Shuhei Imoto

Formation of highly selective and efficient interstrand cross-linking to thymine without photo-irradiation

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyrimidine derivative.

Production of truncated protein by the crosslink formation of mRNA with 2'-OMe oligoribonucleotide containing 2-amino-6-vinylpurine.

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.

DNA-templated click chemistry for creation of novel DNA binding molecules.

We have developed a new methodology for producing new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by the addition of dsDNA. Furthermore, the resulting peptide-acridine conjugate showed a slightly stronger binding to dsDNA. These results indicate that the DNA-templated click chemistry is applicable for screening new binding molecules.

EFFICIENT SYNTHESIS OF 6-SUBSTITUTED PURINE DERIVATIVES USING Pd-CATALYZED CROSS-COUPLING REACTIONS WITH 2'-DEOXYGUANOSINE O6-TOSYLATE

6-Substituted purine analogs function in a variety of biological activities including antiviral pathways. A number of studies have reported on the development of the efficient synthesis of these nucleoside analogs. We previously demonstrated that oligonucleotides containing 2-amino-6-vinylpurine derivatives react with the cytosine at the target site with extreme selectivity. This was the first finding that O 6 -tosylate derivative of guanosine worked as an efficient substrate for Pd(0)-catalyzed cross-coupling reaction with vinyltributylstannane to produce 2-amino-6-vinylpurine. In order to demonstrate usefulness of the tosylate precursor, in this study we investigated transition metal catalysts and ligands in achieving the cross-coupling reaction using boronic acids or Grignard reagents as a coupling partner.

Diastereoselective Synthesis of 6″-(Z)- and 6″-(E)-Fluoro Analogues of Anti-hepatitis B Virus Agent Entecavir and Its Evaluation of the Activity and Toxicity Profile of the Diastereomers

A method for the diastereoselective synthesis of 6″-(Z)- and 6″-(E)-fluorinated analogues of the anti-HBV agent entecavir has been developed. Construction of the methylenecyclopentane skeleton of the target molecules has been accomplished by radical-mediated 5-exo-dig cyclization of the selenides 6 and 15 having the phenylsulfanylethynyl structure as a radical accepting moiety. In the radical reaction of the TBS-protected precursor 6, (Z)-anti-12 was formed as a major product. On the other hand, TIPS-protected 15 gave (E)-anti-12. The sulfur-extrusive stannylation of anti-12 furnished a mixture of geometric isomers of the respective vinylstannane, whereas benzoyl-protected 17 underwent the stannylation in the manner of retention of configuration. Following XeF2-mediated fluorination, introduction of the purine base and deoxygenation of the resulting carbocyclic guanosine gave the target (E)- and (Z)-3 after deprotection. Evaluation of the anti-HBV activity of 3 revealed that fluorine-substitution at the 6″-position of entecavir gave rise to a reduction in the cytotoxicity in HepG2 cells with retention of the antiviral activity.

Fast DNA Interstrand Cross-Linking Reaction by 6-Vinylpurine

Oligonucleotides that incorporate a reactive moiety to form an interstrand cross-link have been widely studied for their potential toward inhibiting gene expression or as basic tools for chemical biology studies. The 6-vinylpurine (2) newly designed in the current study serves well as a new purine-base moiety for increasing cross-link reactivity to target cytosine. Thus, oligonucleotides containing 6-vinylpurine exhibit a more selective and much smoother DNA cross-linking ability to cytosine than the oligonucleotide analogs derived from 2-amino-6-vinylpurine (1) previously explored.

Synthesis of native-like crosslinked duplex RNA and study of its properties

A variety of enzymes have been found to interact with double-stranded RNA (dsRNA) in order to carry out its functions. We have endeavored to prepare the covalently crosslinked native-like duplex RNA, which could be useful for biochemical studies and RNA nanotechnology. In this study, the interstrand covalently linked duplex RNA was formed by a crosslinking reaction between vinylpurine (VP) and the target cytosine or uracil in RNA. We measured melting temperatures and CD spectra to identify the properties of the VP crosslinked duplex RNA. The crosslinking formation increased the thermodynamic stability without disturbing the natural conformation of dsRNA. In addition, a competitive binding experiment with the duplex RNA binding enzyme, ADAR2, showed the crosslinked dsRNA bound the protein with nearly the same binding affinity as the natural dsRNA, confirming that it has finely preserved the natural traits of duplex RNA.

Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives

Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desired N9-substituted products in moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phosphonate derivative 8 was found to exhibit moderated activity (EC50 value of 0.29 μM), but cytotoxicity (CC50 value of 39 μM) against the host cells was also observed.

Design and synthesis of the novel cross-linking agent

Previously, we have developed a highly efficient and selective cross-linking reaction to the cytosine base at the target site of DNA using the oligodeoxynucleotide (ODN) containing 2-amino-6-vinylpurine derivative (1). Based on these results, we have designed the novel cross-linking agents, which are pyrimidine derivatives having two hydrogen bond sites and vinyl group as a reactive moiety. In this paper, we wish to report the results to investigate on the synthesis of the pyrimidine derivatives having potential as novel cross-linking agents.

Synthesis of the Peptide Nucleic Acid (PNA) Incorporating 2-Amino-6-vinylpurine Derivative and Evaluation of the Reactivity

Previously, we established a new strategy of synchronous cross linking reaction activated by hybridization to target genes. In this strategy, the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, is generated from its stable precursors by a hybridization-promoted activation process with selectivity to cytosine. In this paper, we wish to report the synthesis of the peptide nucleic acids (PNAs) incorporating 2-amino-6-vinylpurine derivatives and the evaluation of their reactivity to target DNA.