Synthesis of an Ortho-Functionalized Tetrafluorinated Azobenzene Phosphoramidite for Incorporation into a Tetrafluorinated Azobenzene-Containing siRNA for Photocontrolled Gene Silencing.

This article presents the detailed synthesis and characterization protocols for an ortho-functionalized tetrafluorinated azobenzene containing siRNA, which has photoswitchable properties. To design this tetrafluorinated azobenzene scaffold, several synthetic steps are performed to generate a symmetrical tetrafluorinated azobenzene diol. This diol is treated with dimethoxytrityl chloride (DMT-Cl) to protect one of the alcohols. Next, the DMT-protected tetrafluorinated monoalcohol is phosphitylated to afford the DMT-phosphoramidite building block used for solid-phase synthesis. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of an ortho-functionalized fluorinated azobenzene containing siRNA (F-siRNA), which has photoswitchable properties that can be controlled with visible light. First, the F-siRNA was characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile and melting temperature (Tm ) of the duplexes. Second, biological testing of the F-siRNA is performed in cell culture to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to green light to inactivate the F-siRNA, followed by blue light, which reactivates the F-siRNA. The F-siRNA can be kept inactive for up to 72 hr and reactivated at any time within this 72-hr window. © 2023 Wiley Periodicals LLC.

Synthesis of Dinucleotide Non-Symmetrical Triester Phosphate Phosphoramidites and Their Incorporation Within Oligonucleotides.

In this protocol article, the synthesis of dinucleotide non-symmetrical triester phosphate phosphoramidites will be highlighted. Specifically, we use a selective transesterification starting with tris(2,2,2-trifluoroethyl) phosphate to afford a dinucleotide derivative phosphate ester. Substitution of the final trifluoroethyl group with various alcohols affords a dinucleotide triester phosphate with a hydrophobic group, which can then be deprotected and converted to a phosphoramidite for incorporation within oligonucleotides. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of a DMT- and TBS-protected unsymmetrical dinucleotide Basic Protocol 2: Synthesis of a DMT-protected unsymmetrical dinucleotide phosphotriester monoalcohols Basic Protocol 3: Synthesis of DMT-protected phenylethyl phosphotriester dinucleotide phosphoramidites Basic Protocol 4: Synthesis, purification, and characterization of RNAs containing triester phosphate modifications.

Synthesis, Derivatization and Photochemical Control of an ortho-Functionalized Tetrafluorinated Azobenzene-Modified siRNA.

We report the synthesis of an ortho-functionalized tetrafluorinated azobenzene phosphoramidite for its site-specific incorporation into RNA. The tetrafluorinated azobenzene is embedded within the antisense strand of an siRNA duplex to form an ortho-functionalized tetrafluorinated azobenzene-containing siRNA (F-siRNAzo). The F-siRNAzo is inactivated via trans to cis conversion with green light (530 nm), and reactivated with blue light (470 nm) via cis to trans conversion in cell culture. The long half-life and stability of the tetrafluorinated azobenzene unit allows for reversible control of the F-siRNAzo in cell culture for up 72 hours.

SiRNAs with Neutral Phosphate Triester Hydrocarbon Tails Exhibit Carrier-Free Gene-Silencing Activity.

Short interfering RNAs (siRNAs) show promise as gene-silencing therapeutics, but their cellular uptake remains a challenge. We have recently shown the synthesis of siRNAs bearing a single neutral phenylethyl phosphotriester linkage within the sense strand. Here, we report the synthesis of siRNAs bearing three different hydrophobic phosphate triester linkages at key positions within the sense strand and assess their gene silencing in the absence of a transfection carrier. The best siRNAs bearing hydrophobic phosphate triester tails were not aromatic and exhibited effective gene silencing (IC50 ≈ 56-141 nM), whereas the aromatic derivative with three hydrophobic tails did not exhibit carrier-free gene silencing.

Building siRNAs with Cubes: Synthesis and Evaluation of Cubane-Modified siRNAs.

Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC50 values ranging from 45.4 to 305 pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.

Synthesis and Evaluation of Neutral Phosphate Triester Backbone-Modified siRNAs.

Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2-trifluoroethyl) phosphate. The protected triesters were phosphytilated to generate phosphoramidites for solid-phase oligonucleotide synthesis. Neutral phenylethyl phosphate-modified short-interfering RNAs (siRNAs) were synthesized and evaluated for their gene-silencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many improvements compared to natural unmodified siRNAs.

Selective Transesterification of 2,2,2-Trifluoroethyl Phosphates: Synthesis of Mixed Unsymmetrical Phosphates.

A selective transesterification starting with tris(2,2,2-trifluoroethyl) phosphate has been developed. This method involves a three-step substitution for 2,2,2-trifluoroethoxy groups and enables the facile synthesis of mixed unsymmetric phosphate triesters from three different alcohols. The substitution of the trifluoroethoxy group at the phosphorus proceeds selectively in the presence of DBU or lithium alkoxides. This method can be applied for the preparation of phospholipids.

Synthesis of (±)-myo-inositol 4-methylenephosphonate via Rh-Catalyzed hydrogenation of vinylphosphonate.

Phosphatidylinositol phosphate (PIP) synthetase is a promising target for the development of new anti-mycobacterium compounds. We previously reported that myo-inositol 1-methylenephosphonate showed inhibitory activity against PIP synthetase. Herein, we report the synthesis of unprotected myo-inositol 4-methylenephosphonate, a constitutional isomer of myo-inositol 1-methylenephosphonate and found that the stereoselective hydrogenation of vinylphosphonate proceeded via Rh catalysis.