Cyanine dyes and methods of use
View Patent ↗The present invention provides for cyanine dyes as near IR quenchers. The cyanine dyes have absorption wavelengths in the near-infrared region of about 650-900 nm and are essentially non-fluorescent. The dyes of the invention have at least one linking group. The present invention also provides substantially non-fluorescent, NIR probes. Biological assays based on these novel, substantially non-fluorescent, NIR probes are also provided.
1. An essentially non-fluorescent compound of formula Ib:
wherein:
R 1 and R 2 are each independently selected from the group consisting of hydrogen and optionally substituted (C 1 -C 6 )alkyl; or alternatively, R 1 and R 2 together with the
group to which they are bonded form a 5- to 7-membered ring, said ring being optionally substituted with 1 to 4 substituents selected from the group consisting of halogen, cyano, sulfonate, (C 1 -C 8 )haloalkyl, hydroxy, (C 1 -C 6 )alkoxy and optionally substituted (C 1 -C 8 )alkyl;
R 3 and R 4 are each an optionally substituted (C 1 -C 6 )alkyl, and may optionally join together with the atom to which they are attached to form a 5- to 7-membered carbocyclic ring;
or alternatively the substituents R 3 and R 4 are replaced with the group
wherein B is (C 1 -C 6 )alkyl; or B and R 9a together with the carbon atoms to which they are attached join to form a 5- or 6-membered ring optionally having 1 or 2 heteroatoms and optionally having up to 3double bonds;
R 5 and R 6 are each an optionally substituted (C 1 -C 6 )alkyl, and may optionally join together with the atom to which they are attached to form a ring;
R 7 and R 8 are each independently selected from the group consisting of optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl(C 1 -C 6 )alkyl, optionally substituted heteroaryl(C 1 -C 6 )alkyl, —(CH 2 ) c R 13 , —(CH 2 ) d R 15 , —(CH 2 ) d C(O)OR 15 , and —(CH 2 ) d C(O)NH(CH 2 ) d R 15 , wherein c and d are each independently an integer from 1-50, R 13 is a functional group that does not directly react with a carboxyl, hydroxyl, amino or thio group on a biomolecule, R 15 is a linking group selected from the group consisting of mercapto, amino, haloalkyl, phosphoramidityl, N-hydroxysuccinimidyl ester, sulfo N-hydroxysuccinimidyl ester, isothiocyanato, iodoacetamidyl, maleimidyl and an activated carboxylic acid;
R 9a-9d and R 10c-10d are each independently selected from the group consisting of hydrogen, optionally substituted (C 1 -C 6 )alkyl, —SO 3 Cat, halogen, —C(O)OR 11 , —C(O)NR 11 R 12 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —NR 12 C(O)OR 11 , —(CH 2 ) d R 15 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 and —NR 20 R 21 , wherein Cat is a cation, wherein R 11 and R 12 are each independently selected from the group consisting of hydrogen and optionally substituted (C 1 -C 6 )alkyl; R 20 and R 21 are each independently selected from the group consisting of optionally substituted (C 1 -C 8 )alkyl, CatO 3 S(C 1 -C 50 )alkylene,
a is an integer from 0-3;
b is an integer from 0-2; and
A is selected from the group consisting of hydrogen, halogen, cyano, optionally substituted (C 1 -C 8 )alkyl, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted alkylthio, —(CH 2 ) d R 15 , —R 15 , optionally substituted (C 1 -C 6 )heteroalkyl, phenoxy, and an optionally substituted aryloxy group having the formula
wherein R 36a -R 36e are each independently selected from the group consisting of hydrogen, —SO 3 Cat, —(CH 2 ) d R 15 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 , (C 1 -C 6 )alkyl, carboxyl and NR 20 R 21 ;
wherein said compound contains at least one linking group;
wherein said compound has at least one —NR 20 R 21 group substituted at a position selected from the group consisting of R 10c and R 10d ;
wherein R 14a-14d are each independently selected from the group consisting of hydrogen, optionally substituted (C 1 -C 6 )alkyl, —SO 3 Cat, halogen, —C(O)OR 11 , —C(O)NR 11 R 12 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —NR 12 C(O)OR 11 , —(CH 2 ) d R 15 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 and —NR 20 R 21 ; and
wherein said compound is essentially non-fluorescent and is charge neutral.
2. The compound of claim 1 , wherein R 10c in formula Ib is —NR 20 R 21 .
3. The compound of claim 1 , wherein R 10d in formula Ib is —NR 20 R 21 .
4. The compound of claim 1 , wherein at least one of R 14a to R 14d is —SO 3 Cat.
5. The compound of claim 1 , wherein R 10c and R 10d are both NR 20 R 21 .
6. The compound of claim 1 , of formula Ic:
7. The compound of claim 6 , wherein at least one of R 9b , R 9d and R 10d in formula Ic is an —NR 20 R 21 group.
8. An essentially non-fluorescent compound of formula Id:
wherein:
R 1 and R 2 are each independently selected from the group consisting of hydrogen and optionally substituted (C 1 -C 6 )alkyl; or alternatively, R 1 and R 2 together with the
group to which they are bonded form a 5- to 7-membered ring, said ring being optionally substituted with 1 to 4 substituents selected from the group consisting of halogen, cyano, sulfonate, (C 1 -C 8 )haloalkyl, hydroxy, (C 1 -C 6 )alkoxy and optionally substituted (C 1 -C 8 )alkyl;
R 5 and R 6 are each an optionally substituted (C 1 -C 6 )alkyl, and may optionally join together with the atom to which they are attached to form a ring;
R 7 and R 8 are each independently selected from the group consisting of optionally substituted (C 1 -C 6 )alkyl, optionally substituted aryl(C 1 -C 6 )alkyl, optionally substituted heteroaryl(C 1 -C 6 )alkyl, —(CH 2 ) c R 13 , —(CH 2 ) d R 15 , —(CH 2 ) d C(O)OR 15 , and —(CH 2 ) d C(O)NH(CH 2 ) d R 15 , wherein c and d are each independently an integer from 1-50, R 13 is a functional group that does not directly react with a carboxyl, hydroxyl, amino or thio group on a biomolecule, R 15 is a linking group selected from the group consisting of mercapto, amino, haloalkyl, phosphoramidityl, N-hydroxysuccinimidyl ester, sulfo N-hydroxysuccinimidyl ester, isothiocyanato, iodoacetamidyl, maleimidyl and an activated carboxylic acid;
R 9b-9d and R 10a-10d are each independently selected from the group consisting of hydrogen, optionally substituted (C 1 -C 6 )alkyl, —SO 3 Cat, halogen, —C(O)OR 11 , —C(O)NR 11 R 12 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —NR 12 C(O)OR 11 , —(CH 2 ) d R 15 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 and —NR 20 R 21 , wherein Cat is a cation, wherein R 11 and R 12 are each independently selected from the group consisting of hydrogen and optionally substituted (C 1 -C 6 )alkyl; R 20 and R 21 are each independently selected from the group consisting of hydrogen, optionally substituted (C 1 -C 8 )alkyl, CatO 3 S(C 1 -C 50 )alkylene,
alternatively, any two substituents of R 10a-10d located on adjacent atoms, together with the atroms to which are attached, join to form a 5- or 6-membered ring optionally having 1 or 2 hetroatoms and optionally having up to 3 double bonds; wherein said ring may be further substituted with 1 to 3 substituents selected from the group consisting of optionally substituted (C 1 -C 6 )alky, —SO 3 Cat, halogen, —C(O)OR 11 , —C(O)NR 11 R 12 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —NR 12 C(O)OR 11 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 and —NR 20 R 21 ;
a is an integer from 0-3;
b is an integer from 0-2; and
A is selected from the group consisting of hydrogen, halogen, cyano, optionally substituted (C 1 -C 8 )alkyl, optionally substituted (C 1 -C 6 )dialkylamino, optionally substituted alkylthio, —(CH 2 ) d R 15 , —R 15 , optionally substituted (C 1 -C 6 )heteroalkyl, phenoxy, and an optionally substituted aryloxy group having the formula
wherein R 36a -R 36e are each independently selected from the group consisting of hydrogen, —SO 3 Cat, —(CH 2 ) d R 15 , —C(O)O(CH 2 ) d R 15 , —C(O)NR 11 (CH 2 ) d R 15 , —NR 12 C(O)O(CH 2 ) d R 15 , —S(O) 2 NR 12 (CH 2 ) d R 15 , —R 15 , (C 1 -C 6 )alkyl, carboxyl and NR 20 R 21 ;
wherein said compound contains at least one linking group;
wherein said compound has at least one —NR 20 R 21 group substituted on an aromatic ring; and
wherein said compound is essentially non-fluorescent and is charge neutral.
9. The compound of claim 8 , wherein at least one of R 9b , R 9d , R 10b and R 10d on formula Id is a —NR 20 R 21 group.
10. The compound of claim 1 , 6 or 8 , wherein A is halogen; R 1 and R 2 together with the
group to which they are bonded form a 5- to 7-membered ring; and a and b are each 1.
11. The compound of claim 1 , 6 or 8 wherein A is halogen.
12. The compound of claim 1 , 6 or 8 , wherein A is a substituted aryloxy group, wherein R 36a, R 36b , R 36d and R 36e are each hydrogen, and R 36c is —SO 3 Cat, wherein Cat is a sodium or potassium cation.
13. The compound of claim 1 , 6 or 8 , wherein A is hydrogen; R 1 and R 2 are both hydrogen;
a is 0 or 1; and
b is 0 or 1.
14. The compound of claim 1 , wherein R 7 is —(CH 2 ) d R 15 , wherein d is an integer from 1-10, and wherein R 15 is a N-hydroxy succinimidyl ester, a sulfo N-hydroxysuccinimidyl ester or a maleimide.
15. A compound, wherein said compound is selected from the group consisting of:
16. A compound, wherein said compound is selected from the group consisting of:
17. A compound having the formula: