Synthesis of Deoxynucleoside Triphosphates that Include Proline,Urea, or Sulfonamide Groups and Their Polymerase Incorporation into DNA

To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L ‐proline‐containing residues (dU^tPTP and dU^cPTP), a dipeptide (dU^FPTP), a urea derivative (dU^BpuTP), and a sulfamide residue (dU^BsTP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent (exo^?) and Pwo DNA polymerases, as well as the Klenow fragment of E. coli DNA polymerase I, although they were only acceptable substrates for the 9°N_m polymerase. All of the modified dNTPs, with the exception of dU^BpuTP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX.     

离去基团、非离去基团及中间体性质对取代苯甲酸苯酯氨解反应的影响

X-取代苯甲酸Y-取代苯基酯(X-C6H4COOC6H4-Y)的氨解反应是一个亲核加成-消除过程.为定量评价Um等对非离去基团(X-C6H4-)、离去基团(Y-C6H4O-)及中间体(T±)的性质对氨解反应速率(kN)影响的研究结果,分别用取代基x的Hammett常数σX、Y-C6H4OH的pKa和取代基X、Y的极化效应指数PEI表征上述结构因素,并用其对logkN建立多元线性回归模型,得到了良好的相关结果.进一步分析得出,Y-C6H4OH的pKa对logkN的影响最大(贡献率为90.46%),而取代基X的Hammett常数σX影响最小.     

Anticancer and DNA Binding Activities of Platinum (IV) Complexes; Importance of Leaving Group Departure Rate

The two six-coordinate Pt(IV) complexes, containing bidentate nitrogen donor/methyl ligands with general formula [Pt(X)₂Me₂(^tbu₂bpy)], where ^tbu₂bpy = 4,4′-ditert-butyl-2,2′-bipyridine and X = Cl (C₁) or Br (C₂), serving as the leaving groups were synthesized for evaluation of their anticancer activities and DNA binding properties. To examine anticancer activities of the synthetic complexes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ethidium bromide/acridine orange (EB/AO) staining method were performed. The binding properties of these complexes to DNA and purine nucleotides were examined, using different spectroscopic techniques. These complexes demonstrated significant anticancer activities against three cancer cell lines Jurkat, K562, and MCF-7. On the basis of the results of EB/AO staining, C₁ and C₂ were also capable to induce apoptosis in cancer cells. These complexes comprise halide leaving groups, displaying different departure rates; accordingly, they demonstrated slightly dissimilar anticancer activity and significantly different DNA/purine nucleotide binding properties. The results of DNA interaction studies of these complexes suggest a mixed-binding mode, comprising partial intercalation and groove binding. Overall, the results presented herein indicate that the newly synthesized Pt(IV) complexes are promising class of the potential anticancer agents which can be considered as molecular templates in designing novel platinum anticancer drugs. This study also highlights the importance of leaving group in anticancer activity and DNA binding properties of Pt(IV) complexes.     

A New Approach for the C-Alkylation of Unsaturated Sugars Involving the Benzothiazol-2-Ylthio Leaving Group

ABSTRACT

The leaving ability of the benzothiazol-2-ylthio group was applied to the C-alkylation of a 2,3-enopyranoside model structure. Coordination of the organometallic reagent to the thio-heterocyclic moiety was responsible for a stereospecific syn conjugate alkyl attack.     

Synthetic,Spectroscopic, and Biological Aspects of Triorganosilicon(IV) Complexes of Tridentate Schiff Bases

Abstract

Hexacoordinated organosilicon complexes of type R₃Si(L) (R = ethyl, butyl, phenyl; HL = ligand, obtained by the condensation of 4-aminoantipyrine with 2-hydroxyacetophenone, 2-hydroxybenzophenone, 2-hydroxybenzaldehyde, and 2-hydroxynapthaldehyde) have been synthesized and characterized by elemental analysis, molar conductance, and spectroscopic studies (IR, ¹H, ¹³C, and ²⁹Si NMR). The spectroscopic studies indicated that the ligands acted as tridentate coordinating through azomethine nitrogen, carbonyl oxygen, and oxygen of hydroxyl after deprotonation to the central silicon atom. The ligands and their organosilicon complexes have been evaluated for antimicrobial activities against fungi (Aspergillus niger and Candida albicans), and against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and Gram-negative bacteria (Escherichia coli). The aim of the present work is to synthesize novel eco-friendly fungicides and bactericides and to study the effect of the biological activity of ligands on the complexation with organosilicon moiety. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements to view the free supplemental file.     

Base-Modified DNA Labeled by [Ru(bpy)3]2+ and [Os(bpy)3]2+ Complexes: Construction by Polymerase Incorporation of Modified Nucleoside Triphosphates,Electrochemical and Luminescent Properties,and Applications

Modified 2′-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)₃]²⁺ and [Os(bpy)₃]²⁺ complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru²⁺ or Os²⁺ complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru^2+/3+ or Os^2+/3+. The redox potentials of the Ru²⁺ complexes (1.1–1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os²⁺ complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru²⁺-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for “multicolor” redox labeling of DNA and for DNA minisequencing.     

Impact of the Metal Center and Leaving Group on the Anticancer Activity of Organometallic Complexes of Pyridine-2-carbothioamide

Ru^II(cym)Cl (cym = η⁶-p-cymene) complexes of pyridinecarbothioamides have shown potential for development as orally active anticancer metallodrugs, underlined by their high selectivity towards plectin as the molecular target. In order to investigate the impact of the metal center on the anticancer activity and their physicochemical properties, the Os(cym), Rh- and Ir(Cp*) (Cp* = pentamethylcyclopentadienyl) analogues of the most promising and orally active compound plecstatin 2 were prepared and characterized by spectroscopic techniques and X-ray diffraction analysis. Dissolution in aqueous medium results in quick ligand exchange reactions; however, over time no further changes in the ¹H NMR spectra were observed. The Rh- and Ir(Cp*) complexes were investigated for their reactions with amino acids, and while they reacted with Cys, no reaction with His was observed. Studies on the in vitro anticancer activity identified the Ru derivatives as the most potent, independent of their halido leaving group, while the Rh derivative was more active than the Ir analogue. This demonstrates that the metal center has a significant impact on the anticancer activity of the compound class.     

6-烷基鸟嘌呤与DNA碱基间氢键作用的理论研究

用密度泛函B3LYP方法在6-311＋G**基组水平上对鸟嘌呤及顺(cis-)、反式(anti-)-6-烷基鸟嘌呤(O⁶-AlkylG)与DNA碱基(胸腺嘧啶T、胞嘧啶C、腺嘌呤A、鸟嘌呤G)的氢键二聚体结构进行了优化. 在MP2/cc-pVXZ(X＝D,T)// B3LYP/6-311＋G**水平上, 采用完全基组外推方法校正了氢键二聚体的相互作用能, 并用完全均衡校正法(CP)校正了基组重叠误差(BSSE). 在B3LYP/6-311＋G**水平上计算了各氢键碱基对的全电子波函数, 并用分子中的原子理论(AIM)分析了碱基间的弱相互作用. 计算结果显示, 鸟嘌呤6-O烷基化改变了碱基间的氢键作用模式, 使碱基对发生了明显的螺旋桨式扭转和不同程度的位移, 碱基间的电子密度分布和氢键作用能明显减小. O⁶-AlkylG对DNA碱基间的氢键作用是去稳定化的, 去稳定化影响的顺序为GC＞GG＞GA≈GT. 计算结果与文献给出的实验结论基本一致.     

A Shared Prebiotic Formation of Neopterins and Guanine Nucleosides from Pyrimidine Bases

The prebiotic origins of biopolymers and metabolic co-factors are key questions in Origins of Life studies. In a simple warm-little-pond model, using a drying phase to produce a urea-enriched solution, we present a prebiotic synthetic path for the simultaneous formation of neopterins and tetrahydroneopterins, along with purine nucleosides. We show that, in the presence of ribose and in a formylating environment consisting of urea, ammonium formate, and water (UAFW), the formation of neopterins from pyrimidine precursors is robust, while the simultaneous formation of guanosine requires a significantly higher ribose concentration. Furthermore, these reactions provide a tetrahydropterin–pterin redox pair. This model suggests a prebiotic link in the origin of purine nucleosides and pterin cofactors that provides a possible deep prebiotic temporal connection for the emergence of nucleic acids and metabolic cofactors.     

Thin‐Film Properties of DNA and RNA Bases: A Combined Experimental and Theoretical Study

The structures of the DNA and RNA bases cytosine, uracil, and thymine in thin films with a nominal film thickness of about 20 nm are studied by using X‐ray photoemission spectroscopy (XPS) and Fourier‐transform infrared spectroscopy. The molecules are evaporated in situ from powder on a gold foil. The experimental results indicate that cytosine is composed of two energetically close tautomeric forms, whereas uracil and thymine exist in only one tautomeric form. Additionally, quantum chemical calculations are performed to complement the experimental results. The relative energies of the tautomeric forms of cytosine, uracil, and thymine are calculated using Hartree–Fock (HF), density functional theory (DFT), and post‐HF methods. Furthermore, the assignment of the XPS spectra is supported by using simple model considerations employing Koopmans ionization energies and Mulliken net atomic charges.     

Functional Monomers and Polymers. 145. Synthetic Application Based on the Tautomerism of Polymer-Bound Purine and Pyrimidine Bases

Polymer-bound alkylthiopurine and -pyrimidine bases were prepared and used as the polymeric reagents for the reactions of nitrile, olefin, and enone formation. The corresponding low molecular weight compounds were also prepared for comparison. Differences in the reactivity of these reagents were related to the change in tautomerism of the purine and pyrimidine moieties.     

Cloning and Sequence Analysis of Novel DNA Polymerases from Thermophilic Geobacillus Species Isolated from Hot Springs in Turkey: Characterization of a DNA Polymerase I from Geobacillus kaue Strain NB

The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of G?nen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.     

Tuning the Self‐Assembly of Oligothiophenes on Chemical Vapor Deposition Graphene: Effect of Functional Group,Solvent, and Substrate

Tuning and characterizing the interfacial structure of organic semiconductors on graphene is essential for graphene‐based devices. Regulation of the supramolecular assembling structure of oligothiophenes on graphene by changing functional groups attached to the backbone of oligothiophenes is described and the assembling behavior is compared with that on the basal plane of highly oriented pyrolytic graphite. It reveals that terminal functional groups attached to the conjugated backbone of oligothiophene can entirely change the assembling structures. Significant solvent and substrate effects have also been confirmed by comparing the assembling structures of oligothiophenes deposited from tetrahydrofuran, 1,2,4‐trichlorobenzene, and octanoic acid onto graphene and graphite.     

Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis KIN4/I and Its Application to PCR

A family B DNA polymerase gene from the hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN4/I was highly expressed under the control of T7lac promoter of pET-28ARG in Escherichia coli BL21-CodonPlus(DE3)-RIL cells. The produced I. hospitalis (Iho) DNA polymerase was purified by heat treatment followed by HisTrap? HP column and HiTrap? SP column chromatographies. The molecular mass of the purified Iho DNA polymerase was 88 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH for Iho DNA polymerase activity was 7.0 and the optimal temperature was 70 °C. Iho DNA polymerase was strongly activated by the presence of magnesium ion at an optimum concentration of 3 mM. The optimal concentration of KCl for Iho DNA polymerase activity was 60 mM. The half-life of the enzyme at 94 °C was about 2 h. The optimal conditions for polymerase chain reaction (PCR) were determined. Iho DNA polymerase possesses 3′?→?5′ exonuclease activity, and the fidelity of the Iho DNA polymerase was similar to that of Pfu and Vent DNA polymerases. However, Iho DNA polymerase provided more enhanced efficiency of PCR amplification than Pfu and Vent DNA polymerases. Iho DNA polymerase could successfully amplify a 2-kb λ DNA target with a 10-s extension time and could amplify a DNA fragment up to 8 kb λ DNA.     

Biochemical,Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary

Secretory group V phospholipase A2 (PLA₂-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA₂, the production of PLA₂-V from dromedary. The obtained DrPLA₂-V has an absolute requirement for Ca²⁺ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that K_cat/Km_app is 2.6 ± 0.02 mM⁻¹ s⁻¹. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.     

B—DNA分子中互补碱基对G—C、A—T水合作用的研究

本文通过对B—DNA分子中互补碱基对A—T、G—C水合作用的量子化学计算以及最优化处理求得了它们水合作用的最优配位模式。结果表明,当水分子与互补碱基对A—T、G—C共面且水分子与A—T碱基对中胸腺嘧啶(T)杂环上的O_2原子形成氢键以及与G—C碱基对中胞嘧啶(C)杂环上的O_2原子形成氢键时,它们的水合作用具有最稳定的配位模式。     

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.