Masahide Ishikawa

Unnatural base pairs for specific transcription

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chain-terminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.

Synthesis and properties of 2-azidodeoxyadenosine and its incorporation into oligodeoxynucleotides

Abstract 2-Azidodeoxyadenosine ( 7 ) was conveniently synthesized from deoxyguanosine ( 2 ) by use of a combined reagent of TMSN 3 –BuONO. The structure of the tautomer of the azido derivative was determined by 1 H NMR. Reaction of 7 with i Pr 2 NP(OEt) 2 gave an intermediate 10 of the Staudinger reaction. Incorporation of 7 into a DNA 13mer resulted in a significant decrease of the T m value of the DNA duplex upon hybridization with the complementary strand. The thermal stability was discussed based on the hydrogen bond energy and electrostatic repulsion.

Synthesis of 3-(2-deoxy-β-d-ribofuranosyl)pyridin-2-one and 2-amino-6-(N,N-dimethylamino)-9-(2-deoxy-β-d-ribofuranosyl)purine derivatives for an unnatural base pair

Abstract An unnatural base pair, 2-amino-6-( N , N -dimethylamino)purine (denoted x ) and pyridin-2-one (denoted y ), was designed to prove the structural requirements for base pair formation involving shape complementarity. It was expected that y might satisfy the structural requirements for pairing with x , in which the bulky 6-dimethylamino group may eliminate base pairing with the natural bases. As chemical or biological substrates for DNA synthesis, the phosphoramidite of x and the 2′-deoxy-C3-ribonucleoside triphosphate of y (d y TP) were synthesized, and the incorporation experiment was demonstrated by using the Klenow fragment of Escherichia coli DNA polymerase I .

Chemical synthesis of cytidine-5′-monophosphono-N-acetylneuraminic acid (CMP-neu5ac)

Abstract CMP-Neu5AC was synthesized for the time from the cytidine 5′-phosphoramidite and the Neu5Ac derivative bearing allyl and allyloxycarbonyl groups as the protecting groups.

A facile synthesis of 2-azidoadenosine derivatives from guanosine as photoaffinity probes

Abstract 2-Azidoadenosine ( 1 ) was synthesized in an overall yield of 49% from guanosine via the reaction of 9-(2′,3′,5′-tri- O -acetyl- β -D-ribofuranosyl)-2-amino-6-chloropurine ( 2 ) with isoamyl nitrite and trimethylsilyl azide under neutral and anhydrous conditions. As photoaffinity probes, ATP analogues and the cap structure of eukaryotic mRNA bearing 2-azidoadenosine were synthesized.

Large Scale Synthesis of the Cap Part in Messenger RNA Using a New Type of Bifunctional Phosphorylating Reagent

Abstract Bifunctional phosphorylating reagents 1 and 2 were employed for the synthesis of the cap part, m7G7 pppG, from guanosine 5′-phosphates on a large scale without any protecting groups.

A CONVENIENT METHOD FOR THE SYNTHESIS OF ATP AND AP4A

A bifunctional phosphorylating reagent, O-8-(5-chloroquinolyl) S-phenyl phosphorothioate (1) was employed for the synthesis of adenosine 5′-triphosphate (ATP) and diadenosine 5′-tetraphosphate (Ap4...

One-pot reaction for the synthesis of m7G5′pppG and m7G5′pppA by using an activatable bifunctional phosphorylating reagent

A new bifunctional phosphorylating reagent 1 was prepared and employed for the synthesis of the cap structure, m7G5′pppG and m7G5′pppA in large scale without using any protecting groups starting from the corresponding nucleoside-5′-phosphates.

Synthesis of Fused Oligoribonucleotides with Trideoxyribonucleotide Containing Phosphorothioate to Stabilize Against Nuclease Activity

Abstract Fused oligonucleotides(21mer) consisting of RNA(18mer) and DNA(3mer) were synthesized by combined use of the phosphotriester and phosphoramidite methods. The RNA(18mer) corresponds to the leader sequence of phage fl coat protein mRNA containing initiation codon. The RNA was stabilized against 3′-exonucleases by joining with trideoxy-ribonucleotides containing phosphorothioate linkages and it would be applied to the studies on the initiation complex formation in prokaryotic translation.

Synthesis and Properties of an Initiation Codon Analog Consisting of 2′-O-Methyl Nucleotides

Abstract An initiation codon analog consisting of 2′-O-methyl nucleotides (AmUmG) was synthesized and examined for its binding efficiency to E. coli ribosomes with fMetRNAfMet and also its stability in binding assay systems for comparison with those of r(AUG) and d(ATG). AmUmG was found completely resistant to nucleases under the conditions used.