Hidehito Urata

Synthesis and properties of mirror-image DNA.

We have investigated the conformations of the hexadeoxyribonucleotide, L-d(CGCGCG) composed of L-deoxyribose, the mirror image molecule of natural D-deoxyribose. In this paper, we report the synthesis of four L-deoxynucleosides and the L-oligonucleotide-ethidium bromide interactions. The L-deoxyribose synthon 9 was synthesized from L-arabinose with an over all yield of 28.5% via the Barton-McCombie reaction. The L-deoxynucleosides were obtained by a glycosylation of appropriate nucleobase derivatives with the 1-chloro sugar 9. After derivatization to nucleoside phosphoramidites, L-deoxycytidine and L-deoxyguanosine were incorporated into a hexadeoxynucleotide, L-d(CGCGCG) by a solid-phase beta-cyanoethylphosphoramidite method. This L-hexanucleotide was resistant to digestion with nuclease P1. The conformations of L-d(CGCGCG) were an exact mirror image of that of the corresponding natural one as described previously, and the conformations of the L-d(CGCGCG)-ethidium bromide complex were also the mirror images of those of the D-d(CGCGCG)-ethidium bromide complex under both low and high salt conditions. These results suggest that ethidium bromide prefers not a right-handed helical sense, but the base-base stacking geometry of the B-form rather than that of the Z-form. Thus, L-DNA would be a useful tool for studying DNA-drug interactions.

AgI Ion Mediated Formation of a C–A Mispair by DNA Polymerases†

Silver turns up the A-C: In the presence of Ag(I) ions, a DNA polymerase incorporated deoxyadenosine (from dATP) at the site opposite cytosine in the template strand to afford the full-length product (see scheme), meaning that DNA polymerases prefer a C-Ag(I)-A base pair to the more thermodynamically stable C-Ag(I)-C base pair.

A convenient synthesis of oligonucleotides with a 3′-phosphoglycolate and 3′-phosphoglycaldehyde terminus

Abstract A simple and rapid method for solid phase synthesis of oligonucleotides carrying a 3′-phosphoglycerol terminus was developed. The modified oligonucleotide was readily oxidized by NaIO4 to a 3′-phosphoglycaldehyde. The treatment of this aldehyde with NaClO2 afforded a 3′-phosphoglycolate.

Cellular uptake of Aib-containing amphipathic helix peptide.

Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules.

Thermodynamic study of hybridization properties of heterochiral nucleic acids.

Heterochiral DNA and RNA heptamers, which contained an unnatural L-nucleotide, were synthesized, and thermodynamic analyses of their hybridization properties with complementary DNA and RNA strands were systematically conducted by UV melting experiments. The results clearly demonstrated that the incorporation of an L-ribonucleotide into the RNA strand leads to more significant destabilization of the duplexes than that of an L-deoxyribonucleotide into the DNA strand, regardless of whether the complementary strand is DNA or RNA. The destabilization of the duplexes by the substitution of D-thymidine with L-thymidine in the DNA strand is entropically driven, whereas that by the substitution of D-uridine with L-uridine in the RNA strand is enthalpically driven. The thermodynamic characteristic that the stability of homochiral duplex is far superior to that of heterochiral duplex is much more remarkable in RNA than in DNA. Thus, RNA might have been a self-replicating system superior to DNA to exclude the chiral antipode.

SEQUENCE DEPENDENCE OF THERMODYNAMIC STABILITY OF HETEROCHIRAL DNA

Abstract Several heterochiral dodecadeoxynucleotides ( 2–6 ) containing an unnatural L-enantiomer of D-deoxyribose were synthesized and their thermodynamic stability for duplex formation was investigated. The results suggested that substitution of L-deoxyribose for natural D-deoxyribose somewhat decreases the duplex stability, depending on the site that incorporates the L-deoxynucleoside.

Effect of Ala replacement with Aib in amphipathic cell-penetrating peptide on oligonucleotide delivery into cells.

A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells.

Translocation of an Aib-containing peptide through cell membranes.

The biophysical characteristics and channel-forming activity of peptaibols inserted into artificial membranes have been studied over the last 30 years. However, to our knowledge, no studies have addressed directly their behavior in living cells. In this work, a novel strategy has been employed to precisely assess the living cell membrane-penetrating activity of a fluorescein-labeled Aib (alpha-aminoisobutyric acid)-containing peptide derived from a peptaibol, trichorovin-XIIa (TV-XIIa). We have demonstrated for the first time that the peptide containing an unusual amino acid residue, Aib, is taken up by cells via a non endocytic pathway. The replacement of Aib in the TV-XIIa sequence with Ala inhibits the cellular uptake.

Structural Studies of Heterochiral DNA/DNA, RNA/RNA, AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.

CHIRAL SELECTION IN OLIGOADENYLATE FORMATION IN THE PRESENCE OF A METAL ION CATALYST OR POLY(U) TEMPLATE

The lead ion-catalyzed oligomerization of 5′-phosphorimidazolides of D-, L- or racemic DL-adenosine (D-ImpA, L-ImpA and DL-ImpA) gave oligoadenylates up to a pentamer. The oligomers resulting from racemic ImpA were comparable in yields and length to those from chiral D- or L-ImpA. A complex mixture of homochiral and heterochiral oligomers was formed in the reaction from racemic ImpA. Total dimer product from racemic ImpA by the lead ion catalyst showed homochiral selectivity. The reaction catalyzed by uranyl ion yielded oligoadenylates up to 15mer from chiral D- or L-ImpA in over 95% yield. A complex mixture of isomeric oligoadenylates was formed from racemic DL-ImpA in the presence of uranyl ion catalyst in comparable yields to those from D- or L-ImpA. The analysis of the dimer product from DL-ImpA showed that the homochiral 2′ –5′ linked dimer was selectively formed. D-ImpA polymerized effectively on a poly(U) template, which is exclusively composed of D-uridine, yielding oligoadenylates up to a pentamer. In contrast, L-ImpA or racemic DL-ImpA polymerized far less efficiently on the poly(U) template, demonstrating that chiral selection takes place in the poly(U) template-directed oligoadenylate formation.