Synthesis of novel cationic spermine-conjugated phosphotriester oligonucleotide for improvement of cell membrane permeability

Hayashi, Junsuke; Hamada, Tomoko; Sasaki, Ikumi; Nakagawa, Osamu; Wada, Shun‐ichi; Urata, Hidehito

doi:10.1016/j.bmcl.2015.06.071

Cited by 5 publications

(2 citation statements)

References 18 publications

(12 reference statements)

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“…We first determined the stereochemistry of the Me-PTE-containing T(Me)T based on 31 P-NMR analyses, where the earlier and later eluting T(Me)T-1 and T(Me)T-2 were found to contain the S p and R p diastereomers of the Me-PTE, respectively. A chemical shift difference of 0.16 ppm for the two diastereomers was nearly identical to what was previously reported ( 26 ). We next digested the HPLC-purified Me-PTE-containing 12-mer ODNs with a cocktail of four enzymes, which release Me-PTE as T(Me)T. By comparing the HPLC elution properties of the T(Me)T liberated from the 12-mer ODNs with those of the standards, we demonstrated that the earlier- and later-eluting 12-mer ODNs contained the S p - and R p -Me-PTEs, respectively.…”

Section: Resultssupporting

confidence: 88%

Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Wang

2018

Nucleic Acids Research

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Exposure to many endogenous and exogenous agents can give rise to DNA alkylation, which constitutes a major type of DNA damage. Among the DNA alkylation products, alkyl phosphotriesters have relatively high frequencies of occurrence and are resistant to repair in mammalian tissues. However, little is known about how these lesions affect the efficiency and fidelity of DNA replication in cells or how the replicative bypass of these lesions is modulated by translesion synthesis DNA polymerases. In this study, we synthesized oligodeoxyribonucleotides containing four pairs (Sp and Rp) of alkyl phosphotriester lesions at a defined site, and examined how these lesions are recognized by DNA replication machinery in Escherichia coli cells. We found that the Sp diastereomer of the alkyl phosphotriester lesions could be efficiently bypassed, whereas the Rp counterparts moderately blocked DNA replication. Moreover, the Sp-methyl phosphotriester induced TT→GT and TT→GC mutations at the flanking TT dinucleotide site, and the induction of these mutations required Ada protein, which is known to remove efficiently the methyl group from the Sp-methyl phosphotriester. Together, our study provided a comprehensive understanding about the recognition of alkyl phosphotriester lesions by DNA replication machinery in cells, and revealed for the first time the Ada-dependent induction of mutations at the Sp-methyl phosphotriester site.

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Section: Resultssupporting

confidence: 88%

Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Wang

2018

Nucleic Acids Research

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“…24, 25 In most cases, polyamine conjugation had beneficial effects on target hybridization, 26 nuclease resistance, and cellular uptake. 18, 19 Unfortunately, attachment of a one or more polyamine molecule to either the 5′ or the 3′ end of the ON has certain drawbacks. Clustering of the positive charges at the end of the ONs is not the optimal arrangement for formation of tight ion pairs with the negative charges throughout the ON’s backbone, and can often result in intermolecular aggregations and solubility problems.…”

Section: Introductionmentioning

confidence: 99%

Self-neutralizing oligonucleotides with enhanced cellular uptake

et al. 2017

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There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by Aphosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

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Prodrug‐Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment

Sugimoto,

Hayashi,

Funaki

et al. 2023

ChemBioChem

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Various chemical modifications have been developed to create new antisense oligonucleotides (AONs) for clinical applications. Our previously designed prodrug‐type phosphotriester‐modified oligonucleotide with cyclic disulfides (cyclic SS PTE ON) can be converted into unmodified ON in an intracellular‐mimetic reducing environment. However, the conversion rate of the cyclic SS PTE ON was very low, and the AON with cyclic SS PTE modifications showed much weaker antisense activity than corresponding the fully phosphorothioate‐modified AON. In this study, we synthesized several types of PTE ONs containing linear disulfides (linear SS PTE ONs) and evaluated their conversion rates under reducing conditions. From the results, the structural requirements for the conversion of the synthesized linear SS PTE ONs were elucidated. Linear SS PTE ON with promising promoieties showed a nuclease resistance up to 4.8‐fold compared to unmodified ON and a cellular uptake by endocytosis without any transfection reagent. In addition, although the knockdown activity of the linear SS PTE gapmer AON is weaker than that of the fully phosphorothioate‐modified gapmer AON, the knockdown activity is slightly stronger than that of the cyclic SS PTE gapmer AON. These results suggest that the conversion rates may be related to the expression of the antisense activity.

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Synthesis of novel cationic spermine-conjugated phosphotriester oligonucleotide for improvement of cell membrane permeability

Cited by 5 publications

References 18 publications

Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells

Self-neutralizing oligonucleotides with enhanced cellular uptake

Prodrug‐Type Phosphotriester Oligonucleotides with Linear Disulfide Promoieties Responsive to Reducing Environment

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