Publications by authors named "Hidenori Okamura"

19 Publications

  • Page 1 of 1

Synthesis of C-nucleoside analogues based on the pyrimidine skeleton for the formation of anti-parallel-type triplex DNA with a CG mismatch site.

Bioorg Med Chem 2020 12 23;28(23):115782. Epub 2020 Sep 23.

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

The triplex DNA forming method is an attractive tool as a gene-targeting agent. Using artificial nucleoside analogues based on C-nucleoside, stable and selective triplex DNA can be formed in a specific region of duplex DNA, and its biotechnology applications will greatly expand. In this study, we designed and synthesized novel C-nucleoside analogues based on the pyrimidine skeleton, AP-d(Y-Cl) and AP-d(Y-H), capable of recognizing a CG mismatch site that is not recognized by natural nucleosides. After incorporating them into the oligonucleotides, their triplex forming abilities were evaluated by gel-shift assay. Although it was only one sequence, the 3'-GZG-5' sequence, the stability of the CG mismatch site recognition was greatly improved compared with previous nucleoside analogues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115782DOI Listing
December 2020

Enhancements in the utilization of antigene oligonucleotides in the nucleus by booster oligonucleotides.

Chem Commun (Camb) 2020 Aug 23;56(67):9731-9734. Epub 2020 Jul 23.

Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan.

We recently found the translocation of double-stranded DNA into the nucleus. We herein describe the concept of novel booster oligodeoxynucleotides including 2'-deoxy uridine, which release antigene oligonucleotides in the nucleus by enzymatic digestion. This system exhibited stronger hTERT mRNA expression inhibitory activity than single-stranded antigene oligonucleotides.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0cc04240dDOI Listing
August 2020

Development of novel C-nucleoside analogues for the formation of antiparallel-type triplex DNA with duplex DNA that includes TA and dUA base pairs.

Org Biomol Chem 2020 04;18(15):2845-2851

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Expansion of the triplex DNA forming sequence is required in the genomic targeting fields. Basically, triplex DNA is formed by the interaction between the triplex-forming oligonucleotides and homo-purine region with the target duplex DNA. The presence of the base pair conversion sites hampers stable triplex formation. To overcome this limitation, it is necessary to develop an artificial nucleic acid to recognize the base conversion sites, and the CG and TA base pairs. We describe the synthesis of C-nucleoside analogues and an evaluation of the ability of triplex formation. Consequently, the combined use of the novel C-nucleoside analogues, AY - AY-d(Y-NH2), AY-d(Y-Cl) and IAP-d(Y-Cl), is capable of recognizing duplex DNA including the TA or dUA base pair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0ob00420kDOI Listing
April 2020

Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides.

Science 2019 10;366(6461):76-82

Center for Integrated Protein Science, Department of Chemistry, LMU München, Butenandtstrasse 5-13, 81377 München, Germany.

Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aax2747DOI Listing
October 2019

Proto-Urea-RNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides.

Angew Chem Int Ed Engl 2019 12 30;58(51):18691-18696. Epub 2019 Oct 30.

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU München, Butenandtstr. 5-13, 81377, München, Germany.

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information-carrying RNA oligomers able to self-replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto-RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis-urea (biuret) and tris-urea (triuret) are able to directly react with ribose. The urea-ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides' base-pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea-containing RNA bases are good candidates for a proto-RNA world.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201911746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916321PMC
December 2019

Synthesis of 2'-deoxy-4-aminopyridinylpseudocytidine Derivatives for Incorporation Into Triplex Forming Oligonucleotides.

Curr Protoc Nucleic Acid Chem 2019 06 18;77(1):e80. Epub 2019 Mar 18.

Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan.

This unit describes the detailed synthetic protocol for the preparation of the phosphoramidite units of the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These C-nucleoside derivatives are useful units for the incorporation into triplex forming oligonucleotides (TFOs) to form the stable triplex DNA containing the CG interrupting sites. Commercially available 1-methyl-2'-deoxypseudouridine is prepared from thymidine and 5-iodo-uracil by a simple method, that is, coupling of glycal and 5-iodo-1-methyluracil by the Heck reaction, followed by desilylation and diastereoselective reduction. The carbonyl group at the 4 position of the pseudouridine derivative is activated by 3-nitorotriazole and treated with the corresponding aromatic amine compounds to produce the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These derivatives are then successfully converted to the phosphoramidite units and incorporated into the oligodeoxynucleotides. © 2019 by John Wiley & Sons, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cpnc.80DOI Listing
June 2019

A one-pot, water compatible synthesis of pyrimidine nucleobases under plausible prebiotic conditions.

Chem Commun (Camb) 2019 Feb;55(13):1939-1942

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Germany.

Herein, we report a new prebiotically plausible pathway towards a pyrimidine nucleobase in continuous manner. The route involves simultaneous methylation and carbamoylation of cyanoacetylene-derived α,β-unsaturated thioamide with N-methyl-N-nitrosourea (MNU) in aqueous media. This provides S-methylpyrimidinone in one-pot, which can be converted into a variety of 4-substituted pyrimidine nucleobases including cytosine and uracil.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8cc09435gDOI Listing
February 2019

A Click-Chemistry Linked 2'3'-cGAMP Analogue.

Chemistry 2019 Feb 17;25(8):2089-2095. Epub 2019 Jan 17.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, Munich, Germany.

2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogues with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, the synthesis of a cGAMP analogue with one amide and one triazole linkage is reported. The molecule is best prepared via a first Cu -catalyzed click reaction, which establishes the triazole, while the cyclization is achieved by macrolactamization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201805409DOI Listing
February 2019

Modification of the aminopyridine unit of 2'-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences.

Nucleic Acids Res 2018 09;46(17):8679-8688

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158708PMC
September 2018

Stable and Selective Antiparallel Type Triplex DNA Formation by Targeting a GC Base Pair with the TFO Containing One N-Phenyl-2'-deoxyguanosine.

Chem Pharm Bull (Tokyo) 2018 ;66(6):624-631

Graduate School of Pharmaceutical Sciences, Kyushu University.

The antiparallel triplex DNA is formed by the interaction between purine-rich triplex forming oligonucleotides (TFOs) and the homo-purine region within a duplex DNA. The formation of such a structure with the genome DNA promises to control the gene expression in a living cell. In this study, in an attempt to enhance the stability of the triplex DNAs, we have designed the N-arylated deoxyguanosine derivatives. Among these analogues, we found that the TFOs containing N-phenyl-2'-deoxyguanosine (PhdG) showed a stable and selective triplex DNA formation with the GC base pair as compared to the natural dG/GC triplet. However, the multiple incorporation of PhdG into the TFOs hampered the stable triplex DNA, instead, showed a tendency to form a higher order structure. Therefore, we concluded that the stable and selective triplex DNA formation is expected by the replacement of dG by PhdG in the purine-rich TFO sequence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1248/cpb.c18-00043DOI Listing
July 2018

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth-Generation by Prebiotic Methylations and Carbamoylations.

Angew Chem Int Ed Engl 2018 05 17;57(20):5943-5946. Epub 2018 Apr 17.

Center for Integrated Protein Science (CiPSM) at the Department, of Chemistry, LMU München, Butenandtstrasse 5-13, 81377, München, Germany.

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201801919DOI Listing
May 2018

Wet-dry cycles enable the parallel origin of canonical and non-canonical nucleosides by continuous synthesis.

Nat Commun 2018 01 11;9(1):163. Epub 2018 Jan 11.

Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, 81377, Munich, Germany.

The molecules of life were created by a continuous physicochemical process on an early Earth. In this hadean environment, chemical transformations were driven by fluctuations of the naturally given physical parameters established for example by wet-dry cycles. These conditions might have allowed for the formation of (self)-replicating RNA as the fundamental biopolymer during chemical evolution. The question of how a complex multistep chemical synthesis of RNA building blocks was possible in such an environment remains unanswered. Here we report that geothermal fields could provide the right setup for establishing wet-dry cycles that allow for the synthesis of RNA nucleosides by continuous synthesis. Our model provides both the canonical and many ubiquitous non-canonical purine nucleosides in parallel by simple changes of physical parameters such as temperature, pH and concentration. The data show that modified nucleosides were potentially formed as competitor molecules. They could in this sense be considered as molecular fossils.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-02639-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765019PMC
January 2018

Effect of the 3-halo substitution of the 2'-deoxy aminopyridinyl-pseudocytidine derivatives on the selectivity and stability of antiparallel triplex DNA with a CG inversion site.

Bioorg Med Chem 2017 07 19;25(14):3853-3860. Epub 2017 May 19.

School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Electronic address:

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-CH (AP-ΨdC), 6.1 for 3-H (AP-ΨdC), 4.3 for 3-Cl (AP-ΨdC), 4.4 for 3-Br (AP-ΨdC), and 4.7 for 3-I (AP-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-(AP-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2017.05.035DOI Listing
July 2017

Aminopyridinyl-Pseudodeoxycytidine Derivatives Selectively Stabilize Antiparallel Triplex DNA with Multiple CG Inversion Sites.

Angew Chem Int Ed Engl 2016 09 31;55(40):12445-9. Epub 2016 Aug 31.

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

The sequence-specific formation of triplex DNA offers a potential basis for genome-targeting technologies. In an antiparallel triplex DNA, the sequence-specificity is established by the formation of specific base triplets (G-GC, A-AT, and T-AT) between a triplex-forming oligonucleotide (TFO) and a duplex DNA. However, there are no natural nucleosides that can selectively recognize the inverted CG and TA base pairs. Therefore, the recognition of the CG and TA inversion sites to form a stable triplex DNA has been a long-standing goal for the triplex-forming technology. We now describe the design and synthesis of pseudo-deoxycytidine (ΨdC) derivatives for selective recognition of the CG base pair to expand the triplex-forming sequence. The aminopyridine-bearing ΨdC derivatives showed high selectivity and affinity toward the CG base pair in all neighboring base contexts. Remarkably, 3-methyl-2-aminopyridinyl-ΨdC ((Me) AP-ΨdC) formed a stable triplex with the promoter sequence of the hTERT gene containing four CG inversion sites, and effectively inhibited its transcription in human cancer cells. Thus, (Me) AP-ΨdC is expected to serve as a new starting point of triplex-forming oligonucleotides for a wide variety of genome-targeting applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201606136DOI Listing
September 2016

Enhancement of TFO Triplex Formation by Conjugation with Pyrene via Click Chemistry.

Chem Pharm Bull (Tokyo) 2015 ;63(11):920-6

Graduate School of Pharmaceutical Sciences, Kyushu University.

This paper reports the preparation of 14-mer triplex-forming oligonucleotides (TFOs) containing a 2-O-methyl-1-β-phenyl-α-propargyl-ribose unit, which was conjugated with azide-modified molecules via a click reaction. Modification of these TFOs with pyrene assisted triplex formation, improving the stability of the triplex DNA and the anti-proliferative effects against A549 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1248/cpb.c15-00570DOI Listing
May 2016

An isocytidine derivative with a 2-amino-6-methylpyridine unit for selective recognition of the CG interrupting site in an antiparallel triplex DNA.

Chembiochem 2014 Nov 3;15(16):2374-8. Epub 2014 Sep 3.

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan).

Sequence-specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non-natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N(2)-arylmethyl isodC derivatives and incorporated them into triplex-forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2-amino-6-methylpyridine moiety (AP-isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201402328DOI Listing
November 2014

N-(guanidinoethyl)-2'-deoxy-5-methylisocytidine exhibits selective recognition of a CG interrupting site for the formation of anti-parallel triplexes.

Org Biomol Chem 2013 Jun;11(23):3918-24

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Higashiku Maidashi 3-1-1, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c3ob40472bDOI Listing
June 2013

Properties of oligonucleotide with phenyl-substituted carbocyclic nucleoside analogs for the formation of duplex and triplex DNA.

Nucleosides Nucleotides Nucleic Acids 2012 ;31(12):841-60

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, Japan.

(1S,3S,4R)-1-Phenyl-1-thymidyl-3-hydroxy-4-hydroxymethylcyclopentane (10) and their analogs were synthesized, incorporated into the oligodeoxynucleotides, and their properties were evaluated for the formation of duplex and triplex DNA. The known chiral cyclopentanone derivative was converted into the corresponding ketimine sulfonamide derivative, which was subjected to a stereoselective PhLi addition. The formed sulfonamide was hydrolyzed to afford the primary amino group, on which the thymine moiety was built. The benzyl protecting groups were removed to form the nucleoside analog having a phenyl group and the thymine unit at the 1' position of a carbocyclic skeleton (10). In the estimation of the oligodeoxynucleotides incorporating 10 for duplex and triplex formation, the carbocyclic nucleoside analog 10 did not show the stabilizing effect for duplex formation; on the other hand, it stabilized the triplex. Therefore, the skeleton of the phenyl-substituted carbocyclic nucleoside analog 10 may be a platform for the formation of stable triplex DNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15257770.2012.737970DOI Listing
January 2014

Cloning and characterization of a beta-1,4-mannanase 5C possessing a family 27 carbohydrate-binding module from a marine bacterium, Vibrio sp. strain MA-138.

Biosci Biotechnol Biochem 2009 Jan 7;73(1):109-16. Epub 2009 Jan 7.

Graduate School of Bioresources, Mie University.

The beta-1,4-mannanase 5C gene (man5C) of Vibrio sp. strain MA-138 was cloned and expressed in Escherichia coli. The man5C gene consisted of 2,010 bp nucleotides encoding a protein of 669 amino acids with a predicted molecular weight of 76,309. beta-1,4-Mannanase (Man5C) is a modular enzyme composed of a catalytic module belonging to glycoside hydrolase family 5, a linker region, and a putative carbohydrate-binding module (CBM) belonging to family 27. Recombinant Man5C exhibited maximal activity at 50 degrees C at pH 7.0, and it had a K(m) of 0.6 mg ml(-1) and a V(max) of 556.2 micromol min(-1) mumol(-1) for glucomannan. Binding studies revealed that the C-terminal putative CBM27 had the ability to bind soluble beta-mannans and contributed to increasing the rate of depolymerization by binding to the polymeric substrate. Man5C of Vibrio sp. MA-138 is the first non-extremophile enzyme to be identified as a beta-mannanase possessing CBM27.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1271/bbb.80521DOI Listing
January 2009