next, we describe our efforts to understand selectivity in a series of NHC-catalyzed kinetic resolutions, where we discovered that the electrostatic stabilization of key proton(s) is the common driver of selectivity. Finally, we discuss our breakthrough in understanding asymmetric silylium ion-catalyzed Diels-Alder cycloaddition of cinnamate esters to cyclopentadienes. The endo:exo of these transformations is guided by electrostatic interactions that selectively stabilize the endo-transition state.We conclude with a brief overview of the outstanding challenges and potential roles of computational chemistry in enabling the exploitation of electrostatic interactions in asymmetric organocatalysis.

Impact of a single water molecule on the atmospheric oxidation of thiophene by hydroperoxyl radical

Water as an important assistant can alter the reactivity of atmospheric species. This project is designed to investigate the impact of a single water molecule on the atmospheric reactions of aromatic compounds that have not been attended to comprehensively. In the first part, the atmospheric oxidation mechanisms of thiophene initiated by hydroperoxyl radical through a multiwell-multichannel potential energy surface were studied to have useful information about the chemistry of the considered reaction. It was verified that for the thiophene plus HO2 reaction, the addition mechanism is dominant the same as other aromatic compounds. Due to the importance of the subject and the presence of water molecules in the atmosphere with a high concentration that we know as relative humidity, and also the lack of insight into the influence of water on the reactions of aromatic compounds with active atmospheric species, herein, the effect of a single water molecule on the addition pathways of the title reaction is evaluated. In another word, this research explores how water can change the occurrence of reactions of aromatic compounds in the atmosphere. For this, the presence of one water molecule is simulated by higher-level calculations (BD(T) method) through the main interactions with the stationary points of the most probable pathways. The results show that the mechanism of the reaction with water is more complicated than the bare reaction due to the formation of the ring-like structures. Also, water molecule decreases the relative energies of all addition pathways. Moreover, atoms in molecule theory (AIM) along with the kinetic study by the transition state (TST) and the Rice–Ramsperger–Kassel–Marcus (RRKM) theories demonstrate that the overall interactions of a path determine how the rate of that path changes. In this regard, our results establish that the interactions of water with HO2 (thiophene) in the initial complex 1WHA (1WTA or 1WTB) are stronger (weaker) than the sum of its interactions in transition states. Also, for the water-assisted pathways, the ratio of the partition function of the transition state to the partition functions of the reactants is similar to the respective bare reaction. Therefore, the reaction rates of the bare pathways are more than the water-assisted paths that include the 1WHA complex and are less than the paths that involve the 1WTA and 1WTB complexes.

Scaled σ-functionals for the Kohn-Sham correlation energy with scaling functions from the homogeneous electron gas.

The recently introduced σ-functionals constitute a new type of functionals for the Kohn-Sham (KS) correlation energy. σ-Functionals are based on the adiabatic-connection fluctuation-dissipation theorem, are computationally closely related to the well-known direct random phase approximation (dRPA), and are formally rooted in many-body perturbation theory along the adiabatic connection. In σ-functionals, the function of the eigenvalues σ of the Kohn-Sham response matrix that enters the coupling constant and frequency integration in the dRPA is replaced by another function optimized with the help of reference sets of atomization, reaction, transition state, and non-covalent interaction energies. σ-Functionals are highly accurate and yield chemical accuracy of 1 kcal/mol in reaction or transition state energies, in main group chemistry. A shortcoming of σ-functionals is their inability to accurately describe processes involving a change of the electron number, such as ionizations or electron attachments. This problem is attributed to unphysical self-interactions caused by the neglect of the exchange kernel in the dRPA and σ-functionals. Here, we tackle this problem by introducing a frequency- and σ-dependent scaling of the eigenvalues σ of the KS response function that models the effect of the exchange kernel. The scaling factors are determined with the help of the homogeneous electron gas. The resulting scaled σ-functionals retain the accuracy of their unscaled parent functionals but in addition yield very accurate ionization potentials and electron affinities. Moreover, atomization and total energies are found to be exceptionally accurate. Scaled σ-functionals are computationally highly efficient like their unscaled counterparts.

Mechanistic Basis for the Iridium-Catalyzed Enantioselective Allylation of Alkenyl Boronates.

Iridium(phosphoramidite) complexes catalyze an enantio- and diastereoselective three-component coupling reaction of alkenyl boronic esters, organolithium reagents, and secondary allylic carbonates. The reaction proceeds through an allylation-induced 1,2-metalate shift of the alkenyl boronate to form non-adjacent stereocenters. Mechanistic investigations outline the overall catalytic cycle and reveal trends in reactivity and selectivity. Analysis of relative stereochemistry in products derived from a variety of 1,1-disubtituted alkenyl boronates provides insight into the transition state of the addition and indicates a concerted pathway. Kinetic analysis of the reaction revealed the kinetic order dependence in boronate, the catalyst, and both the slow- and fast-reacting enantiomer of allylic carbonate as well as the turnover-limiting step of the reaction. Determination of nucleophile-specific parameters N and sN for alkenyl boronate complexes enabled comparison to other classes of nucleophiles. DFT calculations indicate the addition of the alkenyl boronate to the cationic Ir(π-allyl) intermediate and the 1,2-metalate shift occur in a concerted mechanism. The stereoselectivity is determined by ligand-substrate steric repulsions and dispersion interactions in the syn addition transition state. Hammett studies supported the computational results with regard to electronic trends observed with both aryl-derived alkenyl boronates and aryl carbonates.

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a β-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1–2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the β-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

Chemical accuracy with σ-functionals for the Kohn-Sham correlation energy optimized for different input orbitals and eigenvalues.

Recently, a new type of orbital-dependent functional for the Kohn-Sham (KS) correlation energy, σ-functionals, was introduced. Technically, σ-functionals are closely related to the well-known direct random phase approximation (dRPA). Within the dRPA, a function of the eigenvalues σ of the frequency-dependent KS response function is integrated over purely imaginary frequencies. In σ-functionals, this function is replaced by one that is optimized with respect to reference sets of atomization, reaction, transition state, and non-covalent interaction energies. The previously introduced σ-functional uses input orbitals and eigenvalues from KS calculations with the generalized gradient approximation (GGA) exchange-correlation functional of Perdew, Burke, and Ernzerhof (PBE). Here, σ-functionals using input orbitals and eigenvalues from the meta-GGA TPSS and the hybrid-functionals PBE0 and B3LYP are presented and tested. The number of reference sets taken into account in the optimization of the σ-functionals is larger than in the first PBE based σ-functional and includes sets with 3d-transition metal compounds. Therefore, also a reparameterized PBE based σ-functional is introduced. The σ-functionals based on PBE0 and B3LYP orbitals and eigenvalues reach chemical accuracy for main group chemistry. For the 10966 reactions from the highly accurate W4-11RE reference set, the B3LYP based σ-functional exhibits a mean average deviation of 1.03 kcal/molcompared to 1.08 kcal/molfor the coupled cluster singles doubles perturbative triples method if the same valence quadruple zeta basis set is used. For 3d-transition metal chemistry, accuracies of about 2 kcal/molare reached. The computational effort for the post-self-consistent evaluation of the σ-functional is lower than that of a preceding PBE0 or B3LYP calculation for typical systems.

Toward chemical accuracy at low computational cost: Density-functional theory with σ-functionals for the correlation energy.

We introduce new functionals for the Kohn-Sham correlation energy that are based on the adiabatic-connection fluctuation-dissipation (ACFD) theorem and are named σ-functionals. Like in the well-established direct random phase approximation (dRPA), σ-functionals require as input exclusively eigenvalues σ of the frequency-dependent KS response function. In the new functionals, functions of σ replace the σ-dependent dRPA expression in the coupling-constant and frequency integrations contained in the ACFD theorem. We optimize σ-functionals with the help of reference sets for atomization, reaction, transition state, and non-covalent interaction energies. The optimized functionals are to be used in a post-self-consistent way using orbitals and eigenvalues from conventional Kohn-Sham calculations employing the exchange-correlation functional of Perdew, Burke, and Ernzerhof. The accuracy of the presented approach is much higher than that of dRPA methods and is comparable to that of high-level wave function methods. Reaction and transition state energies from σ-functionals exhibit accuracies close to 1 kcal/mol and thus approach chemical accuracy. For the 10 966 reactions of the W4-11RE reference set, the mean absolute deviation is 1.25 kcal/mol compared to 3.21 kcal/mol in the dRPA case. Non-covalent binding energies are accurate to a few tenths of a kcal/mol. The presented approach is highly efficient, and the post-self-consistent calculation of the total energy requires less computational time than a density-functional calculation with a hybrid functional and thus can be easily carried out routinely. σ-Functionals can be implemented in any existing dRPA code with negligible programming effort.

Theoretical Investigation of the Mechanism, Performance and Kinetic Experimental Phenomena on Brønsted Acidic Ionic Liquids Catalyzed Dehydration of Sorbitol to Isosorbide

AbstractThe dehydration of sorbitol to isosorbide catalyzed by Brønsted acidic ionic liquids (BILs) have been investigated in detail by performing density functional theory (DFT) calculations. It is found that the double dehydration processes involve an intrinsically consistent molecular mechanism: protonation followed by cyclization, and that throughout most of elementary steps, the sulfonic group in the cation of BIL functions as a Brønsted acid/base catalyst, and the anion participates in the reaction as a nucleophile. For 1‐propylsulfonic acid‐3‐methylimidazolium trifluoromethanesulfonate ([C3SO3Hmim]CF3SO3)‐catalyzed reaction, the calculated free energy barrier of the first dehydration step is higher than that of the second dehydration step, rationalizes well the experimentally observed kinetics that the latter has a larger rate constant than the former. In addition, the association energy of [C3SO3Hmim]CF3SO3 with sorbitol is larger than that with either 1,4‐sorbitan or isosorbide, which is consistent with observed association equilibrium constants. BIL carrying a methyl group at the C2 position of imidazolium ring, [C3SO3Hdmim]CF3SO3, shows much better catalytic performance than other tested BILs. The effectiveness of the catalytic system is attributed to the widely distribution of hydrogen‐bonding interactions in transition states and the superior nucleophilicity of the CF3SO3 anion.

Dynamic and static nature of activated interactions in transition states as elucidated by quantum theory of atoms‐in‐molecules dual functional analysis: A case of ligand exchange at the N of sulfonylimino‐λ3‐bromanes

AbstractA method to elucidate the dynamic and static natures of the activated interactions in transition states (TSs) is proposed using quantum theory of atoms‐in‐molecules (QTAIM) dual functional analysis (QTAIM‐DFA). The natures are determined for the ligand exchange at the N of sulfonylimino‐λ3‐bromanes, RBr‐*‐N(SO2CF3)‐*‐X′R′ (R, R′ = Me or Ph, X′ = Br′ or I′). Asterisks (*) emphasize the presence of bond critical points within the interactions in question. While data from the optimized structures of TSs correspond to the static nature, those from the perturbed and optimized structures represent the dynamic nature. The nature of the interactions in Br‐*‐N‐*‐X′ calculated with QTAIM‐DFA, using the perturbed structures generated using the normal coordinates for the imaginary frequencies (NIV), was very similar to that in trigonal bipyramidal adduct formation through charge transfer. The results with NIV were precisely the same as those obtained based on intrinsic reaction coordinate (IRC). The high applicability of QTAIM‐DFA is demonstrated when analyzing the activated interactions in TSs.

Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (βleavinggroupobs-Enz = -0.33) than the uncatalyzed reaction (βleavinggroupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (ΔβleavinggroupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

The catalytic role of water in the binding site of l,d-transpeptidase 2 within acylation mechanism: A QM/MM (ONIOM) modelling

Mycobacterium tuberculosis is the causative agent of Tuberculosis. Formation of 3 → 3 crosslinks in the peptidoglycan layer of M. tuberculosis is catalyzed by l,d-transpeptidases. These enzymes can confer resistance against classical β-lactams that inhibit enzymes that generate 4 → 3 peptidoglycan crosslinks. The focus of this study is to investigate the catalytic role of water molecules in the acylation mechanism of the β-lactam ring within two models; 4- and 6-membered ring systems using two-layered our Own N-layer integrated Molecular Mechanics ONIOM (B3LYP/6-311++G(2d,2p): AMBER) model. The obtained thermochemical parameters revealed that the 6-membered ring model best describes the inhibition mechanism of acylation which indicates the role of water in the preference of 6-membered ring reaction pathway. This finding is in accordance with experimental data for the rate-limiting step of cysteine protease with the same class of inhibitor and binding affinity for both inhibitors. As expected, the ΔG# results also reveal that the 6-membered ring reaction pathway is the most favourable. The electrostatic potential (ESP) and the natural bond orbital analysis (NBO) showed stronger interactions in 6-membered ring transition state (TS-6) mechanism involving water in the active site of the enzyme. This study could be helpful in the development of novel antibiotics against l,d-transpeptidase.

Folding and binding pathways of BH3-only proteins are encoded within their intrinsically disordered sequence, not templated by partner proteins

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2–like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2–like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder.

A DFT Study on CuH‐Catalyzed Reductive Relay Hydroamination for Synthesis of Remote‐Chiral Amine

AbstractThe reaction mechanism of copper‐hydride‐catalyzed reductive relay hydroamination for remote‐chiral amine was studied. Using density functional theory, we carefully investigated the mechanistic of the generation of R‐ and S‐amines with stereocenters. A series of steps, such as Markovnikov hydrocupration, β‐alkoxide elimination of alkylcopper, insertion of terminal alkene with copper‐hydride catalyst, the electrophile attack, and the formation of C–N bonds have been discussed in the catalytic cycle. The most favorable pathway is obtained by the Re‐attack of the catalyst with the allylic ester. In addition, noncovalent interaction (NCI) analyses show stronger noncovalent interactions in transition state Re‐TS1, frontier molecular orbital (FMO) reveal a larger lowest unoccupied molecular orbital and the highest occupied molecular orbital (LUMO‐HOMO) gap in transition state Si‐TS1, indicating the preference for the S‐configuration chiral amines, which is in good agreement with the experimental observations.

Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and in Vitro-Selected RNA Tertiary Motifs in a Multistep Folding Pathway.

Decades of study of the RNA folding problem have revealed that diverse and complex structured RNAs are built from a common set of recurring structural motifs, leading to the perspective that a generalizable model of RNA folding may be developed from understanding of the folding properties of individual structural motifs. We used single-molecule fluorescence to dissect the kinetic and thermodynamic properties of a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor (TL/TLR). Our results revealed a multistep TL/TLR folding pathway in which preorganization of the ubiquitous AA-platform submotif precedes the formation of the docking transition state and tertiary A-minor hydrogen bond interactions form after the docking transition state. Differences in ion dependences between TL/TLR variants indicated the occurrence of sequence-dependent conformational rearrangements prior to and after the formation of the docking transition state. Nevertheless, varying the junction connecting the TL/TLR produced a common kinetic and ionic effect for all variants, suggesting that the global conformational search and compaction electrostatics are energetically independent from the formation of the tertiary motif contacts. We also found that in vitro-selected variants, despite their similar stability at high Mg2+ concentrations, are considerably less stable than natural variants under near-physiological ionic conditions, and the occurrence of the TL/TLR sequence variants in Nature correlates with their thermodynamic stability in isolation. Overall, our findings are consistent with modular but complex energetic properties of RNA structural motifs and will aid in the eventual quantitative description of RNA folding from its secondary and tertiary structural elements.

Computational Mechanistic Study of Redox-Neutral Rh(III)-Catalyzed C-H Activation Reactions of Arylnitrones with Alkynes: Role of Noncovalent Interactions in Controlling Selectivity.

The mechanism of redox-neutral Rh(III)-catalyzed coupling reactions of arylnitrones with alkynes was investigated by density functional theory (DFT) calculations. The free energy profiles associated with the catalytic cycle, involving C(sp2)-H activation, insertion of alkyne, transfer of O atom, cyclization and protodemetalation, are presented and analyzed. An overwhelming preference for alkyne insertion into Rh-C over Rh-O is observed among all pathways, and the most favorable route is determined. The pivalate-assisted C-H activation step is turnover-limiting, and the cyclization step determines the diastereoselectivity of the reaction, with the stereoselectivity arising mainly from the difference of noncovalent interactions in key transition states. The detailed mechanism of O atom transfer, RhIII-RhI-RhIII versus RhIII-RhV-RhIII cycle, is discussed.

Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes.

Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes.