Gas chromatographic analysis of estrogens. II

Summary A simple method is described for the introduction of samples to a gas Chromatograph by means of a septumless port. The reproducibility and accuracy of the method have been shown by the analysis of ng quantities of estrone, estradiol, and estriol as the heptafluorobutyrates with an electron capture detector.

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Zusammenfassung Die Einbringung von Proben in einen Gaschromatographen durch einen membranlosen Einspritzblock wurde beschrieben. Die Reproduzierbarkeit und Genauigkeit der so erhaltenen Ergebnisse wurde durch die Bestimmung von Nanogrammengen Östron, Östradiol und Östriol als Heptafluorobutyrate mit einem Elektroneneinfangdetektor erwiesen.

     

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.     

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.     

The chemistry of 2'' ,3''-seconucleosides II. Reactions and biological properties of 2'',3''-secopyrimidine ribonucleosides

Reaction of 2',3'-secouridine with acetone gave the 3',5'-$\text{O}$-isopropylidene derivative (1) which upon treatment with mesylchloride gave the 2'-$\text{O}$-mesyl compound (2). Replacement of the mesyl group of 2 with halide could be effected by reaction with a metal halide in DMF. The 3',5'-$\text{O}$-isopropylidene group was removed simultaneously to give a 2'-halogeno-2'-deoxy-2',3'-secouridine. 2',3'-Dichloro-2',3'-dideoxy-2',3'-secouridine upon treatment with base gave 6($\text{R}$)-chloromethyl-2($\text{R}$)-(uracil-1-yl)-1,4-dioxane in addition to O²,2'-anhydro-3'-chloro-3'-deoxy-2',3'-secouridine, as previously reported. 2',3'-Dichloro-2',3'-dideoxy-5'-0-trityl-2',3'-secouridine was converted to 2',3'-dichloro-2',3'-dideoxy-2',3'-secocytidine (16) $\text{via}$ a triazole derivative. Compound 16 was unstable and appeared to form O²,2'-anhydro-3'-chloro-3'-deoxy-2,3'-secocytidine upon standing at room temperature. 5-Vinyl- and 5-($\text{E}$) (2-bromovinyl) uridine dialdehydes have been made, as well as a number of other 5-substituted 2',3'-secouridine derivatives. None of the compounds obtained showed significant activity against a number of virus strains or tumor cell lines, except for 5-($\text{E}$)(2-bromovinyl) uridine dialdehyde, which was inhibitory to the growth of human lymphoblast (Raji, Namalva) cells at a concentration of 28 μ/ml.     

Synthesis of some heterocyclic skeletons via organoiron complexes. Crystal and molecular structure of (5a,6,7,8,9,9a-η6-1,4-benzoxathiino[3,2-b]pyridine)(η5-cyclopentadienyl)iron hexafluorophosphate

Synthesis of the heterocyclic skeletons of some biologically active compounds from (η⁶-o-dichlorobenzene)(η⁵-cyclopentadienyrl)iron hexafluorophosphate in a two step procedure is described. Cyclopentadienyliron hexafluorophosphate complexes of 1,4-benzodioxino[2,3-b]pyridine, 1,4-benzoxathiino[3,2-b]pyridine, 10H-pyrido[3,2-b]benzoxazine, benzo[b]naphtho[2,3-e][1,4]dioxin, 4-methylbenzo[b]benzopyran-2-one[7,6-e][1,4]dioxin and benzo[b]anthracen-9,10-diono[1,2-e][1,4]dioxin were isolated and characterized. Upon pyrolytic sublimation of these complexes the free heterocycles were obtained and characterized. (η⁶-1,4-Benzoxathiino[3,2-b]pyridine)(η⁵-cyclopentadienyl)iron hexafluorophosphate crystalizes in the orthothombic system, space group Pbca; the dihedral angle between the planes of outer rings was found to be 176.8 (1).     

Resonance-enhanced multiphoton ionization (2+1) of the\tilde B and\tilde C' states of ammonia

Resonance-enhanced multiphoton ionization (REMPI) of the \(\tilde B\) and \(\tilde C'\) states of NH₃ in a pulsed supersonic molecular beam was observed at 304–340 nm, using the multiphoton-ionization mass-spectrometer system described herein. The observed features are the result of a 2+1 MPI process and correspond quite well with the reported 3+1 MPI of NH₃ in the literature from the ground state of opposite parity.     

CALCIUM-REGULATED NUCLEAR ENZYMES: POTENTIAL MEDIATORS OF PHYTOCHROME-INDUCED CHANGES IN NUCLEAR METABOLISM?

Calcium ions have been proposed to serve as important regulatory elements in stimulus-response coupling for phytochrome responses. An important test of this hypothesis will be to identify specific targets of calcium action that are required for some growth or development process induced by the photoactivated form of phytochrome (Pfr). Initial studies have revealed that there are at least two enzymes in pea nuclei that are stimulated by Pfr in a Ca(2+)-dependent fashion, a calmodulin-regulated nucleoside triphosphatase and a calmodulin-independent but Ca(2+)-dependent protein kinase. The nucleoside triphosphatase appears to be associated with the nuclear envelope, while the protein kinase co-purifies with a nuclear fraction highly enriched for chromatin. This short review summarizes the latest findings on these enzymes and relates them to what is known about Pfr-regulated nuclear metabolism.     

Theoretical studies on the hydrolysis of mono-phosphate and tri-phosphate in gas phase and aqueous solution

Phosphate hydrolysis by GTPases plays an important role as a molecular switch in signal transduction and as an initiator of many other biological processes. Despite the centrality of this ubiquitous reaction, the mechanism is still poorly understood. As a first step to understand the mechanisms of this process, the nonenzymatic hydrolysis of mono-phosphate and tri-phosphate esters were systematically studied in gas phase and aqueous solution using hybrid density functional methods. The dielectric effect of the environment on the energetics of these processes was also explored. Theoretical results show that for mono-phosphate ester, the dissociative pathway is much more favorable than the associative pathway. However, the reaction barriers for the dissociative and associative pathways of tri-phosphate hydrolysis are very close in aqueous solution, though the dissociative pathway is more favorable in the gas phase. High dielectric solvents, such as water, significantly lower the activation barrier of the associative pathway due to the greater solvation energy of the associative transition states than that of the reactant complex. By contrast, the barrier of the dissociative pathway, with respect to the gas phase, is less sensitive to the surrounding dielectric. In the associative hydrolysis pathway of the tri-phosphate ester, negative charge is transferred from the gamma-phosphate to beta-phosphate through the bridging ester oxygen and results in Pgamma-O bond dissociation. No analogous charge transfer was observed in the dissociative pathway, where Pgamma-O bond dissociation resulted from proton transfer from the gamma-phosphate to the bridge oxygen. Finally, the active participation of local water molecules can significantly lower the activation energy of the dissociative pathway for both mono-phosphate and tri-phosphate.