Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We describe herein topological mRNA capture using branched oligodeoxynucleotides (ODNs) with multiple reactive functional groups. These fragmented ODNs efficiently formed topological complexes on template mRNA in vitro. In cell-based experiments targeting AcGFP mRNA, the bifurcated reactive ODNs showed a much larger gene silencing effect than the corresponding natural antisense ODN.

DOI： 10.1039/d2cc06189a

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Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A:T→G:C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G:C→A:T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

DOI： 10.1002/cbic.202200572

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We herein report a new synthetic method of nucleoside oligophosphates based on an electrophilic activation of 5'-phosphorothioate nucleotides. Treatment of the phosphorothioate with 2,4-dinitrochlorobenzene (DNCB) efficiently afforded the key activated...

DOI： 10.1039/d2ob02260e

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Removing immunogenic uncapped mRNA from in vitro transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide a maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. Herein, we developed hydrophobic photocaged tag-modified cap analogs, which separated capped mRNA from uncapped mRNA by reversed-phase HPLC. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provided 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. The Cap-2-type mRNA showed up to 3 to 4-fold higher translational activity in cultured cells and animals than mRNA prepared by the standard capping method. Notably, the purification process simultaneously removed immunogenic double-stranded mRNA, another major contaminant of in vitro transcribed mRNA, drastically reducing mRNA immunogenicity in cultured cells.

DOI： 10.1038/s41467-023-38244-8

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Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

DOI： 10.1039/d3cc00460k

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mRNA vaccines have attracted considerable attention as a result of the 2019 coronavirus pandemic; however, challenges remain regarding use of mRNA vaccines, including insufficient delivery owing to the high molecular weights and high negative charges associated with mRNA. These characteristics of mRNA vaccines impair intracellular uptake and subsequent protein translation. In the current study, we prepared a minimal mRNA vaccine encoding a tumor associated antigen human gp10025-33 peptide (KVPRNQDWL), as a potential treatment for melanoma. Minimal mRNA vaccines have recently shown promise at improving the translational process, and can be prepared via a simple production method. Moreover, we previously reported the successful use of iontophoresis (IP) technology in the delivery of hydrophilic macromolecules into skin layers, as well as intracellular delivery of small interfering RNA (siRNA). We hypothesized that combining IP technology with a newly synthesized minimal mRNA vaccine can improve both transdermal and intracellular delivery of mRNA. Following IP-induced delivery of a mRNA vaccine, an immune response is elicited resulting in activation of skin resident immune cells. As expected, combining both technologies led to potent stimulation of the immune system, which was observed via potent tumor inhibition in mice bearing melanoma. Additionally, there was an elevation in mRNA expression levels of various cytokines, mainly interferon (IFN)-γ, as well as infiltration of cytotoxic CD8+ T cells in the tumor tissue, which are responsible for tumor clearance. This is the first report demonstrating the application of IP for delivery of a minimal mRNA vaccine as a potential melanoma therapeutic.

DOI： 10.1248/bpb.b22-00746

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The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2' position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2'-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

DOI： 10.1016/j.bmcl.2022.128939

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Site-specific chemical modification of mRNA can improve its translational efficiency and stability. For this purpose, it is desirable to develop a complete chemical synthesis method for chemically modified mRNA. The key is a chemical reaction that introduces a cap structure into the chemically synthesized RNA. In this study, we developed a fast and quantitative chemical capping reaction between 5'-phosphorylated RNA and N7-methylated GDP imidazolide in the presence of 1-methylimidazole in the organic solvent dimethyl sulfoxide. It enabled quantitative preparation of capping RNA within 3 h. We prepared chemically modified 107-nucleotide mRNAs, including N6-methyladenosine, insertion of non-nucleotide linkers, and 2'-O-methylated nucleotides at the 5' end and evaluated their effects on translational activity in cultured HeLa cells. The results showed that mRNAs with non-nucleotide linkers in the untranslated regions were sufficiently tolerant to translation and that mRNAs with the Cap_2 structure had higher translational activity than those with the Cap_0 structure.

DOI： 10.1021/acschembio.1c00996

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Synthetic phosphate-derived functional groups are important for controlling the function of bioactive molecules in vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P-F and P-N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA-gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA-gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti-cancer and anti-virus drugs.

DOI： 10.1002/cmdc.202200188

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Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme's plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

DOI： 10.3390/ijms23073606

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cbic.202100312

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DOI： 10.1002/cbic.202100413

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Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2' sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2' modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2' sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by in vitro transcription.

DOI： 10.1002/cbic.202100004

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Acquired immunodeficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV). Although treatments against HIV infection are available, AIDS remains a serious disease that causes many deaths annually. Although a variety of anti-HIV drugs have been synthesized and marketed to treat HIV-infected patients, nucleoside analogue reverse transcriptase inhibitors (NRTIs), which mimic nucleosides, are used extensively and remain a subject of interest to medicinal chemists. However, HIV has acquired drug resistance against NRTIs, and thus the struggle to find novel therapies continues. In this review, we trace the trajectory of NRTIs, focusing on the synthesis, mechanisms of action and applications of NRTIs that have been developed.

DOI： 10.1002/cmdc.202000695

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DOI： 10.1002/cpz1.43

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Language：Japanese   Publisher：(公社)日本薬学会  

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DOI： 10.1002/anie.202007111

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DOI： 10.1021/acs.jctc.0c00270

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There is great potential for siRNA in the treatment of diseases through the reduction of damaging protein translation by RNA interference. However, the delivery and cell uptake of siRNA pose a serious problem in its therapeutic application. Methods to overcome this issue include chemical modification of the siRNA duplex to improve pharmacokinetics, stability and efficacy, and conjugation to small ligand molecules to enable membrane penetration, targetability and potency. In this review, the most common modifications of siRNA will be discussed, along with ligand conjugates that are believed to be the most promising in advancing the field of targeted siRNA delivery.

DOI： 10.1002/cbic.202000009

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DOI： 10.1039/d0cc02140g

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DOI： 10.1074/jbc.RA119.010921

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DOI： 10.1080/15257770.2019.1671593

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DOI： 10.3987/COM-19-14191

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DOI： 10.1080/15476286.2019.1678364

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DOI： 10.1039/c9cc04872c

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DOI： 10.1248/cpb.c19-00811

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Due to the unique rigid and small steric feature of cyclopropane, cyclopropane nucleosides (CPNs) in which the ribose (deoxyribose) of nucleosides are replaced by a hydroxy-substituted cyclopropane, are of great biological interest. Novel 1,1,2-trisubstituted cyclopropane nucleosides were synthesized in enantiomerically pure forms as potential antiviral agents. In the synthesis, two cyclopropane tosylates, which were prepared from chiral cyclopropane lactones previously reported by us, were used effectively as common intermediates for the CPNs. These CPNs are also potentially useful as nucleoside units to incorporate into oligonucleotides in nucleic acids chemotherapy studies.

DOI： 10.1080/15257770.2019.1625380

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DOI： 10.1080/15257770.2019.1641205

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DOI： 10.1016/j.addr.2019.08.001

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DOI： 10.1002/anie.201900993

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DOI： 10.1002/ange.201900993

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DOI： 10.1002/cbic.201800671

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DOI： 10.1080/15257770.2020.1736772

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<title>Abstract</title>In RNA secondary structure prediction, nearest-neighbor parameters are used to determine the stability of a given structure. We derived the nearest-neighbor parameters for RNAs containing inosine-cytosine pairs. For parameter derivation, we developed a method that combines UV adsorption measurement experiments with free-energy calculations using molecular dynamics simulations. The method provides fast drop-in parameters for modified bases. Derived parameters were compared and found to be consistent with existing parameters for canonical RNAs. A duplex with an internal inosine-cytosine pair is 0.9 kcal/mol more unstable than the same duplex with an internal guanine-cytosine pair, and is as stable as the one with an internal adenine-uracil pair (only 0.1 kcal/mol more stable) on average.

DOI： 10.1101/454124

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DOI： 10.1093/nar/gky744

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DOI： 10.1016/j.redox.2018.04.013

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press Inc.  

This chapter describes a simple and straightforward way to obtain single-stranded circular RNA sequences in vitro. Linear RNA that is phosphorylated at the 5′ end is first prepared by a chemical or enzymatic method, then circularized using ligase. The function of the prepared circular RNA molecule, such as an ability to induce translation, can then be investigated.

DOI： 10.1007/978-1-4939-7562-4_15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Pharmaceutical Society of Japan  

DOI： 10.1248/cpb.c17-00615

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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We herein report the first covalent G-site-binding inhibitor for GST, GS-ESF (1), which irreversibly inhibited the GSTP1-1 function. LC-MS/MS and X-ray structure analyses of the covalently linked GST-inhibitor complex suggested that 1 reacted with Tyr108 of GSTP1-1. The mechanism of covalent bond formation was discussed based on MD simulation results.

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DOI： 10.1021/acs.joc.7b01011

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DOI： 10.1093/nar/gkx459

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DOI： 10.1039/c7tb00846e

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DOI： 10.1093/nar/gkx265

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DOI： 10.1039/C7TB00846E

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<p>In an <i>E. coli</i> cell-free translation system, we found that a circular RNA containing an infinite open reading frame produced more translation product than its linear counterpart by two orders of magnitude, because a ribosome can work more effectively towards the elongation on circular RNA than it can on linear RNA. We then tested circular RNAs containing an infinite open reading frame could be translated in eukaryotic systems, in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. We found that the circular RNAs also produced long peptides in eukaryotic translation systems, possibly owing to the rolling circle amplification mechanism.</p>

DOI： 10.2142/biophys.57.005

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© 2016 American Chemical Society. Lipid chemical mediator resolvins with highly potent anti-inflammatory activity can be leads to develop novel anti-inflammatory drugs; however, they are unstable in oxygen due to their characteristic polyunsaturated structures. To solve the problem, CP-RvE2 has been designed and synthesized in which the cis-olefin of RvE2 was replaced with a cyclopropane. CP-RvE2s were much more stable than RvE2 against autoxidation and equipotent or more potent than RvE2. CP-RvE2s were successfully identified as stable equivalents of RvE2.

DOI： 10.1021/acs.orglett.6b02612

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.

DOI： 10.1021/acs.molpharmaceut.6b00140

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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.

DOI： 10.1021/acs.joc.6b00105

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We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

DOI： 10.1038/srep16435

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© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. We describe a nickel-catalyzed Suzuki-Miyaura arylation of a tertiary iodocyclopropane with arylboronic acids; this is an efficient and convergent strategy for providing various enantioenriched arylcyclopropanes with a quaternary stereogenic center. This is the first metal-catalyzed coupling between a tertiary alkyl electrophile and a wide range of aromatics, including heteroaromatics. We found that the outcome of the Ni-catalyzed coupling with halides as electrophiles was dependent on the stability of the radical species formed during the reaction. The use of tert-butyl alcohol (t-BuOH) as the reaction solvent was very effective, because of its stability under the radical-generating reaction conditions.

DOI： 10.1002/adsc.201401000

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We synthesized three types of nanostructured RNAs that induce RNA interference (RNAi): branched RNAs, dumbbell-shaped RNA, and circular double-stranded RNAs. All three nanostructured RNAs were transformed into double-stranded RNA of approximately 20 base pairs when they were treated with nuclease enzymes such as Dicer. These dsRNA species induced gene silencing when they are were introduced into mammalian cells.

DOI： 10.1007/978-1-4939-1538-5_2

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The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.

DOI： 10.1039/c5ob00199d

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Upon reacting 3',4'-unsaturated cytosine (8 and 9) and adenine nucleosides (13 and 14) with XeF2/BF3 center dot OEt2, the respective novel 3',4'-difluoro-3'-deoxyribofuranosyl nucleosides (10-12 and 15-18) could be obtained. Formation of anti-adducts (11, 16 and 18) revealed that the fluorination involved oxonium ions as incipient intermediates. TBDMS-protected 3',4'-unsaturated adenosine provided the beta-face adducts as sole stereoisomers whereas a-face-selectivity was observed with the TBDPS-protected adenosine 14. The evaluation of the novel 3'-deoxy-3',4'-difluororibofuranosylcytosine-(19-21) and adenine nucleosides (22-25) against antitumor and antiviral activities revealed that 3',4'-difluorocordycepin (24) was found to possess anti-HCV activity. The SI of 24 was comparable to that of the anti-HCV drug ribavirin. However, sofosbuvir, FDA-approved novel anti-HCV drug, showed better SI value. Our finding revealed that the introduction of the fluoro-substituent into the 4'-position of cordycepin derivatives decreased the cytotoxicity to the host cell with retention of the antiviral activity. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2014.08.024

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DOI： 10.1039/C4AN90066A

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On the basis of the three-dimensional diversity-oriented conformational restriction strategy using key chiral cyclopropane units, we previously identified 3 ((2S,3R)-4-amino-3,4-methanobutyric acid) with a chiral trans-cyclopropane structure as a γ-aminobutyric acid (GABA) transporter inhibitor selective for GABA transporter (GAT) subtypes GAT-3 and BGT-1 (betaine/GABA transporter-1). Further conformational restriction of 3 with the rigid bicyclo[3.1.0]hexane backbone led to the successful development of the first highly potent and selective BGT-1 inhibitor 4 (IC50 = 0.59 μM). The bioactive conformation of 3 for BGT-1 was also identified. © 2014 American Chemical Society.

DOI： 10.1021/ml500134k

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DOI： 10.1016/j.actbio.2014.05.034

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Language：Japanese   Publisher：SOC SYNTHETIC ORGANIC CHEM JPN  

Glutathione S-transferase (GST) are phase II enzymes that catalyze the nucleophilic attack of glutathione (GSH) to a wide range of hydrophobic and electrophilic compounds. GST is of high clinical interest in inflammation, pathophysiology and tumor drug resistance. In addition, GST is known to highly express in cancer cells. Therefore, we tried to develop new luminescent probes that can detect and image GST expression in living cells. Recently, we found that the electrophilic centre in the dinitrobenzenesulfonamide derivative is attacked by GSH and that this reaction is catalyzed by GST releasing the strong fluorophore. Based on this knowledge, we synthesized fluorescent probe, F-19 MRI probe, and bioluminogenic probe. The fluorescent probe and kinetic parameters for purified GST were giving a rate enhancement of 10(6) compared with the nonenzymatic reaction. In addition, fluorescent probe successfully image GST in living cells. The fluorescent probe is potentially useful reagent for detection of GST activity in living cells.
F-19 MRI and bioluminogenic probes were synthesized using 4-acetyl-2-nitrobenzenesulfonyl group. F-19 MRI probe exhibited a peak shift of F-19 MRI signal and fluorescence enhancement in a bimodal way in response to GST activity. Bioluminogenic probe provided strong bioluminescence from GST activity. Moreover, both probes were successfully applied to the monitoring of GST expression in living E. coli cells. Successful detection of GST activity in living cells could be very useful for identifying tumor cells that overexpress GSTs.

DOI： 10.5059/yukigoseikyokaishi.72.822

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Here we report a novel method to form a pseudorotaxane architecture using only a pair of reactive oligodeoxyribonucleotides (ODNs), which we designed and synthesized, and then performed the pseudorotaxane formation reaction with both DNA and RNA oligonucleotides. The reaction proceeded smoothly without any extra reagents at 37 degrees C and pH 7.2, leading to the formation of a stable complex on a denaturing polyacrylamide gel. Interestingly, the pseudorotaxane was formed with the cyclized ODN reversibly by the slipping process. This new pseudorotaxane formation represents a promising method for developing new DNA nanotechnologies and antisense oligonucleotides.

DOI： 10.1021/ja5018283

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DOI： 10.1021/ol402832w

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Here we report a new strategy for the buildup reaction of active siRNA species from short RNA fragments in living cells using a chemical ligation reaction. This strategy could decrease undesired immune responses and provide more latitude for RNAi technology in the design and concentration of introduced RNA compared to traditional siRNA methods.

DOI： 10.1039/c3cc47529h

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Protein nanotubes formed by layer-by-layer (LbL) assembly can penetrate cells and act as nanopores for direct transmembrane delivery of chemical compounds.

DOI： 10.1039/c3cc45907a

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.52.771

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Other Link： https://jlc.jst.go.jp/DN/JALC/10041971696?from=CiNii

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We have developed a fluorescence detection system for DNA, assisted by a comb-type cationic polymer (PLL-g-DX), for accelerating the reaction turnover. The combination of fluorogenic DNA probes with a comb-type cationic polymer has been demonstrated to be an effective means of signal amplification during the detection process. The method described herein represents a simple and enzyme-free detection. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2013.10.005

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DOI： 10.1016/j.tet.2013.10.075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

An epidermal growth factor (EGF) derivative with affinity for apatite and titanium surfaces was designed using a peptide moiety derived from salivary statherin, a protein that adheres to hydroxyapatite. Since the active sequence has two phosphoserine residues, the EGF derivative was prepared by organic synthesis, and a 54 residue peptide was successfully prepared using this method. Circular dichroism spectra indicated that the conformation of EGF was not significantly altered by the addition of the affinity peptide sequence and the mitogenic activity was only slightly reduced when compared with the wild-type protein. However, the binding affinity of the modified EGF to hydroxyapatite and titanium was significantly higher than the unmodified EGF. The phosphate groups in the affinity sequence contributed to the affinity of modified EGF to both apatite and titanium. The modified EGF significantly enhanced the growth of cells on hydroxyapatite and titanium. It was also demonstrated that the bound EGF enhanced the signal transduction for longer periods than unbound EGF. In conclusion, the modified EGF had significantly higher binding affinity for apatite and titanium than soluble EGF, and the bound EGF significantly enhanced cell growth by long-lasting activation of intracellular signal transduction. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2013.09.004

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Publisher：ACS Symposium Series  

Polyethylene glycol (PEG) is an amphiphilic polymer that is soluble in both water and organic media and can add many functions to biopolymers by conjugation. Here, PEG was coupled to antibodies or oligonucleotides for solubilization into organic media. The former interacted with antigens and the latter formed specific structures and had catalytic activity in organic media. In addition, we attempted to incorporate PEG genetically into a polypeptide. Although the PEG incorporation ratio decreased as the molecular weight of PEG was increased, PEG with a molecular weight of ≤1000 Da was incorporated successfully. The potential applications of PEGylated biopolymers and the methodology for gene PEGylation are discussed. © 2013 American Chemical Society.

DOI： 10.1021/bk-2013-1144.ch016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Oligonucleotide-templated reactions are powerful tools for the detection of nucleic acid sequences. One of the major scientific challenges associated with this technique is the rational design of non-enzyme-mediated catalytic templated reactions capable of multiple turnovers that provide high levels of signal amplification. Herein, we report the development of a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe underwent a rapid templated reaction without any of the undesired background reactions. The fluorogenic reaction conducted in the presence of a template provided a 223-fold increase in fluorescence after 30 s compared with the nontemplated reaction. The probe provided an efficient level of signal amplification that ultimately enabled particularly sensitive levels of detection. Assuming a simple model for the templated reactions, it was possible to estimate the rate constants of the chemical reaction in the presence and in the absence of the template. From these kinetic analyses, it was possible to confirm that an efficient turnover cycle had been achieved, on the basis of the dramatic enhancement in the rate of the chemical reaction considered to be the rate-determining step. With maximized turnover efficiency, it was demonstrated that the probe could offer a high turnover number of 1500 times to enable sensitive levels of detection with a detection limit of 0.5 pM in the catalytic templated reactions.

DOI： 10.1021/ja404743m

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A pre-type sensitizer for a lanthanide complex on an oligonucleotide was successfully converted to a perfect final structure in a target DNA/RNA-templated reaction, without any chemical reagent or enzyme, under neutral conditions. The final form of the lanthanide oligonucleotide provided a long-lived luminescence signal, appropriate for time-gated luminescence analysis and signal amplification. Target DNA/RNA-assisted time-gated luminescence analysis is a powerful tool for elimination of autofluorescence and detection of target RNA in living bacterial cells.

DOI： 10.1021/ja406724k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Dopamine, an adhesive protein can be covalently deposited onto biomaterials. In this study, we evaluated the ability of dopamine-coated surfaces for small interfering RNA (siRNA) immobilization and release. Dopamine was deposited onto 316L stainless steel discs either as a monolayer at acidic pH or as polydopamine at alkaline pH, following which siRNA was immobilized onto these discs. To investigate the RNA interference ability of immobilized siRNA, reduction of luciferase expression in HeLa, and reduction of Egr-1 expression and cell proliferation in human aortic smooth muscle cells (HAoSMCs) were determined. Dopamine treatment of 316L stainless steel discs under both the acidic and alkaline conditions resulted in the deposition of amino (NH2) groups, which enabled electrostatic immobilization of siRNA. The immobilized siRNA was released from both types of coatings, and enhanced the percent suppression of firefly luciferase activity of HeLa significantly up to similar to 96.5% compared to HeLa on non-dopamine controls (18%). Both the release of siRNA and the percent suppression of firefly luciferase activity were sustained for at least 7 days. In another set of experiments, siRNA sequences targeting to inhibit the activity of the transcription factor Egr-1 were eluted from dopamine-coated surfaces to HAoSMCs. Egr-1 siRNA eluted from dopamine-coated surfaces, significantly reduced the proliferation of HAoSMCs and their protein expression of Egr-1. Therefore, this method of surface immobilization of siRNA onto dopamine-coated surfaces might be effective for nucleic acid delivery from stents. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.actbio.2013.01.007

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Language：Japanese   Publisher：The Pharmaceutical Society of Japan  

DOI： 10.1248/yakushi.12-00239-F

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Other Link： http://orcid.org/0000-0002-2292-8606

Language：Japanese   Publishing type：Research paper (scientific journal)  

RNA interference (RNAi) is a potent and highly specific gene-silencing phenomenon which is initiated or triggered by double-stranded RNAs (dsRNAs). Shortly after the development of RNAi, small interfering RNAs (siRNAs) that are 21 nucleotides in length with a 3′ nucleotide overhang were shown to be very effective in mammalian cells. Much effort has been dedicated to the application of siRNAs, both as biological tools and as therapeutic agents. Currently, synthetic siRNA would be the method of choice for clinical purposes. However, natural RNA strands are quickly degraded in biological fluids. Chemically synthesized unnatural nucleotides have been developed and introduced into the siRNA strand. For example, modification of the ribose moiety with a 2′-deoxy, 2′-O-methyl, or 2′-fluoro group, or modification of the phosphate backbone have been examined. Although these modifications improve the stability of siRNA in serum, they often cause a decrease in RNAi activity. There is also concern that unnatural RNA derivatives are toxic in the human body. A method to stabilize nontoxic natural RNA strands should be very useful for applications in RNAi technology. We came up with an idea that nano-structural design stabilizes natural RNA. We tested several new designs such as dumbbell RNA, double stranded circular RNA, or branched RNA in biological stability and RNA interference activity. Consequently, dumbbell or branched design offered prolonged RNAi effect due to high biological stability. © 2013 The Pharmaceutical Society of Japan.

DOI： 10.1248/yakushi.12-00239-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We have synthesized a 2'-aminomethyl branched-chain sugar nucleoside phosphorodithioate from 2,2'-anhydro uridine and subjected the material to a subsequent cyclization reaction under aqueous conditions using bi-functional linkers. The rate of the cyclization reaction was dependent on the leaving group on the bi-functional linkers. The generation of a chiral phosphorous peak from the achiral precursor, as indicated by P-31 NMR, was identified as a good indicator for potentially probing the local and global features of the DNA structure. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2012.12.028

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：AMER CHEMICAL SOC  

Polyethylene glycol (PEG) is an amphiphilic polymer that is soluble in both water and organic media and can add many functions to biopolymers by conjugation. Here, PEG was coupled to antibodies or oligonucleotides for solubilization into organic media. The former interacted with antigens and the latter formed specific structures and had catalytic activity in organic media. In addition, we attempted to incorporate PEG genetically into a polypeptide. Although the PEG incorporation ratio decreased as the molecular weight of PEG was increased, PEG with a molecular weight of &lt;= 1000 Da was incorporated successfully. The potential applications of PEGylated biopolymers and the methodology for gene PEGylation are discussed.

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We have synthesized a series of 4-substituted-2-nitrobenzene-sulfonyl compounds for caged fluorogenic probes and conducted a Hammett plot analysis using the steady-state kinetic parameters. The results revealed that the glutathione transferase (GST) alpha catalyzed reaction was dependent on the σ value in the same way as the non-enzymatic reaction, whereas the dependence of the σ value of the GST mu and pi was not as pronounced as that of GST alpha. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3an01339a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have developed a system to release a biologically active molecule in response to the sequence of a target gene. The releasing system, which was triggered by the reduction of an azidomethyl group, was successfully applied to protein expression induced by the release of IPTG triggered by endogenous RNA in bacterial cells.

DOI： 10.1039/c2cc37826d

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The one-pot tandem reaction of N-alkyl-N-allyl-2-vinylaniline derivatives with benzo- or naphthoquinones and a ruthenium-alkylidene catalyst leads to isoindolo[2,1-a]quinolines in a variety of colors, which can be altered by exchanging the substituent on the core heterocycle (see scheme). This reaction offers a new synthetic method for π-conjugated small molecules from simple aniline derivatives. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/anie.201206765

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Polymeric micelle prepared through the self-assembly of cationic cholesterol-modified gelatin was tested for siRNA delivery. It exerted the desired effect of gene knockdown in HeLa cells stably expressing the luciferase gene and achieved a two-fold increase in the knockdown ability when compared to Lipofectamine® 2000. It was found that the polymeric micelle exhibited excellent stability and increased the biological stability of the siRNA in serum. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c2mb25424g

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Getting the runaround: Small circular RNA molecules containing an infinite open reading frame were synthesized and tested in an E. coli cell-free translation system. A circular RNA 126 nucleotides in length was found to produce more product than its linear counterpart by two orders of magnitude, because a ribosome can work more effectively towards the elongation on circular RNA than it can on linear RNA in this continuous peptide synthesis. Copyright © 2013 WILEY-VCH Verlag GmbH &amp
Co. KGaA, Weinheim.

DOI： 10.1002/anie.201302044

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Publisher：ECS Transactions  

Certain guanine-rich DNA oligomers that form a G-quadruplex structure and bind hemin inside are called hemin-binding DNA aptamers. A hemin-binding DNA aptamer with the 4c15 sequence forms a parallel G quadraplex and the PS2.M sequence folds and forms an anti-parallel structure. We propose a novel method to examine the peroxidase activity of hemin-binding DNA aptamers through electrochemical measurements. In this study, a polyA chain and thiol were introduced to the 5' terminal of each oligonucleotide. The peroxidase activity of hemin-binding DNA aptamers was determined after modifying a gold electrode with the aptamers. The distance to hemin from the surface of the electrode is most important to determine catalytic activity. Catalytic activity, shown as KM of the examined parallel DNA aptamers, was around 20 μM. 4c15 without a poly A linker showed the highest catalytic activity, and its KM was 19 μM. The KM of the anti-parallel aptamer was almost the same as that of the parallel aptamer. © The Electrochemical Society.

DOI： 10.1149/05028.0001ecst

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Titanium and stainless steel were modified with dopamine for the immobilization of biomolecules, epidermal growth factor (EGF). First, the treatment of metal surfaces with a dopamine solution under different pH conditions was investigated. At higher pH, the dopamine solution turned brown and formed precipitates. Treatment of the metals with dopamine at pH 8.5 also resulted in the development of brown color at the surface of the metals. The hydrophobicity of the surfaces increased after treatment with dopamine, independently of pH. X-ray photoelectron spectroscopy revealed the formation of a significant amount of an organic layer on both surfaces at pH 8.5. According to ellipsometry measurements, the organic layer formed at pH 8.5 was about 1000 times as thick as that formed at pH 4.5. The amount of amino groups in the layer formed at pH 8.5 was also higher than that observed in the layer formed at pH 4.5. EGF molecules were immobilized onto the dopamine-treated surfaces via a coupling reaction using carbodiimide. A greater amount of EGF was immobilized on surfaces treated at pH 8.5 compared with pH 4.5. Significantly higher growth of rat fibroblast cells was observed on the two EGF-immobilized surfaces compared with non-immobilized surfaces in the presence of EGF. The present study demonstrated that metals can become bioactive via the surface immobilization of a growth factor and that the effect of the immobilized growth factor on metals was greater than that of soluble growth factor. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.msec.2012.07.039

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RNA splicing is an important target for basic research of disease mechanisms and for drug discovery. Here, we report a new method for analysis of the in vitro RNA splicing process that produces fluorescence using a reduction-triggered fluorescence (RETF) probe. The fluorescence signal is produced only when the two probes bind side-by-side with a specific RNA target. Precursor messenger RNA and mature messenger RNA originating from the chicken delta-crystallin (CDC) gene were successfully discriminated in solution using an RETF probe with the assistance of helper oligonucleotide strands. Also, we successfully applied RETF probes to the detection of emerging mature mRNA in an in vitro splicing process. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2012.09.033

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We designed and synthesized conformationally restricted analogues and regioisomers of the nonsteroidal anti-inflammatory drug indomethacin. Evaluation of the inhibitory effects of these compounds on COX, P-glycoprotein, and multidrug resistance indicated that NSAIDS modulation of multidrug-resistant P-glycoprotein and multidrug-resistant protein-1 is not associated with COX-1 and COX-2 inhibitory activities. © 2012 American Chemical Society.

DOI： 10.1021/jm301084z

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Language：English   Publisher：AMER CHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/cbic.201200242

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.50.540

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Other Link： https://jlc.jst.go.jp/DN/JALC/10006703539?from=CiNii

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Oligonucleotide-templated reactions are useful for applying nucleic acid sensing. Various chemistries for oligonucleotide-templated reaction have been reported so far. Major scientific interests are focused on the development of signal amplification systems and signal generation systems. We introduce the recent advances of oligonucleotide-templated reaction in consideration of the above two points.

DOI： 10.3390/molecules17032446

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

A photo-responsive peptide aptamer against microbeads immobilized streptavidin was isolated using in vitro selection combined with photo-manipulation. This is the first example of the introduction of a peptide aptamer in the photo-control of dynamic molecular recognition.

DOI： 10.1039/c2cc36618e

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4-exo-trig Cyclization reaction of a 4-pentenyl carbon radical containing the gem-difluoromethylene moiety adjacent to a radical accepting alpha,beta-unsaturated ester was found to proceed efficiently to furnish a novel gem-difluorocyclobutane derivative. The cyclized product could be transformed into a gem-difluoromethylene analogue of oxetanocin T.

DOI： 10.1039/c2cc35876j

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Inc.  

RNA interference (RNAi) is a potent and highly specific gene-silencing phenomenon that was first reported for the nematode Caenorhabditis elegans. It has been discovered that genes could be silenced by introducing double-stranded RNAs (dsRNAs) complementary to the messenger RNA sequences. Since then, RNAi has been shown as an evolutionarily well-conserved process that plays an important role in host defense and in regulation of gene expression. Much effort has been dedicated to the application of the short dsRNA species (short interfering RNAs
siRNAs) as therapeutic agents, as they were shown to be effective in mammalian cells. Recently, we altered the structure of a siRNA molecule and produced dumbbell-shaped nanocircular RNAs. RNA dumbbells were shown to be stabilized in serum compared with its siRNA counterpart, despite their natural RNA strand. It has also been found that RNA dumbbells containing a 23-bp stem and two 9-nt loops exhibit a prolonged RNAi effect in cultured mammalian cells. In this unit, we describe the synthesis of RNA dumbbells from the design, its enzymatic synthesis, and to the purification. © 2012 by John Wiley &amp
Sons, Inc.

DOI： 10.1002/0471142700.nc1604s48

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.201201111

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：DOVE MEDICAL PRESS LTD  

Purpose: Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.
Methods: Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.
Results: It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.
Conclusion: Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.

DOI： 10.2147/IJN.S36350

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DOI： 10.1021/bc2003154

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

RNA interference (RNAi) is one of the most promising new approaches for disease therapy. The design of a dumbbell shaped nanocircular RNA allows it to act as a short interfering RNA (siRNA) precursor. To optimize the,design, we studied the relationship between the nanostructure and RNAi activity by synthesizing various RNA - dumbbells. An RNA dumbbell with a 23 bp stem and 9-nt loops was the most potent. Sequence analysis by mass spectrometry showed that Dicer could edit RNA dumbbells to siRNA species. The reaction offered the slow release of siRNA species, which conferred prolonged RNAi activity. Introduction of DNA into the loop position significantly stabilized the dumbbell in biological fluid without any loss of RNAi activity. In-depth pharmacological evaluation was performed by introducing dumbbells into HeLa cells that stably express : the target luciferase gene. The dumbbells provided a rapid silencing effect and retained this effect for a longer time even at a lower concentration than that at which standard siRNA completely lost RNAi activity. We conclude that an RNA dumbbell with DNA the most promising design for in vivo applications for RNA medicine.

DOI： 10.1021/bc203154

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Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.

DOI： 10.1021/ja205500y

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The synthesis of 4&apos;-ethynyl-2&apos;-deoxy-4&apos;-thioribonucleosides was carried out utilizing an electrophilic glycosidation in which 4-ethynyl-4-thiofuranoid glycal 16 served as a glycosyl donor. Electrophilic glycosidation between 16 and the silylated nucleobases (N(4)-acetylcytosine, N(6)-benzoyladenine, and N(2)-acetyl-O(6)-diphenylcarbamoylguanine) was carried out in the presence of N-iodosuccinimide (NIS), leading to the exclusive formation of the desired beta-anomers 29, 33, and 36. Anti-HIV studies demonstrated that these 4&apos;-thio nucleosides were less cytotoxic to T-lymphocyte (i.e., MT-4 cells) than the corresponding 4&apos;-ethynyl derivatives of 2&apos;-deoxycytidine (44), 2&apos;-deoxyadenosine (45), and 2&apos;-deoxyguanosine (46). Comparison of the selectivity indices (SI) was made between 4&apos;-thionucleosides (32, 41, and 43) and the corresponding 4&apos;-oxygen analogues 44-46 by using the reported CC(50) and EC(50) values. In the case of cytosine and adenine nucleosides, comparable SI values were obtained as follows: 32 (545) and 44 (458); 41 (&gt;230) and 45 (1630). In contrast, 4&apos;-ethynyl-2&apos;-deoxy-4&apos;-thioguanosine 43 was found to possess a SI value of &gt;18200, which is 20 times better than that of 46 (933).

DOI： 10.1021/ml2001054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Antibodies were covalently conjugated with poly(ethylene glycol) (PEG) and the properties of the PEGylated antibodies in organic media were investigated. Two types of monoclonal antibody were used in this study. One was a monoclonal antibody (abzyme) that was prepared against a hapten mimicking a transition state of hydrolysis. Another was a monoclonal antibody against estrogen, which is not soluble in water. By electrophoresis and mass spectral analysis, the covalent conjugation with PEG chains was confirmed. The PEGylated antibodies bound to antigens and the PEGylated abzyme catalyzed a hydrolysis reaction in an aqueous solution. The PEGylated antibodies were soluble in dichloromethane and acetone and interacted with antigen either in dichloromethane or in acetone. In conclusion, PEGylated antibodies can be employed as analytical tools for water-insoluble analytes. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2011.01.001

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Conformationally restricted indomethacin analogues were designed and prepared from the corresponding 2-substituted indoles, which were synthesized by a one-pot isomerization/enamide-ene metathesis as the key reaction. Conformational analysis by calculations, NMR studies, and X-ray crystallography suggested that these analogues were conformationally restricted in the s-cis or the s-trans form due to the 2-substituent as expected. Their biological activities on cyclooxygenase-1 (COX-1) inhibition, cyclooxygenase-2 (COX-2) inhibition, and modulation of MRP-1-mediated multidrug resistance (MDR) are described. Some of these indomethacin analogues enhanced doxorubicin cytotoxicity, although they do not have any COX inhibitory activity, which suggests that the MDR-modulating effect of an NSAID can be unassociated with its COX-inhibitory activity. This may be an entry into the combination chemotherapy of doxorubicin with a MDR modulator. © 2011 American Chemical Society.

DOI： 10.1021/ml100292y

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SOC POLYMER SCIENCE JAPAN  

We developed a new type of oligonucleotide-based catalysts which can be called DNAzyme, ribozyme, or aptazyme. They were in vitro selected from a random sequence library of DNA or RNA for binding hemin. The selected oligonucleotides exhibited peroxidase activity by forming a complex with hemin. In addition, an azobenzene-containing oligonucleotide which bound to hemin was also selected. The binding and the catalytic activity was controlled by photo-irradiation. Subsequently, hybridized biocatalysts with synthetic polymers were synthesized. Polyethylene glycol-conjugated antibodies and oligonucleotides were synthesized and it was demonstrated that they were active in organic media. A photo-responsive polymer was also conjugated with an enzyme and its catalytic activity was controlled in organic media.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

DOI： 10.1002/anie.201104425

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Small circular double-stranded RNAs (dsRNAs) were synthesized. The structural analysis by atomic force microscopy gave direct images for the interpretation of the structural strain present in circular dsRNAs. Finally, we demonstrated that circular dsRNA caused RNAi effect in cells.

DOI： 10.1039/c0cc04551a

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Branched RNAs with three- or four-way junctions were designed by assembling single-stranded RNA for RNA interference. Human Dicer transformed branched RNAs into about 20 base pairs of double-stranded RNA, which is a standard siRNA species. Our tetramer design provides a potent silencing effect over a period of 5 days.

DOI： 10.1039/c1cc11780g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Resistance against anticancer drugs remains a serious obstacle in cancer treatment. Here we used novel strategies to target microsomal glutathione transferase 1 (MGST1) and glutathione transferase pi (GSTP) that are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, including cisplatin and doxorubicin (DOX). By synthetically combining cisplatin with a GST inhibitor, ethacrynic add, to form ethacraplatin, it was previously shown that cytosolic GST inhibition was improved and that cells became more sensitive to cisplatin. Here we show that ethacraplatin is easily taken up by the cells and can reverse cisplatin resistance in MGST1 overexpressing MCF7 cells. A second and novel strategy to overcome GST mediated resistance involves using GST releasable cytostatic drugs. Here we synthesized two derivatives of DOX, 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) and 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) and showed that they are substrates for MGST1 and GSTP (releasing DOX). MGST1 overexpressing cells are resistant to DOX. The resistance is partially reversed by DNS-DOX Interestingly, the less reactive MNS-DOX was more cytotoxic to cells overexpressing MGST1 than control cells. It would appear that, by controlling the reactivity of the prodrug and thereby the DOX release rate, selective toxicity to MGST1 overexpressing cells can be achieved. In the case of V79 cells, DOX resistance proportional to GSTP expression levels was noted. In this case, not only was drug resistance eliminated by DNS-DOX but a striking GSTP-dependent increase in toxicity was observed in the clonogenic assay. In summary, MGST1 and GSTP resistance to cytostatic drugs can be overcome and cytotoxicity can be enhanced in GST overexpressing cells.

DOI： 10.1021/mp2000692

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Publisher：Kobunshi Ronbunshu  

We developed a new type of oligonucleotide-based catalysts which can be called DNAzyme, ribozyme, or aptazyme. They were in vitro selected from a random sequence library of DNA or RNA for binding hemin. The selected oligonucleotides exhibited peroxidase activity by forming a complex with hemin. In addition, an azobenzene-containing oligonucleotide which bound to hemin was also selected. The binding and the catalytic activity was controlled by photo-irradiation. Subsequently, hybridized biocatalysts with synthetic polymers were synthesized. Polyethylene glycol-conjugated antibodies and oligonucleotides were synthesized and it was demonstrated that they were active in organic media. A photo-responsive polymer was also conjugated with an enzyme and its catalytic activity was controlled in organic media. © 2011, The Society of Polymer Science Japan.

DOI： 10.1295/koron.68.405

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We describe herein the synthesis and properties of the novel nucleoside derivative, 4,7-diamino-2-(2-deoxy-beta-D-erythro-pentofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulene (1). The palladium catalyzed cross-coupling reaction of 2,4-diamino-5-iodo-7-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrrolo[2,3-d]pyrimidine (9) with acrylonitrile afforded 2,4-diamino-5-[(E)-1-cyano-2-ethenyl]-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine (10) in 77% yield, which was treated with NaOMe in MeOH in the presence of NaSPh to give the desired 1 in 64% yield. Whereas 1 was stable in concentrated ammonia at room temperature, it was gradually hydrolyzed in water to give 4-amino-2-(2deoxy-beta-D-erythro-pentofuranosyl)-2,6-dihydro-7H-2,3,5,6-tetraazabenzo[cd]azulen-7-one (12). Density functional calculations indicated that 12 was 20 kcal/mol more thermodynamically stable than 1 in a model study. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2010.08.063

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Publisher：ACS Symposium Series  

Aptamers are oligonucleotides that have been engineered through repeated rounds of in vitro selection or by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. Aptamers offer similar molecular recognition properties to the more commonly used antibodies. In addition to their ability to discriminate their target, aptamers offer some advantages over antibodies. For example, aptamers can be engineered completely in a test tube and are readily produced using chemical processes. On the other hand, some aptamers that have been developed exhibit enzyme activity after binding to their target molecule, and function as 'aptazymes'. Numerous aptamers and aptazymes have been used in biotechnology, diagnostics and therapy. In this study, we demonstrate that natural DNA/RNA aptamers produced by selection in vitro, not only bind to hemin, but also show peroxidase activity when in such complexes (i.e., they function as aptazymes). © 2010 American Chemical Society.

DOI： 10.1021/bk-2010-1043.ch009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A photoresponsive RNA aptamer to hemin was selected in vitro from a random sequence library of RNAs with azobenzene residues. The aptamer bound to hemin under visible light irradiation and was released by ultraviolet light. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2010.02.109

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Indole derivatives 3a and 3b of adenophostin A (2) in which the adenine of 2 was replaced with indole or 4-fluoroindole was designed as potential inositol trisphosphate receptor ligands. These target compounds were successfully synthesized from the key disaccharide unit 6. Biological evaluation showed that 3b selectively activates IP3R1, a subtype of IP3 receptors. © 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2009.12.045

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On the basis of the previous results on a histamine H4 receptor agonist 4-methylhistamine and a cyclopropane-based conformationally restricted analog CEIC (3) with potent H3/H4 receptor antagonistic effect, 4-methylhistamine analogs 4 and 5 of CEIC were designed and synthesized. Compound 4 showed strong affinity (Ki = 38.7 nM) for the H3 receptor, which was more potent than a well-known H3 antagonist thioperamide. Stable tautomer and conformation of 3 and 4, which can affect the pharmacological activity, were analyzed by ab initio calculations. © 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2009.12.046

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：AMER CHEMICAL SOC  

Aptamers are oligonucleotides that have been engineered through repeated rounds of in vitro selection or by the process of Systematic Evolution of Ligands by EXponential enrichment (SELEX) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. Aptamers offer similar molecular recognition properties to the more commonly used antibodies. In addition to their ability to discriminate their target, aptamers offer some advantages over antibodies. For example, aptamers can be engineered completely in a test tube and are readily produced using chemical processes. On the other hand, some aptamers that have been developed exhibit enzyme activity after binding to their target molecule, and function as 'aptazymes'. Numerous aptamers and aptazymes have been used in biotechnology, diagnostics and therapy. In this study, we demonstrate that natural DNA/RNA aptamers produced by selection in vitro, not only bind to hemin, but also show peroxidase activity when in such complexes (i.e., they function as aptazymes).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

A new photocaged fluorescent compound, azidomethyl fluorescein, was successfully utilized to monitor the dynamics of oligonucleotides in living human cells.

DOI： 10.1039/b926100a

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Publisher：2009 4th International Conference on Innovative Computing, Information and Control, ICICIC 2009  

For arch aorta surgery, cardiac surgeons approach to the diseased area with median incision and stemotomy according to the operation plan based on patient's medical images. Even using latest medical diagnosis instruments, however, it is impossible to estimate the shape of aneurysm, so that operation planning plan based on medical images is hard to be perfect. As the mentioned above, cardiac surgeons can not recognize the statement of patient's aneurysm before operation. Because of difficulty to completely recognize the statement of aneurysm from patient's medical images, operation plan often has to be changed during the operation. Therefore the coauthor cardiac surgeons highly expect a diagnosis support system that enables them to observe the shape of aneurysm from any angle and to virtually palpate it in order to make more safety operation plan. Then this study aims to develop of a diagnosis assistant system by using virtual reality and haptic interface under considering realtime interaction between them. © 2009 IEEE.

DOI： 10.1109/ICICIC.2009.194

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

We developed a new nucleic acid-based fluorescence probe for protein detection. The method is based on the scission of an aptamer into two probes, which are then attached with a chemically reactive fluorogenic compound. The protein-dependent association of the two probes accelerates a reduction-triggered fluorogenic reaction and indicates the presence of the target protein. which is detected using a fluorescence readout. The fluorescence signal is generated via the deprotection of the azidomethyl group of fluorescein. The arginine-rich motif peptide of the human immunodeficiency virus-1 Rev protein was targeted by this type of probe. Emission was detected at 522 nm and was enhanced by about 19.4-fold in the presence of the target peptide. An oligonucleotide-based reduction-1 triggered fluorescence probe was successfully applied to the defection of the Rev peptide ill solution.

DOI： 10.1248/cpb.57.1223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

A new thiol-reactive electrophilic, disubstituted rhodamine-based fluorogenic probe (bis-2,4-dinitrobenzenesulfonyl rhodamine [BDR]) with very high quantum yield was synthesized and described recently [A. Shibata et al., Bioorg. Med. Chem. Lett. 18 (2008) 2246-2249]. Because hydrophobic electrophiles are often conjugated by glutathione transferases, the BDR or monosubstituted rhodamine derivatives (2,4-dinitrobenzenesulfonyl rhodamine [DR]) were tested with microsomal glutathione transferase 1 (MGST1) and shown to function as substrates. The kinetic parameters for purified enzyme and DR were k(cat) = 0.075 +/- 0.005 s(-1) and K(m) = 21 +/- 3 mu M (k(cat)/K(m) = 3.6 x 10(3) +/- 5.6 x 10(2) M(-1) s(-1)), giving a rate enhancement of 10(6) compared with the nonenzymatic reaction. In cells overexpressing MGST1, the addition of BDR Caused a time-dependent increase of fluorescence compared with control cells. Preincubating the cells with a thiol reagent (N-ethylmaleimide) abolished the fluorescent signal. By using DR, we Could determine the MGST1 activity in whole cell extracts with high sensitivity. In addition, the activity could be increased by thiol reagents (a hallmark of MGST1). Thus, we have identified a new fluorogenic substrate for MGST1 that will be a useful tool in the study of this enzyme and related enzymes. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2009.03.046

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and P-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.

DOI： 10.1021/bc900040t

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Catalytic RNAs with peroxidase activity were obtained by the in vitro selection of RNA aptamer-binding hemin. One of the RNA aptamers selected showed binding affinity to hemin with a dissociation constant of 0.8 mu M and exhibited high peroxidase activity by forming a complex with hemin. The catalytic efficiency of the RNA-hemin complex was 10-fold higher than that of hemin alone. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2009.01.016

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

A novel hemin-binding DNA was synthesized using in vitro selection (SELEX) method. The selected DNA not only bound to the hemin. but also had peroxidase activity when complexed with hemin. A pool of 104-nt single-stranded DNA (ssDNA) molecules containing a randomized sequence of 60nt was synthesized. The DNA pool having random sequences was incubated with a hemin-immobilized affinity column. Bound DNAs were eluted with hemin solution and amplified by PCR with biotin-labeled primers. ssDNAs were isolated from the biotin-labeled double-stranded DNA (dsDNA) molecules after each selection process. After four rounds of selection process, the selected DNAs were cloned and sequenced. Four DNA aptamers were chemically synthesized from the sequenced clones. Two aptamers exhibited binding, affinity to hermin and peroxidase activity. A 21-nt oligonucleotide was designed from the aptamer sequence that formed a complex with hermin and exhibited high peroxidase activity.

DOI： 10.1246/bcsj.82.99

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We have developed an SNAr reaction-triggered fluorescence probe using a new fluorogenic compound derivatized from 7-aminocoumarin for oligonucleotides detection.

DOI： 10.1039/b912896d

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We have developed a new red fluorogenic compound derived from naphthorhodamine for a reduction-triggered fluorescence probe to sense oligonucleotides. The fluorogenic reaction between naphthorhodamine azide derivatives and reducing reagents such as triphenylphosphine (TPP) on the DNA target does not use any enzyme or reagent, and fluoresces at 650 nm. The probes were used for dual color detection of a single nucleotide difference on the leukemia-related bcr/abl gene.

DOI： 10.1039/b817228e

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DOI： 10.1093/nass/nrp033

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DOI： 10.1093/nass/nrp079

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Publisher：Nucleic acids symposium series (2004)  

A new RNA aptamer-binding hemin was synthesized by the in vitro selection (SELEX) method. A pool of 103 bases single strand DNAs containing a randomized sequence of 59 bases was synthesized. The pool was incubated with hemin on hemin-immobilized affinity column. Bound RNAs were eluted off with hemin solution and amplified by PCR. After 3 rounds of selection process, the selected RNAs were cloned and sequenced. Some RNA aptamers, which have affinity to hemin, was selected.

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Publisher：Nucleic acids symposium series (2004)  

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops(1). RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme, and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.

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Publisher：Nucleic acids symposium series (2004)  

A reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from rhodamine 110 was developed for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The reaction proceeds under biological conditions to produce fluorescence signal within 10-20 min in the presence of target DNA or RNA. The probes were successfully applied to the detection of oligonucleotides in solution and endogenous RNA in bacterial cells.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We demonstrate a generation of tandem repeats of a malachite green (MG) RNA aptamer using rolling circle transcription. To keep the higher-order structure of each aptamer on long RNA, we designed a sequence of circular DNA with a 14-base linker. T7 RNA polymerase was superior to Escherichia coli RNA polymerase in the specific transcription of the MG RNA aptamer. Finally. the generation of the fluorescence signal was confirmed from aptamer repeats with MG. (c) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2008.07.040

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonticleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable tinder biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.

DOI： 10.1021/bc800014d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We have developed a new fluorescent probe for biological thiol. The probe was synthesized by the modi. cation of the 2,4-dinitrobenzenesulfonyl group with rhodamine 110. The selective detection of thiol species such as cysteine or glutathione was achieved in biological conditions. Moreover, the probe was successfully applied to the imaging of thiol species in living human cells. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2008.03.014

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Synthesis of W-branched BCA analogues (5) was carried out. Stereospecific construction of the cis-disposed 4'-carbon-substituents and 5'-hydroxymethyl group was secured by employing the bicyclo[3.3.0]lactone 16 as a key intermediate, which was prepared by radical-mediated intramolecular S(H)2' cyclization of the phenylselenomethyl ester 15. After manipulation of the double bond of 16, bis(Boc)adenine was introduced based on the Mitsunobu reaction of the allyl alcohol 24. Transformation of the lactone function of 27 allowed preparation of the 4'-hydroxymethyl (31). the 4'-vinyl (32), the 4'-cyano (34), and the 4'-ethynyl (35) derivatives. Anti-HIV and anti-HCV activities of the free nucleosides 36-38 were also examined. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2007.11.038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

RNA aminoacylation, based on an adenosine transfer mechanism, was achieved by the intermolecular transfer of phenylalaninyl adenosine from a donor probe to an acceptor RNA. This method can be applied to a wide variety of unnatural amino acids for tRNA aminoacylation.

DOI： 10.1246/cl.2008.102

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Language：Japanese   Publisher：SOC SYNTHETIC ORGANIC CHEM JPN  

Despite considerable progress and extensive effort, a general method for highly stereoselective glycosylation particularly for the 1,2-cis-glycosylation has not yet been developed and therefore is required. The alpha/beta-stereo selectivity in glycosylation can be affected by the steric and stereoelectronic (anomeric) effects around the anomeric center, which depend on the conformation of the glycosyl donor substrates. Therefore, we hypothesized that highly alpha- and beta-selective glycosylation can be realized by employing conformationally restricted substrates. We showed that the alpha/beta-stereoselectivity was significantly increased by the conformational restriction and was completely inverted by changing the substrate conformation from the C-4(1)-form into the C-1(4)-form in radical and nucleophilic C-glycosylation reactions as well as in O-glycosylation reactions. The conformational restriction of substrates also effectively facilitates the alpha- and beta-selective radical cyclization reaction at the anomeric position. Using the method, C-glucoside trisphosphates designed as Ca2+-mobilizing agents were successfully,synthesized.

DOI： 10.5059/yukigoseikyokaishi.66.50

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Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.

DOI： 10.1021/bc700244s

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DOI： 10.1093/nass/nrn178

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Language：English   Publishing type：Part of collection (book)   Publisher：Humana Press  

DOI： 10.1007/978-1-60327-040-3_11

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DOI： 10.1093/nass/nrn260

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DOI： 10.1093/nass/nrn256

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops. RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme and are thus transformed into double-stranded RNA in cells, although this RNA is resistant to degradation in serum. The structure was optimized to maximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.

DOI： 10.1021/ja0754453

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DOI： 10.1093/nass/nrm177

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DOI： 10.1093/nass/nrm183

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Previous structure-activity relationship studies of adenophostin A, a potent IP3 receptor agonist, led us to design the novel adenophostin A analogues 5a-c, conjugating an aromatic group at the 5'-position to develop useful IP3 receptor ligands. The common key intermediate, a D-ribosyl alpha-D-glucoside 10 alpha, was stereoselectively synthesized by a glycosidation with the 1-sulfinylglucoside donor 11, which was conformationally restricted by a 3,4-O-cyclic diketal protecting group. After introduction of an aromatic group at the 5-position of the ribose moiety, an adenine base was stereoselectively introduced at the anomeric beta-position to form 7a-c, where the tetra-O-i-butyryl donors 9a-c were significantly more effective than the corresponding O-acetyl donor. Thus, the target compounds 5a-c were synthesized via phosphorylation of the 2', 3", and 4"-hydroxyls. The potencies of compounds 5a-c for Ca2+ release were shown to be indistinguishable from that of adenophostin A, indicating that bulky substitutions at the 5'-position of adenophostin A are well-tolerated in the receptor binding. This biological activity of 5a-c can be rationalized by molecular modeling using the ligand binding domain of the IP3 receptor.

DOI： 10.1021/jm060310d

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DOI： 10.1021/ol0602710

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

5'-Deoxy-5'-phenyladenophostin A (5), designed as a useful IP3 receptor ligand based on the previous structure-activity relationship studies, was successfully synthesized via two key stereoselective glycosidation steps. This compound proved to be a highly potent IP3 receptor agonist.

DOI： 10.1021/o10602710

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beta-C-Glucoside trisphosphates having a C2 side chain (3,7-anhydro-2-deoxy-D-glycero-D-gulo-octitol 1,5,6-trisphosphate, 11) and a C3 side chain (4,8-anhydro-2,3-dideoxy-D-glycero-D-gulo-nonanitol 1,6,7-trisphosphate, 12) were designed as structurally simplified analogues of a potent D-myo-inositol 1,4,5-trisphosphate (IP3) receptor ligand, adenophostin A. Construction of the beta-C-glucosidic structure, which was the key to their synthesis, was achieved by two different methods based on the conformational restriction strategy: (1) radical cyclization with a temporary connecting silicon tether and (2) silane reduction of glyconolactols having an anomeric allyl substituent. Using these methods, the target beta-C-glycoside trisphosphates 11 and 12 were successfully synthesized. A structure-activity relationship was established on a series of C-glucoside trisphosphates, including the previously synthesized related compounds, which were a C-glycosidic analogue 3 of adenophostin A, its uracil congener 5, alpha-C-glucoside trisphosphates 7-9 having a C1, C2, or C3 side chain, and the beta-C-glucoside trisphosphates 10-12 having a C1, C2, or C3 side chain. The O-glycosidic linkage of adenophostin A and its analogues proved to be replaced by the chemically and biologically more stable C-glycosidic linkage. The alpha-C2-glucoside trisphosphate 8 stimulates Call release with a potency similar to that of IP3 in spite of its simplified structure, indicating a better fit to the receptor than the beta-C-glucoside trisphosphates and also the alpha-congeners having a shorter or longer C1 side chain, which was supported by molecular modeling using the ligand binding domain of the IP3 receptor.

DOI： 10.1021/jm051039n

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DOI： 10.1073/pnas.0509938103

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrl013

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

4,8-Anhydro-D-glycero-D-ido-nonanitol 1,6,7-trisphosphate (9), designed as a novel IP3 receptor ligand having an alpha-C-glycosidic\ structure, was synthesized via a radical cyclization reaction with a temporary connecting allylsilyl group as the key-step. Phenyl 2-O-allyldimethylsilyt-3,4-bis-O-TBS-1-seleno- beta-D-glucopyranoside (10a), conformationally restricted in the unusual C-1(4)-conformation, was treated with Bu3SnH/AIBN to form the desired alpha-cyclization product 16a almost quantitatively. On the other hand, when a conformationally unrestricted O-benzyl-protected 2-O-allyldimethylsilyl -l-selenoglucoside 15 was used as the substrate, the radical reaction was not stereoselective and gave a mixture of the alpha-and beta-products. From 16a, the target C-glucoside trisphosphate 9 was synthesized via phosphorylation of the hydroxyls by the phosphoramidite method. During the synthetic study, an efficient procedure for the oxidative C-Si bond cleavage, via a nucleophilic substitution at the silicon with p-MeOPhLi followed by Fleming oxidation, was developed. The C-glycoside 9 was found to be a full agonist for Ca2+ mobilization, although its activity was weaker than that of the natural ligand IP3. Thus, the alpha-C-glucosidic structure was shown to be a useful mimic of the myo-inositol backbone of IP3. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2005.02.025

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DOI： 10.1093/nass/49.1.37

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We previously found that Grignard addition to a C-cyclopropylaldonitrone, C-[cis-2-(N,N-diethyl-carbamoyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (1), stereoselectively gave the antiproduct 3, in which the stereoselectivity was particularly high when MgBr2 was the additive. In this study, the reaction pathway was investigated in detail. The stereoselective addition was initially thought to occur via either a 1,5-chelation-controlled or a bisected s-trans conformation-controlled pathway. However, Grignard addition to a nonchelating silyl ether-type substrate, C-[cis-2-(tert-butyldiphenylsilyloxymethyl)-trans-2-phenylcyclopropyl]-N-benzylaldonitrone (7), also gave the antiproduct 9 with high stereoselectivity suggesting that chelation is not important in the reaction. Theoretical calculations of C-cyclopropylaldonitrones showed that the coordination of Mg2+ at the nitrone oxygen significantly stabilizes the bisected s-trans conformer due to the effective hyper-conjugation between the,pi* of the nitrone C=N bond and the electron-donating cyclopropane orbitals. This kind of orbital interaction is able to stabilize the transition state of the nucleophilic addition and is maximized in the bisected conformation, in which the orbitals of the forming bond and the cyclopropane C-C bond are in an almost planar arrangement. Thus, the high stereoselectivity can be explained by nucleophilic attack on the less hindered side of the C=N bond of the substrates in the Mg2+-coordinated bisected s-trans conformation.

DOI： 10.1021/jo048637e

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DOI： 10.1021/ja046791c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The reduction of glyconolactols having an anomeric carbon substituent by Et3SiH/TMSOTf proceeded with complete stereoselectivity to produce the corresponding beta-C-glycosides when the substrates were conformationally restricted in the C-4(1)-chair form by a 3,4-O-cyclic diketal or a 4,6-O-benzylidene protecting group. Thus, the efficient construction beta-C-glycosides was achieved on the basis of the conformation restriction strategy.

DOI： 10.1021/ol048525+

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DOI： 10.1016/j.tetlet.2004.07.065

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The addition reaction of carbon nucleophiles to cis-substituted cyclopropanecarbaldehydes was systematically investigated. Ab initio calculations of model cyclopropanecarbaldehydes suggested that the bisected s-cis and s-trans conformers are the only two minimum energy conformers, which are stabilized due to the pi-donating stereoelectronic effect of the cyclopropane ring. The experimental results of a series of substrates, that is, cyclopropanecarbaldehydes 1-5 bearing a cis-(tert-butyldiphenyisilyloxy)methyl group, a cis-benzyloxymethyl group, a cis-(p-methoxybenzyloxy)methyl group, cis-N,N-diethylcarbamoyl and trans-phenyl groups, and cis-(tert-butyldiphenylsilyloxy)methyl and trans-phenyl groups, respectively, showed that highly anti-selective Grignard additions could be realized. It turned out that it occurred via an unusual 7-membered 1,4-chelation-controlled pathway. Highly stereoselective Grignard addition via the chelation-controlled pathway occurred even in the reaction of the usually non-chelating silyl ether-type substrate 5. The results have great importance because the 1,4-chelation-controlled stereoselective addition reactions can indeed be realized. Under non-chelation conditions, the syn-products were produced with moderate stereoselectivity, which are likely to be formed via the bisected s-cis conformation-like transition state stabilized by the characteristic orbital interaction. These reactions, especially the chelation-controlled reaction, should be useful because of their t stereoselectivity and stereochemical predictability. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2004.05.063

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at adjacent sites, and "light up" on nucleophilic displacement of the dabsyl probe by the phosphorothioate probe. Four fluorescent dye conjugates were prepared and tested with probes and targets that differ by one nucleotide. Experiments on polymer beads show clear color-based discrimination of DNAs added in solution. Two-color quenched probe pairs were then tested in the discrimination of 16S rRNA sequences in Escherichia coli. Single nucleotide resolution was achieved in the cells with green/red QUAL probes, allowing identification of a one-base sequencing error in the 16S rRNA database. Finally, QUAL probes were successfully applied in live bacterial cells. The method requires only incubation followed by fluorescence imaging, and requires no enzymes, added reagents, cross-linking, fixing, or washes. Because probes must bind side-by-side to generate signal, there is little or no interference from unintended protein binding, which can occur with other probe types. The results suggest that QUAL probes may be of general use in the detection and identification of sequences in solution, on microarrays, and in microorganisms.

DOI： 10.1021/ja038665z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

(1S,2S)-, (1S,2R)-, and (1R,2S)-1-(2,4-Dimethylphenyl)piperazyl-2-phenylcyclopropane (2a, 3, and ent-3, respectively), which were designed as conformationally restricted analogues of haloperidol (1), a clinically effective antipsychotic agent, were synthesized from chiral epichlorohydrins using the Barton reductive radical decarboxylation as the key step. (1S,2R)-1-(tert-Butyldiphenylsilyloxy)methyl-2-carboxy-2-phenylcyclopropane (5), which was prepared from (S)-epichlorohydrin ((S)-7), was converted into its N-hydroxypyridine-2-thione ester 12, the substrate for the reductive radical decarboxylation. When 12 was treated with TMS(3)SiH in the presence of Et(3)B or AIBN, the decarboxylation and subsequent hydride attack on the cyclopropyl radical intermediate from the side opposite to the bulky silyloxymethyl moiety occurred, resulting in selective formation of the corresponding reductive decarboxylation product 4-cis with the cis-cyclopropane structure. From 4-cis, the cis-cyclopropane-type target compound 3 was readily synthesized. Starting from (R)epichlorohydrin ((R)-7), ent-3 was similarly synthesized. Epimerization of the cyclopropanecarboxamide ent-16-cis, a synthetic intermediate for ent-3, on treatment with a base prepared from Bu(2)Mg and i-Pr(2)NH in THF occurred effectively to give the corresponding trans isomer 16-trans, which was converted into 2a with the trans-cyclopropane structure.

DOI： 10.1021/jo0302206

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We previously theorized that, since the stereoselectivity of anomeric radical reactions is significantly influenced by the kinetic anomeric effect, which can be controlled by restricting the conformation of the radical intermediate, the proper conformational restriction of the pyranose ring of the substrates would therefore make highly alpha- and beta-stereoselective anomeric radical reactions possible. This theory was based on our previous results of the anomeric radical reactions with D-xylose derivatives as the substrates. We herein report the anomeric radical deuteration reactions with the conformationally restricted 1-phenylseleno-D-glucose derivatives, 2g and 3g, restricted in a C-4(1)-conformation by an beta-cyclic diketal moiety, and 4g, 5g, 6g, 7g, and 8g, restricted in a C-1(4)-conformation by bulky O-silyl protecting groups. The radical deuterations with Bu3SnD, using the C-4(1)-restricted substrates 2g and 3g, afforded the corresponding alpha-products (alpha/beta = 98:2) highly stereoselectively, whereas the C-1(4)-restricted substrate 6g, having a trigonal (sp(2)) carbon substituent, i.e., -CHO, at the 5-position, selectively gave the beta-products (alpha/beta = 0: 100). Thus, the stereoselectivity was significantly increased by the conformational restriction and was completely inverted by changing the substrate conformation from the C-4(1)-form to the C-1(4)-form. On the other hand, the deuterations with the C-1(4)-restricted substrates 4g and 5g showed that the 1,5-steric effect due to the tetrahedral carbon substituent (-CH2OTIPS or -CH2OH) at the 5-axial position dominantly prevented the hydride transfer from the beta-face competing with the kinetic anomeric effect. This study suggests that, depending on the restricted conformation of the substrates to the C-4(1)- or the C-1(4)-form, the alpha- or beta-products would be obtained highly stereoselectively via anomeric radical reactions of hexopyranoses.

DOI： 10.1021/jo030128+

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The stereoselective hydride reduction of the cis- and trans-substituted cyclopropyl ketones was systematically investigated using a series of structurally simplified substrates, trans-[tert-butyldiphenylsilyloxymethyl]cyclopropyl ketones la-a and trans- (benzyloxymethyl)cyclopropyl methyl ketone (2), and the corresponding cis congeners 3a,b,e and 4. The results showed that, not only in the reduction of the cis-substituted cyclopropyl ketones but also in that of the trans-substituted ketones, high stereoselectivity can be realized when the substrate has a bulky substituent on the cyclopropane ring, even though it is attached to the position trans to the acyl moiety. Ab initio calculations based on the density functional theory (DFT) of cyclopropyl ketones showed that (1) the bisected s-cis and s-trans conformers were the only two minimum energy conformers; while the s-cis conformer was more stable than the s-trans and (2) a bulky alkyl group in the acyl moiety and a cis substituent on the cyclopropane ring made the bisected s-cis conformer much more stable. On the basis of these calculations and experimental results, it is likely that the more stable the bisected s-cis conformer of the substrate, the more stereoselective the hydride reduction. Thus, the stereochemistry can be explained by hydride attack on the bisected s-cis conformation of the substrate from the less-hindered face. The predictability of the stereochemical results is predicated on the bisected s-cis transition-state model, which is very important from the viewpoint of synthetic organic chemistry.

DOI： 10.1021/jo030019v

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/anie.200390261

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Publishing type：Part of collection (book)   Publisher：Elsevier  

DOI： 10.1016/b978-044450951-2/50002-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We hypothesized that, because the stereoselectivity of anomeric radical reactions was significantly influenced by the anomeric effect, which can be controlled by restricting the conformation of the radical intermediate, the proper conformational restriction of the pyranose ring of the substrates would therefore make highly alpha- and beta -stereoselective anomeric radical reactions possible. Thus, the conformationally restricted 1-phenylseleno-D-xylose derivatives 9 and 10, restricted in a C-4(1)-conformation, and 11 and 12, restricted in a C-1(4)-conformation, were designed and synthesized by introducing the proper protecting groups on the hydroxyl groups on the pyranose ring as model substrates for the anomeric radical reactions. The radical deuterations with Bu3SnD and the C-glycosylation with Bu3SnCH2CH=CH2 or CH2 CHCN, using the C-4(1)-restricted substrates 9 and 10, afforded the corresponding alpha -products (alpha/beta = 97:3-85:15) highly stereoselectively, whereas the C-1(4)-restricted substrates 11 and 12 selectively gave the fl-products (alpha/beta = 1:99-0:100). Thus, stereoselectivity was significantly increased by conformational restriction and was completely inverted by changing the substrate conformation from the C-4(1)-form into the C-1(4)-form. Ab initio, calculations suggested that the radical intermediates produced from these substrates possessed the typical C-4(1)- or C-1(4)-Conformation, which was similar to that of the substrates, and that the anomeric effect in these conformations would be the factor controlling the transition state of the reaction. Therefore, the highly alpha- and beta -selective reactions would occur because of the anomeric effect, which could be manipulated by conformational restriction of the substrates, as expected. This would be the first radical C-glycosylation reaction to provide both alpha- and beta -C-glycosides highly stereoselectively.

DOI： 10.1021/ja011321t

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An efficient method for preparing fully O-silylated pyranoses, which are conformationally restricted in the unusual C-1(4)-form, was developed. Thus, successive treatment of pyranosides, such as xylose and glucose derivatives, with NaH and TIPSOTf or TBSOTf in THF at room temperature gave the corresponding fully O-silylated products. (C) 2001 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0040-4039(01)01233-3

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Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-8. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the E-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.

DOI： 10.1021/bc000135u

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DOI： 10.1081/ncn-100002320

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An efficient synthesis of 1-phenyl-2-[(S)-1-aminoethyl]- and 1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamides [1a (PEDC) and 1b (PPDC), respectively], potent NMDA receptor antagonists having a cyclopropane structure, was achieved. We have shown for the first time that C-cyclopropylaldonitrone preferentially exists in the bisected s-trans conformation, due to the characteristic stereoelectronic effects of the cyclopropane ring, by X-ray crystallographic analysis, NMR studies, and theoretical calculations. Based on these findings, the highly stereoselective addition reaction of Grignard reagents to C-cyclopropylaldonitrone 6 was developed, and the reaction was successfully used as the key step for the preparation of the NMDA receptor antagonists 1a and 1b as well as for a newly designed isopropyl-type congener 1c. The facial selectivity of the addition of Grignard reagents can be explained by the attack of the reagents from the less hindered side of the substrate in the predicted bisected s-trans conformation. This Grignard reaction is the first example of a highly stereoselective addition to a nitrone via a non-chelation controlled pathway.

DOI： 10.1039/b008230i

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

An efficient stereoselective synthesis of 4'-beta-thioribonucleosides 14, 15, 27, and 30 using the Pummerer reaction as the key step is described. The Pummerer reaction of 1,4-anhydro-2-0-(2,4-dimethoxybenzoyl)-3,5-O-( 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-3-sulfinyl-D-ribitol (R-10:S-10 = 2.7:1) in the presence of silylated uracil afforded the desired p-anomer of the 4'-thiouridine derivative 11 in 66% yield without formation of its a-anomer. The reaction with R-10 gave 11 in 87% yield, while the one with S-10 resulted in a 27% decrease of the desired product 11 along with a 22% yield of 3,6-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-3-hydroxy-2-hydroxymethylthiophene (12). A likely explanation for the observed difference in the reaction of R-10 and S-10 is that the reaction proceeds via an E2 type pathway, which prefers anti elimination. Thus, R-10 would preferentially afford the alpha-thiocarbocation intermediate 21 via an E2 anti elimination under the reaction conditions. The resulting 21 would be expected to react with silylated uracil stereoselectively to give 11 in good yield. However, formation of the more stable tertiary alpha-thiocarbocation intermediate 23, which would prefer to give 12 and/or to decompose, would compete with the formation of the desired 21 in the reaction with S-10. Consequently, this argument would explain the low yields of the desired product 11 and the poor mass balance in the reaction with S-10. When the sulfoxide 10 (R-10:S-10 = &gt;16:1) prepared by oxidation of 9 with ozone was used for the Pummerer reaction, the desired 11 was obtained in 80% yield. Compound 11 was converted to 4'-beta-thiouridine (14) by treatment of 11 with ammonium fluoride, followed by methanolic ammonia. Similarly, 4'-beta-thiocytidine (15) was prepared when silylated N-4-acetylcytosine was used in the Pummerer reaction. For the Pummerer reactions with purine bases, 6-chloropurine and 2-amino-6-chloropurine were found to be the most suitable. When the reactions were conducted in a mixture of acetonitrile and 1,2-dichloroethane at room temperature, followed by reflux, the desired products 25 and 28 were obtained in 65% and 56% yields, respectively. These compounds were then converted to 4'-beta-thioadenosine (27) and 4'-beta-thioguanosine (30) under the usual conditions. This is therefore the first time that the stereoselective synthesis of 4'-beta-thioribonucleosides has been performed using the neighboring group participation of the Pummerer reaction.

DOI： 10.1021/ja000541o

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Synthesis of the C-glycosidic analogue 9 of adenophostin A, a very potent IP3 receptor agonist, and its uracil congener 10 was achieved via a temporary silicon-tethered radical coupling reaction as the key step. Phenyl 3,4,6-tri-O-(p-methoxybenzyl)-1-seleno-beta-D-glucopyranoside (27) and 3-deoxy3-methylene-1,2-O-isopropylidene-alpha-D-erythro (30) were connected by a dimethylsilyl tether to give the radical coupling reaction substrate 24, which was successively treated with Bu-3-SnH/AIBN in benzene and TBAF in THF to give the coupling product 25 with the desired (3a,1'a)-configuration as the major product. From 25, the targets 9 and 10 were synthesized via introduction of adenine or uracil base by Vorbruggen's method and phosphorylation of the hydroxyls by the phosphoramidite method.

DOI： 10.1021/jo0001333

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

An efficient method for preparing both 1 alpha- and 1 beta-C-glucosides having a 3-hydroxypropyl group at the anomeric position via a radical cyclization reaction with an allylsilyl tether was developed. The stereoselectivity of the radical cyclization can be controlled by the conformation of the pyranose ring, which is effectively manipulated by the hyroxyl protecting groups. (C) 2000 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0040-4039(00)00556-6

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Synthesis of the C-glycosidic analog (3) of adenophostin A, a very potent IP3 receptor agonist, was achieved using a temporary silicon-tethered reductive radical coupling reaction as the key step. Radical reaction of the silaketal substrate 6 with Bu3SnH/AIBN in benzene occurred stereoselectively, and subsequent desilylation gave the desired C-glycosidic disaccharide 7 with the (3 alpha,1'alpha)-configuration as the major product. Compound 7 was converted into the target 3 via the introduction of an adenine base by a Vorbruggen glycosylation reaction. (C) 2000 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0040-4039(00)00171-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A mechanistic study was performed on a novel radical ring-enlargement reaction of (3-oxa-2-silacyclopentyl)methyl radicals into 4-oxa-3-silacyclohexyl radicals. Two pathways, one via a pentavalent silicon bridging radical transition state (or intermediate), the other via beta-elimination to give a ring-opened silyl radical, can be postulated. The radical reactions of 1 and 2, which are precursors for a (3-oxa-2-silacyclopentyl)methyl radical C' and a 4-oxa-3-silacyclohexyl radical D', respectively, showed that the ring-enlargement rearrangement of C' into D' is irreversible. H-1 NMR analysis of the radical reactions of 8a and 8b, which have an asymmetric center at silicon, indicated that the configuration at the silicon atom is retained via a pentavalent silicon-bridging radical transition state (or intermediate) during the ring-enlargement reaction. Furthermore, examination of the radical ring-enlargement reaction with a deuterium-labeled substrate 12D showed that the ring-enlargement reaction did not involve beta-elimination to give a ring-opened silyl radical. Based on these results, we conclude that the ring-enlargement reaction occurs via a pentavalent silicon-bridging radical transition state (or intermediate). This is the first experimental evidence for such a pentavalent silicon radical, which has been previously postulated to understand radical reactions of organic silicon compounds.

DOI： 10.1021/ja993239s

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Dipeptide transporters in small intestine have a very wide substrate specificity, so that the transporter sometimes serves as a carrier for peptide-like compounds. We have synthesized dipeptide analogues conjugated at an E-amino group of Lys in Val-Lys or Lys-Sar with fluorescent compounds such as fluorescein isothiocyanate and coumarin-3-carboxylic acid. Uptakes of these peptide analogues were examined by measuring intracellular accumulations into monolayers of the human intestinal epithelial cell line Caco-2 expressing the dipeptide transporter PEPT1. Kinetic analysis and effects of addition either of uncoupler (protonophore) or by Gly-Sar, one of the good substrates of PEPT1, revealed that fluorescent dipeptides were taken up by passive diffusion. In contrast, these analogues remarkably inhibited the Gly-Sar uptake by Caco-2 cells. Among the fluorescent analogues synthesized in this paper, Vaf-Lys(Flu) was the most potent competitive inhibitor against the Gly-Sar uptake with an inhibition constant of 5 mu M. This value is the smallest among those ever reported: Val-Lys(Flu) has the highest affinity for PEPT1 among chemicals ever reported. The importance of the hydrophobic part of the substrate was pointed out.

DOI： 10.1021/bc980049i

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DOI： 10.1016/j.actbio.2014.05.034

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