rkDNA-graphene oxide as a simple probe for the rapid detection of miRNA21.

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate-containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA-GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.

Synthesis of 2'-Deoxyuridine Modified with a 3,5-Difluoro-4-Methoxybenzylidene Imidazolinone Derivative for Incorporation into Oligonucleotide Probes for Detection of HER2 Breast Cancer Marker.

Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-p-Methoxybenzylidene imidazolinone-1-hexene)-2'-deoxyuridine (dUMBI ) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dUDFMBI as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2'-deoxyuridine.

Synthesis of water-soluble prodrugs of 5-modified 2'-deoxyuridines and their antibacterial activity.

Recently we have synthesized a set of pyrimidine nucleoside derivatives bearing extended alkyltriazolylmethyl substituents at position 5 of the nucleic base, and showed their significant activity against Mycobacterium tuberculosis virulent laboratory strain H37Rv as well as drug-resistant MS-115 strain. The presence of a lengthy hydrophobic substituent leads to the reduction of nucleoside water solubility making their antibacterial activity troublesome to study. A series of water-soluble forms of 5-modified 2'-deoxyuridines 4a-c and 8a-c were synthesized. They appeared at least two orders more soluble compared with the parent compounds 1a and 1b. Their half-hydrolysis time was 5-12 h, which can be considered optimal for prodrugs used in clinics. Obtained compounds showed moderate activity (MIC 48-95 µg·ml-1) against some Gram-positive bacteria including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis and were low cytotoxic for human cell lines (CD50 >> 100 µg·ml-1).

Synthesis and biological assay of new 2'-deoxyuridine dimers containing a 1,2,3-triazole linker. Part I.

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2'-deoxyuridine, thymidine and 5-fluoro-2'-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide-alkyne 1,3-dipolar cycloaddition (click reaction), starting from propargylated 2'-deoxyuridine and 5'-azido-nucleoside derivatives. Their cytotoxic activity was tested in five human cancer cell lines: cervical (HeLa), high grade gliomas (U-118 MG, U-87 MG, T98G), liver (HepG2), and normal human fibroblast cell line (MRC-5) using the sulforhodamine B (SRB) assay. The experiment showed that the obtained dimers with a 1,2,3-triazole moiety were very stable compounds, also in the physiological-like media, and had no anticancer activity.

Fluorogenic "photoclick" labelling of DNA using a Cy3 dye.

Two 2'-deoxyuridines as new building blocks for automated DNA synthesis carry a small aryltetrazole as "photoclickable" group at their 5-positions. The postsynthetic "photoclick" labeling of such presynthesized DNA using a maleimide-modified Cy3 dye shows an up to 17-fold fluorogenicity due to an energy transfer between the pyrazoline moiety and the Cy3 fluorophore in the DNA products. This concept is also applicable to other maleimide-modified dyes.

Design of "Click" Fluorescent Labeled 2'-deoxyuridines via C5-[4-(2-Propynyl(methyl)amino)]phenyl Acetylene as a Universal Linker: Synthesis, Photophysical Properties, and Interaction with BSA.

Microenvironment-sensitive fluorescent nucleosides present attractive advantages over single-emitting dyes for sensing inter-biomolecular interactions involving DNA. Herein, we report the rational design and synthesis of triazolyl push-pull fluorophore-labeled uridines via the intermediacy of C5-[4-(2-propynyl(methyl)amino)]phenyl acetylene as a universal linker. The synthesized nucleosides showed interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) features. A few of them also exhibited dual-emitting characteristics evidencing our designing concept. The HOMO-LUMO distribution showed that the emissive states of these nucleosides were characterized with more significant electron redistribution between the C5-[4-(2-propynyl(methyl)amino)]phenyl triazolyl donor moiety and the aromatic chromophores linked to it, leading to modulated emission property. The solvent polarity sensitivity of these nucleosides was also tested. The synthesized triazolyl benzonitrile (10C), naphthyl (10E), and pyrenyl (10G) nucleosides were found to exhibit interesting ICT and dual (LE/ICT) emission properties. The dual-emitting pyrenyl nucleoside maintained a good ratiometric response in the BSA protein microenvironment, enabling the switch-on ratiometric sensing of BSA as the only protein biomolecule. Thus, it is expected that the new fluorescent nucleoside analogues would be useful in designing DNA probes for nucleic acid analysis or studying DNA-protein interactions via a drastic change in fluorescence response due to a change in micropolarity.

3'-O-Substituted 5-(perylen-3-ylethynyl)-2'-deoxyuridines as tick-borne encephalitis virus reproduction inhibitors.

A series of analogues of potent antiviral perylene nucleoside dUY11 with methylthiomethyl (MTM), azidomethyl (AZM) and HO-C1-4-alkyl-1,2,3-triazol-1,4-diyl groups at 3'-O-position as well as the two products of copper-free alkyne-azide cycloaddition of the AZM derivative were prepared and evaluated against tick-borne encephalitis virus (TBEV). Four compounds (4, 6, 8a, 8b) showed EC50 ≤ 10 nM, thus appearing the most potent TBEV inhibitors to date. Moreover, these nucleosides have higher lipophilicity (clogP) and increased solubility in aq. DMSO vs. parent compound dUY11.

Synthesis and biophysical analysis of modified thymine-containing DNA oligonucleotides.

We report the synthesis of a 5-formyl-2'-deoxyuridine (5fU) phosphoramidite and the preparation of oligonucleotides comprising all known, naturally observed eukaryotic thymidine modifications. Biophysical characterization of the synthetic oligonucleotides indicates that 5fU, but not the other T-derivatives, can alter DNA structures.

Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides.

Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.

Development of ethynyl-2'-deoxyuridine chemical probes for cell proliferation.

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2'-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

Synthesis and anticancer activity of some 5-fluoro-2'-deoxyuridine phosphoramidates.

Two series of novel 4-chlorophenyl N-alkyl phosphoramidates of 3'-O-(t-butoxycarbonyl)-5-fluoro-2'-deoxyuridine (3'-BOC-FdU) (9a-9j) and 5-fluoro-2'-deoxyuridine (FdU) (10a-10j) were synthesized by means of phosphorylation of 3'-BOC-FdU (4) with 4-chlorophenyl phosphoroditriazolide (7), followed by a reaction with the appropriate amine. Phosphoramidates 9a-9j were converted to the corresponding 10a-10j by removal of the 3'-t-butoxycarbonyl protecting group (BOC) under acidic conditions. The synthesized phosphoramidates 9a-9j and 10a-10j were evaluated for their cytotoxic activity in five human cancer cell lines: cervical (HeLa), nasopharyngeal (KB), breast (MCF-7), liver (HepG2), osteosarcoma (143B) and normal human dermal fibroblast cell line (HDF) using the sulforhodamine B (SRB) assay. Two phosphoramidates 9b and 9j with the N-ethyl and N-(methoxy-(S)-alaninyl) substituents, respectively, displayed remarkable activity in all the investigated cancer cells, and the activity was considerably higher than that of the parent nucleoside 4 and FdU. Among phosphoramidates 10a-10j compound 10c with the N-(2,2,2-trifluoroethyl) substituent showed the highest activity. Phosphoramidate 10c was more active than the FdU in all the cancer cell lines tested.

New 3'-O-aromatic acyl-5-fluoro-2'-deoxyuridine derivatives as potential anticancer agents.

New aromatic and aliphatic 3'-O-acyl-5-fluoro-2'-deoxyuridine derivatives were synthesized and evaluated as candidates for prodrugs against various cancer cell lines. As the most promising candidate for antimalignant therapeutics was found a dual-acting acyl derivative 7h, which apparently released not only the known anticancer nucleoside, 5-fluoro-2'-deoxyuridine (FdU), but also an additional active metabolite, acetylsalicylic acid, reinforcing thus therapeutic effect of FdU. Promising therapeutic indices showed also some aromatic dicarboxylic acids derivatives decorated with FdU esters (11 and 12).

Synthesis and evaluation of C-5 modified 2'-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase.

A series of 5'-monophosphates of 5-substituted 2'-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2'-deoxyuridine 5'-monophosphate 3b, displaying an IC50 value against ThyX of 8.32 µM. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX.

Double-headed nucleotides introducing thymine nucleobases in the major groove of nucleic acid duplexes.

Four different double-headed nucleosides each combining two thymine nucleobases with different linkers were synthesised. The 5-position of 2'-deoxyuridine was connected to the N1-position of a thymine through either m- or p-disubstituted phenyl or phenylacetylene linkers by the use of Suzuki or Sonogashira couplings. When introduced into oligonucleotides, the thermal stability of dsDNA and DNA : RNA duplexes were determined and structural information was obtained from CD- and fluorescence spectroscopy. Also the recognition of abasic sites was studied. In general, the more stable duplexes were obtained with m- rather than p-substitution and with phenylacetylene rather than phenyl linkers.

5-Thiocyanato-2'-deoxyuridine as a possible radiosensitizer: electron-induced formation of uracil-C5-thiyl radical and its dimerization.

In this work, we have synthesized 5-thiocyanato-2'-deoxyuridine (SCNdU) along with the C6-deuterated nucleobase 5-thiocyanatouracil (6-D-SCNU) and studied their reactions with radiation-produced electrons. ESR spectra in γ-irradiated nitrogen-saturated frozen homogeneous solutions (7.5 M LiCl in H2O or D2O) of these compounds show that electron-induced S-CN bond cleavage occurs to form a thiyl radical (dU-5-S˙ or 6-D-U-5-S˙) and CN(-)via the initial π-anion radical (SCNdU˙(-)) intermediate in which the excess electron is on the uracil base. HPLC and LC-MS/MS studies of γ-irradiated N2-saturated aqueous solutions of SCNdU in the presence of sodium formate as a OH-radical scavenger at ambient temperature show the formation of the dU-5S-5S-dU dimer in preference to dU by about 10 to 1 ratio. This shows that both possible routes of electron-induced bond cleavage (dUC5-SCN and S-CN) in SCNdU˙(-) and dU-5-S˙ formation are preferred for the production of the σ-type uracilyl radical (dU˙) by 10 fold. DFT/M06-2x/6-31++G(d,p) calculations employing the polarizable continuum model (PCM) for aqueous solutions show that dU-5-S˙ and CN(-) formation was thermodynamically favored by over 15 kcal mol(-1) (ΔG) compared to dU˙ and SCN(-) production. The activation barriers for C5-S and S-CN bond cleavage in SCNdU˙(-) amount to 8.7 and 4.0 kcal mol(-1), respectively, favoring dU-5-S˙ and CN(-) formation. These results support the experimental observation of S-CN bond cleavage by electron addition to SCNdU that results in the formation of dU-5-S˙ and the subsequent dU-5S-5S-dU dimer. This establishes SCNdU as a potential radiosensitizer that could cause intra- and inter-strand crosslinking as well as DNA-protein crosslinking via S-S dimer formation.

Labeling of Cellular DNA with a Cyclosal Phosphotriester Pronucleotide Analog of 5-ethynyl-2'-deoxyuridine.

DNA synthesis is a fundamental biological process central to all proliferating cells, and the design of small molecule probes that allow detection of this DNA is important for many applications. 5-Ethynyl-2'-deoxyuridine, known as EdU, has become a workhorse for metabolic labeling of DNA in mammalian cells, followed by bioconjugation to a small molecule fluorescent azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry, to allow detection. In this study, we demonstrate that a cyclosal phosphotriester pronucleotide analog of EdU is suitable for metabolic incorporation into DNA of proliferating cells and subsequent labeling by CuAAC. This analog has two advantages over EdU; first, by delivering EdU with a preinstalled 5'-monophosphate moiety, it bypasses the need for thymidine kinase processing, and second, the increased lipophilicity compared to EdU may enable passive diffusion across the cell membrane and may circumvent the reliance on nucleoside active transport mechanisms for cellular uptake. These advantages pave the way for the development of additional novel pronucleotides to widen experimental opportunities for future bioconjugation applications involving cellular DNA.

C5-azobenzene-substituted 2'-deoxyuridine-containing oligodeoxynucleotides for photo-switching hybridization.

A new photoisomeric nucleoside dUAz bearing an azobenzene group at the C5-position of 2'-deoxyuridine was designed and synthesized. Photoisomerization of dUAz in oligodeoxynucleotides can be achieved rapidly and selectively with 365 nm (forward) and 450 nm (backward) irradiation. Thermal denaturation experiments revealed that dUAz stabilized the duplex in the cis-form and destabilized it in the trans-form with mismatch discrimination ability comparable to thymidine. These results indicate that dUAz could be a powerful material for reversibly manipulating nucleic acid hybridization with spatiotemporal control.

Synthesis of 6-alkyluridines from 6-cyanouridine via zinc(II) chloride-catalyzed nucleophilic substitution with alkyl Grignard reagents.

6-Cyanouracil derivatives underwent a direct nucleophilic substitution reaction with alkyl Grignard reagents in the presence of zinc(II) chloride as a catalyst to form the corresponding 6-alkyluracils. This methodology is applicable to sugar-protected 6-cyanouridine and 6-cyano-2'-deoxyuridine without the protection at the N(3)-imide and provides a facile and general access to versatile 6-alkyluracil and 6-alkyluridine derivatives.

Effective synthesis of 3'-deoxy-3'-azido nucleosides for antiviral and antisense ribonucleic guanidine (RNG) applications.

Two synthetic routes to 3'-deoxy-3'-azido nucleosides are described, one toward the synthesis of 3'-deoxy-3'-azidouridine and a second toward 3'-deoxy-3'-azidocytidine. The target compounds may serve as precursors to provide building blocks for use in automated synthesis of guanidine-linked RNA analogs (RNG) or oligonucleotide N3'→P5' phosphoramidates. Moreover, the synthetic approaches are adaptable to the general synthesis of 3'-substituted 3'-deoxynucleosides for development of new antiviral drugs.

Synthesis and spectroscopic properties of fluorescent 5-benzimidazolyl-2'-deoxyuridines 5-fdU probes obtained from o-phenylenediamine derivatives.

Fluorescent nucleosides (dU(bmz)) with desirable fluorescence quantum yield (Φ) are synthesized from almost non-fluorescent 5-fdU and o-phenylenediamine derivatives. The fluorescence of these nucleosides is quite sensitive to pH and organic solvents. 4-Methoxybenzene-1,2-diamine was used for the detection of 5-fdU among natural nucleosides.