RESUMEN
6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.
Asunto(s)
Cartilla de ADN/química , Nucleótidos de Desoxiuracil/química , Desoxiuridina/análogos & derivados , Triazinas/química , Tubercidina/análogos & derivados , Reacción de Cicloadición , Cartilla de ADN/síntesis química , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiuracil/síntesis química , Reacción en Cadena de la Polimerasa , Triazinas/síntesis química , Tubercidina/síntesis química , Tubercidina/químicaRESUMEN
Hepatitis C virus (HCV) nucleoside inhibitors display pan-genotypic activity, a high barrier to the selection of resistant virus, and are some of the most potent direct-acting agents with durable sustained virologic response in humans. Herein, we report, the discovery of ß-d-2'-Br,2'-F-uridine phosphoramidate diastereomers 27 and 28, as nontoxic pan-genotypic anti-HCV agents. Extensive profiling of these two phosphorous diastereomers was performed to select one for in-depth preclinical profiling. The 5'-triphosphate formed from these phosphoramidates selectively inhibited HCV NS5B polymerase with no inhibition of human polymerases and cellular mitochondrial RNA polymerase up to 100 µM. Both are nontoxic by a variety of measures and display good stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes. Ultimately, a preliminary oral pharmacokinetics study in male beagles showed that 28 is superior to 27 and is an attractive candidate for further studies to establish its potential value as a new clinical anti-HCV agent.
Asunto(s)
Antivirales/farmacología , Desoxirribonucleósidos/farmacología , Nucleótidos de Desoxiuracil/farmacología , Hepacivirus/efectos de los fármacos , Profármacos/farmacología , Animales , Antivirales/síntesis química , Antivirales/farmacocinética , Línea Celular Tumoral , Desoxirribonucleósidos/síntesis química , Desoxirribonucleósidos/farmacocinética , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/farmacocinética , Perros , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Microsomas Hepáticos/metabolismo , Profármacos/síntesis química , Profármacos/farmacocinética , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
(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.
Asunto(s)
Adenina/análogos & derivados , Aldehídos/química , Citosina/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Uracilo/metabolismo , Adenina/síntesis química , Adenina/química , Aminación , ADN/química , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiuracil/química , Estructura Molecular , Nucleótidos , Uracilo/químicaRESUMEN
In order to establish the Diels-Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4-triazine-modified 2'-deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4-triazine group was attached at the 5-position of 2'-deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2'-deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN-modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4-triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5-tetrazine for postsynthetic labeling by the Diels-Alder reaction with inverse electron demand.
Asunto(s)
ADN/química , Nucleótidos de Desoxiuracil/química , Colorantes Fluorescentes/química , Triazinas/química , Reacción de Cicloadición , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiuracil/síntesis química , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes/síntesis química , Triazinas/síntesis químicaRESUMEN
5-Ethynyl-2'-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Polifosfatos/química , ADN/química , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Reacción en Cadena de la Polimerasa , Técnica SELEX de Producción de AptámerosRESUMEN
Different phosphocholine-cardiolipin-2'-deoxyuridine inclusion complexes were developed, that allowed to compose a water-soluble form of nucleoside analogues with previously defined antituberculosis activity. It was found that the resulting liposomes effectively penetrated to the cells. The increase of cytotoxicity was undoubtedly indicative of accumulation of the nucleoside in the cell culture. The result proved the ability of the liposomes for delivery of the low-soluble compounds to the cells for further investigation of their efficacy. It was shown that treatment of the bacterial cells with the llposomes of the modified nucleosides did not affect the bacterial growth.
Asunto(s)
Antituberculosos , Cardiolipinas , Nucleótidos de Desoxiuracil , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/crecimiento & desarrollo , Fosforilcolina , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Cardiolipinas/química , Cardiolipinas/farmacología , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/farmacología , Liposomas , Fosforilcolina/química , Fosforilcolina/farmacologíaRESUMEN
To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2-5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN , Nucleótidos de Desoxiuracil , Reacción en Cadena de la Polimerasa/métodos , ADN/síntesis química , ADN/química , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/químicaRESUMEN
A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-succinimidyl) carbonate (DSC) and modified 2'-deoxyuridine triphosphate (dUTP); the final product, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained and confirmed by 1H-NMR, 31P-NMR, and HRMS with purity of up to 99%. The synthesized dye-labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved completely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.
Asunto(s)
Nucleótidos de Desoxiuracil/síntesis química , Rodaminas/síntesis química , Análisis de Secuencia de ADN/métodos , Nucleótidos de Desoxiuracil/química , Colorantes Fluorescentes/química , Furanos/síntesis química , Rodaminas/químicaRESUMEN
We herein describe the synthesis of fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate (d(bt)UTP) and primer extension reactions using d(bt)UTP. We also carried out primer extension reactions using the (bt)U template. B family DNA polymerases, such as KOD, Deep Vent (exo-), and 9°N(m) DNA polymerases, were effective for elongation with d(bt)UTP. Deep Vent (exo-) and KOD DNA polymerases have excellent fidelity for incorporating d(bt)UTP only at the site opposite the adenine template and only dATP when using the (bt)U template. Therefore, d(bt)UTP is an excellent fluorescent nucleotide that can be incorporated into DNA by DNA polymerases.
Asunto(s)
Cartilla de ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/química , Colorantes Fluorescentes/química , Oligodesoxirribonucleótidos/química , Uridina Trifosfato/análogos & derivados , Secuencia de Bases , Cartilla de ADN/síntesis química , Cartilla de ADN/metabolismo , Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Modelos Moleculares , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Uridina Trifosfato/síntesis química , Uridina Trifosfato/metabolismoRESUMEN
A series of 5-substituted 2'-deoxyuridine monophosphate analogues has been synthesized and evaluated as potential inhibitors of mycobacterial ThyX, a novel flavin-dependent thymidylate synthase in Mycobacterium tuberculosis. A systematic SAR study led to the identification of compound 5a, displaying an IC(50) value against mycobacterial ThyX of 0.91 µM. This derivative lacks activity against the classical mycobacterial thymidylate synthase ThyA (IC(50) > 50 µM) and represents the first example of a selective mycobacterial FDTS inhibitor.
Asunto(s)
Antituberculosos/síntesis química , Nucleótidos de Desoxiuracil/síntesis química , Flavinas/metabolismo , Mycobacterium tuberculosis/enzimología , Timidilato Sintasa/antagonistas & inhibidores , Antituberculosos/química , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/farmacología , Relación Estructura-Actividad , Timidilato Sintasa/químicaRESUMEN
We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Análisis de Secuencia de ADN/métodos , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/metabolismo , Procesos Fotoquímicos , Reacción en Cadena de la Polimerasa , Moldes Genéticos , Rayos UltravioletaRESUMEN
PURPOSE: A long-lasting, slow-release, crystalline antiviral drug delivery system was initially reported using ganciclovir and cyclic cidofovir as the prototype compounds. The present study was undertaken to investigate the feasibility of applying this system to antiproliferative small molecules. METHODS: The crystalline lipid prodrugs of hexadecyloxypropyl-arabinofuranosylguanine 5'-monophosphate (HDP-P-AraG), hexadecyloxypropyl 5-fluoro-2'-deoxyuridine cyclic 3',5'-monophosphate (HDP-cP-5-F-2dUrd), and hexadecyloxypropyl 5-fluoro-2'-deoxyuridine 5'-monophosphate (HDP-P-5-F-2dUrd) were synthesized from their parent compounds arabinofuranosylguanine (AraG) and 5-fluoro-2'-deoxyuridine (5-F-2dUrd). All three compounds were tested at escalating doses in rabbit eyes. Only one eye of each animal was injected with test compound, and the fellow eye was injected with 5% dextrose as the control. The injected eyes were monitored by slit lamp, a handheld tonometer, indirect ophthalmoscopy, electroretinography (ERG), and histology. The selected doses were used for efficacy study with the rat CNV model or the rabbit PVR model. RESULTS: The highest nontoxic dose for HDP-P-AraG was 75 microg/eye, and was 70 and 210 microg/eye for HDP-P-5-F-2dUrd and HDP-cP-5-F-2dUrd, respectively. All compounds demonstrated a localized depot of crystalline aggregate in the vitreous with a clear view of vitreous and retina elsewhere. The drug depot of HDP-P-AraG was visible for 4 to 5 weeks; HDP-P-5-F-2dUrd, 8 to 10 weeks; and HDP-cP-5-F-2dUrd longer than 14 weeks. The treatment study showed HDP-P-AraG led to 33% reduction in CNV in the rat (P = 0.015), and HDP-cP-5-F-2dUrd provided 100% prevention of trauma-induced PVR in the rabbit (P = 0.046). The pretreatment study demonstrated a significant protection against intraocular proliferation compared with the 5-FU in a parallel study (P = 0.014). CONCLUSIONS: The intravitreous injectable lipid prodrugs of AraG and 5-fluoro-2'-deoxyuridine could be long-lasting, slow-release, antiproliferative compounds to treat unwanted intraocular proliferation.
Asunto(s)
Arabinonucleotidos/administración & dosificación , Nucleótidos de Desoxiuracil/administración & dosificación , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Profármacos/administración & dosificación , Neovascularización Retiniana/prevención & control , Vitreorretinopatía Proliferativa/prevención & control , Animales , Arabinonucleotidos/síntesis química , Arabinonucleotidos/toxicidad , Cristalización , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/toxicidad , Relación Dosis-Respuesta a Droga , Angiografía con Fluoresceína , Inyecciones , Oftalmoscopía , Profármacos/química , Profármacos/toxicidad , Conejos , Ratas , Ratas Endogámicas BN , Retina/efectos de los fármacos , Retina/patología , Neovascularización Retiniana/patología , Tonometría Ocular , Vitreorretinopatía Proliferativa/patología , Cuerpo VítreoRESUMEN
Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Pirenos/química , ADN/biosíntesis , ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/metabolismo , Colorantes Fluorescentes/químicaRESUMEN
We describe the synthesis of 2'-deoxyuridine-5'-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep Vent(R) exo(-) from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2'-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/metabolismo , Polimerasa Taq/metabolismo , Acetileno/química , Aminas/química , Secuencia de Aminoácidos , Secuencia de Bases , Biocatálisis , Dominio Catalítico , Nucleótidos de Desoxiuracil/química , Idoxuridina/química , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , FosforilaciónRESUMEN
UV irradiation of 5-azido-2'-deoxyuridine in water provides up to seven products. All likely result from a pivotal azirene, formed by the intramolecular rearrangement of the initially formed nitrene, that undergoes nucleophilic addition at its C5 position. This study strongly suggests that only nucleophilic amino acid residues in close proximity are cross-linkable in photolabeling experiments by using the 5-azidouracil photophore.
Asunto(s)
Azidas/síntesis química , Química Orgánica/métodos , Nucleótidos de Desoxiuracil/síntesis química , Desoxiuridina/química , Desoxiuridina/síntesis química , Fotoquímica/métodos , Azidas/química , Sitios de Unión , Carbono/química , Reactivos de Enlaces Cruzados/química , ADN/química , Nucleótidos de Desoxiuracil/química , Diseño de Fármacos , Concentración de Iones de Hidrógeno , Luz , Modelos Químicos , Estructura Molecular , Rayos Ultravioleta , Agua/químicaRESUMEN
High levels of androgen receptor (AR) are often indicative of recurrent, advanced, or metastatic cancers. These conditions are also characterized by a high proliferative fraction. 5-Radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine 8 and 5-radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridin-5'-yl monophosphate 13 target AR. They are also degraded intracellularly to 5-radioiodo-2'-deoxyuridine 1 and its monophosphate 20, respectively, which can participate in the DNA synthesis. Both drugs were prepared at the no-carrier-added level. Precursors and methods are readily adaptable to radiolabeling with various radiohalides suitable for SPECT and PET imaging, as well as endoradiotherapy. In vitro and in vivo studies confirm the AR-dependent interactions. Both drugs bind to sex hormone binding globulin. This binding significantly improves their stability in serum. Biodistribution and imaging studies show preferential uptake and retention of 8 and 13 in ip xenografts of human ovarian adenocarcinoma cells NIH:OVCAR-3, which overexpress AR. When these drugs are administered at therapeutic dose levels, a significant tumor growth arrest is observed.
Asunto(s)
Androstanoles/síntesis química , Nucleótidos de Desoxiuracil/síntesis química , Desoxiuridina/análogos & derivados , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Radiofármacos/síntesis química , Receptores Androgénicos/metabolismo , Androstanoles/química , Androstanoles/farmacocinética , Animales , Proteínas Sanguíneas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/farmacocinética , Desoxiuridina/síntesis química , Desoxiuridina/química , Desoxiuridina/farmacocinética , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Radioisótopos de Yodo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Hormono-Dependientes/metabolismo , Unión Proteica , Conejos , Radiofármacos/química , Radiofármacos/farmacocinética , Suero , Globulina de Unión a Hormona Sexual/química , Trasplante HeterólogoRESUMEN
We explore the potential of the Diels-Alder cycloaddition for the functional tagging of DNA strands. A deoxyuridine triphosphate derivative carrying a diene at position 5 of the pyrimidine base was synthesized using a two-step procedure. The derivative was efficiently accepted as substrate in enzymatic polymerization assays. Diene carrying strands underwent successful cycloaddition with maleimide-terminated fluorescence dyes and a polymeric reagent. Furthermore, a nucleotide carrying a peptide via a Diels-Alder cyclohexene linkage was prepared and sequence-specifically incorporated into DNA. The Diels-Alder reaction presents a number of positive attributes such as good chemoselectivity, water compatibility, high-yield under mild conditions and no additional reagents apart from a diene and a dienophile. Furthermore, suitable dienophiles are commercially available in the form of maleimide-derivatives of fluorescent dyes and bioaffinity tags. Based on these advantages, diene- and cyclohexene-based nucleotide triphosphates are expected to find wider use in the area of nucleic acid chemistry.
Asunto(s)
ADN/química , Nucleótidos de Desoxiuracil/química , Colorantes Fluorescentes/química , ADN/metabolismo , Nucleótidos de Desoxiuracil/síntesis química , Nucleótidos de Desoxiuracil/metabolismo , Maleimidas/química , Péptidos/química , Reacción en Cadena de la PolimerasaRESUMEN
Electron paramagnetic resonance (EPR) spectroscopy was applied extensively in studies of nucleic acid structures and dynamics. Two modified 2'-deoxyuridine triphosphates were synthesized bearing a spin label linked to the base by a rigid linker to ensure a tight coupling of spin label dynamics. The incorporation of both spin-labeled nucleotides could be shown in primer extension reactions in presence of DNA polymerases from eukaryotic, prokaryotic, and archaic origin. In further experiments we were able to introduce multiple spin labels during primer extension reactions.
Asunto(s)
Óxidos N-Cíclicos/química , ADN/biosíntesis , Nucleótidos de Desoxiuracil/síntesis química , Marcadores de Spin , Óxidos N-Cíclicos/síntesis química , Óxidos N-Cíclicos/metabolismo , ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Enzimas/metabolismoRESUMEN
Two 5 modified 2'-deoxyuridin triphosphates and a 7 modified 2'-deoxy-7-deazaadenosine were synthesized carrying a terminal azide linked to the base. For probing the sterical influence on incorporation and Staudinger ligation different sized flexible linkers were chosen. All three nucleotides can completely replace their natural counterparts in primer extension as well as polymerase chain reactions (PCR) using Pwo DNA polymerase. For azide labeled primer extension products subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, e.g. for the conjugation of biotin as an affinity tag.