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Replacement of a phosphodiester linkage with an amide linkage can improve the binding affinity of oligonucleotides to complementary RNA and their stability toward nucleases. In addition, restricting the conformation of the sugar moiety and the phosphate backbone in oligonucleotides effectively improves duplex stability. In this study, we designed amide-linked dinucleotides containing a 3′,4′-tetrahydropyran-bridged nucleic acid (3′,4′-tpBNA) with a constrained sugar conformation as well as a torsion angle ε. Phosphoramidites of the designed dinucleotides were synthesized and incorporated into oligonucleotides. Conformational analysis of the synthesized dinucleotides showed that the sugar conformation of the S-isomer of the amide-linked dinucleotide containing 3′,4′-tpBNA was N-type, which has the same conformation as that of the RNA duplex, while that of another R-isomer was S-type. Tmanalysis indicated that the oligonucleotides containing the synthesized S-isomer showed RNA-selective hybridizing ability, although their duplex-forming ability was slightly inferior to that of natural oligonucleotides. Interestingly, the stability of the oligonucleotides toward endonucleases was significantly improved by modification with the two types of amide-linked dinucleotides developed in this study.

In this study, 2′-O,4′-C-methyleneoxy-bridged nucleic acid, a unique consecutive three-acetal-containing nucleic acid (TaNA), was designed. Pyrimidine derivatives of methylated TaNA (Me-TaNA) were also synthesized and introduced into oligonucleotides via solid-phase synthesis. The Me-TaNA-modified oligonucleotides exhibited higher stabilities when forming duplexes with single-stranded RNA or triplexes with double-stranded DNA, relative to the natural oligonucleotides and modified oligonucleotides containing another 2′,4′-bridged 5-methyluridine, such as 2′,4′-BNA/LNA and 2′,4′-ENA. Furthermore, Me-TaNA within oligonucleotides significantly enhanced nuclease resistance.

2′,4′-Bridged nucleic acid (2′,4′-BNA) analogues are used for therapeutic oligonucleotides, owing to their excellent hybridizing ability with complementary RNA and high resistance toward enzymatic degradation. We developed 2′,4′-BNA analogues with oxygen atoms at 6′-positions (e.g., EoNA and EoDNAs) and demonstrated that the presence of 6′-oxygen atoms in the bridge structure could show positive effect on the properties of the modified oligonucleotides. Herein, we designed and synthesized 7′-methyl derivatives of methyleneoxy-bridged 2′-deoxyribonucleic acid (MoDNA), possessing a five-membered bridge with 6′-oxygen atom via radical cyclization for the bridge construction. The synthesized monomers were incorporated into the oligonucleotides by solid-phase oligonucleotide synthesis. The MoDNA-modified oligonucleotides showed high affinity toward single-stranded RNA and double-stranded DNA, as well as excellent resistance toward nuclease compared with the corresponding natural oligonucleotide.

The synthesis of 6′S-Me-2′-O,4′-C-ethylene-bridged 5-methyluridine (6′S-Me-ENA-T) was achieved using visible light-mediated stereoselective radical cyclization as a key step. This is the first example of a method for constructing a 2′,4′-bridged structure from a 4′-carbon radical intermediate. The 6′S-Me-ENA-T monomer was successfully incorporated into oligonucleotides, and their properties were examined. The oligonucleotides containing 6′S-Me-ENA-T exhibited a highly selective hybridization affinity toward single-stranded RNA and an excellent enzymatic stability, compared to the corresponding LNA- and ENA-modified oligonucleotides.

A concise approach for the synthesis of the 5′-carba analogs of nucleoside 5′-phosphates from 2′-deoxy-5′-O-phthalimidonucleosides by a visible-light-mediated deformylative 1,4-addition was developed. This method enabled rapid and facile generation of 4′-carbon radicals of nucleosides. Moreover, this synthetic strategy was applicable to the 5′-carba analogs of nucleoside 5′-phosphates as well as other 5′-carba nucleosides bearing methoxycarbonyl, cyano, and N-methylsulfamoyl groups.

The pentahydrate (a form) of cefazolin sodium (CEZ) exhibited sharp X-ray diffraction peaks, while the dehydrated a form showed weak but distinct diffraction peaks. As expected the amorphous form exhibited a diffuse and halo diffraction pattern. The X-ray procedure to estimate the degree of crystallinity of CEZ was based upon the measurement of the total scattering and the scattering from the crystalline region of the drug. The major difference in the infrared (IR) spectra among the three forms of CEZ was the absence of a spectral band at 1542 cm-1 in the amorphous form. The IR procedure was based upon the measurement of the peak percentage area ratio between the bands at 1542 and 1760 cm-1, where the latter was used as a normalizing peak. The degree of crystallinity of CEZ samples, obtained by either freeze-drying aqueous CEZ solutions or storing the crystalline forms under different humidity conditions, was determined by these two methods. Although the correlation of results by the two methods was good, the X-ray procedure appears to be superior since it can differentiate among the three solid CEZ forms, whereas IR could distinguish between only crystalline and amorphous CEZ, reproducibly.