Dehydrogenative oxidation of primary and secondary alcohols catalyzed by a Cp*Ir complex having a functional C,N-chelate ligand

A new catalytic system for the dehydrogenative oxidation of alcohols using a Cp*Ir complex having a functional C,N-chelate ligand has been developed. With this catalytic system, both primary and secondary alcohols were efficiently converted to aldehydes and ketones, respectively. Mechanistic investigations of this catalytic system have revealed that the catalytically active species is a hydrido iridium complex with a functional C,N-chelate ligand.     

Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: differential scanning calorimetric study

Differential scanning calorimetry (DSC) was used to measure the thermodynamic changes associated with translesion synthesis across major lesion induced in DNA by antitumor oxaliplatin [1,2-d(GG) intrastrand cross-link]. Insertion of matched nucleotides dC at the primer terminus (across unique 3'- or 5'-dG in the unplatinated template) and subsequent extensions resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite either platinated dG in the intrastrand cross-link formed in the template strand and subsequent extensions by one nucleotide resulted only in little changes in thermodynamics. A similar thermodynamic delay was observed for a control template primer containing a dG:dT mismatch across 3'- or 5'-dG in the template and subsequent Watson-Crick primer extensions. The thermodynamic scarcity generated by either the lesion or mismatches was not localized but extended to the 5'-downstream sites, which may be connected with the phenomenon termed "short-term memory" of replication errors retained by some DNA polymerases responding to DNA damages or mismatches. Interestingly, formation of the 1,2-d(GG) intrastrand cross-link of oxaliplatin altered the overall DSC profiles of the dG:dT mismatch template/primers only in a very small extent. While addition of matched nucleotide dC across either dG in the template strand was thermodynamically favored over the presence of a mismatched dT (ΔΔG(0)(310) was 7.6 or 6.8 kJ mol(-1), ΔΔH was 14 or 49 kJ mol(-1)), no such thermodynamic advantage was observed with the 1,2-d(GG) intrastrand cross-link of oxaliplatin at these positions (ΔΔG(0)(310) was 2.8 or -0.3 kJ mol(-1), ΔΔH was 4 or 9 kJ mol(-1)). The equilibrium thermodynamic data also provide insight into the processes associated with misincorporation of incorrect nucleotides during replication bypass across major cross-links of antitumor oxaliplatin. On the other hand, besides thermodynamic effects also kinetic factors play an important role in the processing of the cross-links of antitumor platinum drugs. The impact of the two effects in overall processing DNA adducts by a particular DNA polymerase will depend on its nature.     

Atom-transfer radical addition (ATRA) and cyclization (ATRC) reactions catalyzed by a mixture of [RuCl(2)Cp*(PPh3)] and Magnesium

A new catalytic procedure for atom-transfer radical addition (ATRA) and cyclization (ATRC) reactions is described. The combination of the ruthenium(III) complex [RuCl(2)Cp*(PPh3)] (Cp*: pentamethylcyclopentadienyl) with magnesium allows these reactions to be performed under mild conditions with high efficiency. In most cases, the catalyst concentrations required are significantly lower than those used in previously reported procedures. It is suggested that magnesium acts as a reducing agent that generates and regenerates the catalytically active ruthenium(II) species. The precatalyst [RuCl(2)Cp*(PPh3)] has been analyzed by X-ray crystallography.     

Oxygen-bridged hybrid metallocene-nonmetallocene polymetallic catalysts of group 4 metals for bimodal activity in olefin polymerization: synthesis, characterization, and theoretical investigation

This article describes the syntheses of two covalently linked oxygen-bridged hybrid metallocene-nonmetallocene polymetallic catalysts bearing two different catalytically active group 4 metals. The reactions of Cp*2(Me)Zr(OH) (2) with Ti(NMe2)4 and Hf(NMe2)4 led to the formation of a heterobimetallic compound Cp*2(Me)Zr(micro-O)Ti(NMe2)3 (7) and a trimetallic derivative Cp*2(Me)Zr(micro-O)Hf(NMe2)2(micro-O)Zr(Me)Cp*2 (9), respectively, under the elimination of Me2NH. The crystal data confirm the molecular structures of 7 and 9, which crystallize in the space groups P and P21/n, respectively. 9 is the first example of a crystallographically characterized heterotrimetallic complex having a Zr-O-Hf-O-Zr core. 7 bearing two active catalytic centers, namely, zirconium and titanium, exhibits bimodal activity in olefin polymerization when activated with methylalumoxane (MAO). It produces polyethylene largely controlled by the zirconium center, and polystyrene seems to be formed predominantly by the titanium center. DFT calculations were performed on the supposed cationic intermediates, revealing that a cation generated on the titanium center is sterically more accessible for monomer binding, though it is energetically less-favorable than that generated on the zirconium center.     

Catalytic activity of macroion-porphyrin nanoassemblies

A new type of catalytically active self-assembled nanostructures in aqueous solution is presented. Polyelectrolyte-porphyrin nanoscale assemblies consisting of anionic cylindrical poly(styrene sulfonate) brush molecules and cationic tetravalent meso-tetrakis(4-N-methyl-pyridinium)porphyrin (TMPyP) or meso-tetrakis(4-(trimethyl-ammonium)phenyl)-porphyrin (TAPP), respectively, exhibit up to 8-fold higher catalytic activity with regard to light induced iodide oxidation than the corresponding porphyrins without polymeric template. This is particularly interesting because a general concept rather than a specific binding motif is exploited. The approach introduced here hence is attractive due to its facility and versatility and bears potential, for example, in light harvesting and energy conversion.     

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing

Background: The nonpolar nucleoside of difluorotoluene (F) was previously found to behave similarly to thymidine in single-site deoxynucleoside triphosphate (dNTP) insertion experiments with the Klenow fragment (KF) of DNA polymerase I. Further study was needed, first to see whether F-A base pairs could be replicated in more than one sequence context; second to investigate whether specific base pair replication occurs in the presence of four dNTPs; and third to confirm the presence of F in a replicated DNA strand.Results: A primer bound to a template strand containing eight F residues was extended by KF using the four natural dNTPs at 20 μM. Similarly, the complement (containing eight adenines) was extended using dATP, dGTP, dCTP and dFTP. Comparison of the new strands to authentic strands using standard and ‘inverse’ chemical sequencing showed identical composition within ± 5%.Conclusions: The results confirm that F in a template strand encodes the insertion of dATP and that adenine in a template encodes the insertion of dFTP with good specificity in at least six different nearest neighbor contexts. The results confirm that analog F behaves similarly to thymidine despite its poor hydrogen-bonding ability.     

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

A nucleotide-analogue-induced gain of function corrects the error-prone nature of human DNA polymerase iota

Y-family DNA polymerases participate in replication stress and DNA damage tolerance mechanisms. The properties that allow these enzymes to copy past bulky adducts or distorted template DNA can result in a greater propensity for them to make mistakes. Of the four human Y-family members, human DNA polymerase iota (hpol?ι) is the most error-prone. In the current study, we elucidate the molecular basis for improving the fidelity of hpol?ι through use of the fixed-conformation nucleotide North-methanocarba-2'-deoxyadenosine triphosphate (N-MC-dATP). Three crystal structures were solved of hpol?ι in complex with DNA containing a template 2'-deoxythymidine (dT) paired with an incoming dNTP or modified nucleotide triphosphate. The ternary complex of hpol?ι inserting N-MC-dATP opposite dT reveals that the adenine ring is stabilized in the anti orientation about the pseudo-glycosyl torsion angle, which mimics precisely the mutagenic arrangement of dGTP:dT normally preferred by hpol?ι. The stabilized anti conformation occurs without notable contacts from the protein but likely results from constraints imposed by the bicyclo[3.1.0]hexane scaffold of the modified nucleotide. Unmodified dATP and South-MC-dATP each adopt syn glycosyl orientations to form Hoogsteen base pairs with dT. The Hoogsteen orientation exhibits weaker base-stacking interactions and is less catalytically favorable than anti N-MC-dATP. Thus, N-MC-dATP corrects the error-prone nature of hpol?ι by preventing the Hoogsteen base-pairing mode normally observed for hpol?ι-catalyzed insertion of dATP opposite dT. These results provide a previously unrecognized means of altering the efficiency and the fidelity of a human translesion DNA polymerase.     

2.4 A crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA dodecamer duplex

(1R,2R-Diaminocyclohexane)oxalatoplatinum(II) (oxaliplatin) is a third-generation platinum anticancer compound that produces the same type of inter- and intrastrand DNA cross-links as cisplatin. In combination with 5-fluorouracil, oxaliplatin has been recently approved in Europe, Asia, and Latin America for the treatment of metastatic colorectal cancer. We present here the crystal structure of an oxaliplatin adduct of a DNA dodecanucleotide duplex having the same sequence as that previously reported for cisplatin (Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J. Nature 1995, 377, 649-652). Pt-MAD data were used to solve this first X-ray structure of a platinated DNA duplex derived from an active platinum anticancer drug other than cisplatin. The overall geometry and crystal packing of the complex, refined to 2.4 A resolution, are similar to those of the cisplatin structure, despite the fact that the two molecules crystallize in different space groups. The platinum atom of the [Pt(R,R-DACH)](2+) moiety forms a 1,2-intrastrand cross-link between two adjacent guanosine residues in the sequence 5'-d(CCTCTGGTCTCC), bending the double helix by approximately 30 degrees toward the major groove. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice. The latter is positioned in the minor groove opposite the platinum cross-link. A novel feature of the present structure is the presence of a hydrogen bond between the pseudoequatorial NH hydrogen atom of the (R,R)-DACH ligand and the O6 atom of the 3'-G of the platinated d(GpG) lesion. This finding provides structural evidence for the importance of chirality in mediating the interaction between oxaliplatin and duplex DNA, calibrating previously published models used to explain the reactivity of enantiomerically pure vicinal diamine platinum complexes with DNA in solution. It also provides a new kind of chiral recognition between an enantiomerically pure metal complex and the DNA double helix.     

Photochemistry of benzophenone immobilized in a major groove of DNA: formation of thermally reversible interstrand cross-link

We here report a highly site and sequence selective formation of an interstrand cross-link of (BP)U-containing oligomer duplexes. The cross-link was found spontaneously reverted to original oligomers upon heating, providing a new method for the temporary connection of two DNA strands.     

Enzyme Activation with a Synthetic Catalytic Co‐enzyme Intermediate: Nucleotide Methylation by Flavoenzymes

To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme‐dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin‐methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin‐dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5‐uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.     

Synthesis of an enzyme-like imprinted polymer with the substrate as the template, and its catalytic properties under aqueous conditions

Transition state analogues (TSAs) have long been regarded as ideal templates for the preparation of catalytically active synthetic imprinted polymers. In the current work, however, a new type of molecularly imprinted polymer (MIP) was synthesized with the substrate (homovanillic acid, HVA) as the template and hemin introduced as the catalytic center, with the use of plural functional monomers to prepare the active sites. The MIP successfully mimicked natural peroxidase, suggesting that it may not be imperative to employ a TSA as the template when preparing enzyme-like imprinted polymers and that the imprinted polymer matrix provided an advantageous microenvironment around the catalytic center (hemin), essentially similar to that supplied by apo-proteins in natural enzymes. Significantly, by taking advantage of the special structure of hemin and multiple-site interactions provided by several functional monomers, the intrinsic difficulties for MIPs in recognizing template molecules in polar solutions were overcome. The newly developed polymer showed considerable recognizing ability toward HVA, catalytic activity, substrate specificity and also stability, which are the merits lacked by the natural peroxidase. Meanwhile, the ease of recovery and reuse the MIP implies the potential for industrial application.     

Harnessing the mechanism of glutathione reductase for synthesis of active site bound metallic nanoparticles and electrical connection to electrodes

It is demonstrated herein that the FAD-dependent enzyme glutathione reductase (GR) catalyzes the NADPH-dependent reduction of AuCl4-, forming gold nanoparticles at the active site that are tightly bound through the catalytic cysteines. The nanoparticles can be removed from the GR active site with thiol reagents such as 2-mercaptoethanol. The deep enzyme active site cavity stabilizes very small metallic clusters and prevents them from aggregating in the absence of capping ligands. The behavior of the GR-nanoparticle complexes in solution, and their electrochemical properties when immobilized on graphite paper electrodes are presented. It is shown that the borohydride ion, a known reducing agent for GR, is catalytically oxidized by larger GR-nanoparticle (>or=150 gold atoms) complexes generating catalytic currents, whereas NADPH (the natural reducing agent for GR) is not. It is proposed that the surface of the Toray graphite paper electrode employed here interferes with NADPH binding to the GR-nanoparticle complex. The catalytic currents with borohydride begin at the potential of GR-bound FAD, showing that there is essentially zero resistance to electron transfer (i.e., zero overpotential) from GR-bound FAD through the gold nanoparticle to the electrode.     

Hybridization-triggered isothermal signal amplification coupled with MutS for label-free and sensitive fluorescent assay of SNPs

Combining the specific recognization of MutS protein for mismatched DNA sequences with the target-driven molecular switch that acts as both the template and primer of the polymerization reaction, a new label-free and sensitive fluorescent assay strategy for specific single-stranded DNA sequences or SNPs is proposed.     

Structures of end products resulting from lesion processing by a DNA glycosylase/lyase

DNA glycosylase/lyases initiate the repair of damaged nucleobases in the genome by catalyzing excision of aberrant nucleobases and nicking of the lesion-containing DNA strand. Nearly all of these proteins have the unusual property of remaining tightly bound in vitro to the end products of the reaction cascade. We have taken advantage of this property to crystallize and structurally characterize the end product resulting from complete DNA processing by a catalytically active mutant form of human 8-oxoguanine DNA glycosylase (D268E hOgg1). The resulting structure is consistent with the currently accepted catalytic mechanism for the protein. Unexpectedly, however, soaking of a nucleobase analog into the crystals results in religation of the DNA backbone in situ.     

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.     

Effects of a high-affinity antibody fragment on DNA polymerase reactions near a (6-4) photoproduct site

Pyrimidine (6-4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6-4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41-mer DNA containing a (6-4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases alpha, beta and delta (pol alpha, pol beta and pol delta). None of these DNA polymerases were able to bypass the (6-4) photoproduct and elongation terminated at or near the 3'-pyrimidone of the photoproduct, depending on the dNTP concentration. When a single-chain Fv (scFv) with high affinity for the (6-4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3'-pyrimidone by Klenow fragment, pol alpha, pol beta or pol delta, respectively. These results suggest that the scFv can bind to the template DNA containing a (6-4) photoproduct and inhibit extension reactions by polymerases.