Fumi Nagatsugi

Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages

A novel pH‐sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L‐lysine) (PLL) and a lactosylated poly(ethylene glycol)–antisense ODN conjugate (Lac‐PEG–ODN) containing an acid‐labile linkage (β‐propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH‐7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac‐PEG–ODN) alone, as well as by the lactose‐free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose‐free PIC micelles into HuH‐7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor‐mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid‐labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B‐PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B‐PEI in the endosome compartment, preventing the cleavage of the acid‐labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.

2-Aminopurine derivatives with C6-substituted olefin as novel cross-linking agents and the synthesis of the corresponding β-phosphoramidite precursors

Abstract The 6-vinylated 2-aminopurine nucleoside (1), which was prepared by the Pd(0)-catalyzed cross-coupling reaction using guanosine 6-O-tosylate and vinyl-tributylstannane, has been demonstrated as a potential cross-linking agent. However, attempts for its incorporation into oligonucleotides were unsuccessful because of the high reactivity toward nucleophiles. In this study, new 2′-deoxy nucleoside derivatives with 6-(2-substituted vinyl)-2-aminopurine were designed to diminish the reactivity of the vinyl group. These new nucleosides have been shown to maintain reactivity toward potent nucleophiles such as butylamine and thiols, suggesting that they would form cross-linking with the target nucleobase due to the proximity within the sense-antisense duplex. Thus, the corresponding β-phosphoramidite precursors were successfully prepared, and were applied to an automated oligonucletotide synthesizer.

6-Vinylated guanosine as a novel cross-linking agent and its versatile synthesis from the 6-O-tosylate by Pd(0)-catalyzed cross-coupling

Abstract 6-Vinylated guanosine ( 1 ) was designed as a novel cross-linking agent, and synthesized by a new Pd(0)-catalyzed cross-coupling reaction using guanosine 6-O-tosylate and vinyltributylstannane. Its potential as a cross-linking agent was demonstrated by adduct formation with guanosine and cytidine at the 7-N and 4-N positions, respectively.

Preparation and Electrochemical and Optical Properties of Unsymmetrically Substituted Phthalocyanines with One or Two Trithiole Rings and Related Symmetric Derivatives

4,5-Bis(benzylthio)-3,6-diethylphthalonitrile (1) was mixed with 4 -t-butylphthalonitrile and then treated with lithium alkoxide in n-hexanol to produce the corresponding unsymmetrically substituted phthalocyanines (2) and (3) with two or four benzylthio groups, respectively. Treatment of phthalocyanine (2) with nickel(II) acetate yielded the corresponding metal complex 2-Ni. Two benzyl groups of 2 and 2-Ni were removed with lithium/THF/ammonia at -78 degrees C under argon, and the dithiolate anions generated were then reacted with elemental sulfur to give monotrithiolophthalocyanines (5) and (5-Ni). A similar treatment of 3 produced bistrithiolophthalocyanine (6). Tetrakistrithiolophthalocyanine (7-Ni) was prepared by complexation of phthalocyanine (4) with nickel(II) acetate, followed by a Birch reduction of the resulting nickel(II) complex (4-Ni), and then sulfurization and cyclization of the octathiolate anions that were generated. The structures of the phthalocyanines were determined by (1)H NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The optical and electrochemical properties of the phthalocyanines were examined by UV-vis absorption spectroscopy and cyclic voltammetry. Treatment of 5, 5-Ni, 6, 7, and 7-Ni with trifluoroacetic acid in chloroform generated positively charged species, which were characterized by UV-vis and/or NMR spectroscopy.

Formation of highly selective and efficient interstrand cross-linking to thymine without photo-irradiation

Interstrand cross-linking (ICL) forming oligodeoxynucleotides (ODNs) have been expected to ensure the inhibition of gene expression. In this communication, we report a highly efficient and selective ICL reaction to thymine using a 4-amino-2-vinyl-6-oxopyrimidine derivative.

Design of a novel artificial nucleobase for the selective formation of a triple-complex with a cytosine-guanine base pair

Abstract A new artificial nucleobase (1) was designed for the selective formation of a triple-complex with a cytosine-guanine base pair at the major groove site. It has been proved based on the 1D- and 2D 1H-NMR spectra that the protected 2′-oxy-1 (7) forms a triplex complex with the GC pair selectively.

4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson–Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5′-side of the opposing uridine. In the absence of a 5′-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.

Expansion of triplex recognition codes by the use of novel bicyclic nucleoside derivatives (WNA)

Recently, we have developed new base analogs (WNA) and demonstrated that WNA·T with thymine and WNA·C with cytosine stabilize non-natural antiparallel triplexes with a TA or a CG interrupting site, respectively. However, limitations in recognizable sequences with the WNAcontaining TFO were also found. The objective of this study is to search better WNA analogs for expansion of triplex recognition codes to general duplex sequences. In this study, we designed new WNA analogs by systematic modification of the aromatic part and the recognition part. The new WNA analogs with the benzene ring substituted with bromide or cyanide have determined for selective stabilization of triplexes at a TA interrupting site, and general formation of triplexes having a TA interrupting site has been achieved.

Production of truncated protein by the crosslink formation of mRNA with 2'-OMe oligoribonucleotide containing 2-amino-6-vinylpurine.

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.

Synthesis of ω-fluorinated octanoic acid and its β-substituted derivatives

Abstract Simple syntheses are described for the ω-fluorinated analogs of octanoic acid and its β-substituted derivatives in which insertion of a methyl and dimethyl group, and oxygen substitution at the C-3 position are involved, employing nucleophilic displacement with fluoride ion of the tosylate functions in the later stage of synthesis. The synthetic procedures offer easy and convenient access to the corresponding 18 F-labeled analogs using the readily available [ 18 F]fluoride ion.