1H and 19F NMR relaxation time studies in (NH4)2ZrF6 superionic conductor

1H and 19F spin-lattice relaxation times in polycrystalline diammonium hexafluorozirconate have been measured in the temperature range of 10–400 K to elucidate the molecular motion of both cation and anion. Interesting features such as translational diffusion at higher temperatures, molecular reorientational motion of both cation and anion groups at intermediate temperatures and quantum rotational tunneling of the ammonium group at lower temperatures have been observed. Nuclear magnetic resonance (NMR) relaxation time results correlate well with the NMR second moment and conductivity studies reported earlier.

Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study

Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.

Degradation of 4-fluorobiphenyl in soil investigated by 19F NMR spectroscopy and 14C radiolabelling analysis

The incubation of the model pollutant [U-14C]'-4-fluorobiphenyl (4FBP) in soil, in the presence and absence of biphenyl (a co-substrate), was carried out in order to study the qualitative disposition and fate of the compound using 14C-HPLC and 19F NMR spectroscopy. Components accounted for using the radiolabel were volatilization, CO2 evolution, organic solvent extractable and bound residue. Quantitative analysis of these data gave a complete mass balance. After sample preparation. 14C-HPLC was used to establish the number of 4FBP related components present in the organic solvent extract. 19F NMR was also used to quantify the organic extracts and to identify the components of the extract. Both approaches showed that the composition of the solvent extractable fractions comprised only parent compound with no metabolites present. As the 14C radiolabel was found to be incorporated into the soil organic matter this indicates that metabolites were being generated, but were highly transitory as incorporation into the SOM was rapid. The inclusion of the co-substrate biphenyl was to increase the overall rate of degradation of 4FBP in soil. The kinetics of disappearance of parent from the soil using the data obtained were investigated from both techniques. This is the first report describing the degradation of a fluorinated biphenyl in soil.

A 7Li and 19F NMR relaxation study of LiCF3SO3 in plasticised solid polyether electrolytes

⁷Li and ¹⁹F NMR relaxation time (T₁, T₂, T_1ρ) measurements have been used to probe the dynamics of LiCF₃SO₃ dissolved in an amorphous co-polymer poly(ethylene oxide-co-propylene oxide), and in particular the influence of the plasticising agents propylene carbonate and dimethyl formamide. The changes in relaxation behaviour of ¹⁹F and ⁷Li with increasing plasticiser concentration are very different, as is the effect of each plasticiser. These differences can be explained qualitatively in terms of the interaction between the plasticiser and the ions. Preliminary ⁷Li T_1ρ measurements reveal two components at low temperatures.

NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution 19F NMR

We report high resolution solution 19F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF3-CH2-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6–10 mg), in dodecylmaltoside, were analyzed at 20°C by solution 19F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for 19F labels at positions 67 (−0.2 ppm) and 140 (−0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution 19F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.

Using O2 to probe membrane immersion depth by 19F NMR

A fluorinated detergent, CF3(CF2)5C2H4-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by 19F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on 19F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (R1) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O2 at least 100-fold (i.e., PO2 greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (R120bar − R10) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(N,N-dimethyl-N-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in 19F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by 19F NMR.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR

We have used 19F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of 19F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg2+. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg2+ with an association constant in the range of 100 to 500 M−1, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

Solution 19F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution 19F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution 19F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140–Cys-316, Cys-65–Cys-316, and Cys-139–Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8–10 mg) in dodecylmaltoside and analyzed at 20°C by solution 19F NMR. Distinct chemical shifts were observed for all of the rhodopsin 19F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65–Cys-316 mutant suggests a close proximity between the two residues. When analyzed for 19F-19F NOEs, a moderate negative enhancement was observed for the Cys-65–Cys-316 pair and a strong negative enhancement was observed for the Cys-139–Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140–Cys-316 pair. These NOE effects demonstrate a solution 19F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.

Understanding immobilised enzymes by NMR spectroscopy

Enzyme immobilisation is the conversion of a soluble enzyme molecule into a solid particle form. This allows the recovery of the enzyme catalyst for its re-use and avoids protein contamination of the product streams. A better understanding of immobilised enzymes is necessary for their rational development. A more rational design can help enormously in the applicability of these systems in different areas, from biosensors to chemical industry. Immobilised enzymes are challenging systems to study and very little information is given by conventional biochemical analysis such as catalytic activity and amount of protein. Here, solid-state NMR has been applied as the main technique to study these systems and evaluate them more precisely. Various approaches are presented for a better understanding of immobilised enzymes, which is the aim of this thesis. Firstly, the requirements of a model system of study will be discussed. The selected systems will be comprehensibly characterised by a variety of techniques but mainly by solid-state NMR. The chosen system will essentially be the enzyme α-chymotrypsin covalently immobilised on two functionalised inorganic supports – epoxide silica and epoxide alumina – and an organic support – Eupergit®. The study of interactions of immobilised enzymes with other species is vital for understanding the macromolecular function and for predicting and engineering protein behaviour. The study of water, ions and inhibitors interacting with various immobilised enzyme systems is covered here. The interactions of water and sodium ions were studied by 17O and 23Na multiple-quantum techniques, respectively. Various pore sizes of the supports were studied for the immobilised enzyme in the presence of labelled water and sodium cations. Finally, interactions between two fluorinated inhibitors and the active site of the enzyme will be explored using 19F NMR, offering a unique approach to evaluate catalytic behaviour. These interactions will be explored by solution-state NMR firstly, then by solid-state NMR. NMR has the potential to give information about the state of the protein in the solid support, but the precise molecular interpretation is a difficult task.

Studies of the radiation chemistry and grafting of a fluoropolymer

The radiation chemistry and the grafting of a fluoropolymer, poly(tetrafluoroethylene-coperfluoropropyl vinyl ether) (PFA), was investigated with the aim of developing a highly stable grafted support for use in solid phase organic chemistry (SPOC). A radiation-induced grafting method was used whereby the PFA was exposed to ionizing radiation to form free radicals capable of initiating graft copolymerization of styrene. To fully investigate this process, both the radiation chemistry of PFA and the grafting of styrene to PFA were examined. Radiation alone was found to have a detrimental effect on PFA when irradiated at 303 K. This was evident from the loss in the mechanical properties due to chain scission reactions. This meant that when radiation was used for the grafting reactions, the total radiation dose needed to be kept as low as possible. The radicals produced when PFA was exposed to radiation were examined using electron spin resonance spectroscopy. Both main-chain (–CF2–C.F–CF2-) and end-chain (–CF2–C.F2) radicals were identified. The stability of the majority of the main-chain radicals when the polymer was heated above the glass transition temperature suggested that they were present mainly in the crystalline regions of the polymer, while the end-chain radicals were predominately located in the amorphous regions. The radical yield at 77 K was lower than the radical yield at 303 K suggesting that cage recombination at low temperatures inhibited free radicals from stabilizing. High-speed MAS 19F NMR was used to identify the non-volatile products after irradiation of PFA over a wide temperature range. The major products observed over the irradiation temperature 303 to 633 K included new saturated chain ends, short fluoromethyl side chains in both the amorphous and crystalline regions, and long branch points. The proportion of the radiolytic products shifted from mainly chain scission products at low irradiation temperatures to extensive branching at higher irradiation temperatures. Calculations of G values revealed that net crosslinking only occurred when PFA was irradiated in the melt. Minor products after irradiation at elevated temperatures included internal and terminal double bonds and CF3 groups adjacent to double bonds. The volatile products after irradiation at 303 K included tetrafluoromethane (CF4) and oxygen-containing species from loss of the perfluoropropyl ether side chains of PFA as identified by mass spectrometry and FTIR spectroscopy. The chemical changes induced by radiation exposure were accompanied by changes in the thermal properties of the polymer. Changes in the crystallinity and thermal stability of PFA after irradiation were examined using DSC and TGA techniques. The equilibrium melting temperature of untreated PFA was 599 K as determined using a method of extrapolation of the melting temperatures of imperfectly formed crystals. After low temperature irradiation, radiation- induced crystallization was prevalent due to scission of strained tie molecules, loss of perfluoropropyl ether side chains, and lowering of the molecular weight which promoted chain alignment and hence higher crystallinity. After irradiation at high temperatures, the presence of short and long branches hindered crystallization, lowering the overall crystallinity. The thermal stability of the PFA decreased with increasing radiation dose and temperature due to the introduction of defect groups. Styrene was graft copolymerized to PFA using -radiation as the initiation source with the aim of preparing a graft copolymer suitable as a support for SPOC. Various grafting conditions were studied, such as the total dose, dose rate, solvent effects and addition of nitroxides to create “living” graft chains. The effect of dose rate was examined when grafting styrene vapour to PFA using the simultaneous grafting method. The initial rate of grafting was found to be independent of the dose rate which implied that the reaction was diffusion controlled. When the styrene was dissolved in various solvents for the grafting reaction, the graft yield was strongly dependent of the type and concentration of the solvent used. The greatest graft yield was observed when the solvent swelled the grafted layers and the substrate. Microprobe Raman spectroscopy was used to map the penetration of the graft into the substrate. The grafted layer was found to contain both poly(styrene) (PS) and PFA and became thicker with increasing radiation dose and graft yield which showed that grafting began at the surface and progressively penetrated the substrate as the grafted layer was swollen. The molecular weight of the grafted PS was estimated by measuring the molecular weight of the non-covalently bonded homopolymer formed in the grafted layers using SEC. The molecular weight of the occluded homopolymer was an order of magnitude greater than the free homopolymer formed in the surrounding solution suggesting that the high viscosity in the grafted regions led to long PS grafts. When a nitroxide mediated free radical polymerization was used, grafting occurred within the substrate and not on the surface due to diffusion of styrene into the substrate at the high temperatures needed for the reaction to proceed. Loading tests were used to measure the capacity of the PS graft to be functionialized with aminomethyl groups then further derivatized. These loading tests showed that samples grafted in a solution of styrene and methanol had superior loading capacity over samples graft using other solvents due to the shallow penetration and hence better accessibility of the graft when methanol was used as a solvent.

A new synthetic procedure for the preparation of pyridinium tetrafluoroborate and hexafluorosilicate

Pyridinium tetrafluoroborate (C5H5NHBF4) and pyridinium hexafluorosilicate [(C5H5NH)2SiF6] have been prepared in good yields and high purity by the reaction of pyridinium poly(hydrogen fluoride) with oxides and acids of boron and silicon respectively. The salts have been characterised by melting points, IR, 1H and 19F NMR spectroscopy and chemical analysis.