Capping Reaction Research Articles

Kinetic Study on the Capping Reaction of Living Polyisobutylene with 1,1-Diphenylethylene. 1. Effect of Temperature and Comparison to the Model Compound 2-Chloro-2,4,4-trimethylpentane

The addition reaction of 1,1-diphenylethylene to polymeric and dimeric isobutylene living chain ends in the presence of TiCl4 was investigated in hexane/MeCl 60/40 (v/v) at −80 to −40 °C using on-line UV−vis spectroscopy. The apparent equilibrium constants of capping/decapping, Ke, and the apparent rate constants of capping, kcKi, were directly measured, and from these values, the corresponding rate constants of decapping, kd, were calculated. The activation energy is found to be apparently negative for the capping reaction (kcKi) and positive for the decapping reaction (kd). However, the respective activation parameters for the polyisobutylene differ significantly from those of the dimer; i.e., the chain length affects the kinetics of the capping/decapping reaction.

Efficiency of n-butyllithium/m-diisopropenylbenzene diadduct as a dicarbanion initiator in the making of α,ω-hydroxyl terminated polybutadiene using oxetane as the capping agent

The diadduct made from a 2:1 ratio of n-butyllithium to m-diisopropenylbenzene has been evaluated as a diinitiator for synthesizing α,ω-hydroxyl terminated polybutadiene in cyclohexane solution. While the g.c./m.s. analysis results showed the predominant formation of the diadduct at 20°C, an incomplete reaction at 0°C led to a mixture containing a variety of initiating species of varying number of carbon-lithium ends. The observed narrow monomodal molecular weight distribution resulting from the use of this mixture indicated that this diadduct did not react as a diinitiator but rather as a monoinitiator. Although one of the two carbon-lithium ends of the diadduct was inactive during the polymerization, both ends were able to react with oxetane, thus forming the α,ω-hydroxyl terminated polybutadiene. This monofunctionality during polymerization and difunctionality towards the capping reaction has been corroborated by 1H n.m.r., 13C n.m.r. and hydroxyl titration. The capping reaction of the carbon-lithium end was as efficient for poly(butadienyl)dilithium, synthesized using the diadduct as the initiator, as for poly(butadienyl)lithium, synthesized using n-BuLi as the initiator. The inactivity of one of the two carbon-lithium ends during the polymerization did not prevent the diadduct from being an effective initiator for making telechelic HTPB.

SYNTHESIS AND STRUCTURAL CHARACTERIZATION OF A NOVEL AND STARLIKE C60(PMS)x(CH3)x COPOLYMER

A novel starlike C60(PMS)x(CH3)x (x = 1–3) copolymer was prepared by reaction of a living poly(α-methylstyrene) (PMS) carbanion with C60, followed by a capping reaction with methyl iodine. The characterization techniques used are UV-vis, FT-IR, GPC, DSC, TGA, ESR, 13C NMR, SEM, XRD, cyclic voltammetry (CV) and fluorescence spectroscopy. This starlike polymer has a visibly earthy yellow cast when compared with the unfunctionalized parent polymer (PMS), and is highly soluble in some common organic solvents. Poly(α-methylstyrene) is essentially transparent at wavelengths longer than 280 nm, and its main absorption band is at 250 (vs) nm. Covalent attachment of the multiple PMS arms to the C60 core results in the bathochromic shift of the above band and the appearance of a new absorption band at 328 nm (π→π*). This new band is one of the characteristic absorptions for organofullerenes. The thermal stability and oxidation/reduction activation of pure PMS are enhanced apparently by C60 chemical modifiction. It is also found that in this copolymer there are mainly two kinds of paramagnetic species, whereas no ESR signal in pure poly(α-methylstyrene) is detected, and this indicates the non-existence of unpaired electron or free radical. © 1997 Elsevier Science Ltd

Synthesis and characterization of a soluble and starlike C 60(CH 3) x(PAN) x copolymer

A novel starlike C 60(CH 3) x (PAN) x copolymer was prepared by reaction of the living n Butyl-terminated polyacrylonitrile (PAN) with C 60, followed by a capping reaction with methyl iodine. Multiple PAN arms are attached to the C 60 core. The characterization techniques employed are UV-vis, FT-IR, 13C NMR, TGA, XRD, ESR and SEM. This starlike polymer has a visibly yellow cast when compared with the unfunctionalized polymer. The chemical and physical properties of this polymer depend largely on the concentrations of the fullerene covalently bound to the polymer matrix and on the chain-length of PAN units attached to a C 60 center. A qualitative comparison of the submicro-morphological structures of the copolymer and the pure n Butyl-terminated poly-acrylonitrile is given.

A TOF-SIMS study of an acid-terminated butadiene-acrylonitrile copolymer used as a toughening agent in modern adhesive formulations

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to study one of the abid-terminated butadiene-acrylonitrile copolymers used as toughening agents in adhesive formulations. The capping reaction with diglycidyl ethers of bisphenol-A (DGEBA) was also investigated. At low mass, the positive-ion spectrum of the toughening agent was characterized by the radical butadiene monomer cation; at high mass, the parent ions of an antioxidant in the copolymer dominated the positive- and negative-ion spectra. Purification of the polymer using gel filtration chromatography resulted in little change in the spectra, apart from the total removal of the antioxidant parent ions and associated signals. Spectra containing peaks from a range of cationized oligomers were obtained by depositing sub-monolayer films of the copolymer on silver. The capping reaction resulted in the formation of mono- and di-DGEBA substituted oligomers. The antioxidant also reacted with DGEBA to form the mono- and di-substituted compounds.

New multi-bridged cryptands: Polyazamacrocyclic schiff bases formed in a tetrapode capping reaction

Multi-bridged cryptands were formed in one step by condensation of two moles of a tetraaldehyde with four moles of a triamine. This is the first example of a tetrapode capping reaction.

Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes.

Mutational analysis of yeast mRNA capping enzyme.

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.

Interaction of iron ethynyl and ethynediyl complexes with Cp2Ni2(CO)2: formation of tri- and tetranuclear adducts with a multiply bridging C2H ligand

Reaction of ethynyliron complexes Fp-C{triple_bond} C-H [FP = Fp (1), Fp{lg_bullet}(1{lg_bullet})] with Cp{sub 2}Ni{sub 2}(CO){sub 2} (5) affords the expected adducts Cp{sub 2}Ni{sub 2}({mu}-{eta}{sup 2}:{eta}{sup 2}-FP-C{triple_bond}C-H)[FP = Fp (6), Fp{lg_bullet}(6{lg_bullet})] with a tetrahedral C{sub 2}Ni{sub 2} core together with tetranuclear adducts (CPFe)(CpNi){sub 2}Ni(CO){sub 2} ({mu}-Co)({mu}{sub 4}-C{sub 2}H) [CP = CP (7), Cp{lg_bullet} (7{lg_bullet})] with a quadruply bridging C{sub 2}H ligand. 7 (7{lg_bullet}) is formed by a formal capping reaction of 6 (6{lg_bullet}) with a Ni(CO) fragment. On the other hand, reaction of an ethynediyidiliron complex Fp{lg_bullet}-C{triple_bond} C-Fp{lg_bullet} (2) with 5 affords a dinuclear complex, Cp{lg_bullet}FeCpNi({nu}-CO){sub 2}(CO) (10), without the C{sub 2} ligand, the fate of which is not known. The molecular structures of 6,7, and 10 have been determined by X-ray crystallography. 15 refs., 4 figs., 4 tabs.

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.

Preparation of Quantum-Sized Semiconductor Sulfide-Doped Gels

ABSTRACTUltrasmall semiconductor particles (CdS, ZnS and PbS) are produced either by direct precipitation, γ-radiolysis or in mimetic membranes. A pure cluster powder is then prepared from a chemical capping reaction. The capping of the cluster surface by thiolate complexes permits the separation of aggregates without fusion. These capped clusters can be dispersed in different solvents such as sol-gel precursors allowing to prepare dense and optically transparent xerogels with semiconductor clusters grafted on oxide polymers. The nanocrystallites are characterized by X-ray diffraction, small-angle X-ray scattering and optical spectroscopy.

Synthesis of PS-PEG and PMMA-PEG Branched Block Copolymers by Macroinimers

Abstract A new type of macroinimers were synthesized by the capping reaction of hydroxyl groups of polyazoesters with isocyanato ethyl, methacrylate in the presence of dibutyl tin dilaurate. Macroinimers having PEG-400 and PEG-1500 units were used in the polymerization of methyl methacrylate (MMA) or styrene (S) to obtain PS-PEG or PMMA-PEG branched block copolymers at 60°C. Longer polymerization times or higher macroinimer concentrations led to cross-linked block copolymers. Similarly neat homopolymerization of macroinimers also led to the cross-linked polyethyleneglycols. The products were characterized by IR and NMR spectroscopy, viscosity measurements and fractional precipitation.

Active site localization in a viral mRNA capping enzyme.

Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.

In vitrocapping inTrypanosoma cruziidentifies and shows specificity for the spliced leader RNA and U-RNAs

Messenger RNA maturation in trypanosomes requires a trans-splicing event in which a capped 39 nucleotide leader sequence, the spliced leader (SL), from the 5' terminus of a small RNA (SL-RNA) is joined to the 5' termini of protein coding gene transcripts. We have developed nuclear extracts from Trypanosoma cruzi that label three small endogenous RNAs in the presence of [alpha-32P]GTP. Herein, we have characterized this labelling as 5' capping and shown that the capping activity exhibits an unusual ATP dependence. Moreover, partial sequence analysis identified the three cap-labelled RNAs as the T. cruzi SL-RNA, and two U-RNAs previously uncharacterized in T. cruzi, U2 and Ux. Finally, the capping reaction in the T. cruzi extracts showed apparent specificity for these RNAs--other endogenous or exogenous transcripts were not capped. The apparent specificity of this in vitro capping activity closely reflects the in vivo requirements; i.e., only the SL- and U-RNAs need to be capped since mature mRNAs are capped via trans-splicing. These observations are consistent with the hypothesis that one of the functions of trans-splicing is to supply 5' caps to mature trypanosome mRNAs.

Synthesis of capped oligoribonucleotides by use of protected 7-methylguanosine 5′-diphosphate derivatives

This paper describes the capping reaction of 5′-phosphorylated oligoribonucleotides protected only by a tetrahydropyranyl group at each 2′-position. By use of the conventional capping agent PhSppm7GTMTrmM, m7G5pppGUUA and m7G5′pppGUAUUA were synthesized from the corresponding oligoribonucleotide 5′-monophosphate derivatives. Since the latter was obtained in low yield because of poor solubility of the partially protected pUUA in DMF, the oligomer component was converted into a phosphorimidazolidate species which was found to be soluble in DMF and allowed to react with a newly designed capping agent, ppm7GTMTrmM. This triphosphate bond formation proceeded smoothly to give m7G5′pppGUAUUA in good yield.

The Synthesis and Characterization of Mononuclear and Binuclear Iron(II) Clathrochelate Complexes Derived from 2,3-Butanedione Oxime Hydrazone

The synthesis and characterization of a new series of iron(II) clathrochelate complexes, including the first example of a binuclear covalently linked c1athrochelate complex, is reported. The ligand system is based on the bifunctional chelate 2,3-butanedione oxime hydrazone which forms a tris-complex with iron(II). The c1athrochelate is completed by capping the complex with both a boronic acid/oxime capping reaction and a formaldehyde/hydrazone capping reaction. Electrochemical and spectroscopic characterizations of the complexes are discussed.

Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase.

Transcripts for plant mitochondrial genes are frequently present as multiple size classes. In maize, these differences often result from variation in the 5' noncoding region. To determine where transcription initiates, primary (unprocessed) transcripts were specifically labeled in vitro by the capping reaction catalyzed by guanylyltransferase. Direct mapping of transcription initiation sites was accomplished by hybridization of in vitro-capped RNA with the 5' flanking sequences of mitochondrial genes and subsequent digestion with single-strand-specific RNases. The RNase protection experiments identified three transcription initiation sites for subunit 3 of cytochrome oxidase and at least six transcription initiation sites for subunit 9 of ATP synthase. Thus, transcript size heterogeneity is primarily the result of multiple transcription initiation sites for these genes rather than RNA processing. Primer extension analyses of maize mitochondrial RNA were used to precisely establish the sequences at the initiation sites. Comparison of sequences at transcription initiation sites suggests that some homology exists at these sites, although no highly conserved consensus sequence is obvious.

RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria.

Variation in the length of the 5' non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5' end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5' flanking regions of 26 S, 18 S and 5 S rRNA genes and two protein-coding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5' flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18 + 5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5' hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.

Role of a pertussis toxin substrate in the control of lectin-induced cap formation in human neutrophils.

We have examined the role of GTP-binding proteins and the associated cyclic AMP- and calcium-related transduction mechanisms in the regulation of capping in human neutrophils. Pertussis toxin (PT), a probe for the GTP-binding protein Ni, abolished capping induced by fluorescein isothiocyanate-conjugated concanavalin A (Con-A), whereas cholera toxin, a probe for the GTP-binding protein Ns, was without effect. Consistent with the latter finding, ligands acting at receptors associated with the Ns protein, namely the prostaglandin E1 and beta-adrenergic agonists, were without effect on the capping reaction. The possible role of mobilization of internal calcium was evaluated by using Quin2-loaded cells. Calcium mobilization was observed at concentrations of Con-A which yielded optimal capping (10 micrograms/ml). Treatment with PT, phorbol myristrate acetate or 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) abolished both calcium mobilization and capping. Colchicine, which substantially enhanced capping, had no effect on calcium mobilization. At concentrations of the lectin above those required for capping, superoxide generation and enzyme release were noted. These reactions were less susceptible to inhibition by PT, effects being observed only on the Kact. for Con-A-mediated superoxide generation with little effect on the Vmax. The degree of PT-mediated inhibition for enzyme release with Con-A was much lower than that observed with fMet-Leu-Phe. Our results imply that a step involving Ni-mediated calcium mobilization, sensitive to phorbol myristate acetate, is essential to the regulation of capping; a distinct mechanism may be involved in colchicine-mediated enhancement of capping; and Ni may play a relative minor role in the regulation of lectin-mediated exocytosis.

Tubulin-colchicine complex (TC) inhibits microtubule depolymerization by a capping reaction exerted preferentially at the minus end.

The effects of colchicine and tubulin-colchicine complex (TC) on microtubule depolymerization were studied using the axoneme-subunit system described previously [Bergen LG, Borisy GG; J Cell Biol 84:141-150, 1980]. This system allows the independent analysis of the polymerization kinetics at both the plus and minus ends of a microtubule. Depolymerization was induced by isothermal dilution with 10 volumes of an experimental solution containing colchicine, TC, or buffer alone. Colchicine alone (5-100 microM) blocked depolymerization at the minus end, whereas depolymerization at the plus end occurred at almost control rates. A similar effect was produced by TC (0.4:1-1:1 molar ratio to free tubulin). High molar ratios of TC to tubulin (10:1) blocked depolymerization at both plus and minus ends, and intermediate molar ratios of TC:T allowed depolymerization of the plus ends but at attenuated rates. The blockage was not readily reversible; TC-affected ends neither shortened upon dilution nor grew longer upon incubation with additional tubulin. We conclude that TC at suprastoichiometric ratios to tubulin inhibits microtubule depolymerization by a capping reaction and that this effect is exerted preferentially at the minus end.