A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

Biscans, Annabelle; Rouanet, Sonia; Vasseur, Jean‐Jacques; Dupouy, Christelle

doi:10.1039/c6ob01272h

Cited by 15 publications

(17 citation statements)

References 30 publications

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“…The SILs between Dox and RNA were designed so that the reduction of the disulfide bonds releases both unstable 2'-hemithioacetal RNA and 2-thioethylcarbamate Dox. Then, on one hand, the hemithioacetal evolves spontaneously towards the recovery of 2 -OH RNA by loss of thioformaldehyde as shown previously [31,32]. On the other hand, the release of native doxorubicin takes place through a rearrangement according to two mechanisms [24,33] with either elimination of 1,3-oxathiolan-2-one (Path A) [34], or of ethylene sulfide and carbon dioxide (Path B) [35,36].…”

Section: Introductionmentioning

confidence: 67%

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

et al. 2020

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Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

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Section: Introductionmentioning

confidence: 67%

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

et al. 2020

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“…Several permanent 2’-O-modifications (2’-F, 2’-OMe) have been proposed to increase the nuclease resistance of ONs, but most of them have decreased gene silencing potential. To overcome this drawback, novel prodrug-type RNAs containing a disulfide bridge at the 2’-position have been designed, and in 2016, Urata and our group reported on the synthesis and properties of 2’- O -alkyldithiomethyl-modified RNAs [ 15 – 16 ]. Previously, Urata had described a post-synthetic approach for the synthesis of 2’- O -methyldithiomethyl (MDTM) ONs [ 17 ] that was more practical than the phosphoramidite approach used initially for the chemical synthesis of RNAs using the 2’- O-tert -butyldithiomethyl-protecting group [ 18 ].…”

Section: Reviewmentioning

confidence: 99%

“…Similarly, our group has developed a post-synthetic method on a solid support to introduce various disulfide bond-containing groups at the 2’-OH of RNAs [ 15 ]. Using this versatile method, one precursor, 2’- O -acetylthiomethyl-containing RNA, produces various 2’- O -alkyldithiomethyl (RSSM)-modified RNAs bearing lipophilic or polar groups through a thiol disulfide exchange reaction with alkyldisulfanyl-pyridine derivatives ( Scheme 3 ).…”

Section: Reviewmentioning

confidence: 99%

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Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Debart¹,

Dupouy²,

Vasseur³

2018

Beilstein J. Org. Chem.

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Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought.This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules.

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“…In this case, a 2′‐amino‐2′‐deoxyuridine was introduced as a precursor unit within the ON sequence then the amino group was converted to 2′‐pyridyldithiopropionamide derivative to perform the thiol‐disulfide exchange reaction with cysteine yielding the disulfide linker . Moreover, in a previous work we developed a post‐synthesis strategy based on the thiol‐disulfide exchange reaction for the preparation of siRNA prodrugs 2′‐ O ‐modified by disulfide‐containing groups . We also used this reaction to introduce a disulfide‐bridge between 2′‐ O ‐positions of two adjacent nucleotides in the loop of RNA hairpins .…”

Section: Introductionmentioning

confidence: 99%

Conjugation of Small Molecules to RNA Using a Reducible Disulfide Linker Attached at the 2′‐OH Position through a Carbamate Function

et al. 2019

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A post‐synthesis conjugation method was developed to functionalize oligoribonucleotides with various small molecules of biological interest using a reduction‐sensitive disulfide linker connected to 2′OH via a carbamate function. For this, first we prepared a 2′‐O‐[(N‐(acetylthio)‐ethylcarbamoyl] (2′‐O‐AcSEC) uridine phosphoramidite as the key precursor of the disulfide linker, that was incorporated into 21‐mer RNAs. Next the 2′‐O‐AcSEC group was converted on solid support to the activated 2′‐O‐[N‐(2‐pyridyldisulfanyl)‐ethylcarbamoyl] (2′‐O‐PySSEC) group. Finally, the coupling between 2′‐O‐PySSEC‐modified RNA and diverse small molecules bearing a thiol function was based on a thiol‐disulfide exchange reaction in solution. Several RNA‐small molecule conjugates were obtained with satisfactory conjugation yields. An RNA was successfully double‐conjugated with an anticancer drug (doxorubicin) attesting the efficiency and the robustness of the conjugation method. We have shown that the disulfide‐linked RNA‐small molecule conjugates were cleaved upon 5.6 mm glutathione treatment, featuring the release of the small molecules in cells.

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A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

Cited by 15 publications

References 30 publications

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Conjugation of Small Molecules to RNA Using a Reducible Disulfide Linker Attached at the 2′‐OH Position through a Carbamate Function

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