RESUMO
(3,4-Dihydroxybut-1-ynyl)uracil, -cytosine and -7-deazaadenine 2'-deoxyribonucleoside triphosphates (dNTPs) were prepared by direct aqueous Sonogashira cross-coupling of halogenated dNTPs with dihydroxybut-1-yne and converted to 3,4-dihydroxybutyl dNTPs through catalytic hydrogenation. Sodium periodate oxidative cleavage of dihydroxybutyl-dUTP gave the desired aliphatic aldehyde-linked dUTP, whereas the oxidative cleavage of the corresponding deazaadenine dNTP gave a cyclic aminal. All dihydroxyalkyl or -alkynyl dNTPs and the formylethyl-dUTP were good substrates for DNA polymerases and were used for synthesis of diol- or aldehyde-linked DNA. The aldehyde linked DNA was used for the labelling or bioconjugations through hydrazone formation or reductive aminations.
Assuntos
Adenina/análogos & derivados , Aldeídos/química , Citosina/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Nucleotídeos de Desoxiuracil/síntese química , Uracila/metabolismo , Adenina/síntese química , Adenina/química , Aminação , DNA/química , DNA Polimerase Dirigida por DNA/química , Nucleotídeos de Desoxiuracil/química , Estrutura Molecular , Nucleotídeos , Uracila/químicaRESUMO
DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/química , Transcrição Gênica , Bacillus subtilis/enzimologia , DNA/metabolismo , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/química , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Moldes GenéticosRESUMO
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 MolecularRESUMO
A series of sugar-modified derivatives of cytostatic 7-heteroaryl-7-deazaadenosines (2'-deoxy-2'-fluororibo- and 2'-deoxy-2',2'-difluororibonucleosides) bearing an aryl or heteroaryl group at positionâ 7 was prepared and screened for biological activity. The difluororibonucleosides were prepared by non- stereoselective glycosidation of 6-chloro-7-deazapurine with benzoyl-protected 2-deoxy-2,2-difluoro-D-erythro-pentofuranosyl-1-mesylate, followed by amination and aqueous Suzuki cross-couplings with (het)arylboronic acids. The fluororibo derivatives were prepared by aqueous palladium-catalyzed cross-coupling reactions of the corresponding 7-iodo-7-deazaadenine 2'-deoxy-2'-fluororibonucleoside 20 with (het)arylboronic acids. The key intermediate 20 was prepared by a six-step sequence from the corresponding arabinonucleoside by selective protection of 3'- and 5'-hydroxy groups with acid-labile groups, followed by stereoselective SN 2 fluorination and deprotection. Some of the title nucleosides and 7-iodo-7-deazaadenine intermediates showed micromolar cytostatic or anti-HCV activity. The most active were 7-iodo and 7-ethynyl derivatives. The corresponding 2'-deoxy-2',2'-difluororibonucleoside 5'-O-triphosphates were found to be good substrates for bacterial DNA polymerases, but are inhibitors of human polymeraseâ α.
Assuntos
Adenina/química , Antineoplásicos/farmacologia , Antivirais/farmacologia , Citostáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Ribonucleosídeos/farmacologia , Adenina/análogos & derivados , Antineoplásicos/síntese química , Antineoplásicos/química , Antivirais/síntese química , Antivirais/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citostáticos/síntese química , Citostáticos/química , DNA Polimerase Dirigida por DNA/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Células HeLa , Células Hep G2 , Hepacivirus/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores da Síntese de Ácido Nucleico , Ribonucleosídeos/síntese química , Ribonucleosídeos/química , Relação Estrutura-AtividadeRESUMO
5-(5-Formylthienyl)-, 5-(4-formylphenyl)- and 5-(2-fluoro-5-formylphenyl)cytosine 2'-deoxyribonucleoside mono- (dC(R)MP) and triphosphates (dC(R)TP) were prepared by aqueous Suzuki-Miyaura cross-coupling of 5-iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC(R)TPs were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC(R)MPs with lysine or lysine-containing tripeptide were studied and optimized. In aqueous phosphate buffer (pHâ 6.7) the yields of the reductive aminations with tripeptideâ III were up to 25 %. Bioconjugation of an aldehyde-containing DNA with a lysine-containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer.