Fluorescence quenching azo dyes, their methods of preparation and use
Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition.
1. A method for making an oligonucleotide, comprising introducing the compound of Formula 1 to an automated oligonucleotide synthesizer to chemically incorporate a quencher compound into the oligonucleotide:
wherein R 1-6 on the conjugated ring system are individually an electron withdrawing group, alkyl group, aryl group, hydrogen, heteroaryl group, or a five or six member ring structure formed from the R 1 , R 2 pair, the R 3 , R 4 pair, the R 4 , R 5 pair, or the R 5 , R 6 pair; and wherein R 7 is an anilyl group of Formula 2:
wherein L and L′ are the same or different and are a C 1-10 group, —C 1-10 —O—R 8 , or —C 1-10 —O—R 9 , wherein C 1-10 is a C 1-10 alkyl group, and R 8 and R 9 are independently a hydrogen, trityl, alkoxytrityl, silyl, or phosphoramidite with the proviso that when L or L′ is ethyl, then the other is not hydroxyethyl, and
wherein at least one of L or L′ is —C 1-10 —O—R 8 or —C 1-10 —O—R 9 , and at least one of R 8 and R 9 is a phosphoramidite.
2. The method of claim 1 , wherein the phosphoramidite group is a cyanoethylphosphoramidite.
3. The method of claim 2 , wherein the quencher compound is coupled to the 3′-terminus of the oligonucleotide.
4. The method of claim 3 , wherein the quencher compound is coupled to the 5′-terminus of the oligonucleotide.
5. The method of claim 1 , wherein the synthesis step comprises linking the compound of Formula 1 to a solid support.
6. The method of claim 5 , wherein the solid support is controlled pore glass.
7. The method of claim 1 , wherein the compound of Formula 1 is the compound of Formula 13:
wherein CEP is cyanoethylphosphoramidite.
8. The method of claim 1 , wherein the compound of Formula 1 is the compound of Formula 14:
wherein CEP is cyanoethylphosphoramidite.
9. The method of claim 1 , wherein the oligonucleotide further comprises a fluorophore.