Chemistry of the phenoxathiins and isosterically related heterocycles. XXXI. Synthesis of the 1,3-diazaphenoxathiin ring system and the relationship of the 13C-NMR chemical shift of the C-10a resonances to the molecular dihedral angle determined by X-Ray diffraction: A further investigation

Synthesis of the 1,3-diazaphenoxathiin ring system and the confirmation of its structure by ¹³C-nmr spectroscopy and X-ray crystallography are reported. Implications of the ¹³C-nmr chemical shift of the C-10a resonance and its relationship to the molecular dihedral angle are presented. The molecule crystallizes in the Pbca space group and was found to have a dihedral angle of 165.5(9)°, the structure refining to a final R-factor of T = 0.0427.     

Investigation of water environments in a C18 bonded silica phase using 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy

High resolution 1H magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra have been obtained on typical C18 bonded silicas used in chromatographic solid-phase extraction separations. It has been shown for the first time that water molecules distributed in distinct physico-chemical environments within the chromatographic system can be detected directly using a simple 1H MAS NMR measurement. The resonances assigned to water protons in differing physico-chemical environments have distinct chemical shifts, line widths, relaxation times (T1 and T2) and also exhibit temperature dependent coalescence behaviour. This novel MAS approach may lead to a better understanding of the environments of other analytes in mixtures during such separations.     

Entropy-enthalpy compensations in solutions of dual character molecules with polymeric chromatographic liquid phases

Careful gas chromatographic studies provide thermodynamic data for insights into solution processes in nonvolatile solvents. Using 24 solutes and five stationary phases, several entropy-enthalpy compensation effects in the thermodynamics of solution were identified. Despite solute structure differences, when excess enthalpy and entropy of solutions were examined, entropy-enthalpy compensation effects were found in solvents dominated by single types of interaction: squalane and, to some extent, methoxy poly(ethylene oxide) (PEO). The main reason for the absence of linearity in other solvents is pure solute state interactions in the reference state and the multicharacter nature of solvents. In this study, consideration of solute state interactions was removed through examination of the thermodynamics of transfer between solvent pairs. It was found that solute transfers from squalane to poly[methyl(trifluoropropyl)siloxane] (QF-1) and to poly(methylphenyl) (DC-550) also gave linear relationships. The former system contains a second correlation for ester type solutes. The transfer data for squalane to poly(methylsiloxane) (DC-200) had smaller ranges and were more scattered. The effects of derivatizing groups on the transfer enthalpy and entropy were treated as a summation of hydrocarbon cores with the derivative groups. The group properties of transfer then also show entropy-enthalpy compensation effects. Many solution effects could be explained on the basis of solvent composition and local interactions with solutes.     

Fully imidized soluble polyimides based on a novel dianhydride: 2,2′-dichloro-4,4′,5,5′-benzophenone tetracarboxylic dianhydride

We have synthesized a novel dianhydride, 2,2′-dichloro-4,4′,5,5′-benzophenone tetracarboxylic dianhydride (DCBTDA). Polyimides were synthesized with DCBTDA or 3,3′,4,4′-benzophenone tetracarboxylic dianhydride (BTDA) and several relatively rigid meta- and para- substituted mononuclear diamines. The BTDA based systems were insoluble in dipolar, aprotic solvents whereas the DCBTDA based polymers displayed enhanced solubility in these solvents. The thermal stability of these polyimides was excellent as measured by 5% weight loss decomposition. The T_g's of the polymers were all above 290°C.     

A simple and general chiral silicon Lewis acid for asymmetric synthesis: highly enantioselective [3+2] acylhydrazone-enol ether cycloadditions

A highly diastereo- and enantioselective [3 + 2] acylhydrazone-enol ether cycloaddition mediated by a simple chiral silane Lewis acid is described. The reactions are highly practical, as demonstrated by a larger scale (5 g of the hydrazone) reaction in which the product was obtained after recrystallization in 93% yield and 99% ee. Evidence for a stepwise mechanism and a model for the asymmetric induction are presented, as well.     

Photolithographic synthesis of peptoids

We describe a novel photolithographic approach to the synthesis of peptoids (oligo-N-substituted glycines). This strategy enables the construction of a spatially addressable peptoid microarray, thus providing a potentially powerful tool for the discovery of protein ligands.     

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.     

Carbamoylimidazolium and thiocarbamoylimidazolium salts: novel reagents for the synthesis of ureas, thioureas, carbamates, thiocarbamates and amides

Carbamoylimidazolium salts act as efficient N,N-disubstituted carbamoylating reagents. These salts are readily prepared by the sequential treatment of secondary amines with N,N′-carbonyldiimidazole (CDI) and iodomethane. The carbamoylimidazolium salts are more efficient carbamoyl transfer reagents than the intermediate carbamoylimidazoles, as a result of the ‘imidazolium’ effect. Kinetic studies on the base promoted hydrolysis of both carbamoylimidazoles and carbamoylimidazolium salts reveal over a hundred-fold rate acceleration. The salts react with amines, thiols, phenols/alcohols, and carboxylic acids in high yields, without the need for subsequent chromatographic purification of the products, producing ureas, thiocarbamates, carbamates, and amides, respectively. Analogous thiocarbamoylimidazolium salts were also synthesized from secondary amines and N,N′-thiocarbonyldiimidazole (TCDI), followed by methylation with iodomethane.     

Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been used in conjunction with time-of-flight (TOF) and quadrupole ion trap (IT) mass spectrometry, respectively, to analyze various cytolysin proteins isolated from the sea anemone Stichodactyla helianthus and digested by the protease trypsin. By employing different ionization methods, the subsequent changes in ionization selectivity for the peptides in the digested protein samples resulted in ion abundance variation reflected in the mass spectra. Upon investigation of this variation generated by the two ionization processes, it has been shown in this study that enhanced protein coverage (e.g., >95% for cytolysin III) can be achieved. Additionally, capillary and microbore reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ESI mass spectrometry (MS) as well as flow injection analysis by nanoflow ESI-MS afforded the necessary limit of detection (LOD) for detailed structural information of the cytolysin proteins by tandem mass spectrometry (MS/MS) methods. It can be concluded that cytolysins II and III correspond to sticholysins I and II, that "cytolysin I" is a mixture of modified forms of cytolysins II and III, and that "cytolysin IV" is an incompletely processed precursor of cytolysin III.     

Oxidatively truncated docosahexaenoate phospholipids: total synthesis,generation, and Peptide adduction chemistry

The recent immunological detection of extraordinarily high levels of carboxyethylpyrrole (CEP) modifications of proteins from the retinas of individuals with age-related macular degeneration provided presumptive evidence for the involvement of docosahexaenoate-derived oxidatively truncated phospholipids in retinal pathology. To facilitate the in vivo detection and characterization of the chemistry and biological activities of these postulated naturally occurring molecules, a family of oxidatively truncated phospholipids was prepared by total syntheses. Their formation in oxidation reactions of a docosahexaenoate ester of 2-lysophosphatidylcholine (DHA-PC) was also demonstrated. Free radical-induced oxidative cleavage of DHA-PC promoted by myeloperoxidase or copper ions generates similar mixtures of these phospholipids. The most abundant products were 1-palmitoyl-2-succinoyl-sn-glycero-3-phosphatidylcholine (4.7%) and 2-(6-carboxy-4-oxohex-5-enoyl)-1-palmitoyl-sn-glycero-3-phosphatidylcholine (1.7%). Both of these oxidatively truncated phospholipids are homologues of biologically active arachidonate-derived phospholipids. A minor product from DHA-PC, 2-(4-hydroxy-7-oxohept-5-enoyl)-1-palmitoyl-sn-glycero-3-phosphatidylcholine (0.4% yield), reacted with the epsilon-amino group of a peptide lysyl residue to produce a CEP derivative in 0.7% yield. These observations support the previous conclusion, based on immunological evidence, that CEPs are generated by the reaction of an oxidatively truncated phospholipid with proteins in the retina and further indicate that CEP protein modifications probably represent only a tiny fraction of the products generated upon oxidative damage of DHA-PC in photoreceptor disk membranes.