Radiolabeling of protected tryptophan with [18F]fluoromethyl tosylate: Formation of [18F]fluoromethyl ester of tryptophan instead of 1-N-[18F]fluoromethyl tryptophan methylester.

The reaction of [18F]fluoromethyl tosylate with methyl(tert-butoxycarbonyl)-l-tryptophanate results in formation the O-alkylated ester of the tryptophan instead of alkylation of the indole nitrogen of tryptophan as initially anticipated. Treatment of protected tryptophan with NaH in dimethyl formamide (DMF) along with [18F]fluoromethyl tosylate at 130°C results in the formation of [18F]fluoromethyl(tert-butoxycarbonyl)-l-tryptophanate. Preferential formation of the O-alkylated product is postulated to be due to the hydrolysis of the ester. Confirmation of the O-alkylation was obtained by synthesizing the [19F]fluoromethyl(tert-butoxycarbonyl)-l-tryptophanate insitu and examining its NMR characteristics using multiple NMR techniques. Similar results were also obtained when reacting Boc-tryptophan-N-carboxyanhydride precursor with fluoromethyl tosylate.

Challenges in the automated synthesis of [18F]-1-fluoroethyl tryptophan: Formation of both O- and N-alkylated products.

[18F]Fluoroethyl tosylate was synthesized using an automated "Synthra" module using ethylene di-tosylate and [18F]fluoride/K222/K2CO3 in acetonitrile. [18F]Fluoroethyl tosylate was purified by semi-preparative HPLC followed by reformulation using a C18 Sep-Pak cartridge and eluted with DMF. Using this [18F]fluoroethyl tosylate, we attempted to alkylate protected tryptophan aiming to obtain the N-[18F]fluoroethyl-t-Boc-tryptophan methyl ester. Initial attempts resulted in the formation of the O-alkylated, rather than N-alkylated product. Manual removal of the cartridge from the automated module, followed by an extended drying of the cartridge under high flow nitrogen, was required to form the desired N-alkylated product. This demonstrates that the drying process in automated modules requires modification for sensitive N-alkylation of compounds and may be essential for compounds like tryptophan methyl ester that have multiple potential sites of alkylation in their chemical structure.

Heteronuclear NMR spectroscopic investigations of hydrogen bonding in 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines.

The 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines have previously revealed the presence of a strong intramolecular hydrogen bond. This in turn gives rise to a more complicated multiplet for the protons attached to the carbon adjacent to the amino group. This intramolecular hydrogen bond was investigated by a deuterium exchange experiment using heteronuclear NMR spectroscopy (1H, 13C, 15N and 2H). We observed changes in the multiplet structure and chemical shifts providing further evidence that the deuterium replaces the hydrogen in the intramolecular hydrogen bond. A time course study of the D2O exchange confirmed the presence of a strong hydrogen bond. The comparison of the structures obtained by X-ray crystallography showed a very small difference in planarity between the two-substituted and four-substituted amino compounds. In both the cases, the phenyl ring is not absolutely coplanar with the thiazole unit. The existence of this intramolecular hydrogen bond in 2-(benzo[d]thiazole-2'-yl)-N-alkylanilines was further confirmed by single crystal X-ray crystallography.

Synthesis, characterization and (11) C-radiolabeling of aminophenyl benzothiazoles: structural effects on the alkylation of amino group.

Several aminophenyl benzothiazoles were prepared with a view to using them as amyloid binding agents for imaging ß-amyloid in Alzheimer's disease. These precursors were radiolabeled with (11) C-positron-emitting radioisotope using an automated synthesizer and selected radiolabeled compounds were further purified by HPLC. Our results demonstrate that changes in structure have a major influence on the radioactive yield and the ease with which the radiolabel can be introduced. Aminophenyl benzothiazoles with an attached isopropyl group resisted dialkylation perhaps due to steric hindrance caused by this group. Straight chain attachment of methyl, ethyl, butyl, and crotyl groups in the structure decreased the radiochemical yield. Notably, the o-aminophenyl benzothiazole derivatives were difficult to alkylate despite stringent experimental conditions. This reactivity difference is attributed to the hydrogen bonding characteristics of the o-amino group with the nitrogen atom of the thiazole ring.

A comparative study between para-aminophenyl and ortho-aminophenyl benzothiazoles using NMR and DFT calculations.

Ortho-substituted and para-substituted aminophenyl benzothiazoles were synthesised and characterised using NMR spectroscopy. A comparison of the proton chemical shift values reveals significant differences in the observed chemical shift values for the NH protons indicating the presence of a hydrogen bond in all ortho-substituted compounds as compared to the para compounds. The presence of intramolecular hydrogen bond in the ortho amino substituted aminophenyl benzothiazole forces the molecule to be planar which may be an additional advantage in developing these compounds as Alzheimer's imaging agent because the binding to amyloid fibrils prefers planar compounds. The splitting pattern of the methylene proton next to the amino group also showed significant coupling to the amino proton consistent with the notion of the existence of slow exchange and hydrogen bond in the ortho-substituted compounds. This is further verified by density functional theory calculations which yielded a near planar low energy conformer for all the o-aminophenyl benzothiazoles and displayed a hydrogen bond from the amine proton to the nitrogen of the thiazole ring. A detailed analysis of the (1)H, (13)C and (15)N NMR chemical shifts and density functional theory calculated structures of the compounds are described.

Synthesis and characterization of 1- and 2-cinnamoyloxyacetonaphthones.

The synthesis of 1- and 2-cinnamoyloxyacetonaphthones was achieved in one step using hydroxyl acetonaphthones and substituted cinnamic acids in the presence of a catalytic amount of phosphoroxychloride. Structural characterization was accomplished using high-resolution nuclear magnetic resonance (NMR) spectroscopy. Chemical shifts of the compounds were compared and the change in the chemical shifts relative to electron-donating and -withdrawing groups is presented. Introduction of a thiophene ring instead of phenyl-substituted analogs caused shielding of the olefinic proton.

A study of the binding requirements of calyculin A and dephosphonocalyculin A with PP1, development of a molecular recognition model for the binding interactions of the okadaic acid class of compounds with PP1.

The interactions of the okadaic acid class of compounds, with special emphasis on the solution structures of calyculin A and dephosphonocalyculin A with PP1 are reported. After examination of the interactions of all docked structures, a receptor based pharmacophore model for the interactions of the protein phosphatase inhibitors has been developed. Calyculin A or dephosphonocalyculin A can interact with the enzyme in either a manner similar to the reported crystal structure, or in an extended form. The inhibitors require two essential regions interacting with the hydrophobic region and the central metal binding regions of the enzyme. This simplified model is consistent with previously published models of the okadaic acid class of compounds with PP1.

The solution structures of calyculin A and dephosphonocalyculin A by NMR.

The NMR solution structure of calyculin A (1) in chloroform exhibits intramolecular interactions, resembling the original crystal structure. In methanol, calyculin A has the hydrogen bonding moieties solvent exposed. Dephosphonocalyculin A in chloroform resembles calyculin A in chloroform and the crystal structure of calyculin A. Dephosphonocalyculin A in methanol resembles calyculin A in methanol.

Small molecular probes for G-protein-coupled C5a receptors: conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a.

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by 1H NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH. OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH.OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

Conformations of human apolipoprotein E(263-286) and E(267-289) in aqueous solutions of sodium dodecyl sulfate by CD and 1H NMR.

Structures of apoE(263-286) and apoE(267-289) have been determined in aqueous solution containing 90-fold molar excess of perdeuterated sodium dodecyl sulfate by CD and 1H NMR. Conformations were calculated by distance geometry based on 370 and 276 NOE distance restraints, respectively. RMSD for superimposing the region 265-284 from an ensemble of 41 structures for apoE(263-286) is 0.64 +/- 0.17 A for backbone atoms (N, C alpha, C = O) and 1.51 +/- 0.13 A for all atoms. The backbone RMSD for an ensemble of 37 structures for apoE(267-289) is 0.74 +/- 0.21 A for the region 268-275 and 0.34 +/- 0.10 A for the region 276-286. A two-domain structure was found for apoE(267-289) with the C-terminal half adopting a very well defined helix and the N-terminal segment 268-275 a less well defined helix, suggesting that the N-terminus may weakly bind to SDS. For apoE(263-286), an amphipathic helix-bend-helix structural motif was found with all hydrophobic side chains on the concave face. The existence of a bend around residues Q273 to G278 is consistent with their temperature coefficients of amide protons as well as secondary shifts of alpha-protons. Comparison of the structures of the two peptides revealed that the enhanced binding of apoE(263-286) to lipid could be attributed to the formation of a hydrophobic cluster consisting of residues W264, F265, L268, and V269. Aromatic side chains are proposed to be especially important in anchoring apolipoprotein fragments to micelles.

Conformational studies of an amphipathic peptide corresponding to human apolipoprotein A-II residues 18-30 with a C-terminal lipid binding motif EWLNS.

A peptide was designed and synthesized to enhance the lipid binding properties of a 13-residue fragment of apolipoprotein A-II. The peptide, VTDYGKDLMEKVKEWLNS [apoA-II(18-30)+], contains a five-residue amphipathic motif, EWLNS, at the C-terminus of apolipoprotein A-II residues 18-30. The lipid binding properties of apoA-II(18-30)+ were assessed using optical spectroscopy in the presence of sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), tetradecyltrimethyl ammonium chloride (TMA) and dimyristoylphosphatidylcholine (DMPC). The fluorescence emission spectra and the circular dichroism data suggested that apoA-II(18-30)+ interacted most strongly with SDS and most weakly with DMPC. An ensemble of structures for apoA-II(18-30)+ in aqueous solution containing SDS was calculated using distance geometry/simulated annealing methods from 308 NOE-based distance restraints. The backbone (N-C-C = O) RMSD from the average structure of an ensemble of 15 out of 20 calculated structures was 0.54 +/- 0.16 A. Apart from some dynamic fraying at both termini, the distance geometry and simulated annealing calculations showed that apoA-II(18-30)+ adopted a well defined amphipathic helix with distinct hydrophobic and hydrophilic faces.

Pore geometry information via pulsed field gradient NMR.

Studies of echo attenuation at long diffusion times in pulsed field gradient NMR experiments on a variety of rock core samples are interpreted in the light of recent theoretical analysis of the effect of pore geometry and surface relaxation. This study is motivated by the need to test the applicability of that theory to real rock systems.

3D autocorrelation for the determination of large pore sizes.

A data analysis methodology is used to process 3D NMR image data acquired for porous systems. The method extracts the mean size of those repeating elements in the image data which are largely compared with the image voxel dimensions. In this work we extend the two-dimensional (2D) image analysis method described by others to three spatial dimensions (3D). 3D image data were acquired at a magnetic field strength of 7 T using NMR microscopy hardware. The 3D autocorrelation function obtained from the data reveals a characteristic pore size in each dimension.

Strategies for overcoming linewidth limitations in quantitative petrophysical NMR measurements.

The simple pulse-acquire experiment has been used to evaluate the level of accuracy and precision achievable in NMR fluid saturation measurements for a range of rock core samples saturated with either brine or hydrocarbons. For a set of more than 70 cores measured at 0.66 T the mean error in the NMR measurement is only 0.35% porosity when the sample linewidths are less than 50 ppm. However, for a significant portion of cores, those with very broad NMR linewidths (> 50 ppm), difficulties associated with nonuniform excitation are encountered. The magnetic susceptibility difference between pore fluid and rock matrix translates into relatively broad NMR linewidths, and this feature of petrophysical samples is the major difficulty in performing quantitative NMR experiments. Numerical simulations are used to complement the experimental results in order to develop strategies for obtaining accurate NMR results with these difficult samples.

Quantitative longitudinal fluid saturation profiles with a slice-selected CPMG sequence.

A technique for obtaining quantitative longitudinal saturation/porosity profiles of rock cores which are longer than the NMR coil has been developed. The slice-selected experiment uses a prefocused pulse in conjunction with a magnetic field gradient for the localization and a CPMG sequence to sample the data. A variety of rock core samples has been studied ranging from limestones to shaly sandstones. Comparison of the relaxation decay curves obtained from these experiments and the bulk experiments show that reliable localized relaxation data are obtained.