Akihiro Ohkubo

2-(Azidomethyl)benzoyl as a new protecting group in nucleosides ☆

Abstract A new protecting group, 2-(azidomethyl)benzoyl (AZMB), which can be easily removed by treatment with MePPh 2 in dioxane–H 2 O or reduction with HCOONH 4 –Pd/C in dioxane–MeOH, was developed for protection of the hydroxy and amino functions of deoxyribonucleosides.

Controlling the Fluorescence of Benzofuran‐Modified Uracil Residues in Oligonucleotides by Triple‐Helix Formation

We developed fluorescent turn‐on probes containing a fluorescent nucleoside, 5‐(benzofuran‐2‐yl)deoxyuridine (dUBF) or 5‐(3‐methylbenzofuran‐2‐yl)deoxyuridine (dUMBF), for the detection of single‐stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dUMBF achieved superior fluorescence enhancement than that containing dUBF. NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3‐methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3‐methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex‐induced fluorescence enhancement of the oligonucleotide probe containing dUMBF.

Access to a novel near-infrared photodynamic therapy through the combined use of 5-aminolevulinic acid and lanthanide nanoparticles

BACKGROUND There have been considerable efforts to develop photodynamic therapy (PDT) for cancer, in which photoirradiation of a sensitizer delivered near cancer cells results in the conversion of oxygen into active species, causing cell destruction. Aiming at the best cancer selectivity, one PDT method employed protoporphyrin IX (PPIX), which selectively accumulated in cancer cells after oral administration of 5-aminolevulinic acid (ALA). The drawback, however, is that blue incident lights are required to excite PPIX, resulting in low tissue penetrability, and therefore limiting its application to surface cancers. METHODS To overcome the low penetrability of the incident light, we employed a light energy upconverter, lanthanide nanoparticle (LNP), which, upon irradiation with highly penetrative near-infrared (NIR) radiation, emits visible light within the Q-band region of PPIX absorbance allowing its sensitization. To discover the optimum conditions for the LNP-assisted PDT, the cytotoxicity and PPIX-sensitizability of LNPs were first studied. Then, the LNP-assisted PDT was validated using the MKN45 cell line: cells were pretreated with ALA and LNP, irradiated with a 975-nm diode laser, and subjected to MTT assay to measure cell viability. RESULTS The singlet oxygen generation on NIR-irradiation of the PPIX-LNP mixture was proved, indicating that the emission from LNP could excite the PPIX sensitizer. An intermittent NIR-irradiation for 32 min of MKN45, pretreated with LNP (1mg/mL) and ALA (2mM), caused 87% cell destruction. CONCLUSIONS The potential applicability of the NIR-irradiation PDT with ALA- and LNP-pretreated cancer cells was demonstrated.

Fluorescence Properties of Pyrimidopyrimidoindole Nucleoside dCPPI Incorporated into Oligodeoxynucleotides

A series of oligodeoxynucleotides labeled by a pyrimidopyrimidoindole deoxynucleoside (1a: dC(PPI)) and its derivatives 2a and 3a substituted with electron-donating and -withdrawing groups, respectively, were synthesized according to the phosphoramidite approach. The photophysical properties and quenching efficiencies of oligonucleotides incorporating dC(PPI) derivatives were studied in detail. The thermal denaturation experiments and molecular dynamics simulation of DNA duplexes incorporating dC(PPI) suggested that a modified base of dC(PPI) could form base pairs with guanine and adenine in canonical Watson-Crick and reverse-wobble geometries, respectively. The fluorescence of oligonucleotides incorporating dC(PPI) derivatives increased upon binding to the counter strands, except when dC(PPI) and guanine formed a base pair. It was revealed that dGMP quenched the fluorescence of the cyano derivative 3a most effectively, whereas it affected that of the methoxy derivative 2a least effectively. The involvement of the electron transfer from guanine to the dC(PPI) derivatives in the fluorescence quenching was supported by energy considerations.

New nucleotide pairs for stable DNA triplexes stabilized by stacking interaction.

New nucleotide pairs applicable to formation of DNA triplexes were developed. We designed oligonucleotides incorporating 5-aryl deoxycytidine derivatives (dC5Ars) and cyclic deoxycytidine derivatives, dCPPP and dCPPI, having an expanded aromatic area, as the second strand. As pairing partners, two types of abasic residues (C3: propylene linker, phi: abasic base) were chosen. It was concluded that, when the 5-aryl-modified cytosine bases paired with the abasic sites in TFOs in a space-fitting manner, the stability of the resulting triplexes significantly increased. The recognition of C3 toward dC5Ars was selective because of the stacking interactions between their aromatic part and the nucleobases flanking the abasic site. These results indicate the potential utility of new nucleotide triplets for DNA triplex formation, which might expand the variety of structures and sequences and might be useful for biorelated fields such as DNA nanotechnologies.

Linear Relationship between Deformability and Thermal Stability of 2'-O-Modified RNA Hetero Duplexes

We describe the relationship between the experimentally determined melting temperatures of 2′-O-modified-RNA/RNA duplexes and their deformability estimated from molecular dynamics simulations. To clarify this relationship, we synthesized several fully modified oligoribonucleotides such as 2′-O-cyanoethyl RNAs and 2′-O-methoxyethyl RNAs and compared the actual melting temperatures of the duplexes with their calculated deformabilities. An increase of the melting temperatures by 2′-O-modifications was found to correlate strongly with an increase of the helical elastic constants in U14/A14, (CU)7/(AG)7, and (GACU)3/(AGUC)3 sequences. Linear regression analyses could be used to estimate the melting temperature with an accuracy of ±2.0 °C in our model case. Although the strong correlation was observed in the same base sequence, the linear regression functions were different from each base sequence. Our results indicated the possibility of predicting the thermal stability of 2′-O-modified duplexes at the computer-aided molecular design stage.

Synthesis and Triplex Formation of Oligonucleotides Containing 8-Thioxodeoxyadenosine

For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides containing 8-thioxodeoxyadenosine (s(8)dA) residues in place of the protonated deoxycytidines required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)dA exhibited much stronger hybridization ability than dC under neutral conditions when four s(8)dA bases were arranged in a consecutive sequence.

Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs

A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2’-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (dgC) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2’-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2’-OMegC), 2’-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2’-OMegCs), dgC and 4S-(2-guanidoethyl)-4-thiothymidine (gsT). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine (DANac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing DANac, dgC, 2’-OMe-2-thiothymidine (2’-OMesT) and 2’-OMe-8-thioxoadenosine (2’-OMesA) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.

Chemical synthesis of U1 snRNA derivatives.

U1 snRNA is an interesting biological tool for splicing correction and regulation of gene expression. However, U1 snRNA has never been chemically synthesized. In this study, the first chemical synthesis of U1snRNA and its analogues was carried out. Moreover, it was found that the binding affinity of the modified U1 snRNA with an ethylene glycol linkage to snurportin 1 (nuclear import adaptor) was as high as that of the unmodified RNA.

Prediction of the stability of modified RNA duplexes based on deformability analysis: oligoribonucleotide derivatives modified with 2′-O-cyanoethyl-5-propynyl-2-thiouridine as a promising component

We describe a method to predict the stability of a modified RNA duplex. Ten unique modified RNA duplexes showed a linear relationship between the calculated and experimentally determined duplex stabilities.