Radical Chain Reduction of Alkylboron Compounds with Catechols

The conversion of alkylboranes to the corresponding alkanes is classically performed via protonolysis of alkylboranes. This simple reaction requires the use of severe reaction conditions, that is, treatment with a carboxylic acid at high temperature (>150 degrees C). We report here a mild radical procedure for the transformation of organoboranes to alkalies. 4-tert-Butylcatechol, a well-established radical inhibitor and antioxidant, is acting as a source of hydrogen atoms. An efficient chain reaction is observed due to the exceptional reactivity of phenoxyl radicals toward alkylboranes. The reaction has been applied to a wide range of organoboron derivatives such as B-alkylcatecholboranes, trialkylboranes, pinacolboronates, and alkylboronic acids. Furthermore, the so far elusive rate constants for the hydrogen transfer between secondary alkyl radical and catechol derivatives have been experimentally determined. Interestingly, they are less than 1 order of magnitude slower than that of tin hydride at 80 degrees C, making catechols particularly attractive for a wide range of transformations involving C-C bond formation.

Synthesis Of Triblock Copolymers By Radical Trap Assisted-Atom Transfer Radical Coupling

Monobrominated diblock copolymers composed of poly(styrene) (PSt), poly(methylacrylate) (PMA), or poly(methyl methacrylate) (PMMA) were synthesized by consecutive atom transfer radical polymerizations (ATRP). The brominated diblocks were utilized in atom transfer radical coupling (ATRC) and radical trap-assisted ATRC (RTA-ATRC) reactions to form ABA type triblock copolymers. Once PMMA-PStBr and PSt-PMABrBr were produced by ATRP, the synthes of PSt-PMA-PSt and PMMA-PSt- PMMA by ATRC and also by RTA-ATRC were attempted. The coupling methods were compared and it was found that RTA-ATRC succeeded in synthesizing PSt-PMA-PSt where ATRC could not, and that RTA-ATRC improved coupling over ATRC for PMMAPSt- PMMA. Incorporation of the radical trap 2-methyl-2-nitrosopropane (MNP) midchain allowed for simple thermal cleavage of the triblock to confirm the RTA-ATRC pathway occurred in preference over the head to head radical coupling pathway of ATRC. Triblocks made by ATRC did not cleave under our conditions, as no MNP was present and thus no labile C-O bond was incorporated. The RTA-ATRC pathway allowed for lower catalyst amounts (2 molar equivalents of copper(I)bromide and 2 molar equivalents of copper metal) and a high degree of coupling at lower temperatures (40°C). The RTA-ATRC improved upon ATRC because of its ability to generate a persistent radical and proceed by first order kinetics with respect to the chain end radical.

Polymerization of Styrene and Cyclization to Macrocyclic Polystyrene in a One-Pot, Two-Step Sequence

Dibrominated polystyrene (BrPStBr) was produced by atom transfer radical polymerization (ATRP) at 80 degrees C, using the bifunctional initiator benzal bromide to afford the telechelic precursor. The ATRP reaction was stopped around 40% monomer conversion and directly converted into an radical trap-assisted atom transfer radical coupling (RTA-ATRC) reaction by lowering the temperature to 50 degrees C, and adding the radical trap 2-methyl-2-nitrosopropane (MNP) along with additional catalyst, reducing agent, and ligand to match ATRC-type reaction conditions. In an attempt to induce intramolecular coupling, rather than solely intermolecular coupling and elongation, the total reaction volume was increased by the addition of varying amounts of THF. Cyclization, along with intermolecular coupling and elongation, occurred in all cases, with the extent of ring closure a function of the total reaction volume. The cyclic portion of the coupled product was found to have a (G) value around 0.8 by GPC analysis, consistent with the reduction in hydrodynamic volume of a cyclic polymer compared to its linear analog. Analysis of the sequence by H-1 NMR confirmed that propagation was suppressed nearly completely during the RTA-ATRC phase, with percent monomer conversion remaining constant after the ATRP phase. (C) 2013 Elsevier Ltd. All rights reserved.

Hierarchical self-assembly of the DNA-grafted supramolecular polymers

Conjugation of functional entities with a specific set of optical, mechanical or biological properties to DNA strands allows engineering of sophisticated DNA-containing architectures. Among various hybrid systems, DNA-grafted polymers occupy an important place in modern materials science. In this contribution we present the non-covalent synthesis and properties of DNA-grafted linear supramolecular polymers (SPs), which are assembled in a controllable manner from short chimeric DNA-pyrene oligomers. The synthetic oligomers consist of two parts: a 10 nucleotides long DNA chain and a covalently attached segment of variable number of phosphodiester-linked pyrenes. The temperature-dependent formation of DNA-grafted SPs is described by a nucleation-elongation mechanism. The high tendency of pyrenes to aggregate in water, leads to the rapid formation of SPs. The core of the assemblies consists of stacked pyrenes. They form a 1D platform, to which the DNA chains are attached. Combined spectroscopic and microscopic studies reveal that the major driving forces of the polymerization are π-stacking of pyrenes and hydrophobic interactions, and DNA pairing contributes to a lesser extent. AFM and TEM experiments demonstrate that the 1D SPs appear as elongated ribbons with a length of several hundred nanometers. They exhibit an apparent helical structure with a pitch-to-pitch distance of 50±15 nm. Since DNA pairing is a highly selective process, the ongoing studies are aimed to utilize DNA-grafted SPs for the programmable arrangement of functional entities. For example, the addition of non-modified complementary DNA strands to the DNA-grafted SPs leads to the cooperative formation of higher-order assemblies. Also, our experiments suggest that the fluorescent pyrene core of 1D ribbons serves as an efficient donor platform for energy transfer applications.

Hierarchical self-assembly of the DNA-grafted supramolecular polymers

Conjugation of functional entities with a specific set of optical, mechanical or biological properties to DNA strands allows engineering of sophisticated DNA-containing architectures. Among various hybrid systems, DNA-grafted polymers occupy an important place in modern materials science. In this contribution we present the non-covalent synthesis and properties of DNA-grafted linear supramolecular polymers (SPs), which are assembled in a controllable manner from short chimeric DNA-pyrene oligomers. The synthetic oligomers consist of two parts: a 10 nucleotides long DNA chain and a covalently attached segment of variable number of phosphodiester-linked pyrenes. The temperature-dependent formation of DNA-grafted SPs is described by a nucleation-elongation mechanism. The high tendency of pyrenes to aggregate in water, leads to the rapid formation of SPs. The core of the assemblies consists of stacked pyrenes. They form a 1D platform, to which the DNA chains are attached. Combined spectroscopic and microscopic studies reveal that the major driving forces of the polymerization are π-stacking of pyrenes and hydrophobic interactions, and DNA pairing contributes to a lesser extent. AFM and TEM experiments demonstrate that the 1D SPs appear as elongated ribbons with a length of several hundred nanometers. They exhibit an apparent helical structure with a pitch-to-pitch distance of 50±15 nm. Since DNA pairing is a highly selective process, the ongoing studies are aimed to utilize DNA-grafted SPs for the programmable arrangement of functional entities. For example, the addition of non-modified complementary DNA strands to the DNA-grafted SPs leads to the cooperative formation of higher-order assemblies. Also, our experiments suggest that the fluorescent pyrene core of 1D ribbons serves as an efficient donor platform for energy transfer applications.

Identification of the block in targeted retroviral-mediated gene transfer

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

Identification of the proton pathway in bacterial reaction centers: Replacement of Asp-M17 and Asp-L210 with Asn reduces the proton transfer rate in the presence of Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule QB (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, kAB(2) [QA⨪QB⨪ + H+ → QA(QBH)−] in native and mutant RCs in the absence and presence of Cd2+. Previous work has shown that the binding of Cd2+ decreases kAB(2) in native RCs ≈100-fold. The preceding paper shows that bound Cd2+ binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with QB⨪ by searching for mutations that greatly affect kAB(2) (≳10-fold) in the presence of Cd2+, where kAB(2) is limited by the proton transfer rate (kH). Upon mutation of Asp-L210 or Asp-M17 to Asn, kH decreased from ≈60 s−1 to ≈7 s−1, which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (kH ≈ 7 s−1) with that in native RCs in the absence of Cd2+ (kH ≥ 104 s−1), we conclude that alternate proton transfer pathways, which have been postulated, are at least 103-fold less effective.

On the role of the K-proton transfer pathway in cytochrome c oxidase

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the “peroxy” state, Pr) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate proton transfer by “shutting off” the protonic connectivity through the K-pathway after initiation of the O2 reduction chemistry. This “shutoff” prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Three Drought-Responsive Members of the Nonspecific Lipid-Transfer Protein Gene Family in Lycopersicon pennellii Show Different Developmental Patterns of Expression1

Genomic clones of two nonspecific lipid-transfer protein genes from a drought-tolerant wild species of tomato (Lycopersicon pennellii Corr.) were isolated using as a probe a drought- and abscisic acid (ABA)-induced cDNA clone (pLE16) from cultivated tomato (Lycopersicon esculentum Mill.). Both genes (LpLtp1 and LpLtp2) were sequenced and their corresponding mRNAs were characterized; they are both interrupted by a single intron at identical positions and predict basic proteins of 114 amino acid residues. Genomic Southern data indicated that these genes are members of a small gene family in Lycopersicon spp. The 3′-untranslated regions from LpLtp1 and LpLtp2, as well as a polymerase chain reaction-amplified 3′-untranslated region from pLE16 (cross-hybridizing to a third gene in L. pennellii, namely LpLtp3), were used as gene-specific probes to describe expression in L. pennellii through northern-blot analyses. All LpLtp genes were exclusively expressed in the aerial tissues of the plant and all were drought and ABA inducible. Each gene had a different pattern of expression in fruit, and LpLtp1 and LpLtp2, unlike LpLtp3, were both primarily developmentally regulated in leaf tissue. Putative ABA-responsive elements were found in the proximal promoter regions of LpLtp1 and LpLtp2.

The joining (J) chain is present in invertebrates that do not express immunoglobulins.

Joining (J) chain is a component of polymeric, but not monomeric, immunoglobulin (Ig) molecules and may play a role in their polymerization and transport across epithelial cells. To date, study of the J chain has been confined to vertebrates that produce Ig and in which the J chain displays a considerable degree of structural homology. The role of the J chain in Ig polymerization has been questioned and, since the J chain can be expressed in lymphoid cells that do not produce Ig, it is possible that the J chain may have other functions. To explore this possibility, we have surveyed J-chain gene, mRNA, and protein expression by using reverse transcriptase-coupled PCR, Northern blot analysis, and immunoblot analysis in invertebrate species that do not produce Ig. We report that the J-chain gene is expressed in invertebrates (Mollusca, Annelida, Arthropoda, Echinodermata, and Holothuroidea), as well as in representative vertebrates (Mammalia, Teleostei, Amphibia). Furthermore, J-chain cDNA from the earthworm has a high degree of homology (68-76%) to human, mouse, and bovine J chains. Immunohistochemical studies reveal that the J chain is localized in the mucous cells of body surfaces, intestinal epithelial cells, and macrophage-like cells of the earthworm and slug. This study suggests that the J chain is a primitive polypeptide that arose before the evolution of Ig molecules and remains highly conserved in extent invertebrates and vertebrates.