Cyanine dyes

Patent metadata

Field	Value
Publication number	US-9441270-B2
Application number	US-201414489143-A
Country	US
Kind code	B2
Filing date	Sep 17, 2014
Priority date	Aug 25, 2010
Publication date	Sep 13, 2016
Grant date	Sep 13, 2016

Abstract

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

First claim

What is claimed is:

1. A cyanine compound having the structure of Formula (I) or (II):

wherein
n is an integer selected from 1, 2 and 3;
e is an integer selected from 0, 1 and 2;
R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl, or a bond to M;
R c , and R d are independently selected from a bond to M, and alkyl substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, phosphoric acid and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, phosphoric acid;
R a and R e , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O)NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond to M,
wherein
R 9 is a member selected from OR 10 , and a bond to M
wherein
R 10 is a member selected from H and substituted or unsubstituted alkyl; and

R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond to M,

wherein
M comprises a linker bound to a carrier molecule, and at least one of R a , R c , R d , R e , R f , R g , R h , R i , R 9 , R 12 and R 13 is a bond to M.

2. The cyanine compound according to claim 1 , wherein said linker bound to a carrier molecule is a linker bound to a moiety having the structure of Formula III:

wherein
u is an integer selected from 1, 2, 3, 4, 5, 6, 7 and 8;
r is 0 or 1; and
Y is a nucleobase.

3. The cyanine compound according to claim 1 , said compound having the structure of Formula IV:

4. The cyanine compound of claim 1 , wherein the linker of the M moiety comprises the structure C(O)NH(CH 2 ) t , wherein the index t is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

5. The cyanine compound of claim 1 , wherein the linker of the M moiety comprises a polyvalent scaffold.

6. The cyanine compound according to claim 5 , wherein said polyvalent scaffold is a polyvalent arylene scaffold.

7. The cyanine compound of claim 5 , wherein the polyvalent scaffold comprises a second cyanine moiety bound thereto, said second cyanine moiety having a structure according to Formula I or Formula II.

8. The cyanine compound of claim 1 , wherein the linker of the M moiety comprises biotin bound to streptavidin.

9. A cyanine compound according to one of the following formulae:

wherein M comprises a linker bound to a carrier molecule.

10. A cyanine compound according to one of the following formulae:

wherein Y is a nucleobase.

11. The cyanine compound according to claim 1 , having the formula:
M-(R D ) 2

wherein
each R D has a structure independently selected from Formula I and Formula II.

12. The cyanine compound according to claim 6 , wherein M comprises the polyvalent arylene scaffold moiety:

13. The cyanine compound according to claim 12 , having the formula:

wherein
each R D has a structure independently selected from Formula I and Formula II.

14. The cyanine compound according to claim 2 , having at least one Y moiety comprising a natural nucleobase.

15. The cyanine compound according to claim 1 , wherein the carrier molecule comprises a member selected from a nucleic acid, a protein, a glycoprotein, a peptide, a saccharide, a hormone, a receptor, an antigen, a substrate, metabolite, a transition state analog, a cofactor, an inhibitor, a drug, a dye, a nutrient, and a growth factor.

16. The cyanine compound according to claim 1 , wherein the carrier molecule comprises a member selected from a solid support, a virus and a microorganism.

17. A method of monitoring an enzyme reaction, said method comprising:
(a) forming a reaction mixture by contacting said enzyme with a cyanine compound according to claim 1 , wherein said cyanine compound is a substrate for said enzyme under conditions sufficient for said enzyme and said cyanine compound to react; and
(b) monitoring fluorescence of said reaction mixture, thereby monitoring said enzyme reaction.

18. The method according to claim 17 , wherein said enzyme is a DNA polymerase and said cyanine compound comprises a nucleic acid moiety, which is said substrate for said enzyme.

19. The method according to claim 17 , wherein said enzyme reaction is template directed DNA synthesis.

20. The method according to claim 17 , wherein said reaction is a component of a single molecule DNA sequencing analysis.

21. A cyanine compound according to formula II:

wherein
n is an integer selected from 1, 2 and 3;
e is an integer selected from 0, 1 and 2;
R f , R g , R h , and R i are members independently selected from halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heteroalkyl, or a bond to M;
R c , and R d are independently selected from a bond to M, and alkyl substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, phosphoric acid and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, phosphoric acid;
R a and R e , are independently selected from C(O)R 9 , OR 12 , NR 12 R 13 , CR 12 C(O)R 13 , NR 12 C(O) 2 R 13 , SO 3 H, and C(O)NR 12 R 13 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a bond to M,
wherein
R 9 is a member selected from OR 10 , and a bond to M
wherein
R 10 is a member selected from H and substituted or unsubstituted alkyl; and

R 12 and R 13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or uns

Assignees

• Pacific Biosciences California Inc

Inventors

• Yue Stephen
• Shen Gene
• Sun Wei-Chuan (David)

Classifications

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C09B23/0033

  the substituent being bound through a sulfur atom · CPC title

• C09B23/164

  containing one nitrogen atom · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07D209/14

  Radicals substituted by nitrogen atoms, not forming part of a nitro radical · CPC title