Nuclear Magnetic Resonance Time Scale Research Articles

Solution Nuclear Magnetic Resonance Structure of the GATase Subunit and Structural Basis of the Interaction between GATase and ATPPase Subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

The Denatured State Ensemble Contains Significant Local and Long-Range Structure under Native Conditions: Analysis of the N-Terminal Domain of Ribosomal Protein L9

The denatured state ensemble (DSE) represents the starting state for protein folding and the reference state for protein stability studies. Residual structure in the DSE influences the kinetics of protein folding, the propensity to aggregate, and protein stability. The DSE that is most relevant for folding is the ensemble populated under native conditions, but the stability of proteins and the cooperativity of their folding normally prevent direct characterization of this ensemble. Indirect experiments have been used to infer residual structure in the DSE under nondenaturing conditions, but direct characterization is rare. The N-terminal domain of ribosomal protein L9 (NTL9) is a small mixed α-β domain that folds cooperatively on the millisecond time scale. A destabilized double mutant of NTL9, V3A/I4A-NTL9, populates the DSE in the absence of denaturant and is in slow exchange with the native state on the nuclear magnetic resonance time scale. The DSE populated in buffer was compared to the urea-induced DSE. Analysis of (1)H and (13)C chemical shifts reveals residual secondary structure in the DSE in buffer, which is stabilized by both local and long-range interactions. (15)N R2 relaxation rates deviate from random coil models, suggesting hydrophobic clustering in the DSE. Paramagnetic relaxation enhancement experiments show that there are transient long-range contacts in the DSE in buffer. In contrast, the urea-induced DSE has significantly less residual secondary structure and markedly fewer long-range contacts; however, the urea-induced DSE deviates from a random coil.

The Coil-to-Helix Transition in IlvN Regulates the Allosteric Control of Escherichia coli Acetohydroxyacid Synthase I

The solution structure of IlvN, the regulatory subunit of Escherichia coli acetohydroxyacid synthase I, in the valine-bound form has been determined using high-resolution multidimensional, multinuclear nuclear magnetic resonance (NMR) methods. IlvN in the presence or absence of the effector molecule is present as a 22.5 kDa dimeric molecule. The ensemble of 20 low-energy structures shows a backbone root-mean-square deviation of 0.73 ± 0.13 Å and a root-mean-square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, more than 98% of the backbone φ and ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map, which is indicative of the fact that the structures are of high stereochemical quality. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain seen in metabolic enzymes. In the valine-bound form, IlvN exists apparently as a single conformer. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR time scale. Thus, a large shift in the conformational equilibrium is observed upon going from the free form to the bound form. The structure of the valine-bound form of IlvN was found to be similar to that of the ACT domain of the unliganded form of IlvH. Comparisons of the structures of the unliganded forms of these proteins suggest significant differences. The structural and conformational properties of IlvN determined here have allowed a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis.

NMR investigations of structural and dynamics features of natively unstructured drug peptide - salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H(α) chemical shifts and three bond H(N) -H(α) coupling constants indicated that most of the residues of the peptide are populating the α-helical region of the Ramachandran (ϕ, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10(2) -10(3) s(-1) ), inferred by the multiple chemical shift assignments in the region Leu4-Leu12 and around Pro23 (for residues Gln20-Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self-association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The (15) N NMR-relaxation data revealed (i) the presence of slow (microsecond-to-millisecond) timescale dynamics in the N-terminal region (Cys1-Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond-to-picosecond) motions in the C-terminal arm. Put together, the various results suggested that (i) the flexible C-terminal of sCT (from Thr25-Thr31) is involved in identification of specific target receptors, (ii) whereas the N-terminal of sCT (from Cys1-Gln20) in solution - exhibiting significant amount of conformational plasticity and strong bias towards biologically active α-helical structure - facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs.

Synthesis and Stereodynamics of N-Formyl-1,2,3- Selenadiazolopyridines

The 4,6-diaryl-6,7-dihydro-[1,2,3]selenadiazolo[5,4-c]pyridine-5(4H)-carbalde hydes were synthesized from 2,6-diarylpiperidin-4-ones and characterized using infrared, 1H, 13C Nuclear Magnetic Resonance (NMR), and mass spectroscopic techniques. On the NMR time scale, the compounds exist in syn and anti isomeric forms. Separate signals were obtained for isomers in the NMR spectra. The compounds’ stereodynamic nature was studied based on the intensity and position of NMR signals. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.

Analysis of electrostatic interactions in the denatured state ensemble of the N‐terminal domain of L9 under native conditions

The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.

A Comparison of Complexation of Li+ Ion with Macrocyclic Ligands 15-Crown-5 and Benzo-derivatives in Binary Nitromethane–Acetonitrile Mixtures by Using Lithium-7 NMR Technique and Ab Initio Calculation

Lithium-7 nuclear magnetic resonance (NMR) measurements were used to investigate the stoichiometry and stability of Li+ complexes with 15-crown-5 (15C5), benzo-15-crown-5 (B15C5) and dibenzo-15-crown-5 (DB15C5) in a number of nitromethane (NM)–acetonitrile (AN) binary mixtures. In all cases, the exchange between the free and complexed lithium ion was fast on the NMR time scale and a single population average resonance was observed. While all crown ethers form 1:1 complexes with Li+ ion in the binary mixtures used, stepwise formation constants of the 1:1 (ligand/metal) complexes were evaluated from computer fitting of the NMR-mole ratio data to equations which relate the observed metal ion chemical shifts to formation constants. There is an inverse linear relationship between the logarithms of the stability constants and the mole fraction of AN in the solvent mixtures. The stability order of the 1:1 complexes was observed to be 15C5.Li+ > B15C5.Li+ > DB15C5.Li+. The optimized structures of the free ligands and their 1:1 complexes with the Li+ ion were predicted by ab initio theoretical calculations using the Gaussian 98 software. The results of calculations are discussed. .

Functional Identification of Toxin-Antitoxin Molecules from Helicobacter pylori 26695 and Structural Elucidation of the Molecular Interactions

Bacterial toxin-antitoxin (TA) systems are associated with many important cellular processes including antibiotic resistance and microorganism virulence. Here, we identify and structurally characterize TA molecules from the gastric pathogen, Helicobacter pylori. The HP0894 protein had been previously suggested, through our structural genomics approach, to be a putative toxin molecule. In this study, the intrinsic RNase activity and the bacterial cell growth-arresting activity of HP0894 were established. The RNA-binding surface was identified at three residue clusters: (Lys(8) and Ser(9)), (Lys(50)-Lys(54) and Glu(58)), and (Arg(80) and His(84)-Phe(88)). In particular, the -UA- and -CA- sequences in RNA were preferentially cleaved by HP0894, and residues Lys(52), Trp(53), and Ser(85)-Lys(87) were observed to be the main contributors to sequence recognition. The action of HP0894 could be inhibited by the HP0895 protein, and the HP0894-HP0895 complex formed an oligomer with a binding stoichiometry of 1:1. The N and C termini of HP0894 constituted the binding sites to HP0895. In contrast, the unstructured C-terminal region of HP0895 was responsible for binding to HP0894 and underwent a conformational change in the process. Finally, DNA binding activity was observed for both HP0895 and the HP0894-0895 complex but not for HP0894 alone. Taken together, it is concluded that the HP0894-HP0895 protein couple is a TA system in H. pylori, where HP0894 is a toxin with an RNase function, whereas HP0895 is an antitoxin functioning by binding to both the toxin and DNA.

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4′-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4′-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface.

ChemInform Abstract: Solid State Proton Spin Relaxation in Ethylbenzenes: Methyl Reorientation Barriers and Molecular Structure

We have investigated the dynamics of the ethyl groups and their constituent methyl groups in polycrystalline ethylbenzene (EB), 1,2‐diethylbenzene (1,2‐DEB), 1,3‐DEB, and 1,4‐DEB using the solid state proton spin relaxation (SSPSR) technique. The temperature and Larmor frequency dependence of the Zeeman spin‐lattice relaxation rate is reported and interpreted in terms of the molecular dynamics. We determine that only the methyl groups are reorienting on the nuclear magnetic resonance time scale. The observed barrier of about 12 kJ/mol for methyl group reorientation in the solid samples of EB, 1,2‐DEB, and 1,3‐DEB is consistent with that of the isolated molecule, implying that in the solid state, intermolecular electrostatic interactions play a minor role in determining the barrier. The lower barrier of 9.3±0.2 kJ/mol for the more symmetric 1,4‐DEB suggests that the crystal structure is such that the minimum in the anisotropic part of the intramolecular potential is raised by the intermolecular interactions leading to a 3 kJ/mol decrease in the total barrier. We are able to conclude that the methyl group is well away from the plane of the benzene ring (most likely orthogonal to it) in all four molecules, and that in 1,2‐DEB, the two ethyl groups are in the anticonfiguration. Our SSPSR results are compared with the results obtained by microwave spectroscopy and supersonic molecular jet laser spectroscopy, both of which determine molecular geometry better than SSPSR, but neither of which can determine ground electronic state barriers for these molecules.

Nuclear Magnetic Resonance Spectroscopy Demonstrating Dynamic Stabilization of CdSe Quantum Dots by Alkylamines

The exchange kinetics of octylamine between a free state and a state bound to the surface of CdSe quantum dots is analyzed using nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1D 1H and DOSY and NOESY spectroscopy, we find that all octylamine molecules present interact with the CdSe quantum dot surface, although the octylamine resonances and diffusion coefficient resemble those of free octylamine. This indicates that these NMR observables are a weighted average of a free and a bound state, implying that the overall octylamine exchange rate is fast as compared to the intrinsic NMR time scale. A lower limit on the first-order desorption rate constant of 50 s−1 follows from DOSY measurements as a function of the diffusion delay. This result is compared with literature data based on the quenching of the CdSe photoluminescence upon alkylamine desorption.

The influence of sodium chloride on the self-association and chromonic mesophase formation of Edicol Sunset Yellow

We have investigated the effect of an inorganic electrolyte (sodium chloride) on the aggregation behaviour and liquid crystals of Edicol Sunset Yellow. Edicol self-aggregates in aqueous solution to form single molecule stacks, which then become ordered to form nematic and hexagonal (columnar) mesophases at high concentrations. We have employed changes in the 1H nuclear magnetic resonance (NMR) chemical shifts to monitor the aggregate formation in solution. A single spectrum is observed at all concentrations because the exchange between Edicol monomers in solution and those in stacks is fast on the NMR time scale. The results show that at low Edicol concentrations (<1 wt%) the concentration of aggregates is small, but at high concentrations (20 wt%) the fraction of monomers is tiny. At low Edicol concentrations, low levels of salinity appear to alter aggregate shape and size, resulting in a disaggregation/aggregation effect occurring over four orders of magnitude of added electrolyte. However, little alteration is seen in the fraction of aggregates. At high electrolyte levels, when the Debye length is comparable to the stack lengths (a few nanometres), the fraction of aggregates increases, presumably because of the reduced intra-stack electrostatic repulsion. Importantly, we have also shown that the isodesmic theory of aggregation (equal K) is too simple to describe accurately the aggregation process from the monomer to the pre-nematic phase concentrations. NMR quadrupole splittings indicate that there is no specific Na+ ion binding to the stacks. At the very highest concentrations of Edicol and sodium chloride the aggregates and mesophases are destabilised. The reason for this has yet to be elucidated.

A proton spin-lattice relaxation rate study of methyl and t-butyl group reorientation in the solid state

We have measured the solid state nuclear magnetic resonance (NMR) 1H spin-lattice relaxation rate from 93 to 340 K at NMR frequencies of 8.5 and 53 MHz in 5- t-butyl-4-hydroxy-2-methylphenyl sulfide. We have also determined the molecular and crystal structures from X-ray diffraction experiments. The relaxation is caused by methyl and t-butyl group rotation modulating the spin–spin interactions and we relate the NMR dynamical parameters to the structure. A successful fit of the data requires that the 2-methyl groups are rotating fast (on the NMR time scale) even at the lowest temperatures employed. The rotational barrier for the two out-of-plane methyl groups in the t-butyl groups is 14.3±2.7 kJ mol −1 and the rotational barrier for the t-butyl groups and their in-plane methyl groups is 24.0±4.6 kJ mol −1. The uncertainties account for the uncertainties associated with the relationship between the observed NMR activation energy and a model-independent barrier, as well as the experimental uncertainties.

Dominant Conformation of Valsartan in Sodium Dodecyl Sulfate Micelle Environment

The interaction of valsartan (VST), a novel antihypertensive drug, with sodium dodecyl sulfate (SDS) micelles has been investigated using Nuclear Magnetic Resonance (NMR) spectroscopy and Molecular Dynamics (MD) simulation. VST has two conformations in solution, exchanging slowly on the NMR time scale via the trans/cis (conformer A/B) isomerization of the amide bond. It is suggested that drugs in the sartan class incorporate and diffuse into biological membranes before they interact with AT(1) receptors. SDS is used to mimic the membrane environment to characterize two VST conformers. (1)H NMR chemical shift analysis, proton relaxation rates, and self-diffusion coefficient measurements suggest that conformer A has a higher binding affinity to SDS and is the dominant conformer distributed in the SDS micelles. The location of VST in the micelles is determined by NOE measurements and by the MD simulation, showing that the butyl chain and biphenyl groups of VST interact with the alkyl group of SDS through hydrophobic interactions. Preferable binding free energy is found for conformer A by the MD simulation, which demonstrates that the relatively concentrated hydrophobic surface of conformer A is responsible for its higher affinity to the micelles. Our results are in good agreement with a recent simulation of VST bound onto the AT(1) receptor by Potamitis et. al (J. Chem. Inf. Model. 2009) who demonstrate that conformer A (trans conformation in their definition) is the one binding to the receptor. The results presented in our study suggest that the biological membrane plays an essential role in stabilization of the active state of VST. Thus, understanding the interactions between the sartan drugs and the membrane environment should facilitate the studies of the functional mechanism of these compounds with their receptor and provide insight on the development of new approaches for drug discovery.

13C NMR investigation of fullerene–cubane C60·C8H8 cocrystals

AbstractThe rotor–stator molecular cocrystal C60·C8H8 (fullerene–cubane) has been investigated by 13C nuclear magnetic resonance (NMR). The room‐temperature spectrum obtained using 1H–13C cross‐polarization technique shows two lines with chemical shifts identical with the shifts of the original molecular constituents demonstrating the lack of a strong electronic interaction between C60 and C8H8. The temperature dependence of the spin‐lattice relaxation time of the fullerene component confirms the existence of a first‐order orientational ordering transition around 145 K. The activation energies of large‐angle C60 reorientations above and below the ordering transitions are 260 K and 570 K, respectively. The transition temperature and the activation energies are significantly lower than in other C60 compounds. The 13C spectrum remains narrow down to 115 K indicating that similarly to pristine C60, the molecular reorientational motion is still fast in the ordered phase on the NMR time scale.

Peptidyl-Prolyl Isomerase FKBP52 Controls Chemotropic Guidance of Neuronal Growth Cones via Regulation of TRPC1 Channel Opening

Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase-independent mechanisms, FKBPs are most highly expressed in the nervous system, where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino-terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1alpha to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.

Substituted β-Cyclodextrin and Calix[4]arene As Encapsulatory Vehicles for Platinum(II)-Based DNA Intercalators

The encapsulation of three platinum(II)-based anticancer complexes, [(5,6-dimethyl-1,10-phenanthroline)(1 S,2 S-diaminocyclohexane)platinum(II)] (2+) ( 56MESS), [(5,6-dimethyl-1,10-phenanthroline)(1 R,2 R-diaminocyclohexane)platinum(II)] (2+) ( 56MERR), and [(5,6-dimethyl-1,10-phenanthroline)(ethylenediamine)platinum(II)] (2+) ( 56MEEN), with carboxylated-beta-cyclodextrin (c-beta-CD) and p-sulfonatocalix[4]arene (s-CX[4]) has been examined by one- and two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, pulsed gradient spin-echo NMR, ultraviolet spectrophotometry, glutathione degradation experiments, and growth inhibition assays. Titration of any of the three metal complexes with c-beta-CD resulted in 1:1 encapsulation complexes with the cyclodextrin located over the intercalating ligand of the metal complexes, with a binding constant of 10 (4)-10 (5) M (-1). In addition to binding over the phenanthroline ligand of 56MEEN, c-beta-CD was also found to portal bind to the ethylenediamine ligand, with fast exchange kinetics on the NMR timescale between the two binding sites. In contrast, the three metal complexes all formed 2:2 inclusion complexes with s-CX[4] where the two metal complexes stacked in a head-to-tail configuration and were capped by the s-CX[4] molecules. Interestingly, the 56MEEN-s-CX[4] complex appeared to undergo a thermodynamically controlled rearrangement to a less soluble complex over time. Encapsulation of the metal complexes in either c-beta-CD or s-CX[4] significantly decreased the metal complexes' rate of diffusion, consistent with the formation of larger particle volumes. Encapsulation of 56MESS within s-CX[4] or c-beta-CD protected the metal complex from degradation by reduced L-glutathione, with a reaction half-life greater than 9 days. In vitro growth inhibition assays using the LoVo human colorectal cancer cell line showed no significant change in the cytotoxicity of 56MESS when encapsulated by either s-CX[4] or c-beta-CD.

Dynamics of Silica-Supported Catalysts Determined by Combining Solid-State NMR Spectroscopy and DFT Calculations

The molecular dynamics of a series of organometallic complexes covalently bound to amorphous silica surfaces is determined experimentally using solid-state nuclear magnetic resonance (NMR) spectroscopy and density functional theory calculations (DFT). The determination is carried out for a series of alkylidene-based catalysts having the general formula [([triple bond]SiO)M(ER)(=CH(t)Bu)(R')] (M = Re, Ta, Mo or W; ER = C(t)Bu, NAr or CH2(t)Bu; R' = CH2(t)Bu, NPh2, NC4H4). Proton-carbon dipolar coupling constants and carbon chemical shift anisotropies (CSA) are determined experimentally by solid-state NMR. Room-temperature molecular dynamics is quantified through order parameters determined from the experimental data. For the chemical shift anisotropy data, we validate and use a method that integrates static values for the CSA obtained computationally by DFT, obviating the need for low-temperature measurements. Comparison of the room-temperature data with the calculations shows that the widths of the calculated static limit dipolar couplings and CSAs are always greater than the experimentally determined values, providing a clear indication of motional averaging on the NMR time scale. Moreover, the dynamics are found to be significantly different within the series of molecular complexes, with order parameters ranging from <S(z)> = 0.5 for [([triple bond]SiO)Ta(=CH(t)Bu)(CH2(t)Bu)2] and [([triple bond]SiO)Re([triple bond]C(t)Bu)(=CH(t)Bu)(CH2(t)Bu)] to <S(z)> = 0.9 for [([triple bond]SiO)Mo([triple bond]NAr)(=CH(t)Bu)(R') with R' = CH2(t)Bu, NPh2, NC4H4. The data also show that the motion is not isotropic and could be either a jump between two sites or more likely restricted librational motion. The dynamics are discussed in terms of the molecular structure of the surface organometallic complexes, and the orientation of the CSAs tensor at the alkylidene carbon is shown to be directly related to the magnitude of the alpha-alkylidene CH agostic interation.