Reductively cleavable linkers for incorporation into oligonucleotides
Azo dye-based cleavable linkers address the instability and complexity issues of existing linkers by enabling stable and selective cleavage of oligonucleotides under mild reductive conditions, allowing controlled release of functional molecules.
Patent Information
- Application Number
- DE202025003413
- Authority / Receiving Office
- DE · DE
- Patent Type
- Utility models
- Current Assignee / Owner
- Filing Date
- 2025-11-10
- Publication Date
- 2026-01-22
- Estimated Expiration
- 2035-11-30
AI Technical Summary
Existing cleavable linkers for oligonucleotides, such as photocleavable and disulfide linkers, are either complex to operate or unstable under standard synthesis conditions, necessitating the development of a more stable and easily cleavable alternative.
Incorporation of azo dye-based cleavable linkers into oligonucleotides, designed with one hydroxyl group blocked and one phosphitylated, allowing for selective cleavage under mild reductive conditions using tris(2-carboxyethyl)phosphine or sodium dithionite.
Enables easy and selective cleavage of oligonucleotides and conjugates under defined conditions, facilitating the release of functional molecules for effect or removal.
Abstract
Description
[0001] Many chemical, biochemical, and biological applications require stable molecular linkages that can be cleaved mildly, rapidly, and selectively under certain conditions. For this purpose, cleavable linkers are incorporated into the respective molecules. In the case of oligonucleotides, phosphoramidite building blocks are suitable for the direct incorporation of these linkers during automated solid-phase synthesis on a DNA synthesizer.
[0002] Commonly used cleavable linkers include, for example, so-called PC linkers (photocleavable linkers) or disulfide linkers. In the first case, cleavage requires more complex equipment (irradiation apparatus), while in the second case, the linker is often not sufficiently stable under standard synthesis conditions.
[0003] One solution to the problem is the use of cleavable linkers based on azo dyes. Azo groups can be easily cleaved reductively under mild conditions, e.g., by treatment with tris(2-carboxyethyl)phosphine (TCEP) or dithionite (e.g., sodium dithionite, Na₂S₂O₄).
[0004] This splitting is accompanied by a decolorization and can therefore be easily observed visually.
[0005] Dinuclear or polynuclear azo dyes are designed and synthesized such that a hydroxyl group is attached to an alkyl or PEG chain on each side of the azo bridge(s). One of these hydroxyl groups is blocked with a protecting group suitable for solid-phase oligonucleotide synthesis, ideally dimethoxytrityl, while the other is phosphitylated to form phosphoramidite. The resulting building blocks can then be incorporated into an oligonucleotide strand or at the end of a solid-phase oligonucleotide synthesis. Furthermore, these building blocks can be inserted between an oligonucleotide and other detection or reactive molecules (e.g., dyes, haptens, peptides, proteins, antibodies, antibiotics, lipids, sugar molecules, reactive linkers with functional groups such as amino, carboxy, alkyne, azide, thiol).
[0006] Oligonucleotides or oligonucleotide conjugates, which were modified during synthesis with appropriate cleavable linkers based on azo compounds, can now be easily and selectively cleaved under reductive conditions. In this way, parts of an oligonucleotide or molecules coupled to an oligonucleotide can be released under defined conditions and thus either exert their effect or be removed from the system.
Claims
[1] Phosphoramidite compounds characterized by The following structures: m, n, k, l each independently of each other: 0 to 20 R1: Trityl, Dimethoxytrityl, Monomethoxytrityl, Acetyl, Trifluoroacetyl, Benzoyl R2 to R 17 Each independently of each other: H, alkyl, substituted alkyl, alkoxy, halogen, nitro [2] Phosphoramidite compounds characterized by the structures of claim 1, wherein alkyl chains may be wholly or partially replaced by polyethylene glycol chains (PEG). [3] Oligonucleotide compounds with reductively cleavable linkers, characterized by The following structures: m, n, k, l each independently of each other: 0 to 20 Oligo1, Oligo2: each oligonucleotide with 2 to 100 nucleotide units R2 to R 17 Each independently of each other: H, alkyl, substituted alkyl, alkoxy, halogen, nitro [4] Oligonucleotide conjugates with reductively cleavable linkers, characterized byThe following structures: m, n, k, I each independently of each other: 0 to 20 Oligo: oligonucleotide with 2 to 200 nucleotide units R2 to R 17 Each independently of each other: H, alkyl, substituted alkyl, alkoxy, halogen, nitro R 18 : Dye, hapten, peptide, protein, antibody, antibiotic, lipid, sugar, reactive linker