Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate‐controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition‐state structure and bonding optimally requires the use of theoretical models. In this work, we apply density‐functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′‐O‐transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well‐studied series of phosphate and phosphorothioate mono‐, di‐ and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non‐enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.     

Ab initio rotation-vibration spectra of HCN and HNC

We have calculated an ab initio HCN/HNC linelist for all transitions up to J= 25 and 18000 cm(-1) above the zero point energy. This linelist contains more than 200 million lines each with frequencies and transition dipoles. The linelist has been calculated using our semi-global HCN/HNC VQZANO + PES and dipole moment surface, which were reported in van Mourik et al. (J. Chem. Phys. 115 (2001) 3706). With this linelist we synthesise absorption spectra of HCN and HNC at 298 K and we present the band centre and band transition dipoles for the bands which are major features in these spectra. Several of the HCN bands and many of the HNC bands have not been previously studied. Our line intensities reproduce via fully ab initio methods the unusual intensity structure of the HCN CN stretch fundamental (00(0)1) for the first time and also the forbidden (02(2)0) HCN bending overtone. We also compare the J = 1-->0 pure rotational transition dipole in the HCN/HNC ground and vibrationally excited states with experimental and existing ab initio results.     

Resolving Component Overlap in Multiwavelength Kinetic Spectroscopy: Application to Raman Scattering from Intermediates in Triplet-State Photoreactions

Abstract— Understanding of reaction mechanisms in photochemistry is advanced both by kinetic measurements to determine rates of reaction and by spectroscopic studies of the structures of precursors and photoproducts. When kinetics and spectroscopy are combined in a time-resolved, multiwavelength spectroscopic measurement on a reacting system, spectra and structures of intermediates can also be determined. In this paper, the application of multidimensional least-squares and factor analysis techniques for resolving overlapped spectra from intermediates in photochemical kinetics are discussed. The methods are employed specifically to resolve Raman spectra of intermediates in triplet-state photoreactions. By varying excitation intensity, spectra of excited-triplet states are resolved from ground states and solvent. By varying the concentration of a triplet quencher in the sample, the Raman spectrum of a sensitizer excited state (benzophe-none) can be resolved from those of acceptor-triplet states or radical photoproducts. Applications of these concepts in other areas of photochemical kinetics are addressed.     

Exploiting in situ NMR to monitor the formation of a metal–organic framework

The formation processes of metal–organic frameworks are becoming more widely researched using in situ techniques, although there remains a scarcity of NMR studies in this field. In this work, the synthesis of framework MFM-500(Ni) has been investigated using an in situ NMR strategy that provides information on the time-evolution of the reaction and crystallization process. In our in situ NMR study of MFM-500(Ni) formation, liquid-phase ¹H NMR data recorded as a function of time at fixed temperatures (between 60 and 100 °C) afford qualitative information on the solution-phase processes and quantitative information on the kinetics of crystallization, allowing the activation energies for nucleation (61.4 ± 9.7 kJ mol⁻¹) and growth (72.9 ± 8.6 kJ mol⁻¹) to be determined. Ex situ small-angle X-ray scattering studies (at 80 °C) provide complementary nanoscale information on the rapid self-assembly prior to MOF crystallization and in situ powder X-ray diffraction confirms that the only crystalline phase present during the reaction (at 90 °C) is phase-pure MFM-500(Ni). This work demonstrates that in situ NMR experiments can shed new light on MOF synthesis, opening up the technique to provide better understanding of how MOFs are formed.

A new in situ NMR methodology for studying the formation processes of MOFs is reported, supported by SAXS and PXRD experiments. Synthesis of a phosphonate-based MOF is described, from molecular aggregation through to nucleation and crystallisation.     

Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.

Sulfonyl-triazole probes modified with a kinase recognition element are developed for live cell activity-based profiling to identify tyrosine sites located in catalytic and regulatory domains that are important for kinase function.     

ThH5: An Actinide-Containing Superhalogen Molecule

Thorium and its compounds have been widely investigated as important nuclear materials. Previous research focused on the potential use of thorium hydrides, such as ThH₂, ThH₄, and Th₄H₁₅, as nuclear fuels. Here, we report studies of the anion, ThH₅⁻, by anion photoelectron spectroscopy and computations. The resulting experimental and theoretical vertical detachment energies (VDE) for ThH₅⁻ are 4.09 eV and 4.11 eV, respectively. These values and the agreement between theory and experiment facilitated the characterization of the structure of the ThH₅⁻ anion and showed its neutral counterpart, ThH₅ to be a superhalogen. ThH₅⁻, which exhibits a C_4v structure with five Th−H single bonds, possesses the largest known H/M ratio among the actinide elements, M. The adaptive natural density partitioning (AdNDP) method was used to further analyze the chemical bonding of ThH₅⁻ and to confirm the existence of five Th−H single bonds in the ThH₅⁻ molecular anion.     

Synthesis,Antibacterial, and Antioxidant Evaluation of Novel Series of Condensed Thiazoloquinazoline with Pyrido,Pyrano, and Benzol Moieties as Five- and Six-Membered Heterocycle Derivatives

A novel synthesis of thiazolo[2,3-b]quinazolines 4(a–e), pyrido[2′,3′:4,5]thiazolo[2,3-b]quinazolines {5(a–e), 6(a–e), and 7(a–e)}, pyrano[2′,3′:4,5]thiazolo[2,3-b]quinazolines 8(a–e), and benzo[4,5]thiazolo[2,3-b]quinazoloine9(a–e) derivatives starting from 2-(Bis-methylsulfanyl-methylene)-5,5-dimethyl-cyclohexane-1,3-dione 2 as efficient α,α dioxoketen dithioacetal is reported and the synthetic approaches of these types of compounds will provide an innovative molecular framework to the designing of new active heterocyclic compounds. In our study, we also present optimization of the synthetic method along with a biological evaluation of these newly synthesized compounds as antioxidants and antibacterial agents against the bacterial strains, like S. aureus, E. coli, and P. aeruginosa. Among all the evaluated compounds, it was found that some showed significant antioxidant activity at 10 μg/mL while the others exhibited better antibacterial activity at 100 μg/mL. The results of this study showed that compound 6(c) possessed remarkable antibacterial activity, whereas compound 9(c) exhibited the highest efficacy as an antioxidant. The structures of the new synthetic compounds were elucidated by elemental analysis, IR, ¹H-NMR, and ¹³C-NMR.