Fluorine Label Research Articles

Equilibrium concentration profiles of physically end tethered polystyrene molecules at the air-polymer interface

Deuteriopolystyrene of molecular weight ca 32 000 g mol −1 has been functionalized at one end with a fluorocarbon. On mixing this polymer with an unfunctionalized hydrogenous polystyrene a surface excess layer of the deuteriopolymer forms on annealing at 423 K. The surface excess as a function of deuteriopolymer content has been obtained using nuclear reaction analysis and neutron reflectometry, and the shape of the profile from neutron reflectometry alone. From previous secondary ion mass spectral data and by comparison with predictions of surface enrichment theory, it is concluded that the deuteriopolystyrene is tethered by the fluorine label to the air surface. The parameters of this polymer ‘brush’ obtained experimentally are the surface volume fraction of labelled polymer, the brush height, the surface excess and the layer thickness. The surface excess and surface volume fraction of the deuteriopolymer and the adsorption isotherm (surface excess at a function of equilibrium bulk volume fraction) have been compared to the predictions of a self-consistent field theory. Best agreement is obtained with a sticking energy of 1.6 k B T; however, there appear to be some disparities when compared to the limiting ‘dry’ brush predictions. There is evidence from neutron reflectometry of brush formation before annealing of the polymer films.

Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy.

After polymerization filamentous actin (F-actin) still shows a number of rather narrow 1H NMR signals in its Mg2+ form which are quenched when Mg2+ is replaced by Ca2+. These resonances originate from mobile residues in F-actin. For assignment of these resonances three different strategies were used, the fluorine labeling of Cys-374 by 4-(perfluoro-tert-butyl)phenyliodoacetamide, binding studies with antibodies (Fab) against the seven N-terminal amino acids of actin, and two-dimensional 1H NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by 1H NMR spectroscopy, 19F NMR spectroscopy of actin labeled at its C-terminal cysteine shows no significant spectral changes in dependence on the divalent ion present. In its G- (globular) form a strong, narrow 19F resonance can be observed at 15.06 ppm (relative to the external standard trifluoroacetic acid) which is broadened substantially after polymerization of actin. At 283 K the corresponding transverse relaxation time T2 decreases from 16.7 ms to approximately 0.6 ms. These data suggest that the highly mobile residues observed by 1H NMR spectroscopy do not originate from the C-terminus. Binding of Fab directed against the N-terminal amino acids of actin to Mg-F-actin leads to the disappearing of the 1H NMR resonances assigned to a mobile domain in F-actin. This indicates that the mobile region probably comprises the N-terminal amino acids. By homonuclear two-dimensional 1H NMR spectroscopy it was finally possible to sequentially assign the resonances of the mobile domain of F-actin. It turned out that amino acids 1-22 are in a highly mobile state in Mg-F-actin. The nuclear Overhauser effect data indicate that, rather surprisingly, in this high mobility state some of the beta-pleated structure is still conserved. The population of F-actin protomers in the M- (mobile) state can be obtained from the NMR spectra and was determined under different experimental conditions. In the presence of 150 mM KCl approximately half of the protomers in Mg-F-actin are in the M-state. This number is largely independent of the pH in the range studied (pH 7.2-7.8) and of the temperature in range studied (283-310 K). The equilibrium constant KMI for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.

An efficient radiosynthesis of [ 18F]fluoromisonidazole

An efficient preparation of the hypoxic cell tracer [ 18F]3-fluoro-1-(2′-nitro-1′-imidazolyl)-2-propanol ([ 18F]fluoromisonidazole) is reported. This radiopharmaceutical is of interest to probe hypoxic tissue in infarcts and tumors. One-step radiolabeling and rapid protection group removal provided 55–80% yield in 50 min. The process is similar to common fluorine labeling procedures, simplifying the procedure for most laboratories, and offers an improvement over more difficult previous methods. The labeling precursor was prepared in five steps from readily available materials in a straightforward reaction scheme.

The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR.

Escherichia coli citrate synthase is strongly and specifically inhibited by NADH, but this inhibition can be prevented by reacting the enzyme with Ellman's reagent. We have now labeled the single reactive cysteine covalently with monobromobimane and isolated and sequenced the bimane-containing cyanogen bromide peptide and identified the cysteine as Cys-206. Modeling studies suggest that this residue is on the subunit surface, 25-30 A from the active site. Mutation of Cys-206 to serine (C206S), or of Gly-207 to alanine (E207A), weakened NADH binding and inhibition; when these mutations were present together, NADH binding was weaker by 18-fold and inhibition by 250-fold. The mutations also had small effects on substrate binding at the active site. Cys-206 of wild type enzyme and of the mutant E207A was alkylated with 1,1,1-trifluorobromoacetone and the environment of the fluorine nuclei studied by 19F NMR. With wild type enzyme, the NMR spectrum consisted of two peaks of about equal intensity but different line widths, at -8.65 ppm (line width 11.2 +/- 0.5 Hz) and -7.6 ppm (line width 57 +/- 4 Hz). As the labeled wild type citrate synthase was titrated with KCl, the narrow peak converted to the broad one. The same range of KCl concentrations was needed for this conversion as for the allosteric activation of E. coli citrate synthase. The E207A mutant gave the broader NMR peak almost exclusively. We propose that the fluorine label in wild type citrate synthase exists in two conformational states with different mobilities, exchanging slowly on the NMR time scale, and that treatment with KCl, or truncation of the Glu-207 side chain by mutagenesis, stabilizes one of these states. Consistent with this explanation is the finding that Cys-206 reacts more quickly with Ellman's reagent in the presence of KCl, and that this rate is faster yet in the E207A mutant.

19F nuclear magnetic resonance as a probe of the spatial relationship between the heme iron of cytochrome P-450 and its substrate

The distance between the heme iron of ferrous cytochrome P-450-CAM and a fluorine label attached to the 9-methyl carbon of its substrate, (1R)-(+)-camphor, has been determined using 19F NMR. This investigation uses the Solomon-Bloembergen equation to measure the distance from a paramagnetic heme iron to a fluorine probe incorporated into a substrate that is not in fast exchange. The structural identity of the substrate analogue, 9-fluorocamphor, has been established using one- and two-dimensional NMR methods and mass spectrometry. The relaxation rate of 9-fluorocamphor bound to high-spin paramagnetic ferrous P-450-CAM has been studied at 188, 282, and 376 MHz, and the correlation time has been directly determined from the frequency dependence of the relaxation rate. When the substrate analogue was bound to the low-spin diamagnetic ferrous-CO derivative of the enzyme, the relaxation rate was found to be 100 times slower and was therefore neglected in the distance calculation. The relaxation data for the paramagnetic system and the correlation time have been used to calculate a distance of 3.8 A between the heme iron and the C-9 fluoride. A fit of the distance and the chemical shift data to the pseudocontact shift equation predicts an angle of approximately 52 degrees between the heme normal and the Fe-F vector. The solution state Fe-F distance is somewhat shorter and the angle between the heme normal and the Fe-F vector slightly larger for the substrate-bound ferrous enzyme reported herein than the analogous values for the substrate-bound ferric enzyme determined in the solid state by x-ray crystallography. These differences may reflect a structural change at the substrate-binding site upon reduction of the iron.

19F-NMR of fluorine labeled acyl chains in phospholipid bilayer vesicles

19F-labeled phospholipids, L-α-bis-(ω-fluoro palmitoyl)phosphatidylcholine, L-α-bis-(12,12-difluoro stearoyl)phosphatidylcholine and L-α-bis-(6,6-difluoro palmitoyl)phosphatidylcholine, were incorporated in phospholipid vesicles by sonication of aqueous lipid emulsions. Vesicles were prepared both from the pure fluorine substituted phospholipids as well as from lipid mixtures obtained by combining the fluorine substituted lipids with the synthetic phospholipids, L-α-dilauroyl-, L-α-dimyristoyl-, L-α-dipalmitoyl- and L-α-distearoyl-phosphatidylcholine. Characterization by gel permeation chromatography showed that stable unilamellar vesicles with diameters of ≌200 Å could be obtained with a minimum or absence of multilamellar material. The vesicles give rise to two 19F resonances in most cases as observed previously [K.J. Longmuir and F.W. Dahlquist, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2716]. The chemical shift differences undergo systematic changes that confirm the interpretations that the dual 19F resonances arise from the inner and outer halves of the vesicle bilayer. The shift separation increases systematically as the fluorine label is positioned closer to the phospholipid headgroup and decreases systematically with increasing temperature. Both observations agree with what is currently known about phospholipid vesicle structure. Anomalous results are obtained with DSPC as host vesicle since only a single resonance of inbedded fluorinated phospholipids is found.

93] Fluorine-labeled retinals and rhodopsins

Publisher Summary This chapter discusses the fluorine (F)-labeled retinals and rhodopsins. The use of F as a label to probe for structural information of complex biomolecules by nuclear magnetic resonance (nmr) spectroscopy is an established technique. Its advantages over other NMR active nuclei are several. Among them is the absence of background signals from proteins or detergents. The disadvantages associated with the method are the synthetic difficulties sometimes encountered in introducing the fluorine labels at strategic positions and the possibility that the highly electronegative fluorine atom might change the overall properties of the biomolecules of interest. The absorption maxima of isomers of several fluorinated rhodopsins are listed in the accompanying table along with those of the corresponding retinals. That the selectivity of fluororetinal isomers in their reaction with cattle opsin is virtually the same as the parent retinals suggests that the fluorine substituents do not exert a vastly different steric effect.

Synthesis and quality control of aliphatic and aromatic 18F-labelled compounds for probing metabolism in-vivo

Biomolecules tagged with fluorine-18, a positron emitter with a half-life of 110 min, are gaining importance in diagnostic nuclear medicine for measuring regional functions in-vivo by means of positron emission tomography. Procedures for introducing 18 F into organic compounds, however, are limited due to the short half-life. In addition, the toxicity of many fluorine compounds requires practically carrier-free products. Hence, fast syntheses have to be carried out using fluorinating agents in the micro- or nanogram scale. On the other hand, the tracer provides unique possibilities for studying metabolic functions of toxic or centrally active fluorine compounds in-vivo. We have synthesized several aliphatic and aromatic fluorine-18 labelled compounds by nucleophilic 18 F-for-halogen exchange: For the study of regional metabolism in heart and liver of mice 16- 18 F-hexadecanoic acid, 17- 18 F-heptadecanoic acid, 2- 18 F-, and (9,10)- 18 F-stearic acid were prepared in a mixture of molten acetamide and the corresponding bromofatty acid ester followed by hydrolysis and purification by high pressure liquid chromatography. Variation of temperature, reaction time, and KF- carrier finally led to an optimum radiochemical yield of about 30% [1]. The biochemical effects of the fluorine label, as expected on the basis of β-oxidation, is clearly reflected in the pharmacokinetics and the chemical fate of the fluorine label observed in mice: The odd-numbered compound, 17- 18 F- heptadecanoic acid, is catabolized to β- 18 F-propionic acid while the even-numbered 16- 18 F-hexadecanoic acid ends up with 18 F-fluoroacetic acid entering the citric cycle. Further degradation, i.e. dehalogenation, only occurs in the case of 17- 18 F-heptadecanoic acid yielding free 18 F- fluoride which can be detected in high yield among its metabolites [2]. With respect to diagnostic methods for the localisation of thrombi, the protein urokinase labelled with 18 F- fluoroacetic acid could be a useful compound, since it is expected that it will be concentrated in the thrombus thus giving the possibility of localisation by positron- emission tomography. 18 F-fluoroacetic acid has therefore been prepared carrier-free in order to prevent the occupation of active sites in urokinase. For the study of regional metabolism in brain, 2- 18 F- nicotinic acid diethylamide is potentially useful. The nonhalogenated compound is known to be a centrally acting pharmaceutical. Preparation of the 18 F-labelled compound was carried out by a similar procedure as in the case of the fatty acids starting from the corresponding chloro- compound. By this method optimum radiochemical yields up to 46% could be obtained. Preliminary results in experiments with mice show a fast accumulation of 18 F-activity in the brain within the first seconds after injection, followed by a slower decrease [3].

On the mechanism for loss of CO from aromatic oximes. Evidence for a nucleophilic attack from fluorine labelling

AbstractThe mehanisms for loss of CO from benzaldoxime and acetophenone oxime have been investigated with the aid of both fluorine and deuterium labelling as well as field free region metastable peak shapes. Kinetic evergy release data and the metastable ion abundance raion test were used to obtain information about the product ions. It is proposed that loss of CO from both are generated via the nucleophilic attack of the oxygen lone pair eletrons at the ortho position of the phenyl ring as the first step in the reaction sequence.

Electron spin resonance of phosphorescent fluorine-substituted aromatics

The lowest excited triplet state of diphenyl and of six fluorinated derivatives has been investigated by optical and E.S.R. spectroscopy in ethanol solutions at 77 K. Phosphorescence spectra, lifetimes, zero field energies and hyperfine splittings of Δ m =2 and Δ m =1 stationary peaks are reported. The principal axes of the zero field splitting tensors were determined by magnetophotoselection. The data are interpreted using a model for the effects of fluorine substitution introduced in Part I and the fluorine hyperfine coupling tensor derived in that paper. The results are: (i) a clear indication of the planarity of phosphorescent diphenyl; (ii) a spin density map of the diphenyl triplet state. The further application of the fluorine labelling method to determine simply the electronic structure of phosphorescent hydrocarbons is outlined.

Aromatic Fluorine as a Chemical Label for Detecting Reaction Mechanism

AbstractThis article explains how nucleophilically activated fluorine can be used for the detection of ion‐pair formation during the decomposition of various aryl radical generators. Thus a concurrent dual mechanism for the breakdown of certain aryl radical sources can be experimentally demonstrated by fluorine labeling. The bearing of this information on the mechanism of diazo coupling is also discussed.In addition, labeling with aromatic fluorine is applied to the thermolysis of aryl azides, the aminolysis of 3‐aryl‐4‐bromosydnones, and the hydrolysis of 3‐arylsydnone imines. Finally some mechanistic problems are posed which may possibly be solved by this labeling technique.

Syntheses of heterocyclic compounds. Part XVIII. Aminolysis of 3-aryl-4-bromosydnones, and acid hydrolysis of 3-arylsydnoneimines

Reaction of various 3-aryl- and 3-heteroaryl-4-bromosydnones with piperidine gives the corresponding piperidino-glycyl-piperidines. Fluorine labelling has confirmed the postulated course of fission induced by acids in 3-aryl-sydnoneimines.

The Chemical Behavior of Fluorine 18 Produced by the O16(H3,n) Nuclear Reaction

F/sup 18/ atoms produced by the Li/sup 6/(n, alpha )H/sup 3/, O/sup 16/ (H/sup 3/ ,n)F/dup 18/ nuclear reactions were found to substitute hydrogen on organlc molecules. The extent of labeling of a given organic molecule was found to be proportional to the number of hydrogens per molecule. In solid lithium salts, irradiated by slow neutrons, the yield of organically bound F/sup 18/ was 0.6 to 1.0% per aliphatic hydrogen, as compared to 0.3 to 0.6% per aromatic hydrogen. Compounds were also labeled in solutions and the yleld was dependent on the concentration of the solute. The yield of labeling of aliphatic solutes was higher than that of aromatic. The addition of aromatic compounds to an aliphatic solvent diminished the labeling yield of the solvent, however no appreciable labeling of the aromatic solute was observed. Aliphatic solutes, on the other hand, did not affect the yield of labeling of aliphatic solvents. Formic acid and nitrite ions were found to diminish considerably the yield of labeling in aqueous or alcoholic solutions. The results suggest that the active chemical species in the fluorine labeling reactions are non-excited fluorine atoms. Interactions with electron donors are of primary importance in the behavior of fluorinemore » atoms in organic media.« less