Functionalized Nucleic Acids Nuclease Stability
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Solution Overview
Problem
Oligonucleotides face instability against nucleases, poor cell penetration, and distribution due to their natural sequences, limiting their therapeutic, diagnostic, and research applications.
Innovation Solution
The synthesis of novel functionalized nucleic acids and nucleic acid prodrugs with chiral phosphorous moieties, specifically phosphorothiotriesters, is described, involving processes such as reacting H-phosphonates with silylating and thiosulfonate reagents to enhance stability and binding affinity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If natural sequences of DNA or RNA are used for therapeutics, then the oligonucleotides are simple and easy to manufacture, but they show instability against extra and intracellular nucleases and poor cell penetration
Solution Approach 1:
The patent modifies the chemical parameters of the oligonucleotide backbone by replacing natural phosphodiester linkages with phosphorothioate linkages (substituting oxygen with sulfur). This parameter change fundamentally alters the chemical properties of the backbone, providing resistance to nuclease degradation while maintaining the ability to be synthesized through adapted chemical protocols
Solution Approach 2:
The invention creates composite oligonucleotide structures by combining modified nucleotide units with phosphorothioate backbones. These composite structures integrate the sequence-specific recognition capability of natural nucleic acids with the enhanced stability of chemically modified backbones, achieving both therapeutic efficacy and nuclease resistance
2Device complexity
If natural sequences of DNA or RNA are used for therapeutics, then the oligonucleotides are simple in structure, but they show poor cell penetration and distribution
Solution Approach 1:
The patent changes the chemical parameters of the oligonucleotide backbone by introducing phosphorothioate linkages, which alter the electrostatic properties and hydrophobicity of the molecule. These parameter changes enhance cell membrane penetration capability while maintaining relatively simple synthetic procedures adapted from standard oligonucleotide synthesis
3Reliability
If modified oligonucleotides with chiral phosphorous moieties are synthesized, then stability towards nucleases and binding affinity are improved, but the synthesis process becomes more complex
Solution Approach 1:
The patent employs preliminary action by incorporating chiral phosphorous reagents during the oligonucleotide synthesis process itself, rather than attempting to modify the backbone after synthesis. This approach integrates the chirality induction step into the standard synthesis protocol, reducing overall process complexity compared to post-synthesis modification methods
Solution Approach 2:
The invention uses chiral phosphorous intermediates as mediators during the coupling reactions of oligonucleotide synthesis. These intermediates facilitate the formation of phosphorothioate linkages with defined stereochemistry, enabling controlled introduction of chirality without requiring complex multi-step synthesis procedures
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These modified oligonucleotides demonstrate improved stability, increased cell penetration, and bio-distribution, addressing the limitations of natural oligonucleotides in therapeutic and diagnostic contexts.
Implementation Method 1
reacting an H-phosphonate of structure Ia with an silylating reagent to provide a silyloxyphosphonate
Implementation Method 2
reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIa to provide a phosphorothiotriester of structure IIIa
Data Source
AI summary
The present application, among other things, provides technologies, e.g., reagents, methods, etc. for preparing oligonucleotides comprising phosphorothiotriesters linkages. In some embodiments, provided methods comprise reacting an H-phosphonate of structure Ia or Ib with a silylating reagent to provide a silyloxyphosphonate, and reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIa or IIb to provide an oligonucleotide of structure IIIa or IIIb. In some embodiments, provided methods comprise reacting an H-phosphonate of structure Ic with a silylating reagent to provide a silyloxyphosphonate, reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent of structure IVc to provide a phosphorothiotriester comprising a thiosulfonate group of structure Vc, and then reacting the phosphorothiotriester comprising a thiosulfonate group of structure Vc with a nucleophile of structure VIc to provide an oligonucleotide of structure IIIc. In some embodiments, the present application provides a thiosulfonate reagent of structure IIa: