Modified carbocyanine dyes and their conjugates
View Patent ↗Chemically reactive carbocyanine dyes incorporating an indolium ring moiety that is substituted at the 3-position by a reactive group or by a conjugated substance, and their uses, are described. Conjugation through this position results in spectral properties that are uniformly superior to those of conjugates of spectrally similar dyes wherein attachment is at a different position. The invention includes derivative compounds having one or more benzo nitrogens.
1. A method of staining a biological sample, comprising:
combining a dye solution comprising a compound having the formula:
and its salts, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 3 is carboxyalkyl or —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a carboxylic acid, a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide; d is 0-5; e is 1-5; and z′ is 0 or 1;
R 4 is alkyl;
R 13 is alkyl or sulfoalkyl;
R 14 is alkyl or sulfoalkyl;
R 6 is selected from hydrogen or sulfo;
R 7 is selected from hydrogen or sulfo;
R 8 is selected from hydrogen or sulfo;
R 9 is selected from hydrogen or sulfo;
R 16 is selected from hydrogen or sulfo;
R 17 is selected from hydrogen or sulfo;
R 18 is selected from hydrogen or sulfo;
R 19 is selected from hydrogen or sulfo;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2, or 3;
with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.
2. The method of claim 1 , wherein R 2 is sulfoalkyl.
3. The method of claim 1 , wherein R 12 is alkyl.
4. The method of claim 1 , wherein R 12 is ethyl.
5. The method of claim 1 , wherein R 4 is methyl.
6. The method of claim 1 , wherein R 14 is methyl.
7. The method of claim 1 , wherein each of the sulfoalkyl groups are sulfopropyl groups.
8. The method of claim 1 , wherein R 7 and R 17 are sulfo.
9. The method of claim 1 , wherein n is 1.
10. The method of claim 1 , wherein n is 2.
11. The method of claim 1 , wherein R 3 is —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a succinimidyl ester of a carboxylic acid.
12. The method of claim 10 , wherein; d is 3 or 5 and z′ is 0.
13. The method of claim 1 , wherein R 3 is —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a maleimide.
14. The method of claim 12 , wherein d is 3 or 5; e is 2; and z′ is 1.
15. The method of claim 1 , wherein R 16 is alkyl.
16. The method of claim 1 , wherein the salt is sodium.
17. The method according to claim 1 , wherein the compound has the formula:
and its salts, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 3 is carboxyalkyl or —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a carboxylic acid, a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide; d is 0-5; e is 1-5; and z′ is 0 or 1;
R 13 is sulfoalkyl;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2 or 3.
18. The method according to claim 1 , wherein the compound has the formula:
or a salt thereof, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 13 is alkyl or sulfoalkyl;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2 or 3.
19. The method according to claim 1 , wherein the sample comprises cells, proteins, or nucleic acid polymers.
20. The method according to claim 1 , wherein the sample is contained in or on a solid or semi-solid matrix that is a membrane, an electrophoretic gel, silicon chip, a glass slide, a microwell plate or a microfluidic chip.
21. A method for forming a dye-conjugate of a protein, peptide, or a nucleic acid polymer, said method comprising:
combining a dye solution comprising a compound of the formula:
and its salts, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 3 is carboxyalkyl or —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a carboxylic acid, a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide; d is 0-5; e is 1-5; and z′ is 0 or 1;
R 4 is alkyl;
R 13 is alkyl or sulfoalkyl;
R 14 is alkyl or sulfoalkyl;
R 6 is selected from hydrogen or sulfo;
R 7 is selected from hydrogen or sulfo;
R 8 is selected from hydrogen or sulfo;
R 9 is selected from hydrogen or sulfo;
R 16 is selected from hydrogen or sulfo;
R 17 is selected from hydrogen or sulfo;
R 18 is selected from hydrogen or sulfo;
R 19 is selected from hydrogen or sulfo;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2, or 3;
with a sample comprising a protein, peptide, or a nucleic acid polymer whereby a dye conjugate is formed.
22. A kit for fluorescent labeling of a biological sample, comprising:
a dye solution comprising a compound having the formula:
and its salts, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 3 is carboxyalkyl or —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a carboxylic acid, a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide; d is 0-5; e is 1-5; and z′ is 0 or 1;
R 4 is alkyl;
R 13 is alkyl or sulfoalkyl;
R 14 is alkyl or sulfoalkyl;
R 6 is selected from hydrogen or sulfo;
R 7 is selected from hydrogen or sulfo;
R 8 is selected from hydrogen or sulfo;
R 9 is selected from hydrogen or sulfo;
R 16 is selected from hydrogen or sulfo;
R 17 is selected from hydrogen or sulfo;
R 18 is selected from hydrogen or sulfo;
R 19 is selected from hydrogen or sulfo;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2, or 3;
and a buffer.
23. The kit of claim 21 , further comprising instructions for fluorescent labeling a biological sample.
24. A composition comprising a compound having the formula:
and its salts, wherein,
R 2 is alkyl or sulfoalkyl;
R 12 is alkyl or sulfoalkyl;
R 3 is carboxyalkyl or —(CH 2 ) d (CONH(CH 2 ) e ) z′ —R x , wherein R x is a carboxylic acid, a succinimidyl ester of a carboxylic acid, a hydrazide, or a maleimide; d is 0-5; e is 1-5; and z′ is 0 or 1;
R 4 is alkyl;
R 13 is alkyl or sulfoalkyl;
R 14 is alkyl or sulfoalkyl;
R 6 is selected from hydrogen or sulfo;
R 7 is selected from hydrogen or sulfo;
R 8 is selected from hydrogen or sulfo;
R 9 is selected from hydrogen or sulfo;
R 16 is selected from hydrogen or sulfo;
R 17 is selected from hydrogen or sulfo;
R 18 is selected from hydrogen or sulfo;
R 19 is selected from hydrogen or sulfo;
wherein sulfo is sulfonic acid or sulfonate; and
n is 1, 2, or 3;
and a protein, peptide, or a nucleic acid polymer.