Carborhodamine compounds and methods of preparation thereof
The carborhodamine dyes disclosed herein are novel reagents suitable for automated incorporation of carborhodamine dyes into oligonucleotides that can be used in detection methods for nucleic acid targets. This disclosure provides an efficient and simple process for the preparation of carborhodamine compounds and introduces previously unknown reagents for the automated synthesis of oligonucleotide-carborhodamine conjugates.
1. A method for preparing carborhodamine compounds having a structure of
comprising reacting a compound having a structure of
with a compound having a structure of
in a reaction mixture comprising a solvent and a Lewis acid,
wherein:
R 1 and R 2 are independently (C 1 -C 20 )alkyl or (C 1 -C 20 )heteroalkyl, or together R 1 and R 2 form a ring structure comprising one or more rings,
R 3 , R 6 , R 7 , and R 10 are independently selected from H, (C 1 -C 20 )alkyl, (C 1 -C 20 )alkoxy, (C 1 -C 20 )alkenyl, (C 1 -C 20 )alkynyl, aryl, heteroaryl, a linking group, halo, —OR′, —OC(O)R′, —NR′R″, —SR′, —OC(O) NR′R″, —NR″C(O)R′, —NR″C(O)OR′, —NR′C(O)NR″R′″, and —SO 2 H, wherein R′, R″ and R′″ are independently selected from H, (C 1 -C 8 )alkyl, (C 1 -C 8 )heteroalkyl, aryl, heteroaryl and a linking group, and wherein (C 1 -C 20 )alkyl, aryl, and heteroaryl groups are unsubstituted or substituted,
R 4 , R 5 , R 8 and R 9 are independently selected from H, (C 1 -C 20 )alkyl, (C 1 -C 20 )alkoxy, (C 1 -C 20 )alkenyl, (C 1 -C 20 )alkynyl, aryl, heteroaryl, and a linking group, wherein (C 1 -C 20 )alkyl, aryl, and heteroaryl groups are unsubstituted or substituted,
or
R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 form one or more 4- to 7-member ring systems by bridging between one or more of R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 ,
R 11 , R 12 , R 13 and R 14 are independently selected from H, (C 1 -C 20 )alkyl, (C 1 -C 20 )alkoxy, (C 1 -C 20 )alkenyl, (C 1 -C 20 )alkynyl, aryl, heteroaryl, a linking group, halo, —OR iv , —OSO 2 R iv , —OC(O) R iv , —NR iv R v , —SR iv , —R iv , —CN, —NO 2 , —CO 2 R iv , —CONR iv R v , —C(O) R iv , —SO 2 R iv , —SO 2 NR iv R v , —N 3 , —CH(Ph) 2 , perfluoro(C 1 -C 4 )alkoxy, and perfluoro(C 1 -C 4 )alkyl, wherein Ph is phenyl, wherein R iv and R v are independently selected from H, (C 1 -C 20 )alkyl perfluoro(C 1 -C 4 )alkyl, perfluoro(C 1 -C 4 )heteroalkyl, aryl and heteroaryl, and wherein aryl and heteroaryl groups are unsubstituted or substituted,
or
R 11 , R 12 , R 13 and R 14 form one or more 4- to 7-member ring systems by bridging between one or more of R 11 , R 12 , R 13 and R 14 , and
the linking group is selected from (C 1 -C 20 )alkyl, aryl, (C 1 -C 20 )heteroalkyl, heteroaryl and combinations thereof, wherein at least one hydrocarbon hydrogen of the linking group is replaced with a protected or unprotected carboxyl or a hydroxyl group.
2. The method of claim 1 , wherein the solvent is 1,2-dichloroethane and the Lewis acid is aluminum chloride.
3. The method of claim 1 , wherein one or more hydrocarbon hydrogens of R 1 or R 2 is replaced with a linking group.
4. The method of claim 1 , wherein R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 form one or more 4- to 7-member ring systems by bridging between one or more of R 3 with R 4 , R 4 with R 5 , R 5 with R 6 , R 7 with R 8 , R 8 with R 9 , and R 9 with R 10 .
5. The method of claim 1 , wherein R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 form one or more 4- to 7-member ring systems linked to one or more further 4- to 7-member ring systems, and wherein the one or more further 4- to 7-member ring systems comprise up to 3 double bonds.
6. The method of claim 5 , wherein the one or more further 4- to 7-member ring systems comprise heteroatoms.
7. The method of claim 1 , wherein R 11 , R 12 , R 13 and R 14 form one or more 4- to 7-member ring systems by bridging between one or more of R 11 with R 12 , R 12 with R 13 , and R 13 with R 14 .
8. The method of claim 1 , wherein R 11 , R 12 , R 13 and R 14 form one or more 4- to 7-member ring systems linked to one or more further 4- to 7-member ring systems, and wherein the one or more further 4- to 7-member ring systems comprise up to 3 double bonds.
9. The method of claim 8 , wherein the one or more further 4- to 7-member ring systems comprise heteroatoms.
10. The method of claim 1 , wherein at least one hydrocarbon hydrogen of the linking group is replaced with a protected carboxyl or a protected hydroxyl group, wherein the protected carboxyl or the protected hydroxyl group further comprises a protecting group, and wherein the protecting group comprises an amide, an ester, or an ether group.