13C-H coupling constants and maximum overlap orbitals in some three-membered heterocyclic compounds

The criterion of maximum total overlap, modified to permit inclusion of ionic structures, has been employed to determine the hybridized bonding orbitals for ethyleneimine, ethylene oxide, ethylene sulfide, and cyclopropane. ¹³C-H coupling parameters obtained from the NMR spectra of these compounds, which are linear functions of s-character in the appropriate carbon orbitals, served as experimental criteria for adjusting ionic content. The results appear reasonable as judged by calculated HCH angles and the order of heteroatom electronegativities.

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Zusammenfassung Das Kriterium maximaler Überlappung wurde in einer Modifikation, die den Einschluß ionischer Strukturen gestattet, zur Bestimmung der Hybridzustände von Äthylenimin, Äthylenoxyd, Äthylensulfid und Cyclopropan herangezogen. Die ¹³CH-Kopplungskonstanten der NMR-Spektren, die linear vom s-Charakter der entsprechenden Kohlenstoffzustände abhängen, dienten als experimentelles Kriterium für die Bestimmung der Anteils an ionischen Strukturen. Nach den berechneten HCH-Winkeln sowie der Reihenfolge der Elektronegativitäten der Heteroatome zu schließen, scheinen die Ergebnisse befriedigend zu sein.

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Résumé Le critère de recouvrement maximum total, modifié pour permettre l'inclusion de structures ioniques, a été utilisé pour déterminer les orbitales hybrides de l'éthylèneimine, l'oxide d'éthylène, le sulfure d'éthylène et le cyclopropane. Les paramètres de couplage ¹³C-H obtenus des spectres RNM de ces composés, qui sont des fonctions linéaires du caractère s des orbitaux de carbone correspondants, servaient de critère expérimental pour l'ajustage du contenu ionique. Le calcul des angles HCH et l'ordre des électronégativités des hétéroatomes conduisent à conclure que les résultants sont raisonnables.

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We wish to thank the National Institutes of Health for a Grant in partial support of this work. One of us (P. R. Certain) thanks the National Science Foundation for support through the Undergraduate Research Participants program.     

Positron annihilation lifetime evaluation of thermal cycling effects in atactic polystyrene

Positron annihilation lifetime spectroscopy (PALS) was used to investigate the thermal cycling characteristics of two different molecular weight atactic polystyrene resins. Annealed compression molded samples were thermally cycled over the temperature range 253 K to 393 K with PAL spectra taken in 10 K increments upon both cooling and heating. The longest lived component lifetime and intensity, indicative of orthopositronium pick-off, exhibit thermal dependencies that can be interpreted in a manner consistent with anticipated free volume changes associated with structural transitions.     

Multielectron redox chemistry of iron porphyrinogens

Iron octamethylporphyrinogens were prepared and structurally characterized in three different oxidation states in the absence of axial ligands and with sodium or tetrafluoroborate as the only counterions. Under these conditions, the iron- and ligand-based redox chemistry of iron porphyrinogens can be defined. The iron center is easily oxidized by a single electron (E(1/2) = -0.57 V vs NHE in CH(3)CN) when confined within the fully reduced macrocycle. The porphyrinogen ligand also undergoes oxidation but in a single four-electron step (E(p) = +0.77 V vs NHE in CH(3)CN); one of the ligand-based electrons is intercepted for the reduction of Fe(III) to Fe(II) to result in an overall three-electron oxidation process. The oxidation equivalents in the macrocycle are stored in C(alpha)-C(alpha) bonds of spirocyclopropane rings, formed between adjacent pyrroles. EPR, magnetic and Mossbauer measurements, and DFT computations of the redox states of the iron porphyrinogens reveal that the reduced ligand gives rise to iron in intermediate spin states, whereas the fully oxidized ligand possesses a weaker sigma-donor framework, giving rise to high-spin iron. Taken together, the results reported herein establish a metal-macrocycle cooperativity that engenders a multielectron chemistry for iron porphyrinogens that is unavailable to heme cofactors.     

Exo- und endo-Tricarbonyleisenkomplexe von bicyclischen 2,3-Dimethylidenverbindungen

Exo- and endo-Tricarbonyliron Complexes of Bicyclic 2,3-Dimethylidene Compounds. The preparation of exo- and endo-tricarbonyliron complexes (exo- and endo- 5 , -6 , -8 , and 9 ) of 2,3-dimethylidene-5-bicyclo[2.2.1]heptene( 1 ), -bicyclo[2.2.1]-heptane ( 2 ), -5-bicyclo[2.2.2]octene ( 3 ) and -bicyclo[2.2.2]octane ( 4 ) is described. The complexes are obtained by thermal reaction of the bicyclic butadienes with di-ironenneacarbonyl in hexane solution. exo- and endo- 5 are also formed photochemically from ironpentacarbonyl and 1 in pentane solution at ?35°. The structural assignment of exo- and endo -5 and -6 is based on their mass-spectra and on coordination shifts in ¹H- and ¹³C-NMR.-spectra exo- and endo -6 are correlated with exo- and endo -5 , respectively, by hydrogenation. Hydrogenation of the uncomplexed double bond in exo- and endo -5 occurs in both complexes from the exo side as shown by deuteration experiments. The free ligand 1 reacts in the same stereospecific manner.     

A new type of tweezer complex involving a rhenium-rhenium multiple bond that enforces an unusual structure in a dipalladium(II) unit

The substitution of the mu-acetato ligands in cis-Re(2)(mu-O(2)CCH(3))(2)Cl(2)(mu-dppm)(2) (1, dppm = Ph(2)PCH(2)PPh(2)) and trans-Re(2)(mu-O(2)CCH(3))(2)Cl(2)(mu-dppE)(2) (2, dppE = Ph(2)PC(=CH(2))PPh(2)) by [4-Ph(2)PC(6)H(4)CO(2)](-) occurs with retention of stereochemistry to give cis-Re(2)(mu-O(2)CC(6)H(4)-4-PPh(2))(2)Cl(2)(mu-dppm)(2) (3) and trans-Re(2)(mu-O(2)CC(6)H(4)-4-PPh(2))(2)Cl(2)(mu-dppE)(2) (6), respectively. The uncoordinated phosphine groups in complexes 3 and 6 have been used to form mixed-metal assemblies with Au(I) and Pd(II), including the Re(2)Pd(2) complex cis-Re(2)(mu-O(2)CC(6)H(4)-4-PPh(2))(2)Cl(2)(mu-dppm)(2)(Pd(2)Cl(4)) (5), in which the planar [(P)ClPd(mu-Cl)(2)PdCl(P)] unit has the unusual cis structure. The crystal structures of 3 and 5 have been determined.     

EF-Tu binding peptides identified,dissected, and affinity optimized by phage display

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.     

Quantitative high-performance liquid chromatography of nucleosides in biological materials

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.     

Model systems for flavoenzyme activity: flavin-functionalised SAMs as models for probing redox modulation through hydrogen bonding

We report the fabrication of flavin-functionalised self-assembled monolayers upon gold electrodes and their subsequent redox modulation via hydrogen bonding to 2,6-diethylamidopyridine.     

A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and 15N-metabolic labelling

We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.     

Directed evolution studies with combinatorial libraries of T4 lysozyme mutants

Summary Gene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of >10⁷ mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection inlacZ orpheA deficient strains ofE. coli to investigate whether they sample sufficient diversity to contain mutants with acquired -galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novelE. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4–6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min^– for thelacZ orpheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from futureE. coli complementation schemes.