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1.
Food Chem Toxicol ; 119: 222-230, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29653181

RESUMO

The purpose of this study was to investigate the inhibitory effect of ZH-1 ((6S,9aS,6aR,9bR)-6-(phenylcarbonyl)-6,6a,9a,9b-tetrahydro-8H-azolidino[3,4-a]b enzo [e]indolizine-7,9-dione) and its potential interaction with gemcitabine in A549 cells. MTT assay showed that the combined use of gemcitabine and ZH-1 presented a significant inhibition effect on A549 cell growth with the cell viability from 82.3 ±â€¯5.6% to 51.0 ±â€¯6.6%. The CI value was 0.60 suggesting a synergistic effect between these two drugs. HPLC-MS/MS data indicated that combined treatment with gemcitabine and ZH-1 induced a significant decrease in deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate levels compared with use of gemcitabine alone. Five RNs as well as seven dRNs were considered to be significantly contributive to the discrimination of samples. Furthermore, western blot analysis revealed that the combination treatment caused A549 cell apoptosis via the intrinsic pathway by up-regulating Bax/Bcl-2 ratio, activating caspase-9, caspase-3 and poly-ADP-ribose polymerase, and promoting caspase-7, caspase-9 and poly-ADP-ribose polymerase cleavage. Collectively, the combined treatment with gemcitabine and ZH-1 exerted a strong synergistic action on anticancer activity through growth inhibition, perturbations in ribonucleotides and deoxyribonucleotides and the activation of intrinsic apoptotic signaling pathway.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxirribonucleotídeos/síntese química , Células A549 , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Humanos
2.
Org Biomol Chem ; 14(43): 10123-10133, 2016 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-27714238

RESUMO

This article reviews the different possibilities towards progression of the formation of DNA/RNA in the chemical world, before life, in enzyme-free conditions. The advent of deoxyribo- and ribopentose-sugars, nucleosides, nucleotides and oligonucleotides in the prebiotic soup is briefly discussed. Further, the formation of early single stranded oligomers, base-pairing possibilities and information transfer based on the stability parameters of the derived duplexes is reviewed. Each theory has its own merits and demerits which we have elaborated upon. Lastly, using clues from this literature, a possible explanation for the specific 3'-5'-linkages in RNA is proposed.


Assuntos
DNA/química , RNA/química , Pareamento de Bases , Desoxirribonucleotídeos/síntese química , RNA/síntese química , Precursores de RNA/química
3.
ACS Synth Biol ; 5(7): 672-8, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-26914388

RESUMO

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.


Assuntos
Desoxirribonucleotídeos/síntese química , Reação em Cadeia da Polimerase/métodos , Biologia Sintética/métodos , Técnicas de Química Sintética , Desoxirribonucleotídeos/metabolismo , Ligação de Hidrogênio , Pirimidinas/química , Pirimidinas/metabolismo , Pirróis/química , Pirróis/metabolismo , Taq Polimerase/genética , Taq Polimerase/metabolismo
4.
ACS Chem Biol ; 8(11): 2452-65, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23992753

RESUMO

Nucleoside analogs are an important class of anticancer agent that historically show better efficacy against hematological cancers versus solid tumors. This report describes the development and characterization of a new class of nucleoside analog that displays anticancer effects against both hematological and adherent cancer cell lines. These new analogs lack canonical hydrogen-bonding groups yet are effective nucleotide substrates for several high-fidelity DNA polymerases. Permutations in the position of the non-hydrogen-bonding functional group greatly influence the kinetic behavior of these nucleosides. One particular analog designated 4-nitroindolyl-2'-deoxynucleoside triphosphate (4-NITP) is unique as it is incorporated opposite C and T with high catalytic efficiencies. In addition, this analog functions as a nonobligate chain terminator of DNA synthesis, since it is poorly elongated. Consistent with this mechanism, the corresponding nucleoside, 4-nitroindolyl-2'-deoxynucleoside (4-NIdR), produces antiproliferative effects against leukemia cells. 4-NIdR also produces cytostatic and cytotoxic effects against several adherent cancer cell lines, especially those that are deficient in mismatch repair and p53. Cell death in this case appears to occur via mitotic catastrophe, a specialized form of apoptosis. Mass spectroscopy experiments performed on nucleic acid isolated from cells treated with 4-NIdR validate that the non-natural nucleoside is stably incorporated into DNA. Xenograft mouse studies demonstrate that administration of 4-NIdR delays tumor growth without producing adverse side effects such as anemia and thrombocytopenia. Collectively, the results of in vitro, cell-based, and animal studies provide evidence for the development of a novel nucleoside analog that shows enhanced effectiveness against solid tumors.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/farmacologia , Nucleosídeos/síntese química , Nucleosídeos/farmacologia , Animais , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxirribonucleotídeos/química , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias/tratamento farmacológico , Nucleosídeos/química
5.
Bioconjug Chem ; 24(6): 1081-93, 2013 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-23682869

RESUMO

Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.


Assuntos
Primers do DNA/biossíntese , Endonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Oligonucleotídeos/biossíntese , Reação em Cadeia da Polimerase , Azidas/química , Química Click , Primers do DNA/genética , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Corantes Fluorescentes/química , Estrutura Molecular
6.
J Org Chem ; 78(7): 3021-9, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23506239

RESUMO

5-(α-Thyminyl)-5,6-dihydrothymine, also called spore photoproduct or SP, is commonly found in the genomic DNA of UV-irradiated bacterial endospores. Despite the fact that SP was discovered nearly 50 years ago, its biochemical impact is still largely unclear due to the difficulty of preparing SP-containing oligonucleotide in high purity. Here, we report the first synthesis of the phosphoramidite derivative of dinucleotide SP TpT, which enables successful incorporation of SP TpT into oligodeoxyribonucleotides with high efficiency via standard solid-phase synthesis. This result provides the scientific community a reliable means to prepare SP-containing oligonucleotides, laying the foundation for future SP biochemical studies. Thermal denaturation studies of the SP-containing oligonucleotide found that SP destabilizes the duplex by 10-20 kJ/mol, suggesting that its presence in the spore-genomic DNA may alter the DNA local conformation.


Assuntos
Desoxirribonucleotídeos/síntese química , Timina/análogos & derivados , Desoxirribonucleotídeos/química , Estrutura Molecular , Processos Fotoquímicos , Timina/química
7.
Bioorg Med Chem ; 21(3): 703-11, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23280146

RESUMO

A system for delivery of analogues of 2'-deoxyribonucleoside triphosphate (dNTP) based on SiO(2) nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO(2)-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO(2)-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO(2)-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO(2)-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO(2)-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei.


Assuntos
Desoxirribonucleotídeos/farmacocinética , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Polifosfatos/farmacocinética , Dióxido de Silício/química , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/química , Células HeLa , Humanos , Microscopia Confocal , Estrutura Molecular , Polimerização , Polifosfatos/síntese química , Polifosfatos/química
8.
Molecules ; 17(11): 13569-91, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23154273

RESUMO

Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.


Assuntos
Desoxirribonucleotídeos/síntese química , Sequência de Bases , DNA/síntese química , DNA/química , Adutos de DNA/síntese química , Adutos de DNA/química , DNA Catalítico , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos/química , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleotídeos/síntese química , Nucleotídeos/química , Ligação Proteica , Técnica de Seleção de Aptâmeros
9.
Chem Biodivers ; 9(9): 2050-63, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22976990

RESUMO

Deletion of the substituted pyrimidine ring in purine-2'-deoxynucleoside 5'-monophosphates leads to the artificial nucleotide analog dImMP(2-). This analog can be incorporated into DNA to yield, upon addition of Ag(+) ions, a molecular wire. Here, we measured the acidity constants of H(2)(dImMP)(±) having one proton at N(3) and one at the PO(3)(2-) group by potentiometric pH titrations in aqueous solution. The micro acidity constants show that N(3) is somewhat more basic than PO(3)(2-) and, consequently, the (H·dImMP)(-) tautomer with the proton at N(3) dominates to ca. 75%. The calculated micro acidity constants are confirmed by (31)P- and (1)H-NMR chemical shifts. The assembled data allow many quantitative comparisons, e.g., the N(3)-protonated and thus positively charged imidazole residue facilitates deprotonation of the P(O)(2)(OH)(-) group by 0.3 pK units. Information on the intrinsic site basicities also allows predictions about metal-ion binding; e.g., Mg(2+) and Mn(2+) will primarily coordinate to the phosphate group, whereas Ni(2+) and Cu(2+) will preferably bind to N(3). Macrochelate formation for these metal ions is also predicted. The micro acidity constant for N(3)H(+) deprotonation in the (H·dImMP·H)(±) species (pk(a) 6.46) and the M(n+)-binding properties are of relevance for understanding the behavior of dImMP units present in DNA hairpins and metalated duplexes.


Assuntos
Desoxirribonucleotídeos/química , Imidazóis/química , Nucleotídeos/química , Desoxirribonucleotídeos/síntese química , Concentração de Íons de Hidrogênio , Imidazóis/síntese química , Estrutura Molecular
10.
J Inorg Biochem ; 105(9): 1212-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21723807

RESUMO

Copper(II) complexes with a new chelator-type nucleoside-histidine modified 2'-deoxyriboadenosine (N-[(9-ß-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine) were studied by potentiometric and spectroscopic (UV-visible, CD, EPR) techniques, in conjunction with computer modeling optimization. The ligand can act as bidentate or tridentate depending on pH range. In acidic pH a very stable dimeric complex Cu(2)L(2) predominates with coordination spheres of both metal ions composed of oxygen atoms from carboxylic groups, one oxygen atom from ureido group and two nitrogen atoms derived from purine base and histidine ring. Above pH 5, deprotonation of carbamoyl nitrogens leads to the formation of CuL(2), Cu(2)L(2)H(-1) and Cu(2)L(2)H(-2) species. The CuL(2)H(-1) and CuL(2)H(-2) complexes with three or four nitrogens in Cu(II) coordination sphere have been detected in alkaline medium. Our findings suggest that N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine chelates copper(II) ions very efficiently. The resulting complex might be used as an alternative base-pairing mode in which hydrogen-bonded base pairs present in natural DNA are replaced by metal-mediated ones.


Assuntos
Adenosina/síntese química , Quelantes/síntese química , Complexos de Coordenação/síntese química , Cobre/metabolismo , Sondas de DNA/síntese química , DNA/metabolismo , Desoxirribonucleotídeos/síntese química , Histidina/metabolismo , Adenosina/análise , Adenosina/metabolismo , Pareamento de Bases , Quelantes/análise , Quelantes/metabolismo , Dicroísmo Circular , Complexos de Coordenação/análise , Complexos de Coordenação/metabolismo , Cobre/química , DNA/química , Sondas de DNA/análise , Sondas de DNA/metabolismo , Desoxirribonucleotídeos/análise , Desoxirribonucleotídeos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Modelos Moleculares , Potenciometria , Prótons , Espectrofotometria Ultravioleta
11.
Nucleic Acids Res ; 39(7): 2995-3004, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21300642

RESUMO

To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.


Assuntos
Adenina/análogos & derivados , Óxidos N-Cíclicos/química , Citosina/análogos & derivados , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Adenina/química , Adenina/metabolismo , Pareamento de Bases , Óxidos N-Cíclicos/metabolismo , Citosina/química , Citosina/metabolismo , DNA Polimerase I/metabolismo , Primers do DNA , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Especificidade por Substrato , Temperatura , Moldes Genéticos
12.
Nucleic Acids Res ; 39(4): 1623-37, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20952399

RESUMO

This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.


Assuntos
Dano ao DNA , Replicação do DNA , DNA/biossíntese , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , Elétrons , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Nucleotídeos/química
13.
J Org Chem ; 75(12): 3945-52, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20364862

RESUMO

Template-directed primer extension usually requires a polymerase, nucleoside triphosphates, and magnesium ions as cofactors. Enzyme-free, chemical primer extensions are known for preactivated nucleotides at millimolar concentrations. Based on a screen of carbodiimides, heterocyclic catalysts, and reactions conditions, we now show that near-quantitative primer conversion can be achieved at submillimolar concentration of any of the four deoxynucleotides (dAMP, dCMP, dGMP and dTMP). The new protocol relies on in situ activation with EDC and 1-methylimidazole and a magnesium-free buffer that was tested successfully for different sequence motifs. The method greatly simplifies chemical primer extension assays, further reduces the cost of such assays, and demonstrates the potential of the in situ activation approach.


Assuntos
Carbodi-Imidas/química , Desoxirribonucleotídeos/síntese química , Sequência de Bases , Primers do DNA , Desoxirribonucleotídeos/química , Microquímica , Dados de Sequência Molecular , Estrutura Molecular
14.
Nucleic Acids Res ; 38(8): 2617-23, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20211841

RESUMO

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.


Assuntos
Desoxirribonucleotídeos/síntese química , Análise de Sequência de DNA/normas , Software , Sequência de Bases , DNA/química , Desoxirribonucleotídeos/química , Controle de Qualidade
15.
Biochemistry ; 49(9): 1814-21, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20143879

RESUMO

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.


Assuntos
Pareamento Incorreto de Bases/genética , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/genética , Guanosina/análogos & derivados , Mutagênicos/síntese química , Catálise , Adutos de DNA/síntese química , Adutos de DNA/genética , Adutos de DNA/metabolismo , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/genética , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Glicosilação , Guanosina/síntese química , Guanosina/genética , Guanosina/metabolismo , Humanos , Testes de Mutagenicidade , Mutagênicos/metabolismo , Estereoisomerismo , Moldes Genéticos
16.
Org Biomol Chem ; 7(21): 4369-77, 2009 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-19830285

RESUMO

We previously reported the synthesis of a borononucleotide analogue of thymidine monophosphate and its association towards the formation of a new borono-linked dinucleotide. Here we describe the completion of the set of four 2'-deoxyborononucleotide analogues of natural nucleotide monophosphates, namely the previously unknown dCbn, dGbn and dAbn. These analogues were all prepared from the respective 5'-aldehydic nucleosides through a homologation/reduction sequence. The borononucleotides were subsequently obtained by either borylation (dCbn and dGbn) or cross-metathesis (CM) in the presence of the Hoveyda-Grubbs catalyst (dAbn). The reversible formation of the corresponding dinucleotides between these new analogues and uridine was studied by (1)H NMR, and semi-empirical calculations were carried out to provide bond length and electrostatic information that assess the structural similarities existing between these bioisosteres and their natural counterparts.


Assuntos
Boro/química , Nucleotídeos de Desoxiadenina/química , Desoxicitidina Monofosfato/química , Nucleotídeos de Desoxiguanina/química , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Uridina/química
17.
Nucleic Acids Symp Ser (Oxf) ; (52): 259-60, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776352

RESUMO

Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.


Assuntos
Desoxirribonucleotídeos/química , Temperatura Alta , Reação em Cadeia da Polimerase , Bacteriófago lambda/genética , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/metabolismo , HIV-1/genética , Humanos , Cinética
18.
Nucleic Acids Symp Ser (Oxf) ; (52): 281-2, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776363

RESUMO

Here, we report the synthesis of 3'-deoxyapionucleoside 3''-triphosphates (apioNTPs) and the analysis of their property as substrate of enzymatic polymerization. We established the large scale synthetic route of 3-deoxy-D-apiose from D-galactose, and regio- and stereo-selective glycosylation procedures. Resulting 3'-deoxyapionucleosides were then converted into their 3''-triphosphates. We carried out primer-extension reactions and found that Therminator polymerase, a variant of 9 degrees N DNA polymerase, was an efficient enzyme for incorporation of apioNTPs to synthesize DNA-dependent 3'-deoxy-D-apiose nucleic acids (apioNAs).


Assuntos
DNA/química , Desoxirribonucleotídeos/síntese química , Pentoses/química , DNA/biossíntese , Primers do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Glicosilação
19.
Nucleic Acids Symp Ser (Oxf) ; (52): 385-6, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776415

RESUMO

A new linker bearing two orthogonal precursor groups - an amino group for the reaction with electrophilic agents, and also a terminal triple bond for the reaction with an aliphatic azido group (1,3-dipolar cycloaddition reaction) for the introduction of the different residues into nucleosides is designed and synthesized. 5'-Triphosphates of various nucleosides bearing proposed linker group have been obtained.


Assuntos
Desoxirribonucleosídeos/química , Éteres/química , Ftalimidas/química , Acetamidas/síntese química , Acetamidas/química , Nucleotídeos de Desoxicitosina/química , Desoxirribonucleotídeos/síntese química , Desoxirribonucleotídeos/química , Didesoxinucleosídeos/química , Éteres/síntese química , Ressonância Magnética Nuclear Biomolecular , Ftalimidas/síntese química
20.
Nucleic Acids Symp Ser (Oxf) ; (52): 537-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776491

RESUMO

Commercially available trans-4-hydroxy-L-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with N-phosphonomethyl moiety attached to the nitrogen atom of prolinol ring. The synthetic methodology based on the inversion of configuration at both 1- and 4- positions led, in result, to all diastereoisomeric O-protected 4-mesyloxyprolinol-N-methylphosphonates. Alkylation of nucleobases using the synthons afforded the nucleotide analogues corresponding to alpha- and beta-nucleotides in both L- and D-series. The NMR-based conformational study of alpha- and beta-nucleotides in aqueous solution performed at two different pH values securing either N-fully protonated or deprotonated forms revealed in both cases occurrence of the same mostly populated conformer. All final prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found.


Assuntos
Desoxirribonucleotídeos/síntese química , Organofosfonatos/síntese química , Pirrolidinas/química , Desoxirribonucleotídeos/química , Nucleosídeos/química , Organofosfonatos/química
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