RESUMEN
Coronaviruses are a group of enveloped viruses with non-segmented, single-stranded, and positive-sense RNA genomes. It belongs to the 'Coronaviridae family', responsible for various diseases, including the common cold, SARS, and MERS. The COVID-19 pandemic, which began in March 2020, has affected 209 countries, infected over a million people, and claimed over 50,000 lives. Significant efforts have been made by repurposing several approved drugs including antiviral, to combat the COVID-19 pandemic. Molnupiravir is found to be the first orally acting efficacious drug to treat COVID-19 cases. It was approved for medical use in the UK in November 2021 and other countries, including USFDA, which granted approval an emergency use authorization (EUA) for treating adults with mild to moderate COVID-19 patients. Considering the importance of molnupiravir, the present review deals with its various synthetic strategies, pharmacokinetics, bio-efficacy, toxicity, and safety profiles. The comprehensive information along with critical analysis will be very handy for a wide range of audience including medicinal chemists in the arena of antiviral drug discovery especially anti-viral drugs against any variant of COVID-19.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , COVID-19 , Citidina , Hidroxilaminas , SARS-CoV-2 , Humanos , Antivirales/farmacología , Antivirales/química , Antivirales/uso terapéutico , Antivirales/síntesis química , Hidroxilaminas/uso terapéutico , Hidroxilaminas/química , Hidroxilaminas/farmacología , COVID-19/virología , SARS-CoV-2/efectos de los fármacos , Citidina/análogos & derivados , Citidina/uso terapéutico , Citidina/farmacología , Citidina/química , Citidina/síntesis química , Uridina/farmacología , Uridina/análogos & derivados , Uridina/síntesis química , Uridina/química , Uridina/uso terapéutico , Pandemias , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológicoAsunto(s)
Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Desarrollo de Medicamentos/tendencias , Pandemias , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/uso terapéutico , Alanina/administración & dosificación , Alanina/análogos & derivados , Alanina/uso terapéutico , Animales , Antivirales/farmacología , Aves/virología , COVID-19/virología , Ensayos Clínicos como Asunto , Coronavirus/clasificación , Coronavirus/efectos de los fármacos , Infecciones por Coronavirus/epidemiología , Citidina/administración & dosificación , Citidina/análogos & derivados , Citidina/síntesis química , Citidina/uso terapéutico , Industria Farmacéutica/tendencias , Europa (Continente) , Humanos , Hidroxilaminas/administración & dosificación , Hidroxilaminas/síntesis química , Hidroxilaminas/uso terapéutico , Indoles/administración & dosificación , Indoles/uso terapéutico , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Leucina/administración & dosificación , Leucina/uso terapéutico , Orthomyxoviridae/efectos de los fármacos , Pirrolidinonas/administración & dosificación , Pirrolidinonas/uso terapéutico , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Síndrome Respiratorio Agudo Grave/epidemiología , Reserva Estratégica , Estados UnidosRESUMEN
The structure of life's first genetic polymer is a question of intense ongoing debate. The "RNA world theory" suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2',3'-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.
Asunto(s)
Citidina/síntesis química , Ácidos Nucleicos/síntesis química , Tetrosas/síntesis química , Conformación de Carbohidratos , Citidina/química , Ácidos Nucleicos/química , Procesos Fotoquímicos , Tetrosas/químicaRESUMEN
Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4'-Azido-thymidine/4'-Azido-2'-deoxy-5-methylcytidine derivatives (6, 10-15) were synthesized, and their anti-HBV activities evaluated. Compounds 10-15 were synthesized via an SNAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11-15 showed no antiviral activity. However, 4'-Azido thymidine (6) and 4'-Azido-2'-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC50 = 0.63 and 5.99 µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100 µM.
Asunto(s)
Antivirales/farmacología , Citidina/análogos & derivados , Zidovudina/análogos & derivados , Antivirales/síntesis química , Antivirales/química , Citidina/síntesis química , Citidina/química , Citidina/farmacología , Células Hep G2 , Virus de la Hepatitis B/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Estereoisomerismo , Zidovudina/síntesis química , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
A 109-membered library of 5'-substituted cytidine analogs was synthesized, via funding through the NIH Roadmap Initiative and the Pilot Scale Library (PSL) Program. Reaction core compounds contained -NH2 (2) and -COOH (44 and 93) groups that were coupled to a diversity of reactants in a parallel, solution phase format to produce the target library. The assorted reactants included -NH2, -CHO, -SO2Cl, and -COOH functional groups, and condensation with the intermediate core materials 2 and 44 followed by acidic hydrolysis produced 3-91 in good yields and high purity. Linkage of the amino terminus of d-phenylalanine methyl ester to the free 5'-COOH of 44 and NaOH treatment led to core library -COOH precursor 93. In a libraries from libraries approach, compound 93 served as the vital building block for our unique library of dipeptidyl cytidine analogs 94-114 through amide coupling of the -COOH group with numerous commercial amines followed by acidic deprotection. Initial screening of the complete final library through the MLPCN program revealed a modest number of hits over diverse biological processes. These hits might be considered as starting points for hit-to-lead optimization and development studies.
Asunto(s)
Citidina/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Aminas/química , Ácidos Carboxílicos/química , Citidina/análogos & derivados , Hidrólisis , Estructura Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Relación Estructura-ActividadRESUMEN
Epigenetic information is encoded in the mammalian genome in the form of cytosines methylated at the 5 position. Cytosine methylation has multiple biological effects, but our understanding of these effects has lagged because extant methods for mapping methylation sites genome-wide have severe shortcomings. For instance, the gold standard bisulfite sequencing approach suffers from the use of harsh reaction conditions resulting in DNA cleavage and incomplete conversion of unmethylated cytosine to uracil. We report here on a new photochemical method in which a DNA (cytosine-5)-methyltransferase can be used to covalently attach reactive functionalities which upon irradiation at â¼350 nm initiate photoinduced intramolecular reactions that convert modified C to T analogues. We synthesized a model compound, a cinnamyl ether-containing cytidine derivative, and demonstrated its conversion to a thymidine analogue using mild conditions and a DNA-compatible wavelength (â¼350 nm), enabled by the use of a triplet sensitizer, thioxanthone. Transfer of a cinnamyl ether or comparable reactive functionality from an AdoMet analog to cytosine followed by the use of this photoconversion method would require only small amounts of DNA and allow complete methylation profiling on both long and short read sequencing platforms.
Asunto(s)
Citidina/química , Timidina/química , Islas de CpG , Reacción de Cicloadición , Citidina/síntesis química , ADN/química , ADN/metabolismo , Metilación de ADN , Rayos Láser , Espectroscopía de Resonancia Magnética , Fotólisis , Espectrofotometría Ultravioleta , Tioxantenos/química , Xantonas/químicaRESUMEN
The possible role of DNA i-motif structures in telomere biology and in the transcriptional regulation of oncogene promoter regions is supported by several recent studies. Herein we investigate the effect of four cytidine nucleosides (and combinations thereof) on i-motif structure and stability, namely 2'-deoxycytidine (dC), 2'-deoxy-5-methyl-cytidine (5-Me-dC), 2'-deoxy-2'-fluoro-arabinocytidine (2'F-araC), and 2'-deoxy-2'-fluoro-5-methyl-arabinocytidine (5-Me-2'F-araC). The base pair 5-Me-2'F-araC:2'F-araC produced i-motifs with a pH1/2 ("pKa ") value that closely matches physiological pH (7.34±0.3). NMR analysis of the most stable telomeric sequence (HJ-2) at pHâ 7.0 indicated that the structure is stabilized by hybrid 5-Me-dC:2'F-araC hemiprotonated base pairs and therefore highlights the significance of the interplay between base and sugar modifications on the stability of i-motif structures.
Asunto(s)
Citarabina/análogos & derivados , Citarabina/química , Citidina/análogos & derivados , Citidina/química , Metilación de ADN , ADN/química , Motivos de Nucleótidos , Citarabina/síntesis química , Citidina/síntesis química , Halogenación , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Telómero/químicaRESUMEN
The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors.
Asunto(s)
ARN/química , Uridina/análogos & derivados , Emparejamiento Base , Secuencia de Bases , Citidina/síntesis química , Citidina/química , Citidina/metabolismo , Temperatura de Transición , Uracilo/química , Uracilo/metabolismo , Uridina/síntesis química , Uridina/químicaRESUMEN
Triplex formation against a target duplex DNA has the potential to become a tool for the genome research. However, there is an intrinsic restriction on the duplex DNA sequences capable of forming the triplex DNA. Recently, we demonstrated the selective formation of the stable antiparallel triplexes containing the CG inversion sites using the 2'-deoxy-1-methylpseudocytidine derivative (ΨdC), whose amino group was conjugated with the 2-aminopyridine at its 5-position as an additional hydrogen bonding unit (AP-ΨdC). The 1-N of 2-aminopyridine was supposed to be protonated to form the hydrogen bond with the guanine of the CG inversion site. In this study, to test the effect of the 3-substitution of the 2-aminopyridine unit of AP-ΨdC on the triplex stability, we synthesized the 3-halogenated 2-aminopyridine derivatives of AP-ΨdC. The pKa values 1-N of the 2-aminopyridine unit of AP-ΨdC as the monomer nucleoside were determined to be 6.3 for 3-CH3 (MeAP-ΨdC), 6.1 for 3-H (AP-ΨdC), 4.3 for 3-Cl (ClAP-ΨdC), 4.4 for 3-Br (BrAP-ΨdC), and 4.7 for 3-I (IAP-ΨdC), suggesting that all the halogenated AP-ΨdCs are not protonated under neutral conditions. Interestingly, although the recognition selectivity depends on the sequence context, the TFO having the sequence of the 3'-G-(IAP-ΨdC)-A-5' context showed the selective triplex formation with the CG inversion site. These results suggest that the protonation at the 1-N position plays an important role in the stable and selective triplex formation of AP-ΨdC derivatives in any sequences.
Asunto(s)
Citidina/análogos & derivados , ADN/química , Aminopiridinas/química , Secuencia de Bases , Citidina/síntesis química , Citidina/química , ADN/metabolismo , Halógenos/química , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Conformación de Ácido NucleicoRESUMEN
Oligonucleotides containing 2'-O-methylated 5-methylisocytidine (3) and 2'-O-propargyl-5-methylisocytidine (4) as well as the non-functionalized 5-methyl-2'-deoxyisocytidine (1b) were synthesized. MALDI-TOF mass spectra of oligonucleotides containing 1b are susceptible to a stepwise depyrimidination. In contrast, oligonucleotides incorporating 2'-O-alkylated nucleosides 3 and 4 are stable. This is supported by acid catalyzed hydrolysis experiments performed on nucleosides in solution. 2'-O-Alkylated nucleoside 3 was synthesized from 2'-O-5-dimethyluridine via tosylation, anhydro nucleoside formation and ring opening. The corresponding 4 was obtained by direct regioselective alkylation of 5-methylisocytidine (1d) with propargyl bromide under phase-transfer conditions. Both compounds were converted to phosphoramidites and employed in solid-phase oligonucleotide synthesis. Hybridization experiments resulted in duplexes with antiparallel or parallel chains. In parallel duplexes, methylation or propargylation of the 2'-hydroxyl group of isocytidine leads to destabilization while in antiparallel DNA this effect is less pronounced. 2'-O-Propargylated 4 was used to cross-link nucleosides and oligonucleotides to homodimers by a stepwise click ligation with a bifunctional azide.
Asunto(s)
Emparejamiento Base , Citidina/química , Citidina/síntesis química , ADN/química , ARN/química , Técnicas de Química Sintética , Hibridación de Ácido NucleicoRESUMEN
The Lewis acid MgCl2 allows control of the metalation regioselectivity of uracils and uridines. In the absence of the Lewis acid, metalation of uracil and uridine derivatives with TMPMgCl·LiCl occurs at the position C(5). In the presence of MgCl2, zincation using TMP2Zn·2LiCl·2MgCl2 occurs at the position C(6). This metalation method provides easy access to functionalized uracils and uridines. Using TMP2Zn·2LiCl·2MgCl2 also allows to functionalize cytidine derivatives at the position C(6).
Asunto(s)
Citidina/química , Ácidos de Lewis/química , Cloruro de Magnesio/química , Uracilo/química , Uridina/química , Técnicas Químicas Combinatorias , Citidina/síntesis química , Estructura Molecular , Compuestos Organometálicos/química , Estereoisomerismo , Uracilo/síntesis química , Uridina/síntesis química , Zinc/químicaRESUMEN
Sialylconjugates on cell surfaces are involved in many biological events such as cellular recognition, signal transduction, and immune response. It has been reported that aberrant sialylation at the nonreducing end of glycoconjugates and overexpression of sialyltransferases (STs) in cells are correlated with the malignance, invasion, and metastasis of tumors. Therefore, inhibitors of STs would provide valuable leads for the discovery of antitumor drugs. On the basis of the transition state of the enzyme-catalyzed sialylation reaction, we proposed that the cyclopentane skeleton in its two puckered conformations might mimic the planar structure of the donor (CMP-Neu5Ac) in the transition state. A series of cyclopentane-containing compounds were designed and synthesized by coupling different cyclopentane α-hydroxyphosphonates with cytidine phosphoramidite. Their inhibitory activities against recombinant human ST6Gal-I were assayed, and a potent inhibitor 48l with a Ki of 0.028 ± 0.006 µM was identified. The results show that the cyclopentanoid-type compounds could become a new type of sialyltransferase inhibitors as biological probes or drug leads.
Asunto(s)
Ciclopentanos/síntesis química , Ciclopentanos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Sialiltransferasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Citidina/análogos & derivados , Citidina/síntesis química , Humanos , Cinética , Conformación Molecular , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Chemically-modified derivatives of cytidine, bearing a 5-(N-substituted-carboxamide) functional group, are new reagents for use in aptamer discovery via the SELEX process (Systematic Evolution of Ligands by EXponential enrichment). Herein, we disclose a practical synthesis of 5-(N-benzylcarboxamide)-2'-deoxycytidine, and the corresponding 5-(N-1-naphthylmethylcarboxamide)- and 5-(N-3-phenylpropylcarboxamide)-2'-deoxycytidine analogs, as both the suitably-protected 3'-O-cyanoethylphosphoramidite reagents (CEP; gram scale) and the 5'-O-triphosphate reagents (TPP; milligram-scale). The key step in the syntheses is a mild, palladium(0)-catalyzed carboxyamidation of an unprotected 5-iodo-cytidine. Use of the CEP reagents for solid-phase oligonucleotide synthesis was demonstrated and incorporation of the TPP reagents by KOD polymerase in a primer extension assay confirmed the utility of these reagents for SELEX. Finally, the carboxyamidation reaction was also used to prepare the nuclease-resistant sugar-variants: 5-(N-benzylcarboxamide)-2'-O-methyl-cytidine and 5-(N-3-phenylpropylcarboxamide)-2'-deoxy-2'-fluoro-cytidine.
Asunto(s)
Citidina/síntesis química , Oligonucleótidos/síntesis química , Citidina/análogos & derivados , Citidina/química , Oligonucleótidos/química , Técnicas de Síntesis en Fase SólidaRESUMEN
Novel 4 'α-trifluoromethyl-2 'ß-methyl carbocyclic nucleoside analogs have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell cultures. Construction of cyclopentene intermediate 10a was achieved via sequential Johnson-Claisen orthoester rearrangement and ring-closing metathesis starting from the α-trifluoromethyl-α,ß-unsaturated ester 5. Stereoselective dihydroxylation and desilylation yielded the target carbodine analogs. The synthesized nucleoside analogs mentioned above (18 and 19) were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides showed neither significant antiviral activity nor toxicity up to 50 µM.
Asunto(s)
Antivirales/síntesis química , Citidina/análogos & derivados , Hepatitis C/tratamiento farmacológico , Línea Celular Tumoral , Ciclopentanos/química , Citidina/síntesis química , Ésteres , Humanos , Estructura Molecular , Nucleósidos/química , ARN Viral/química , Proteínas no Estructurales Virales/química , Replicación ViralRESUMEN
Fluorescent gold/silver nanoclusters templated by DNA or oligonucleotides have been widely reported since DNA or oligonucleotides could be designed to position a few metal ions at close proximity prior to their reduction, but nucleoside-templated synthesis is more challenging. In this work, a novel type of strategy taking cytidine (C) as template to rapid synthesis of fluorescent, water-soluble gold and silver nanoclusters (C-AuAg NCs) has been developed. The as-prepared C-AuAg NCs have been characterized by UV-vis absorption spectroscopy, fluorescence, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), and inductively coupled plasma mass spectroscopy (ICP-MS). The characterizations demonstrate that C-AuAg NCs with a diameter of 1.50 ± 0.31 nm, a quantum yield â¼9%, and an average lifetime â¼6.07 µs possess prominent fluorescence properties, good dispersibility, and easy water solubility, indicating the promising application in bioanalysis and biomedical diagnosis. Furthermore, this strategy by rapid producing of highly fluorescent nanoclusters could be explored for the possible recognition of some disease-related changes in blood serum. This raises the possibility of their promising application in bioanalysis and biomedical diagnosis.
Asunto(s)
Aleaciones/química , Citidina/síntesis química , Fluorescencia , Oro/química , Nanoestructuras/química , Plata/química , Citidina/química , Tamaño de la Partícula , Solubilidad , Propiedades de Superficie , Agua/químicaRESUMEN
2'-Deoxyzebularine and its α-anomer have been efficiently synthesized with relatively high stereoselectivity by a modified procedure of the silyl method of the N-glycosidic bond formation. An SnCl4-catalyzed condensation of silylated pyrimidin-2-one with 1-α-chloro-3,5-di-O-p-toluoyl-2-deoxy-d-ribofuranose under kinetic control condition (-33°C, 1,2-dichloroethane) led to the mixture of ß- and α-anomeric nucleosides in 3:1 ratio. Analogous condensation at +35°C (thermodynamic control conditions) provided mainly p-toluoyl protected α-2'-deoxyzebularine (α:ß=4:1), easily separated by crystallization from the anomeric mixture. The structures of both 2'-deoxyzebularine anomers were confirmed by X-ray analysis of the crystals and conformational studies in solution performed using an NMR method.
Asunto(s)
Técnicas de Química Sintética , Citidina/análogos & derivados , Conformación de Carbohidratos , Cristalografía por Rayos X , Citidina/síntesis química , Citidina/química , Glicosilación , Modelos Moleculares , Soluciones , Estereoisomerismo , Especificidad por SustratoRESUMEN
The development of novel nucleoside analogues for the formation of triplex DNA containing pyrimidine-purine inversion sites has been a challenging field. In this paper, we describe the design and synthesis of non-natural nucleoside analogues, N-substituted-2'-deoxy-5-methylisocytidine derivatives, and their evaluation for triplex formation. It has been shown that N-(guanidinoethyl)-2'-deoxy-5-methylisocytidine exhibits selective recognition of a CG interrupting site and potentiates the formation of anti-parallel triplexes.
Asunto(s)
Citidina/metabolismo , ADN/química , ADN/metabolismo , Desoxicitidina/análogos & derivados , Guanidinas/metabolismo , Secuencia de Bases , Citidina/síntesis química , Desoxicitidina/síntesis química , Desoxicitidina/metabolismo , Guanidinas/síntesis química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
A series of 4'-[1,2,3]triazole-2'-deoxy-2'-fluoro-ß-d-arabinofuranosylcytosines (9-17) were prepared by Cu(I)-mediated [3 + 2] cycloaddition reactions (CuAAC) of 1-(4'-azido-2'-deoxy-2'-fluoro-ß-d-arabinofuranosyl)cytosine (1) with appropriate alkynes in good yields. Their structures were fully established by (1)H NMR, (13)C NMR, HRMS, and elemental analysis. Most of these nucleoside analogs exhibited potent anti-HIV-1 activity with no cytotoxicity observed at the highest tested concentration up to 25 µM. Among them, compounds 9, 10 and 13 exhibited extremely potent antiviral activity, thus had a great potential for further development as novel nucleoside reverse transcriptase inhibitors (NRTIs) for the treatment of HIV-1 infection. Besides, the anti-HBV activity of compounds 10, 11 and 17 had been investigated.
Asunto(s)
Antivirales/farmacología , Citidina/farmacología , VIH-1/efectos de los fármacos , Triazoles/farmacología , Antivirales/síntesis química , Antivirales/química , Supervivencia Celular/efectos de los fármacos , Citidina/síntesis química , Citidina/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/metabolismo , Humanos , Modelos Químicos , Estructura Molecular , Triazoles/síntesis química , Triazoles/químicaRESUMEN
Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimental technique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modified nucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfully synthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based on the biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Analogue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via "click" chemistry with fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC was efficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-ethynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and the rate of metabolism of EC was much faster than that of EU.
Asunto(s)
Citidina/análogos & derivados , Microscopía Fluorescente , ARN/análisis , Alquinos/química , Animales , Azidas/química , Línea Celular , Química Clic , Citidina/síntesis química , Citidina/química , Colorantes Fluorescentes/química , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Intestino Grueso/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Ratones , ARN/metabolismoRESUMEN
An efficient synthetic route to biologically relevant (-)-5-fluorocarbodine 6 was developed. Direct coupling of N(6)-protected 5-fluorouracil 15 with cyclopentenyl intermediate 13, followed by formation of a macrocycle between the base and the carbocyclic sugar moiety, via ring-closing metathesis, allowed for a facial selective hydrogenation of the sugar double bond to give, exclusively, the desired 4'-ß stereoisomer.