Frontiers in Organic Chemistry - Current Issue

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.

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.

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.

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.

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.

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'β-hydrogen abstraction that gives rise to the Z-form-specific 2'a-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 π-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 π-stacks in Z-DNA.

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-βT with a thymine and WNA-βC 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.

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.

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.

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.

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 α-helical or β-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.

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(γ-L-glutamic acid) and poly(α-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 γ-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 γ-PRNA oligomers with an isopoly(L-glutamic acid) backbone and poly the α-PRNA with poly(α-L-glutamic acid) backbone can form a tight complex with complementary DNA and/or RNA further the recognition of DNA with γ-PRNA oligomers and α-PRNA are controlled by the borate added as an external factor.

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.

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.