Laminated morphology of nontwisting beta-sheet fibrils constructed via peptide self-assembly

A synthetic peptide has been de novo designed that self-assembles into beta-sheet fibrils exhibiting a nontwisted, stacked morphology. The stacked morphology is constituted by 2.5 nm wide filaments that laterally associate to form flat fibril laminates exceeding 50 nm in width and micrometers in length. The height of each fibril is limited to the length of exactly one peptide monomer in an extended beta-strand conformation, approximately 7 nm. Once assembled, these highly ordered, 2-D structures are stable over a wide range of pH and temperature and exhibit characteristics similar to those of amyloid fibrils. Furthermore, the rate of assembly and degree of fibril lamination can be controlled with kinetic parameters of pH and temperature. Finally, the presence of a diproline peptide between two beta-sheet-forming strands in the peptide sequence is demonstrated to be an important factor in promoting the nontwisting, laminated fibril morphology.     

Determination of titanium and zirconium wear debris in blood serum by means of HNO3/HF pressurized digestion using ICP – optical emission spectrometry

A ‘one bottle’ method to determine particulate debris of titanium and zirconium in blood serum was developed. Inductively coupled plasma – optical emission spectrometry (ICP-OES) was used to simultaneously detect both elements at concentrations above 50 ng/mL. Pressurized digestion by means of nitric and hydrofluoric acid in PTFE^TM-containers in a specific time-heat-pressure protocol apparatus was applied to assure complete solvation of particles including oxides. Total decomposition of the matrix was achieved and reasonable detection limits were accomplished. The amount of remaining carbon did not cause any matrix problems during measurement. Received: 10 October 1997 / Revised: 5 December 1997 / Accepted: 9 December 1997     

A novel bis-phenanthridine triamine with pH controlled binding to nucleotides and nucleic acids

The new bis-phenanthridine triamine is characterised by three pK(a) values: 3.65; 6.0 and >7.5. A significant difference in the protonation state of at pH = 5 (four positive charges) and at pH = 7 (less than two positive charges) accounts for the strong dependence of -nucleotide binding constants on nucleotide charge under acidic conditions, whereas at neutral pH all -nucleotide complexes are of comparable stability. All experimental data point at intercalation as the dominant binding mode of to polynucleotides. However, there is no indication of bis-intercalation of the two phenanthridine subunits in binding to double stranded polynucleotides, the respective complexes being most likely mono-intercalative. Thermal stabilisation of calf thymus DNA (ct-DNA) and poly A-poly U duplexes upon addition of is significantly higher at pH = 5 than at neutral conditions. This is not the case with poly dA-poly dT, indicating that the specific secondary structure of the latter, most likely the shape of the minor groove, plays a key role in complex stability. At pH = 5 acts as a fluorimetric probe for poly G (emission quenching) as opposed to other ss-polynucleotides (emission increase), while at neutral conditions this specificity is lost. One order of magnitude higher cytotoxicity of compared to its "monomer" can be accounted for by cooperative action of two phenanthridinium units and the charged triamine linker. The results presented here are of interest to the development of e.g. sequence-selective cytostatic drugs, and in particular for the possibility to control the drug activity properties over binding to DNA and/or RNA by variation of the pH of its surrounding.     

Cationic and neutral diphenyldiazomethanerhodium(I) complexes as catalytically active species in the C-C coupling reaction of olefins and diphenyldiazomethane

Cationic rhodium(I) complexes cis-[Rh(acetone)2(L)(L')]+ (2: L = L'=C8H14; 3: L=C8H14; L'=PiPr3; 4: L=L'=PiPr3), prepared from [RhCl(C8H14)2]2] and isolated as PF6 salts, catalyze the C-C coupling reaction of diphenyldiazomethane with ethene, propene, and styrene. In most cases, a mixture of isomeric olefins and cyclopropanes were obtained which are formally built up by one equivalent of RCH=CH2 (R = H, Me, Ph) and one equivalent of CPh2. The efficiency and selectivity of the catalyst depends significantly on the coordination sphere around the rhodium(I) center. Treatment of 4 with Ph2CN2 in the molar ratio of 1:1 and 1:2 gave the complexes trans-[Rh(PiPr3)2(acetone)(eta1-N2CPh2)]PF6 (8) and trans-[Rh(PiPr3)2(eta1-N2CPh2)2]PF6 (9), of which 8 was characterized by X-ray crystallography. Since 8 and 9 not only react with ethene but also catalyze the reaction of C2H4 and free Ph2CN2, they can be regarded as intermediates (possibly resting states) in the C-C coupling process. The lability of 8 and 9 is illustrated by the reactions with pyridine and NaX (X=Cl, Br, I, N3) which afford the mono(diphenyldiazomethane)rhodium(I) compounds trans-[Rh(PiPr3)2(py)(eta1-N2CPh2)]PF6 (10) and trans-[RhX(eta1-N2CPh2)(PiPr3)2] (11-14), respectively. The catalytic activity of the neutral complexes 11 - 14 is somewhat less than that of the cationic species 8, 9 and decreases in the order Cl > Br> I > N3.     

Plutonium isotopic composition measurement by high resolution gamma-spectrometry

Plutonium isotopic composition and the activities of non-separate transuranic elements:²³⁷Np and²⁴¹Am, are determined in the presence of long lived-fission products traces, by gamma-spectrometric measurements with a Ge-ultrapure detector. The quantitative values from the gamma-spectra analysis, are presented comparatively to mass-spectrometric data. This work has been done under AIEA, UNPD 7455/Rom. program.     

Temperature inducible beta-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of beta-structure with rising temperatures in all activation domain peptides. The high stability of beta-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce alpha-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L78E79-->D78L79) showed wild-type structure, the M32 mutant peptide (L78L81L83-->A78A81A83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a beta-structural element is involved in binding to a cellular cofactor.     

New flexible synthesis of pyrazoles with different,functionalized substituents at C3 and C5

Syntheses of pyrazoles featuring a functionalized side chain attached to carbon 3 and varying alkyl and aryl substituents attached to carbon 5 are presented. Installation of R = methyl, isopropyl, tert-butyl, adamantyl, or phenyl groups at C5 is reported here, starting by coupling protected alkynols with acid chlorides RCOCl, forming alkynyl ketones, which are reacted with hydrazine to form the pyrazole nucleus. Alcohol deprotection and conversion to a chloride gave 5-substituted 3-(chloromethyl)- or 3-(2-chloroethyl)pyrazoles. This sequence can be done within 2 d on a 30 g scale in excellent overall yield. Through nucleophilic substitution reactions, the chlorides are useful precursors to other polyfunctional pyrazoles. In the work here, derivatives with side chains LCH(2)- and LCH(2)CH(2)- at C3 (L = thioether or phosphine) were made as ligands. The significance of the ligands made here is that by placing a ligating side chain on a ring carbon (C3), rather than on a ring nitrogen, the ring nitrogen not bound to the metal and its attached proton will be available for hydrogen bonding, depending on the steric environment created by R at C5.     

Synthesis of D-seco-13α-Androst-5-ene Derivatives

Summary. 3β-Hydroxy-16,17-seco-13α-androsta-5,16-dien-17-al was obtained from 3β-acetoxyandrost-5-en-17-one in six steps with a Grob fragmentation as the key step. This seco-steroid, containing a formyl group and an unsaturated side-chain in a sterically favourable cis position, is a useful synthon for the synthesis of novel heterocycles condensed to the 3β-hydroxy-13α-androst-5-en-17-one skeleton.     

Matrix-assisted laser desorption mass spectrometry of homopolymer oligodeoxyribonucleotides. Influence of base composition on the mass spectrometric response

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has the potential for providing a rapid alternative to gel electrophoresis for DNA sequence analysis provided that an intense mass spectrometric response can be obtained from mixtures of DNA fragments containing up to 300 nucleotides. MALDI-MS has not yet proved viable for such analyses because the MS response falls off rapidly for mixed-base DNA fragments containing more than 20–30 nucleotides. Previous studies have demonstrated that base composition is a critical factor in the MALDI-MS response of oligodeoxyribonucleotides. This paper describes an investigation of the physical roots of the observed influence of base composition on the mass spectrometric response, focusing on homopolymer oligodeoxyribonucleotides (dT₇, dT₁₀, dT₁₈, dT₃₆, dG₇, dG₁₀, dG₁₈, dI₁₈ and dU₁₈) and dT₅G₅. Forty-eight different matrix compounds were tested for their ability to produce laser desorption masses spectra from such homopolymer oligodeoxyribonucleotides. Considerably stronger mass spectrometric responses were obtained from polydeoxythymidines than from polydeoxyguanosines, polydeoxycytidines and polydeoxyadenosines. Although mass spectral peaks corresponding to dT₁₈ were observed from 20 of the matrices studied, no discernible response was observed for dG₁₈ from any of these matrices. To elucidate the physical basis for origins of the observed differences in response, a number of factors were investigated including the ionization efficiency, the tendency towards fragmentation and the extent to which the oligodeoxyribonucleotides were incorporated into the matrix crystals. The results of these experiments indicate that low ionization efficiency is not a likely main contributor to the low response to polydeoxyguanosines, fragmentation is a likely main contributor to the low response to polydeoxyguanosines, the overall incorporation of polydeoxyguanosines into matrix crystals is comparable to that for polydeoxythymidines and the exocyclic amino group of guanosine, adenosine and cytidine has a strong inhibitory effect on the mass spectrometric response.     

Hydroxo hydrido complexes of iron and cobalt (Sn-Fe-Sn, Sn-Co-Sn): probing agostic Sn...H-M interactions in solution and in the solid state

Bis(toluene)iron 9 reacts with Lappert's stannylene [Sn[CH(SiMe3)2]2] (4) to form the paramagnetic bis-stannylene complex [[(eta6-toluene)Fe-Sn-[CH(SiMe3)2]2]2] (10). Compound 10 reacts with H2O to form the hydroxo hydrido complex [(eta6-C7H8)(mu-OH)(H)-Fe-[Sn[CH(SiMe3)2]2]2] (12) in high yield; its solid-state structure has been elucidated by X-ray and neutron diffraction analysis. In agreement with the 1H NMR results, 12 contains a hydridic ligand whose exact coordination geometry could be determined by neutron diffraction. The 1H and 119Sn NMR analysis of 12 suggested a multicenter Sn/Sn/H/Fe bonding interaction in solution, based on significantly large values of J(Sn,H,Fe) = 640+/-30 Hz and J(119Sn,119Sn) = 4340+/-100 Hz. In solution, complex 12 exists as two diastereomers in a ratio of about 2:1. Neutron diffraction analysis has characterized 12 as a classical metal hydride complex with very little Sn...H interaction and a typical Fe-H single bond (1.575(8) A). This conclusion is based on the fact that the values of the Sn...H contact distances (2.482(9) and 2.499(9) A) are not consistent with strong Fe-H...Sn interactions. This finding is discussed in relation to other compounds containing M-H...Sn units with and without strong three-center interactions. The neutron diffraction analysis of 12 represents the first determination of a Sn-H atomic distance employing this analytical technique. The cobalt analogues [(eta5-Cp)(mu-OH)(H)Co-[Sn[CH(SiMe3)2]2]2] (15) and [(eta5-Cp)(OD)(D)Co-[Sn[CH-(SiMe3)2]2]2] [D2]15, which are isolobal with 12, were prepared by the reaction of [(eta5-Cp)Co-Sn[CH(SiMe3)2]2] (14) with H2O and D2O, respectively. The magnitude of J(Sn,H) (539 Hz) in 15 is in the same range as that found for 12. The molecular structure of 15 has been determined by X-ray diffraction which reveals it to be isostructural with 12. The coordination geometries of the Co(Fe)-Sn1-O-Sn2 arrangements in 12 and 15 are fully planar within experimental error. Compounds 10 and 15 are rare examples of fully characterized complexes obtained as primary products from water activation reactions.