Light-controllable cell-membrane disturbance for intracellular delivery.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

Synthesis of Coumarin-Conjugated Oligonucleotides via Knoevenagel Condensation to Prepare an Oligonucleotide Library.

DNA-encoded libraries (DELs) are attracting attention as a screening tool in the early stages of drug discovery. In the development of DELs, drug candidate compounds are chemically synthesized on barcode DNA. Therefore, it is important to perform the synthesis under mild conditions so as to not damage the DNA. On the other hand, coumarins are gaining increasing research focus not only because they possess excellent fluorescence properties, but also because many medicines contain a coumarin skeleton. Among the various reactions developed for the synthesis of coumarins thus far, Knoevenagel condensation followed by intramolecular cyclization under mild conditions can yield coumarins. In this study, we developed a new synthetic method for preparing a coumarin-conjugated oligonucleotide library via Knoevenagel condensation. The results showed that coumarins substituted at the 5-, 6-, 7-, or 8-positions could be constructed on DNA to afford a total of 26 coumarin-conjugated DNAs. Moreover, this method was compatible with enzymatic ligation, demonstrating its utility in DEL synthesis. The developed strategy for the construction of coumarin scaffolds based on Knoevenagel condensation may contribute to the use of DELs in drug discovery and medicinal chemistry.

Synthesis, Duplex-Forming Ability, and Enzymatic Stability of Oligonucleotides Modified with Amide-Linked Dinucleotides Containing a 3',4'-Tetrahydropyran-Bridged Nucleic Acid.

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. Tm analysis 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.

Post-Synthetic Nucleobase Modification of Oligodeoxynucleotides by Sonogashira Coupling and Influence of Alkynyl Modifications on the Duplex-Forming Ability.

Recently, it was reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex-forming ability of oligonucleotides (ONs) is unclear. In this study, post-synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl-modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl-modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl-modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl-modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method.

Synthesis and biophysical properties of tetravalent PEG-conjugated antisense oligonucleotide.

This study was aimed at developing a novel platform for tetravalent conjugation of 4-arm polyethylene glycol (PEG) with an antisense oligonucleotide (ASO). The ASO technology has several limitations, such as low cellular uptake, poor nuclease stability, and short half-life. PEG-conjugated ASOs may result in an improvement in the pharmacokinetic behavior of the drug. Moreover, PEGylation can reduce enzymatic degradation and renal excretion of the conjugates, thereby, increasing its blood stability and retention time. In this study, we successfully synthesized PEG-ASO conjugate consisting of 4-arm-PEG and four molecules of ASO (4-arm-PEG-tetra ASO). Its hybridization ability with complementary RNA, enzymatic stability, and in vitro gene silencing ability were evaluated. No significant difference in hybridization ability was observed between 4-arm-PEG-tetra ASO and the parent ASO. In addition, gene silencing activity of the 4-arm-PEG-tetra ASO was observed in vitro. However, the in vitro activity of the 4-arm-PEG-tetra ASO was slightly reduced as that of the parent ASO. Moreover, the 4-arm-PEG-tetra ASO showed appreciable stability in cellular extract, suggesting that it hybridizes with mRNA in its intact form, without being cleaved in the cell, and exhibits ASO activity.

Development of Phosphoramidite Reagents for the Synthesis of Base-Labile Oligonucleotides Modified with a Linear Aminoalkyl and Amino-PEG Linker at the 3'-End.

Oligonucleotides with an amino linker at the 3'-end are useful for the preparation of conjugated oligonucleotides. However, chemically modified nucleosides, which are unstable under basic conditions, cannot be incorporated into oligonucleotides using the conventional method entailing the preparation of oligonucleotides bearing a 3'-amino linker. Therefore, we designed Fmoc-protected phosphoramidites for the synthesis of base-labile oligonucleotides modified with a 3'-amino linker. The resultant phosphoramidites were then successfully incorporated into oligonucleotides bearing a 3'-amino linker. Various basic solutions were investigated for protecting group removal. All the protecting groups were removed by treating the oligonucleotides with 40% aqueous methylamine at room temperature for 2 h. Thus, the deprotection time and temperature were significantly reduced compared to the conventional conditions (28% NH3 aq., 55 °C, 17 h). In addition, the oligonucleotide protecting groups could be removed using a mild base (e.g., 50 mM potassium carbonate methanol solution). Furthermore, base-labile oligonucleotides bearing an amino linker at the 3'-end were successfully synthesized using the developed phosphoramidite reagents, highlighting the utility of our strategy.

Synthesis, Duplex-Forming Ability, and Nuclease Resistance of Oligonucleotides Containing a Thymidine Derivative with a 1-Oxaspiro[4.5]decane Skeleton.

Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6'-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6'-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6'-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6'-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6'-C-spiro-thymidine on the 3'-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.

Synthesis and physical and biological properties of 1,3-diaza-2-oxophenoxazine-conjugated oligonucleotides.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tCO) and its derivative G-clamp strongly bind to guanine and, when incorporated into double-stranded DNA, significantly increase the stability of the latter. As the phenoxazine skeleton is a constituent of major pharmaceuticals, we hypothesized that oligonucleotides (ONs) containing phenoxazine bases would induce property changes related to intracellular uptake and migration in tissues. In this study, we designed and synthesized a novel G-clamp-linker antisense oligonucleotide (ASO) in which a G-clamp base with a flexible linker was introduced into the 5'-end of an ASO targeting mouse long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (mMALAT1). Compared to unconjugated ASO, the G-clamp-linker ASO induced significantly more effective knockdown of mMALAT1 in mouse skeletal muscle. The ASOs conjugated with 2'-deoxyribonucleotide(s) bearing a tCO nucleobase at the 5'-end exhibited a similar knockdown effect in skeletal muscle. Thus, it may be possible to improve therapeutic effects against skeletal muscle diseases, such as muscular dystrophy, by using ONs with incorporated phenoxazine nucleobases.

Synthesis and Properties of Oligonucleotides Containing 2'-O,4'-C-Methyleneoxy-Bridged Pyrimidine Derivatives.

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.

Conjugation of oligonucleotides with activated carbamate reagents prepared by the Ugi reaction for oligonucleotide library synthesis.

The DNA-encoded library (DEL) is a powerful tool for drug discovery. As a result, to obtain diverse DELs, many DNA-compatible chemical reactions have been developed over the past decade. Among the most commonly used reactions in medicinal chemistry, multicomponent reactions (MCRs) can lead to the generation of various compounds in a one-step reaction. In particular, the Ugi reaction can easily provide a peptoid library. Thus, we herein report a solution-phase DEL synthesis based on the Ugi reaction. Using 6-(4-nitrophenoxycarbonylamino)hexanoic acid and N-4-nitrophenoxycarbonylglycine as carboxylic acids, peptoids with activated carbamate moieties were obtained as the products of the Ugi reaction. These peptoids were then treated with oligonucleotides bearing a 5'- or 3'-terminal aminohexyl linker to give various oligonucleotide-tagged peptoids in good yields. Moreover, the obtained peptoids could be substituted by a Suzuki cross-coupling reaction and by hydrolysis of the carboxylate ester, followed by condensation with amines. These advances should therefore promote pharmaceutical and medicinal research using DELs.

Synthesis of Sugar and Nucleoside Analogs and Evaluation of Their Anticancer and Analgesic Potentials.

Chemical modification of sugars and nucleosides has a long history of producing compounds with improved selectivity and efficacy. In this study, several modified sugars (2-3) and ribonucleoside analogs (4-8) have been synthesized from α-d-glucose in a total of 21 steps. The compounds were tested for peripheral anti-nociceptive characteristics in the acetic acid-induced writhing assay in mice, where compounds 2, 7, and 8 showed a significant reduction in the number of writhes by 56%, 62%, and 63%, respectively. The compounds were also tested for their cytotoxic potential against human HeLa cell line via trypan blue dye exclusion test followed by cell counting kit-8 (CCK-8) assay. Compound 6 demonstrated significant cytotoxic activity with an IC50 value of 54 µg/mL. Molecular docking simulations revealed that compounds 2, 7, and 8 had a comparable binding affinity to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. Additionally, the bridged nucleoside analogs 7 and 8 potently inhibited adenosine kinase enzyme as well, which indicates an alternate mechanistic pathway behind their anti-nociceptive action. Cytotoxic compound 6 demonstrated strong docking with cancer drug targets human cytidine deaminase, proto-oncogene tyrosine-protein kinase Src, human thymidine kinase 1, human thymidylate synthase, and human adenosine deaminase 2. This is the first ever reporting of the synthesis and analgesic property of compound 8 and the cytotoxic potential of compound 6.

Synthesis and properties of oligonucleotides bearing thymidine derivatives with 1,6-dioxaspiro[4.5]decane skeleton.

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.

Impact of Plasma Donepezil Concentration on Behavioral and Psychological Symptoms of Dementia in Patients with Alzheimer's Disease.

BACKGROUND/AIMS: The behavioral and psychological symptoms of dementia (BPSD) detract from the quality of life of not only dementia patients but also their family members and caregivers. Donepezil is used to treat Alzheimer's disease and is metabolized via cytochrome P450 (CYP) 2D6 and CYP3A4/5. It is controversial whether donepezil improves or exacerbates BPSD. This study investigated the relationships among BPSD, the pharmacokinetics of donepezil including its metabolite, 6-O-desmethyl donepezil, genetic polymorphisms of CYPs and P-glycoprotein, and patient backgrounds in 52 patients with Alzheimer's disease. METHODS: BPSD were assessed using the Neuropsychiatric Inventory (NPI), with scores ≥20 points defined as severe BPSD. Plasma donepezil and 6-O-desmethyl donepezil concentrations were measured using liquid chromatography-tandem mass spectrometry. RESULTS: Although significant relationships between NPI scores and plasma donepezil concentrations were not seen, none of the 15 patients (29%) with high plasma donepezil concentrations (≥60 ng/mL) developed severe BPSD. Polymorphisms of CYP2D6, CYP3A5, and ABCB1 did not influence NPI scores. There were no significant relationships between NPI and patient background factors such as dosing regimen, concomitant use of other drugs, or laboratory test results. Two patients who underwent multiple blood samplings over 2 years showed an inverse correlation between plasma donepezil concentrations and NPI scores. DISCUSSION/CONCLUSIONS: These results indicate that higher plasma concentrations of donepezil contribute to preventing or alleviating rather than developing or deteriorating BPSD.

Synthesis of Oligonucleotides Containing 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) Moieties Using Post-Synthetic Modification Strategy.

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2'-N-pentafluorophenoxycarbonyl-2'-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2'-N-alkylaminocarbonyl-2'-amino-LNA (2'-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2'-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2'-amino-LNA. Thus, 2'-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.

Synthesis of 2'-C,4'-C-Methyleneoxy-Bridged Thymidine Derivatives and Properties of Modified Oligonucleotides.

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.

Synthesis of the Methyl Analog of 2'-O,4'-C-Ethylene-Bridged 5-Methyluridine via Intramolecular Radical Cyclization and Properties of Modified Oligonucleotides.

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.

2'-C,4'-C-Ethyleneoxy-Bridged 2'-Deoxyribonucleic Acids (EoDNAs) with Thymine Nucleobases: Synthesis, Duplex-Forming Ability, and Enzymatic Stability.

This chapter describes procedures for (1) the synthesis of six 2'-C,4'-C-ethyleneoxy-bridged thymidine phosphoramidites, i.e., methylene-EoDNA-T, (R)-Me-methylene-EoDNA-T, (S)-Me-methylene-EoDNA-T, EoDNA-T, (R)-Me-EoDNA-T, and (S)-Me-EoDNA-T phosphoramidites, (2) the introduction of the phosphoramidites into oligonucleotides, (3) UV-melting experiments of the duplexes of the modified oligonucleotides and complementary RNA, and (4) nuclease degradation experiments of the modified oligonucleotides.

Photoredox-Catalyzed Deformylative 1,4-Addition of 2'-Deoxy-5'- O-phthalimidonucleosides for Synthesis of 5'-Carba Analogs of Nucleoside 5'-Phosphates.

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.

Synthesis and hybridizing properties of isoDNAs including 3'-O,4'-C-ethyleneoxy-bridged 5-methyluridine derivatives.

3',4'-Ethyleneoxy-bridged 5-methyluridine derivatives with methyl groups in the bridge, (R)-Me-3',4'-EoNA-T and (S)-Me-3',4'-EoNA-T, were synthesized, and these two analogs and unsubstituted 3',4'-EoNA-T were successfully incorporated into a 2',5'-linked oligonucleotide (isoDNA). Their duplex-forming ability with complementary DNA and complementary RNA, and triplex-forming ability with double-stranded DNA, were evaluated by UV-melting experiments. The results indicated that isoDNAs, including these 3',4'-EoNA analogs, could hybridize exclusively with complementary RNA. In particular, 3',4'-EoNA-T and (R)-Me-3',4'-EoNA-T modifications within isoDNA could stabilize the duplexes with complementary RNA compared with unmodified or 3',4'-BNA-modified isoDNAs.

Triplex- and Duplex-Forming Abilities of Oligonucleotides Containing 2'-Deoxy-5-trifluoromethyluridine and 2'-Deoxy-5-trifluoromethylcytidine.

A facile synthesis of 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine phosphoramidites from commercially available 2'-deoxyuridine and 2'-deoxycytidine was achieved, respectively. The obtained phosphoramidites were incorporated into oligonucleotides, and their binding affinity to double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) was evaluated by UV-melting experiments. The triplex-forming abilities of oligonucleotides including 5-trifluoromethylpyrimidine nucleobases with dsDNA were decreased. Especially, the stability of the triplex containing a trifluoromethylcytosine (CF3C)-GC base triplet was low, likely due to the low pKa of protonated CF3C by the electron-withdrawing trifluoromethyl group. A slight decrease in stability of the duplex formed with ssRNA by oligonucleotides including 5-trifluoromethylpyrimidine nucleobases was only observed, suggesting that they might be applicable to various ssRNA-targeted technologies using features of fluorine atoms.