Cyanine dye activating group with improved coupling selectivity

• US 6,002,003 A
• Filed: 04/14/1998
• Issued: 12/14/1999
• Est. Priority Date: 04/14/1998
• Status: Expired due to Fees

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Inventors: Dobashi, Thomas S., Shen, Gene G.-Y.
• Primary Examiner(s): Richter, Johann
• Assistant Examiner(s): KEATING, DOMINIC J
• Application Number:
  US09/059,900
• Time in Patent Office: 609 Days
• Field of Search: 544/232, 544/277, 544/317, 546/23, 546/24, 548/113, 548/547, 548/548, 548/549, 548/579, 548/565, 548/453, 549/473
• US Class Current: 544/232
• CPC Class Codes: C07D 209/08 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C09B 23/08 more than three >CH- groups, e.g. polycarbocyanines

Abstract

Activating groups for cyanine dyes used to label chain terminators in nucleotide sequencing, based on N-hydroxyphthalimide, are disclosed. From these activating groups, activated dyes of the present Invention are prepared which react with the derivitized nucleotide chain terminators to give a labeled chain terminator of the present Invention. The activating groups of the present Invention allow the dye-chain terminator reaction to occur at a much higher yield and with much greater selectivity for the mono-substituted product, compared with the prior art.

Claims

1. An activated dye having the formula:

##STR1## wherein X is selected from the group consisting of --CH═

CH--CH═

CH--CH═

, and --CH═

CH--CH═

CH--CH═

CH--CH═

; and

wherein R₁ is ##STR2## or --H; and

wherein R₂ is ##STR3## or --H; and

further wherein R₁ and R₂ are not both --H.

2. An activated dye having the formula:

##STR4## wherein X is selected from the group consisting of --CH═

CH--CH═

CH--CH═

, and --CH═

CH--CH═

CH--CH═

CH--CH═

; and

wherein R₁ is ##STR5## or --H; and

wherein R₂ is ##STR6## or --H; and

further wherein R₁ and R₂ are not both --H.

3. A dye-labeled chain terminator having the formula:

space="preserve" listing-type="equation">A--X--B

wherein A is a cyanine dye selected from the group consisting of ##STR7## wherein Y is selected from the group consisting of --CH═

CH--CH═

CH--CH═

, and --CH═

CH--CH═

CH--CH═

CH--CH═

; and

wherein B is selected from the group consisting of ##STR8## wherein Z is ##STR9## and wherein X is a linking group that results from reacting A and N-hydroxyphthalimide to produce an activated dye; and

thereafter reacting said activated dye with B.

20. A method for preparing a dye-labeled chain terminator of claim 3 comprising:

combining a suitable activated dye with a dideoxynucleotide phosphate to form a mixture.

21. The method of claim 20 wherein said combining step occurs in the presence of an HCO₃^- /CO₃^2- buffer.

22. The method of claim 21 wherein said combining step occurs at room temperature for between about 1 and about 16 hours.

23. The method of claim 20 comprising the additional step of isolating said chain terminator from said mixture by preparative thin-layer chromatography on silica gel using a CH₂ Cl₂ /CH₃ OH mixture in an approximately 1:

1 ratio.

24. The method of claim 23 comprising the additional step of further purifying said chain terminator by reverse phase high performance liquid chromatography.

25. The dye-labeled chain terminator of claim 3, wherein X is an aminopropynyl group.

4. A method for preparing an activated dye having a formula selected from the group consisting of:

##STR10## wherein X is selected from the group consisting of --CH═

CH--CH═

CH--CH═

, and --CH═

CH--CH═

CH--CH═

CH--CH═

; and

wherein R₁ is ##STR11## or --H; and

wherein R₂ is ##STR12## or --H; and

further wherein R₁ and R₂ are not both --H;

which comprises;

combining a suitable cyanine dye with carbonyliimidazole to form a mixture; and

adding to said mixture N-hydroxyphthalimide to form a final mixture.

5. The method of claim 4 wherein said dye and said carbonyliimidazole are combined in the presence of N,N-dimethyl formamide.

6. The method of claim 4 wherein said mixture is reacted for 2 hours.

7. The method of claim 4 wherein N-hydroxyphthalimide is added to said mixture at a concentration of about 2 equivalents.

8. The method of claim 4 comprising the additional step of stirring said final mixture for between about 5 and 12 hours.

9. The method of claim 8 comprising the additional step of combining said final mixture with ethyl acetate.

10. The method of claim 9 comprising the additional step of separating a resulting precipitate which comprises the desired activated dye.

11. The method of claim 10 wherein said separating step is performed by filtration and involves the additional step of drying said isolated precipitate.

12. The method of claim 4 wherein said dye is Cy5.

13. The method of claim 12 wherein the first combining step occurs in the presence of DMF, and said dye and said carbonyliimidazole are stirred at ambient for approximately 2 hours.

14. The method of claim 13 wherein said dye is present in said combining step at about 0.256 mmol, and said carbonyliimidazole is present in said combining step at about 0.64 mmol.

15. The method of claim 14 comprising the addition step, subsequent to said adding step, of stirring said final mixture for between about 5 and about 12 hours.

16. The method of claim 15 comprising the additional step of pouring said final mixture into a solution comprising about 150 ml of ethyl acetate.

17. The method of claim 16 comprising the additional step of isolating a resulting solid from said final mixture by vacuum filtration.

18. The method of claim 17 comprising the additional step of washing said resulting solid after vacuum filtration with ethyl acetate.

19. The method of claim 18 comprising the additional step of drying said resulting solid after washing by storing in a vacuum dessicator.