Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

Expansion of Antibacterial Spectrum of Muraymycins toward Pseudomonas aeruginosa.

It is urgent to develop novel anti-Pseudomonas agents that should also be active against multidrug resistant P. aeruginosa. Expanding the antibacterial spectrum of muraymycins toward P. aeruginosa was investigated by the systematic structure-activity relationship study. It was revealed that two functional groups, a lipophilic side chain and a guanidino group, at the accessory moiety of muraymycins were important for the anti-Pseudomonas activity, and analogue 29 exhibited antibacterial activity against a range of P. aeruginosa strains with the minimum inhibitory concentration values of 4-8 µg/mL.

An efficient approach to the discovery of potent inhibitors against glycosyltransferases.

We describe a standardized approach for searching potent and selective inhibitors of glycosyltransferases by high throughput quantitative MALDI-TOFMS-based screening of focused compound libraries constructed by 1,3-dipolar cycloaddition of the desired azidosugar nucleotides with various alkynes. An aminooxy-functionalized reagent with a stable isotope was conjugated with oligosaccharides to afford glycopeptides as acceptor substrates with improved ion sensitivity. Enhanced ionization potency of new substrates allowed for MALDI-TOFMS-based facile and quantitative analysis of enzymatic glycosylation in the presence of glycosyl donor substrates. A non-natural synthetic sugar nucleotide was identified to be the first highly specific inhibitor for rat recombinant alpha2,3-(N)-sialyltransferase (alpha2,3ST, IC(50) = 8.2 microM), while this compound was proved to become a favorable substrate for rat recombinant alpha2,6-(N)-sialyltransferase (alpha2,6ST, K(m) = 125 microM). Versatility of this strategy was demonstrated by identification of two selective inhibitors for human recombinant alpha1,3-fucosyltransferase V (alpha1,3-FucT, K(i) = 293 nM) and alpha1,6-fucosyltransferase VIII (alpha1,6-FucT, K(i) = 13.8 microM).

Synthesis of novel 2'-deoxy type trans-3',4'-bridged nucleic acid.

We newly designed and synthesized a 2'-deoxy type trans-3',4'-bridged nucleic acid (trans-3',4'-BNA) analogues bearing a 4,7-dioxabicyclo[4.3.0]nonane structure. The synthesis of the trans-3',4'-BNA was carried out successfully from thymidine over 21 steps. The structure of trans-3',4'-BNA was confirmed by x-ray crystallographic analysis, indicating that the furanose ring has a typical S-type conformation with C(3')-exo puckering.

Synthesis and properties of trans-3',4'-bridged nucleic acids having typical S-type sugar conformation.

The synthesis of nucleoside analogues with a conformationally restricted sugar moiety is of great interest. The present research describes the synthesis of BNA (bridged nucleic acid) monomers 1 and 2 bearing a 4,7-dioxabicyclo[4.3.0]nonane skeleton and a methoxy group at the C2' position. Conformational analysis showed that the sugar moiety of these monomers is restricted in a typical S-type conformation. It was difficult to synthesize the phosphoramidite derivative of the ribo-type monomer 1, while the phosphoramidite of the arabino-type monomer 2 was successfully prepared and incorporated into oligodeoxynucleotides (ODNs). The hybridization ability of the obtained ODN derivatives containing 2 with complementary strands was evaluated by melting temperature (T(m)) measurements. As a result, the ODN derivatives hybridized with DNA and RNA complements in a sequence-selective manner, though the stability of the duplexes was lower than that of the corresponding natural DNA/DNA or DNA/RNA duplex.

Synthesis and properties of a novel bridged nucleic acid analogue, 5'-amino-3',5'-BNA.

An oligonucleotide P3'-->N5' phosphoramidate (5'-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3'-->N5' phosphoramidate linkage and a constrained sugar moiety, 5'-amino-3'-C,5'-N-methylene bridged nucleic acid (5'-amino-3',5'-BNA). The binding affinity of the 5'-amino-3',5'-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5'-amino-3',5'-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5-amino-DNA.

Synthesis and properties of a novel bridged nucleic acid analogue bearing a P3'-->N5' phosphoroamidate linkage, 5'-amino-3', 5'-BNA.

We have designed and successfully synthesized a novel bridged nucleic acid analogue with a P3'-->N5' phosphoramidate linkage, 5'-amino-3',5'-BNA. X-ray crystallographic analysis demonstrated that the 5'-amino-3',5'-BNA had C1-exo-O4-endo sugar puckering and a constrained gamma dihedral angle of 22.8 degrees. The oligonucleotides containing 5'-amino-3',5'-BNA exhibited strong binding affinity towards complementary strands. In addition, the 5'-amino-3',5'-BNA oligonucleotide was easily hydrolyzed at its phosphoramidate linkage under mild acidic conditions.

trans-3',4'-BNAs, novel nucleic acid analogues with an S-type conformation: synthesis and incorporation into oligonucleotides.

We designed and successfully synthesized novel nucleic acid analogues with a C3'-C4' trans-fused six-membered ring, trans-3',4'-BNAs from D-glucose. A 1H-NMR experiment and X-ray crystallographic analysis demonstrated that the sugar puckering of trans-3',4'-BNA was restricted in an S-type conformation. We also achieved its incorporation into oligonucleotides using automated DNA synthesizer.

Stable oligonucleotide-directed triplex formation at target sites with CG interruptions: strong sequence-specific recognition by 2',4'-bridged nucleic-acid-containing 2-pyridones under physiological conditions.

A sequence of double-stranded DNA (dsDNA) which can be recognized by a triplex-forming oligonucleotide (TFO) is limited to a homopurine-homopyrimidine sequence. To develop novel nucleoside analogues which recognize CG interruption in homopurine-homopyrimidine dsDNA, we synthesized a novel 2'-O,4'-C-methyleneribonucleic acid (2'-O,4'-C-methylene bridged nucleic acid; 2',4'-BNA) that bears the unnatural nucleobases, 2-pyridone (PB) or its 5-methyl congener (mPB); these analogues were introduced into pyrimidine TFOs using a DNA synthesizer. A TFO with a 2'-deoxy-beta-D-ribofuranosyl-2-pyridone (P) or 2',4'-BNA abasic monomer (HB) was also synthesized. The triplex-forming ability of various synthesized 15-mer TFOs and the corresponding homopurine-homopyrimidine dsDNA, which contained a single pyrimidine-purine (PyPu) interruption, was examined in UV melting experiments. It was found that PB and mPB in the TFOs successfully recognized CG interruption under physiological conditions (7 mM sodium phosphate, 140 mM KCl, 5 mM spermine, pH 7.0). Furthermore, triplex formation between the dsDNA target which contained three CG interruptions and the TFO with three PB units was also confirmed. Additional four-point 2',4'-BNA modifications of the TFO containing three PB units significantly enhanced its triplex-forming ability towards the dsDNA and had a Tm value of 43 degrees C under physiological conditions. These results indicate that a critical inherent problem of TFOs, namely, the sequence limitation of the dsDNA target, may be overcome to a large extent and this should promote antigene applications of TFOs in vitro and in vivo.

A 2',4'-Bridged Nucleic Acid Containing 2-Pyridone as a Nucleobase: Efficient Recognition of a C⋠G Interruption by Triplex Formation with a Pyrimidine Motif.

Significantly enhanced binding affinity to C⋅G base pairs without loss of sequence selectivity is achieved by using a nucleotide containing a 2-pyridone and a 2'-O,4'-C-methylene-bridged nucleic acid analogue (PB , see picture). The degree of stabilization of the triplex formed enables C⋅G interruptions in a homopurine⋅homopyrimidine double-stranded DNA to be detected.