Frontiers in Organic Chemistry

ISSN: 1574-0900 - 1 Issue in 2006

Frontiers in Organic Chemistry, Volume 1, No. 1, 2005

Contents

Editorial: Pp.v-vi
Yoshihiro Hayakawa
[Abstract]


Contributors Pp.ix-xii
[Abstract]


Strategies Useful for the Chemical Synthesis of Oligonucleotides and Related Compounds Pp.3-40
Masaki Tsukamoto and Yoshihiro Hayakawa
[Abstract]


Stereocontrolled Synthesis of Phosphorothioate DNA by the Oxazaphospholidine Approach Pp.41-61
Takeshi Wada
[Abstract]


Oligonucleotides Bearing a 5-Substituted Pyrimidine Nucleoside: Their Synthesis, Properties, and Application Pp.63-78
Hiroaki Ozaki, Masayasu Kuwahara and Hiroaki Sawai
[Abstract]


4’-Thionucleic Acids: Chemistry, Properties, and Applications for Developing Functional Oligonucleotides Pp.79-102
Noriaki Minakawa, Shuichi Hoshika, Naonori Inoue, Yuka Kato and Akira Matsuda
[Abstract]


Creation of Conformationally Rigid Bent and Linear Nucleic Acids by 3-Dimensional Fixation of Conformation of Mono- and Di-nucleotide Building Blocks Pp.103-128
Mitsuo Sekine, Koh-ichiroh Shohda and Kohji Seio
[Abstract]


Chemical Approach to Probing Different DNA Structures Pp.129-144
Yan Xu and Hiroshi Sugiyama
[Abstract]


Molecular Design for Specific Recognition and Reaction in Genome-Targeting Chemistry Pp.145-162
Shigeki Sasaki and Fumi Nagatsugi
[Abstract]


Base and Backbone Modified Oligonucleotides and their Application As Antisense Agent Pp.163-194
Tomohisa Moriguchi and Kazuo Shinozuka
[Abstract]


Artificial Ribonucleases as Antisense Compounds Pp.195-207
Hideo Inoue
[Abstract]


Sugar-Modified Nucleic Acid Analogues as Potential Materials for Genomic Technologies Pp.209-228
Takeshi Imanishi and Satoshi Obika
[Abstract]


Synthesis, Biological Properties and Antisense Effects of Oligonucleotide-Petide Conjugates Pp.229-241
Takanori Kubo, Ruminana Bakalova, Zhivko Zhelev, Hideki Ohba and Masayuki Fujii
[Abstract]


Functional Artificial Nucleic Acids: Peptide Ribonucleic Acids (Prnas) - Novel Strategy for Active Control of DNA Recognition by External Factors
Pp.243-277
Takehiko Wada, Hirofumi Sato and Yoshihisa Inoue
[Abstract]


Nucleic Acid Seqeuence Analysis Using Oligonucleotide Probes Pp.279-296
Mitsunobu Nakamura, Kenji Kanaori and Kazushige Yamana
[Abstract]


Reaction of NO with Nucleic Acid Bases and its Biological Implication
Pp.343-355
Seung Pil Pack, Toshinori Suzuki, Hiroshi Ide, Tsutomu Kodaki and Keisuke Makino
[Abstract]

Abstracts

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Editorial
Yoshihiro Hayakawa

In recent years, important biological activities of nucleic acids, and in particular, those of oligonucleotides, which are small fragments of nucleic acids, have been identified. The importance of nucleic acids has thus been shown not only as genes but also as biologically functional molecules. As a result, it has become important to understand the mechanisms by which these biological functions are expressed at the molecular level. Furthermore, the creation of functionally useful artificial nucleic acids (nucleic acids having new functions or improved functions that are not possessed by natural nucleic acids in biological molecular assemblies) has also become an important research theme. This type of research is now being carried out extensively.
In order to carry out such research effectively and precisely, an approach using various chemical strategies/techniques in addition to biological strategies/techniques is necessary. For example, it is essential to use organic chemical structural analysis to understand the function of nucleic acids at the molecular level. This analysis can be conducted using various chemical instrument methods, including nuclear magnetic resonance (NMR). Simulations with various chemical calculations will be of great help for the design of artificial nucleic acids with useful functions. The creation of artificial nucleic acids may be achieved only by chemical synthesis; it is extremely difficult or generally impossible to synthesize artificial nucleic acids by biological methods. To date, it has been perceived that biological researchers in the fields of molecular biology, pharmaceutical science and medical science would play a primary role in the research of nucleic acids. The role of chemists was considered a secondary or largely supporting role for biological researchers. That might have been true when nucleic acid research was started decades ago, but this concept is no longer valid. In recent years, chemistry has been able to play the main role in much nucleic acid research. There is no doubt that further nucleic acid research will only be accomplished with the active participation of chemists. In the pursuit of the above research, namely the clarification of nucleic acid function at the molecular level and the creation of artificial nucleic acids with novel functions, it is vital that chemists and biological researchers collaborate on equal terms.
In such collaboration, chemists should recognize that nucleic acid research is a theme of chemical research, and chemists should realize how they can contribute to nucleic acid research. It is therefore imperative to let chemists know the importance of chemistry and its necessity in nucleic acid research. However, there are few books in which the nucleic acid research is introduced from a chemistry viewpoint. We thus planned the publication of this book in order to let chemists know the importance of chemistry in nucleic acid science, particularly with regard to the necessity of chemical approaches in understanding the functions of functional nucleic acids, in creating them and in discovering new drugs. This book contains 14 articles written by distinguished Japanese organic chemistry researchers who are active at the cutting edge of this field. They have each introduced their own research and the background of this research in an easily understandable manner. We are proud of the completed book and of the fact that its unique contents are unparalleled. We hope that this book will enlighten numerous chemical researchers, especially young researchers, so that they will actively join this field and become the leaders of the next generation.
Although the contents of this book are extremely chemistry-oriented, we believe that the various strategies and techniques introduced here will prove to be useful for the execution of a range of bio-related research. This would include research in molecular biology, medical science and pharmaceutical science, in which artificial modification/alteration of DNA and RNA is necessary. We would also be pleased if this book is widely read by non-chemist researchers and if it contributes to improvements in their research strategies. In addition, we hope to inspire new mutual exchanges and joint studies between non-chemists and chemists. The time for molecular biologists, pharmaceutical scientists, medical scientists and chemists to individually conduct nucleic acid research has passed. It is time for numerous researchers in various fields to actively collaborate and conduct interdisciplinary research. We sincerely hope that this book will become a “mediator” between them.
Lastly, we would like to express our heartfelt appreciation to Prof. Atta-ur-Rahman, NI, HI, SI, TI at the University of Karachi and Mrs. Asma Taha at Bentham Science Publishers. They have made extraordinary efforts in all stages of producing this book, from planning through to proofreading.


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Contributors

Masaki Tsukamoto Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Yoshihiro Hayakawa Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Takeshi Wada Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702, Kashiwa 277-8562, Japan.
Hiroaki Ozaki Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan.
Masayasu Kuwahara Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan.
Hiroaki Sawai Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan.
Noriaki Minakawa Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Shuichi Hoshika Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Naonori Inoue Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Yuka Kato Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Akira Matsuda Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Mitsuo Sekine Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
CREST, JST (Japan Science and Technology Agency), 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Koh-ichiroh Shohda Department of Life Science, Tokyo Institute of Technology and 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Kohji Seio CREST, JST (Japan Science and Technology Agency), 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan.
Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226- 8503, Japan.
Yan Xu Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo, Kyoto, 606- 8502, Japan.
Hiroshi Sugiyama Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo, Kyoto, 606- 8502, Japan.
Shigeki Sasaki Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Fumi Nagatsugi Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Tomohisa Moriguchi Department of Chemistry, Faculty of Engineering, Gunma University, 376-8515, Kiryu, Japan.
Kazuo Shinozuka Department of Chemistry, Faculty of Engineering, Gunma University, 376-8515, Kiryu, Japan.
Hideo Inoue Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8100%, Japan.
Takeshi Imanishi Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Satoshi Obika Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Takanori Kubo aDepartment of Biological and Environmental Chemistry, School of Science and Technology, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan.
Ruminana Bakalova Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology, (Aist-Shikoku), Ministry of International Trade and Industry, 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
Zhivko Zhelev Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology, (Aist-Shikoku), Ministry of International Trade and Industry, 2217- 14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
Hideki Ohba Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology, (Aist-Shikoku), Ministry of International Trade and Industry, 2217-14 Hayashi, Takamatsu, Kagawa 761-0395, Japan.
Masayuki Fujii Department of Biological and Environmental Chemistry, School of Science and Technology, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan.
Molecular Engineering Institute, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan.
Takehiko Wada PRESTO/JST and Graduate School of Engineering, Osaka University, Yamada-oka, Suita 565-0871, Japan, ICORP Entropy Control Project, JST, 4-6-3 Kamishinden, Toyonaka 565-0085, Japan.
Hirofumi Sato PRESTO/JST and Graduate School of Engineering, Osaka University, Yamada-oka, Suita 565-0871, Japan, ICORP.
Entropy Control Project, JST, 4-6-3 Kamishinden, Toyonaka 565-0085, Japan.
Yoshihisa Inoue PRESTO/JST and Graduate School of Engineering, Osaka University, Yamada-oka, Suita 565-0871, Japan, ICORP.
Entropy Control Project, JST, 4-6-3 Kamishinden, Toyonaka 565-0085, Japan.
Mitsunobu Nakamura Department of Materials Science and Chemistry, Himeji Institute of Technology, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
Kenji Kanaori Department of Applied Biology, KyotoInstitute of Technology, Matsugasaki, Sakyo-ku, Kyoto 605-8100%, Japan.
Kazushige Yamana Department of Materials Science and Chemistry, Himeji Institute of Technology, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan.
Seung Pil Pack International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Toshinori Suzuki Department of Biological Pharmacy, School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan.
Hiroshi Ide Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Tsutomu Kodaki Institute of Advanced Energy, Kyoto University, Gokasho, Uji 611-0011, Japan.
CREST, JST (Japan Science and Technology Agency), Gokasho, Uji 611-0011, Japan.
Keisuke Makino International Innovation Center, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-8501, Japan.


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Strategies Useful for the Chemical Synthesis of Oligonucleotides and Related Compounds
Masaki Tsukamoto and Yoshihiro Hayakawa

This review summarizes recent progress in useful strategies for the chemical synthesis of nucleic acids and related compounds surrounding the core of present author’s works. Among the methods employed for internucleotide-bond formation, the phosphoramidite method is superior in many respects, including coupling efficiency, stability of building blocks, ease of automation, purity of the product, and synthetic applicability. The original phosphoramidite method has several drawbacks, and thus a great amount of effort has been focused on the development of promoters for internucleotide-bond formation, oxidation of the phosphite intermediate, protecting groups, and linkers and solid supports in order to broaden the synthetic applicability of this extremely useful method. The author’s work to date, including the development of acid/azole complexes as promoters, anhydrous/non-basic oxidation methods, an AOC/allyl-protected strategy, will be highlighted, together with a discussion of the applicability of these methods for the synthesis of biologically important compounds.


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Stereocontrolled Synthesis of Phosphorothioate DNA by the Oxazaphospholidine Approach
Takeshi Wada

Stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates (PS-ODNs) using nucleoside 3’-O-oxazaphospholidine derivatives as monomer units is described. A series of dialkyl(cyanomethyl)ammonium salts were developed and used as new activators for the condensation reactions of the diastereopure nucleoside 3’-O-oxazaphospholidines with 3’-O-protected nucleosides. In the presence of the new activators, the condensation reactions proceeded rapidly to give the corresponding dinucleoside phosphite triesters with high deastereoselectivity. After sulfurization and deprotection, diastereo-pure (Rp)- and (Sp)-dinucleoside phosphorothioates were obtained in excellent yields. The present methodology was also applied to the solid-phase synthesis of stereoregulated PSODNs. In addition, ab initio molecular orbital calculations were carried out to elucidate the mechanism of these diastereoselective phosphitylation and condensation reactions.


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Oligonucleotides Bearing a 5-Substituted Pyrimidine Nucleoside: Their Synthesis, Properties, and Application
Hiroaki Ozaki, Masayasu Kuwahara and Hiroaki Sawai

5-Substituted 2'-deoxyuridine derivatives and 5-substituted arabinofuranosyluracil derivatives were synthesized from 2,2'-anhydro-5-methoxycarbonylmethyluridine. Introduction of the 5-substituted 2’-deoxyuridine analog into DNAs was carried out using conventional phosphoramidite chemistry with a DNA synthesizer. Modified ODNs bearing the nucleoside analogs were prepared either by a pre-synthetic modification method or a post-synthetic modification method. Effect of the 5-substituent on thermal stability of duplexes was investigated by measuring the melting behaviors. The modified ODNs could induce RNase H activity and then would be useful as an antisense agent. 5- Substituted 2’-deoxyuridine analog triphosphates could serve as substrates of thermophilic family B DNA polymerases in a primer extension reaction or PCR, to give the corresponding modified ODNs. The 5-methoxycarbonylmethyl-2’- deoxyuridine residues incorporated into DNA by PCR could be used to the postsynthetic derivatization. The modified DNA prepared by PCR is useful for in vitro selection of the functionalized DNA.


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4’-Thionucleic Acids: Chemistry, Properties, and Applications for Developing Functional Oligonucleotides
Noriaki Minakawa, Shuichi Hoshika, Naonori Inoue, Yuka Kato and Akira Matsuda

This comprehensive review summarizes 1) the synthesis of 4’- thionucleosides with emphasis on their stereoselectivity; 2) the physical and physiological properties of 4’-thionucleic acids; and 3) the applications of 4’- thioRNA for developing functional oligonucleotides. Full details of the stereoselective and practical syntheses of 4’-thioribonucleosides and 2’-deoxy-4’- thionucleosides are described. Since 4’-thionucleic acids have high hybridization and nuclease resistance properties, these modified nucleic acids are expected to be instrumental in the development of a new generation of functional oligonucleotides. Our preliminary studies concerning SELEX and RNAi are also described.


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Creation of Conformationally Rigid Bent and Linear Nucleic Acids by 3-Dimensional Fixation of Conformation of Mono- and Di-nucleotide Building Blocks
Mitsuo Sekine, Koh-ichiroh Shohda and Kohji Seio

In this article, two kinds of cyclic systems to construct sterically locked pU and UpU derivatives were reviewed. One is the bridged structure between the 5-postion of the uracil moiety and the 5’-phosphate group. In this case, the use of a propylene alkyl chain as the bridged structure resulted in induction of the C3’-endo conformation in the ribose moiety which is observed in the typical A-type RNA duplex. Incorporation of this structural motif (pc3U) into the 3’-downstream U of UpU shows that its C3’-endo conformation was preserved but the sugar puckering of the 5’-upstream U was orientated to a C2’-endo form. The use of UBNA and pc3U as the 5’-upstream and 3’-downstream uridine components, respectively, resulted in formation of two P-chiral diastereoisomers, namely, linear- and benttype dimers of UBNApc3U. Oligonucleotides incorporating the linear-type dimer showed strong hybridization affinity for the complementary strand. Oligonucleotides incorporating the bent-type dimer exhibited no significant hybridization affinity for the complementary RNA strand. However, the bent structural motif showed significant thermal stability when incorporated into the U-turn region of the tRNA anticodon stem and loop. Another cyclic UpU system having a largemembered ring structure was introduced to create the conformationally locked Uturn structure. The bridged structures having amide and urethane linkages were used. Oligonucleotides having these cyclic structural UpU motifs showed significant rigidity but the conformation of the two uridine sugar moieties was not fixed in the C3’-endo form. Finally, a cyclic UpU derivative was designed as an ideal U-turn mimic. This dimer has 3’-deoxy-3’-aminouridine and 2’-deoxy-2’- fluorouridine derivatives as the 5’-upstream and 3’-downstrem components. The bridged structure was constructed between the 2’-hydroxyl group of the 5’- upstream U and the 5-position of the 3’-downstream U via a propyrene chain. The detailed studies of the cyclic UpU revealed that this compound has a U-turn bent structure having a rigid CD spectrum.


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Chemical Approach to Probing Different DNA Structures
Yan Xu and Hiroshi Sugiyama

DNA is polymorphic and exists in a variety of distinct conformations. Duplex DNA can adopt a variety of sequence-dependent secondary structures, which range from the canonical right-handed B form through to the left-handed Z form. Triplex and tetraplex structures also exist. All of these unique conformations are assumed to play important biological roles in processes such as DNA replication, and gene expression and regulation. However, the biological roles associated with the different structural conformations of DNA are not well understood because of the short lifetime of appearance of each structure and the difficulty in creating a system to demonstrate the DNA local structure. A stable Zform DNA under physiological salt conditions is needed to investigate the properties of Z-form DNA. To obtain stable Z-DNA, we synthesized various modified guanine derivatives and introduced these into oligonucleotides to evaluate their capacity to stabilize Z-form DNA. We found that incorporation of 8- methyl-2´-deoxyguanosine (m8G) and 8-methylguanosine (m8rG) into DNA dramatically stabilized the Z form, and facilitated the B–Z transition, even for ATcontaining sequences. Developing a Z-stabilizing monomeric unit, the Z stabilizer, allowed us to understand the solution structure of Z-DNA and to reveal the specific 2´b-hydrogen abstraction that gives rise to the Z-form-specific 2´_-hydroxylation of the IU-containing Z-form under UV irradiation. We also investigated the photoreaction of 5-halouracil in the A-form, B-form, G-quartet, and proteininduced DNA kinks. Hydrogen abstraction by 2´-deoxyuridin-5-yl generated from 5-halouracil under irradiation was atom specific and highly dependent on the DNA structure. In addition, DNA-mediated charge transport chemistry was sensitive to the DNA structure and base pair p-stacking. Ab initio molecular orbital calculation shows that the 5´–GG–3´ sequence possessed the smallest vertical IP and that about 70% of the HOMO is localized on the 5´–G of 5´–GG–3´ in B-form DNA. We propose that the electronic properties of DNA are highly dependent on the orientation of p-stacking (i.e., A-, B-, and Z-form DNA have different electronic properties). Furthermore, experimental studies show that bromouracil-containing Z-DNA has a unique electronic property, and that charge-transfer from G to BrU occurs efficiently within the four-base p-stacks in Z-DNA.


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Molecular Design for Specific Recognition and Reaction in Genome-Targeting Chemistry
Shigeki Sasaki and Fumi Nagatsugi

Molecules that can target DNA or RNA with high efficiency and specificity are of great interest because of potential applications to modulation of gene expression at a specific site. Our approach in genome-targeting chemistry has been focused on development of reactive molecules with high base- as well as sequence selectivity. This paper summarizes our contributions in this field. At first, a general concept of reactive molecules in genome-targeting chemistry is introduced. In the following section, molecular design to achieve specific and efficient reactions in the living system is described. Two new reactive molecules, 2-amino-6-vinylpurine derivative as an efficient cross-linking agent, and the Snitroso-6-thioguanosine as a selective S to N nitoroso group transfer agent are summarized. These two molecules demonstrate successful examples of reactive agents that are activated only in the complementary hybrids. In the third section, new nucleoside analogs to expand recognition codes of triplex formation are discussed. We have recently developed non-natural nucleoside analogs (W-shaped nucleoside analog: WNA) having a heterocyclic ring as a recognition unit and an aromatic ring as a stacking part on the bicyclo[3.3.0]octane skeleton. It has been shown that the two analogs, WNA-bT with a thymine and WNA-bC with a cytosine, exhibit stable formation of non-natural triplexes containing a TA and a CG interrupting site with high stability, respectively. These new genome-targeting molecules will be applied to artificial modulation of gene expression in vitro as well as in vivo.


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Base and Backbone Modified Oligonucleotides and their Application As Antisense Agent
Tomohisa Moriguchi and Kazuo Shinozuka

The synthesis, some basic physicochemical properties and the application as an antisense molecule of modified oligonucleotides will be discussed in this article. The oligonucleotides covered in this section are base and/or backbone modified oligonucleotides. The influence of the modifications on the thermal stability of the duplexes consisting of the modified oligonucleotides is discussed. At the same time, the influence of the modifications on the nucleaseresistant property of the modified oligonucleotides is also discussed. The antisense activity of some modified oligonucleotides is included with available data. The relevant references are presented at the end of this article.


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Artificial Ribonucleases as Antisense Compounds
Hideo Inoue

Antisense oligonucleotides can be used to inhibit gene expression. It is generally accepted that most of the antisense oligonucleotides developed so far act by an RNase H-mediated mechanism, and thereby cleavage of specific mRNAs occurs. Recently, chemical agents that cleave RNA site-specifically, without enzymatic assistance, have been developed. Such artificial chemical nucleases, which are conjugates of an antisense oligonucleotide and an RNA-cleaving catalyst(s), are potentially important in antisense chemotherapy and would also be useful for molecular biology, including RNA engineering. Metal complexes, oligoamines and imidazoles have been used as RNA cleavage catalysts. This review highlights the recent developments in the field of artificial RNases, with metal complexes cleaving RNA via the transesterification and/or hydrolysis of the target phosphodiester linkage.


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Sugar-Modified Nucleic Acid Analogues as Potential Materials for Genomic Technologies
Takeshi Imanishi and Satoshi Obika

Chemical modifications of natural nucleic acid architecture are well known to be potential for development of highly functional oligonucleotide analogues applicable to various genomic technologies, e.g. antisense and antigene methodologies, and numerous kinds of artificial nucleic acids have been developed to date. Chemical modifications of natural nucleic acids can be classified into three categories: nucleobase, internucleoside linkage and sugar modifications. Among these categories, sugar modifications have been received as the most promising way to develop practical oligonucleotide analogues for various genomic technologies in recent years. This review focused on sugar-modified nucleosides and their oligonucleotide derivatives.


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Synthesis, Biological Properties and Antisense Effects of Oligonucleotide-Petide Conjugates
Takanori Kubo, Ruminana Bakalova, Zhivko Zhelev, Hideki Ohba and Masayuki Fujii

In order to improve the biological and pharmacological properties of antisense oligonucleotides, we have been recently focussed our efforts on synthesis of DNA-peptide conjugates and biological evaluation of them. Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by SPFC [1]. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed cationic a-helical or b-sheet peptides with amphipathic character. Some polyamines and sugars were also conjugated with oligonucleotides by SPFC in good yields. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugates-RNA hybrids could activate RNase H as effective as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into cell nucleus, (f) the conjugates with NES could be localized in cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.


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Functional Artificial Nucleic Acids: Peptide Ribonucleic Acids (Prnas) - Novel Strategy for Active Control of DNA Recognition by External Factors
Takehiko Wada, Hirofumi Sato and Yoshihisa Inoue

The effect of adding borax and boric acids on the nucleobase orientation and recognition behavior of novel mono- and oligomeric peptide ribonucleic acids (PRNAs) has been investigated. The base orientation of 5’- amino-5’-deoxyuridine and 5’-amino-5’-deoxycytidine was shown by CD and NOE difference spectral studies to switch from anti to syn in borate buffer or upon addition of borax. The origin of this phenomenon is elucidated to be the cooperative effect of the cyclic borate esterification of sugar’s cis-2’,3’-diol and the hydrogen bonding interaction between the sugar’s 5’-amino proton and the base’s 2-carbonyl oxygen. Because this new strategy for switching the base orientation through the addition of borate is potentially applicable to the recognition control of nucleic acids if the sugar’s 5’-proton and cis-2’,3’-diol remain unmodified, we synthesized a series of PRNAs, in which the 5’-amino- 5’-deoxypyrimidine ribonucleoside moiety was appended to a mono- or oligo(g-L-glutamic acid) and poly(a-L-glutamic acid) backbone through the 5’-amino group. The synthetic routes and procedures were established for all of the four fluorenemethyloxycarbonyl-protected PRNA monomers carrying uracil, N-benzoylcytosine, hypoxanthine, and N-benzoyladenosine nucleobases. Furthermore, a couple of PRNA 12-mers with desired purine-pyrimidine mixed sequences were prepared in high yields by the solid-phase synthesis. The orientation switching through the addition of borate was also confirmed with the monomeric model, and the switching efficiency was enhanced for oligomeric g-PRNA. In case of the PRNA oligomer containing pyrimidinepurine mixed sequence, efficient orientational switching of nucleobases induced by added borates was observed. Finally, it was unambiguously demonstrated that the g-PRNA oligomers with an isopoly(L-glutamic acid) backbone and poly the a-PRNA with poly(a-L-glutamic acid) backbone can form a tight complex with complementary DNA and/or RNA further the recognition of DNA with g-PRNA oligomers and a-PRNA are controlled by the borate added as an external factor.


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Nucleic Acid Seqeuence Analysis Using Oligonucleotide Probes
Mitsunobu Nakamura, Kenji Kanaori and Kazushige Yamana

We have designed and synthesized oligonucleotides possessing a pyrene or a bis-pyrene at the defined position using phosphoramidite chemistry. The pyrene probes exhibit strongly enhanced fluorescence upon binding to specific sequences of DNA/RNA. The attractive features of our probes are that the pyrene fluorescence is sensitive to local base sequences and structures of probe-nucleic acid complexes around the pyrene modification. The pyrene probes would thus be useful in fluorescence recognition of nucleic acid sequences and structures.


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Reaction of NO with Nucleic Acid Bases and its Biological Implication
Seung Pil Pack, Toshinori Suzuki, Hiroshi Ide, Tsutomu Kodaki and Keisuke Makino

Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. We have explored, therefore, DNA damage caused by NO (or slightly acidic HNO2). Before our study, only oxidative deamination was established as a major pathway to convert dGuo to dXao, dAdo to dIno and dCyd to dUrd. In our study, another major pathway initiated by the attack to the exo-amino groups has been demonstrated. For dGuo, 2'-deoxyoxanosine (dOxo) production through the dGuodiazoate (intermediate) formation has been determined: The dOxo yield is 1/3 of that for dXao, and the glycosylic bond is as stable as that of dGuo. DNA polymerases recognize dOxo as both dGuo and dAdo, indicative of G:C to A:T conversion. Also it has been found that both dOxo and the intermediate show high reactivity with amino groups, and that a stable diazoate with the similar high reactivity is produced from dCyd. So we have investigated DNA-protein crosslinks (DPCs) induced by dOxo. When a DNA duplex containing dOxo at the sitespecific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between dOxo and protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing dOxo. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis.