Unusual bond activation processes in the reaction of group 4 cyclopentadienyl alkyne complexes with azobenzene

The alkyne complexes [Cp′₂M(L)(η²-Me₃SiC₂SiMe₃)] (Cp′ = substituted or unsubstituted cyclopentadienyl; M = Ti, Zr, Hf; L = Py, THF) can serve as metallocene precursors by substitution of the alkyne molecule with other ligands. The reactions of the unsubstituted cyclopentadienyl complexes [Cp₂Zr(THF)(η²-Me₃SiC₂SiMe₃)] (1) and [Cp₂Ti(η²-Me₃SiC₂SiMe₃)] (2) with azobenzene were investigated. In the first case the diazene complex [Cp₂Zr(THF)(N₂Ph₂)] (3) was obtained by alkyne exchange. In the reaction of the titanium complex 2 a NN bond cleavage of azobenzene and a C-H activation of the cyclopentadienyl ligand were observed and the dinuclear imido bridged compound 4 was formed. This mixed valence complex is bridged additionally by a cyclopentadienyl ligand in a η¹:η⁵-coordination mode which is very unusual for titanium complexes. The molecular structures of both compounds were confirmed by X-ray crystallography and compared to former structural data shown in literature.     

A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36

Transmembrane protein CD36 is considered to bind its distinct ligands such as long-chain fatty acids primarily by recognizing their terminal carboxyl moiety. In this study, we provide evidence that long-chain fatty aldehydes, such as oleic aldehyde, can be recognized by CD36. We suggest that a single aldehyde group may also serve as one of the structural elements recognizable by CD36.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Infrared spectroscopy as a tool for chemotaxonomic investigations within the Achillea millefolium group

An FT-IR-based method for the rapid characterization and identification of Achillea species was developed. With the IR spectra of the CH2Cl2 extracts of A. aspleniifolia, A. collina, A. millefolium, A. millefolium ssp. sudetica, A. pannonica, A. pratensis, A. roseoalba, A. setacea, and A. styriaca, a spectrum library was created. This database allowed the identification of the respective taxa by comparison of IR spectra. Moreover, information concerning dominant structural elements of sesquiterpene constituents was obtained, providing hints about the composition of a plant sample of unknown origin.     

Cross-linking and mass spectrometry as a tool for studying the structural biology of ribonucleoproteins

1. Download : Download high-res image (138KB)
2. Download : Download full-size image

     

A framework for assessing the quality of democratic deliberation: enhancing deliberation as a tool for bioethics

The goal of democratic deliberation (DD) bioethics research is to elicit informed and considered opinions on ethically controversial issues. But the trustworthiness of DD outcomes depends on the quality of deliberations. We provide a framework to evaluate the quality of deliberations and apply that framework to a DD project on surrogate consent for dementia research involving randomly selected samples of the older general public. Using a mixed method approach, we found that participants were very satisfied with the sessions, learned and used new information, were respectful and collaborative, and were able to "reason together" to arrive at societal policy recommendations. Implications and limitations of the paper are also discussed.     

Amino-functionalized zirconocene (C5H4CH(Me)NMe2)2ZrCl2, a catalyst for the dehydropolymerization of PhSiH3: Probing of peripheral substituent effects on catalyst dehydropolymerization activity

The amino-functionalized metallocene (C₅H₄CH(Me)NMe₂)₂ZrCl₂, [(Cp^N)₂ZrCl₂] was synthesized by salt metathesis of ZrCl₄ and 2 equiv. of C₅H₄CH(Me)NMe₂Li. The metallocene was obtained in good yield as a mixture of rac and meso diastereomers as established by NMR spectroscopy. The addition of 2 equiv. of n-BuLi to the metallocene (Cp^N)₂ZrCl₂ produced a co-catalyst system which was active, at a 1.0 mol% loading, in the dehydropolymerization of PhSiH₃ to poly(phenylsilane), PPSi. The PPSi was obtained as a 9:1 linear–cyclic mixture (M_w=3850, M_n=2300) as established by GPC analysis; ²⁹Si{¹H} NMR spectroscopy revealed an atactic polymer microstructure.     

Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice

Transposable elements (TEs) are DNA fragments that have the ability to move from one chromosomal location to another. The insertion of TEs into gene-rich regions often affects changes in the expression of neighboring genes. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome. It has been found to show exceptionally active transposition in a few japonica rice varieties, including Gimbozu, where mPing insertion rendered adjacent genes stress-inducible. In the Gimbozu population, it is highly possible that several genes with modified expression profiles are segregating due to the de novo mPing insertions. In our study, we utilized a screening system for detecting de novo mPing insertions in the upstream region of target genes and evaluated the effect of mPing on the stress response of the target genes. Screening for 17 targeted genes revealed five genes with the mPing insertion in their promoters. In most cases, the alteration of gene expression was observed under stress conditions, and there was no change in the expression levels of those five genes under normal conditions. These results indicate that the mPing insertion can be used as a genetic tool to modify an expression pattern of a target gene under stress conditions without changing the expression profiles of those under natural conditions.     

Palladium(II) as a versatile template for the formation of tetraaza macrocycles via Mannich-type reactions

The versatility of palladium(II) as a template for Mannich-type macrocyclization is illustrated. Reaction of (bis(3-aminopropyl)piperazine)palladium(II) with formaldehyde and nitroethane in basic aqueous solution yields the ‘reinforced’ macrocycle 7-methyl-7-nitro-1,5,9,13-tetraazabicyclo[11.2.2]heptadecane as its palladium(II) complex. The crystal structure shows the palladium ion lies in a slightly tetrahedrally distorted square plane of four nitrogen donors, with distances to the two tertiary donors [av. 2.059(3) Å] slightly shorter than those to the secondary amines [av. 2.066(3) Å]. The 3-methyl-3-nitro-1,5,9,13-tetraazacyclohexadecane as its palladium(II) complex was prepared by an analogous route. In a separate reaction based on the [Pd(en)(chxn)]²⁺ (en = ethane-1,2-diamine; chxn = cyclohexane-1,2-diamine) intermediate, an unsymmetrical macrocycle with a fused cyclohexane ring, 4,11-dimethyl-4,11-dinitro-2,6,9,13-tetraazabicyclo[12.4.0]octadecane was isolated as its palladium(II) complex. Accessibility to an isolable mixed-ligand precursor is a key to this reaction, provided by using palladium(II) as the templating metal. Reaction of (4,8-diazaundecane-1,11-diamine)palladium(II) with formaldehyde and diethyl malonate in basic aqueous solution yields, with ester hydrolysis and decarboxylation, the carboxylate-pendant macrocycle 1,5,9,13-tetraazacyclohexadecane-3-carboxylic acid as its palladium(II) complex. The crystal structure is comprised of hydrogen-bonded dimers {[Pd(L)][Pd(L-H)]}³⁺ where the pair of inversion related square-planar complexes share a single proton between their pendant carboxylates. Bis(3-aminopropyl)(piperazine)palladium(II) yields the macrocyclic complex ion (1,5,9,13-tetraazabicyclo[11.2.2]heptadecane-7-carboxylic acid)palladium(II), in a similar reaction.     

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase

A transgenic mouse strain with early and uniform expres sion of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies     

Peroxidase and laccase as catalysts for removal of the phenylhydrazide protecting group under mild conditions

A new method is proposed for the removal of the phenylhydrazide protecting group by the action of peroxidase or laccase, the enzymes attributed to the class of oxidoreductases. The deblocking procedure is performed under mild oxidative conditions, i.e., aqueous solution and neutral or close to neutral pH. Such mild oxidizing agents as 1 mM H(2)O(2) and air oxygen are used for unmasking. The method is available for the deblocking of both alpha- and gamma-carboxyl groups. The enzyme-catalyzed removal of the phenylhydrazide protecting group causes no oxidative modification nor destruction of methionine or tryptophan side chains. (c) 1993 John Wiley & Sons, Inc.     

CZE-ICP-MS as a tool for studying the hydrolysis of ruthenium anticancer drug candidates and their reactivity towards the DNA model compound dGMP

Elucidating the mode of action and thereby opening the way to the design of chemotherapeutic agents is one of the major goals of metal-based anticancer research. Hydrolysis and DNA binding play an important role for pharmaceutical formulation and for exerting anticancer activity. Herein, for the first time the application of capillary zone electrophoresis–inductively-coupled plasma mass spectrometry (CZE–ICP-MS) for studying the hydrolytic stability and the binding of the ruthenium anticancer drug candidates KP418, KP1019, and RAPTA-C to dGMP is described. RAPTA-C was found to hydrolyze fastest and showed the highest reactivity toward the DNA model compound, whereas KP418 was the most stable compound in both these respects.     

The amphipathic peptide mellitin as a tool to study the membrane-dependent activation of tissue transglutaminase

Summary The role of membrane phospholipids on the cross-linking activity of guinea pig liver (tissue) transglutaminase has been investigated using the amphipathic model peptide melittin as glutaminyl substrate and the primary amine monodansylcadaverine as extrinsic amine donor. A marked increase of transglutaminase catalytic activity was observedin vitro assays in the presence of neutral membrane phospholipids. In contrast, activation was abolished in the presence of membranes containing pure anionic lipids. Enzyme activation could be ascribed to a direct binding of the lipid to the protein as demonstrated in enzymatic assays using a non membrane-interacting peptide (Cbz-Gln-Gly). The data obtained with model peptides suggest that the cross-linking activity of tissue transglutaminase could be modulated by the local microenvironment composition of the lipid bilayer and indicate that membrane phospholipids should be taken into account for further experiments directed to asess, the still poor understood, physiological role of tissue transglutaminase.     

The amphipathic peptide mellitin as a tool to study the membrane-dependent activation of tissue transglutaminase

The role of membrane phospholipids on the cross-linkingactivity of guinea pig liver (tissue) transglutaminase hasbeen investigated using the amphipathic model peptidemelittin as glutaminyl substrate and the primary aminemonodansylcadaverine as extrinsic amine donor. A markedincrease of transglutaminase catalytic activity was observedin vitro assays in the presence of neutral membranephospholipids. In contrast, activation was abolished in thepresence of membranes containing pure anionic lipids. Enzyme activation could be ascribed to a direct binding ofthe lipid to the protein as demonstrated in enzymatic assaysusing a non membrane-interacting peptide (Cbz-Gln-Gly). Thedata obtained with model peptides suggest that the cross-linking activity of tissue transglutaminase could bemodulated by the local microenvironment composition of thelipid bilayer and indicate that membrane phospholipidsshould be taken into account for further experimentsdirected to assess, the still poor understood, physiologicalrole of tissue transglutaminase.     

Supported organometallic complexes part 39: cationic diamine(ether-phosphine)ruthenium(II) complexes as precursors for the hydrogenation of trans-4-phenyl-3-butene-2-one

Treatment of RuCl₂(η¹-Ph₂PCH₂CH₂OCH₃)₂(diamine) (1L₁-1L₇) with one equivalent of AgX (X=OTf, BF₄) in CH₂Cl₂ results in the formation of the monocationic ruthenium(II) complexes [RuCl(η¹-Ph₂PCH₂CH₂OCH₃)(η²-Ph₂PCH₂CH₂OCH₃)(diamine)]⁺X⁻ (2L₁-2L₇). These complexes were characterized by NMR, and mass spectroscopy as well as by elemental analyses, 2L₁ additionally by an X-ray structural analysis. Complex 2L₁ crystallizes in the monoclinic space group C2/c with Z=8. The monocationic and neutral complexes were applied as catalysts in the selective hydrogenation of trans-4-phenyl-3-butene-2-one. With the exception of 1L₃/1L₇ and 2L₃/2L₇ all catalysts showed high activities and selectivities toward the hydrogenation of the carbonyl group under mild conditions. However, the activity of the cationic catalysts is only half of that of their neutral congeners.     

Chemical modification of the small intestinal Na+/d-glucose cotransporter by amino group reagents: Evidence for a role of amino group(s) in the binding of the sugar

Some amino group reagents inactivate the small-intestinal Na⁺/d-glucose cotransporter, as measured either as a catalyst of Na⁺-dependent d-glucose transport or as a Na⁺-dependent phlorizin ligand. The amino group(s) studied in this paper are not identical with those investigated previously (Biber, J., Weber, J. and Semenza, G. (1983) Biochim. Biochim. Acta 728, 429–437): these are protected from inactivation by the simultaneous presence of Na⁺ plus sugar substrates. They are thus likely to be located within the substrate-binding site.     

Secretomers as a new tool for the monitoring of CTL responses

Efforts to follow tumor-specific immune responses in patients are often thwarted by lack of knowledge of the appropriate tumor antigens and the CTL epitopes of those antigens. There is, therefore, a growing need for techniques to monitor tumor-specific immune responses in settings where tumor antigens, and antigenic epitopes, remain unidentified. Here we describe a novel system to follow tumor-specific CTL immune responses. A truncated, soluble murine class I MHC (H-2D^b) molecule was fused with a rat IgG_2a Fc, in order to allow secretion of the complex. Tumor-specific CTL could then be detected as a result of the complex fastening to specific T cell receptors (TCR). These constructs were inserted into the genome of a recombinant adenovirus vector. Infection of tumor cells with these adenovirus constructs results in the secretion of the complexes into the culture supernatant. These soluble divalent class I MHC molecules were used to detect and activate specific CTL populations.     

Synthesis, spectroscopic and magnetostructural evidence for the formation of Cu(II) complexes of pyridyl-2-carboxylate (2-pca) and quinolyl-2-carboxylate (2-qca) as a result of a novel oxidative P-dealkylation reaction of diethyl 2-pyridylmethylphosphonate (2-pmpe) and diethyl 2-quinolylmethylphosphonate (2-qmpe) ligands

The interaction of diethyl 2-pyridylmethylphosphonate (2-pmpe) ligand with CuX₂ salts unexpectedly leads to the formation of compounds of the formula Cu(2-pca)₂ [X=Cl⁻ (1), CH₃COO⁻ (3)], and Cu(2-pca)Cl [X=Cl⁻ (2)] (2-pca=pyridine-2-carboxylate ion). The diethyl 2-quinolylmethylphosphonate ligand (2-qmpe) reacts with CuX₂ salts to similarly yield compounds of stoichiometry Cu(2-qca)₂ · H₂O (X=ClO₄ ⁻ (4)], and for X=Cl Cu(2-qca)₂ · H₂O (5) and Cu(2-qca)Cl (6), (2-qca=quinoline-2-carboxylate ion). These compounds are products of a novel oxidative P-dealkylation reaction, which takes place on 2-pmpe and 2-qmpe ligands under the used conditions. The compounds were characterized by infrared, ligand field, EPR spectroscopy and magnetic studies. Cu(2-pca)₂ exists in two crystalline forms, a blue form (1) and a violet form (3). For 3 the single-crystal structure was determined. The copper atom is four-coordinated in a square-planar geometry. The stack between related (and hence parallel) pca moieties involves interatomic distances of 3.27 Å. Cu(2-qca)₂ · H₂O also exists in two forms, a green (4) and a blue-green (5). Both these complexes are five coordinated, involve the same CuN₂O₃ chromophore and are examples of the distortion isomers. Variable-temperature magnetic susceptibility measurements (1.9-300 K) have shown that the antiferromagnetic coupling observed is much stronger in 6 than in 1, 3, 4 and 5. For 2 a ferromagnetic exchange occurs.     

H NMR spectroscopy as a tool to determine accurate photoisomerization quantum yields of stilbene-like ligands coordinated to rhenium(I) polypyridyl complexes

In this work, the use of proton nuclear magnetic resonance, ¹H NMR, was fully described as a powerful tool to follow a photoreaction and to determine accurate quantum yields, so called true quantum yields (Φ_true), when a reactant and photoproduct absorption overlap. For this, Φ_true for the trans-cis photoisomerization process were determined for rhenium(I) polypyridyl complexes, fac-[Re(CO)₃(NN)(trans-L)]⁺ (NN = 1,10-phenanthroline, phen, or 4,7-diphenyl-1,10-phenanthroline, ph₂phen, and L = 1,2-bis(4-pyridyl)ethylene, bpe, or 4-styrylpyridine, stpy). The true values determined at 365 nm irradiation (e.g. Φ_NMR = 0.80 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺) were much higher than those determined by absorption spectral changes (Φ_UV-Vis = 0.39 for fac-[Re(CO)₃(phen)(trans-bpe)]⁺). Φ_NMR are more accurate in these cases due to the distinct proton signals of trans and cis-isomers, which allow the actual determination of each component concentration under given irradiation time. Nevertheless when the photoproduct or reactant contribution at the probe wavelength is negligible, one can determine Φ_true by regular absorption spectral changes. For instance, Φ_313 nm for free ligand photoisomerization determined both by absorption and ¹H NMR variation are equal within the experimental error (bpe: Φ_UV-Vis = 0.27, Φ_NMR = 0.26; stpy: Φ_UV-Vis = 0.49, Φ_NMR = 0.49). Moreover, ¹H NMR data combined with electronic spectra allowed molar absorptivity determination of difficult to isolate cis-complexes.     

Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16. To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M_r 44 000 and M_r 12000 polypeptides can be precipitated from both red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human β₂-microglobulin (β₂-m) specific antibody confirmed a class-I-like structure associated with β₂-m on M' positive red cells and the absence of such a structure on M' negative red cells. Sequential precipitations gave analogous results. Proteolytic degradation by papain and V8 protease did not reveal any substantial difference between red and white M'-A16 positive cells, but a slight difference in the pI of the immunoprecipitable components of red and white cells was observed. All together, this indicates that either the blood group antigen M' is the BoLA-A16 class I antigen or M' and BoLA-A16 are two different class I polypeptides with the same relative mass, sharing identical epitopes and both associated with β₂-m. Comparable results were obtained with M₁ and BoLA-A24.