Fluorescent dyes

Patent metadata

Field	Value
Publication number	US-11807643-B2
Application number	US-202117374514-A
Country	US
Kind code	B2
Filing date	Jul 13, 2021
Priority date	Jul 16, 2020
Publication date	Nov 7, 2023
Grant date	Nov 7, 2023

Abstract

Automated oligonucleotide synthesis-compatible fluorescent dye phosphoramidite compounds, solid supports, and labeled polynucleotides incorporating the compounds are provided. The compounds allow universal incorporation of the fluorescent label into any position of the polynucleotide.

First claim

We claim:

1. A compound represented by Formula I:

or a stereoisomer, a salt, or a tautomer thereof,
wherein:
X is H, halogen, or C 1 -C 5 alkyl;
R 1 , R 2 , R 3 , and R 4 are independently H or optionally substituted C 1 -C 6 alkyl;
R 5 , R 6 , R 7 , R 8 , and R 9 are independently H, halogen, or optionally substituted C 1 -C 6 alkyl;
L 1 is an optionally substituted C 2 -C 10 alkylene or optionally substituted C 2 -C 50 heteroalkylene;
L 2 and L 3 are independently an optionally substituted C 2 -C 10 alkylene or optionally substituted C 2 -C 30 heteroalkylene;
Q 1 is a hydroxyl protecting group;
Z is CH, N, NHC(O)N, or OC(O)N;
Y is OH, OP(OCH 2 CH 2 CN)NR 10 R 11 or a solid support; and
R 10 and R 11 are independently optionally substituted C 1 -C 6 alkyl,
wherein optionally substituted alkyl refers to alkyl optionally substituted with F, Cl, Br, I, ═O, ═N—CN, ═N—OR, ═NR, OR, NR 2 , SR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR 2 , OC(O)R, C(O)R, or NO 2 , wherein each R is independently H, C 1 -C 8 alkyl, C 2 -C 8 heteroalkyl, C 1 -C 8 acyl, C 2 -C 8 heteroacyl, C 2 -C 8 alkenyl, C 2 -C 8 heteroalkenyl, C 2 -C 8 alkynyl, C 2 -C 8 heteroalkynyl, C 6 -C 10 aryl, or C 5 -C 10 heteroaryl, and each R is optionally substituted with F, Cl, Br, I, ═O, ═N—CN, ═N—OW, ═NR′, OR′, NR′ 2 , SR′, SO 2 R′, SO 2 NR′ 2 , NR′SO 2 R′, NR′CONR′ 2 , NR′C(O)OR′, NR′C(O)R′, CN, C(O)OR′, C(O)NR′ 2 , OC(O)R′, C(O)R′, and NO 2 , wherein each R′ is independently H, C 1 -C 8 alkyl, C 2 -C 8 heteroalkyl, C 1 -C 8 acyl, C 2 -C 8 heteroacyl, C 6 -C 10 aryl, or C 5 -C 10 heteroaryl,
wherein optionally substituted heteroalkylene refers to heteroalkylene optionally substituted with C 1 -C 6 alkyl, C 2 -C 6 heteroalkyl, alkynyl, F, Cl, Br, I, N 3 , OR, NR 2 , SR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR 2 , OC(O)R, C(O)R, oxo, and NO 2 , wherein each R is independently H, C 1 -C 6 alkyl, or C 2 -C 6 heteroalkyl,
wherein heteroalkylene, heteroalkyl, heteroalkenyl, heteroalkynyl, and heteroacyl, refer to the corresponding hydrocarbon wherein one or more chain carbon atoms have been replaced by a heteroatom selected from the group consisting of N, O, S, and P, and
wherein heteroaryl refers to a monocyclic or fused bicyclic aromatic ring system which contains as ring members one or more heteroatoms selected from the group consisting of N, O, and S.

2. The compound of claim 1 , wherein X is Cl, Br, or F.

3. The compound of claim 1 , wherein X is Cl.

4. The compound of claim 1 , wherein the solid support is controlled pore glass or polystyrene.

5. The compound of claim 1 , wherein the compound is compound of formula (IA):

or a stereoisomer, a salt, or a tautomer thereof.

6. The compound of claim 1 , wherein R 1 is H.

7. The compound of claim 1 , wherein R 2 is methyl.

8. The compound of claim 1 , wherein R 3 is methyl.

9. The compound of claim 1 , wherein R 4 is methyl.

10. The compound of claim 1 , wherein the compound is a compound of formula (IB):

or a stereoisomer, a salt, or a tautomer thereof.

11. The compound of claim 1 , wherein L 1 is C 2 -C 6 alkylene or —(CH 2 CH 2 O) m CH 2 CH 2 —, wherein m is an integer from 1 to 10.

12. The compound of claim 1 , wherein L 1 is C 2 alkylene.

13. The compound of claim 1 , wherein Z is OC(O)N.

14. The compound of claim 1 , wherein the compound is a compound of formula (IC):

or a stereoisomer, a salt, or a tautomer thereof, wherein m is an integer from 1 to 10.

15. The compound of claim 1 , wherein the compound is a compound of formula (ID):

a stereoisomer, a salt, or a tautomer thereof, wherein W is NH or O, and L 4 is an substituted C 2 -C 10 alkylene or optionally substituted C 2 -C 50 heteroalkylene.

16. The compound of claim 1 , wherein Q 1 is trimethylsilyl, TBDMS, acetyl, trityl, or trityl.

17. The compound of claim 1 , wherein R 10 and R 11 are isopropyl.

18. The compound of claim 1 , wherein R 5 is methyl.

19. The compound of claim 1 , wherein R 7 is methyl.

20. The compound of claim 1 , wherein R 8 is methyl.

21. The compound of claim 1 , wherein R 6 and R 9 are H.

22. The compound of claim 1 , wherein the compound is a compound of formula (IE):

or a stereoisomer, a salt, or a tautomer thereof.

23. A labeled polynucleotide prepared using automated phosphoramidite synthesis formed from the compound of claim 1 .

24. The labeled polynucleotide of claim 23 , wherein the labeled polynucleotide further comprises a fluorescence quencher.

25. The labeled polynucleotide of claim 23 , wherein the labeled polynucleotide is attached to a solid support.

26. The labeled polynucleotide of claim 25 , wherein the solid support is a controlled pore glass bead, polystyrene bead, magnetic bead, or microwell plate.

27. A method for preparing a labeled conjugate of a biomolecule, a synthetic polymer, or a solid support comprising contacting the biomolecule, the synthetic polymer, or the solid support with a compound of claim 1 , wherein Y is OP(OCH 2 CH 2 CN)NR 10 R 11 and R 10 and R 11 are independently optionally substituted C 1 -C 6 alkyl, in a suitable solvent under conditions sufficient to covalently attach the compound to the biomolecule, the synthetic polymer, or the solid support thereby forming the labeled conjugate.

28. The method of claim 27 , wherein the solid support is controlled pore glass or polystyrene.

29. The method of claim 27 , wherein the biomolecule is a polynucleotide.

30. The method of claim 27 , wherein the conditions sufficient to covalently attach the compound to the polynucleotide are automated phosphoramidite oligonucleotide synthesis conditions.

31. An automated molecular diagnostics cartridge comprising the labeled polynucleotide of claim 23 .

32. The cartridge of claim 31 , wherein the cartridge is a PCR diagnostic cartridge.

33. A compound represented by Formula I:

or a stereoisomer, a salt, or a tautomer thereof,
wherein:
X is H, halogen, or C 1 -C 5 alkyl;
R 1 , R 2 , R 3 , and R 4 are independently H or C 1 -C 6 alkyl;
R 5 , R 6 , R 7 , R 8 , and R 9 are independently H, halogen, or C 1 -C 6 alkyl;
L 1 is C 2 -C 10 alkylene or C 2 -C 50 heteroalkylene;
L 2 and L 3 are independently C 2 -C 10 alkylene or C 2 -C 30 heteroalkylene;

Assignees

• Cepheid

Inventors

• Lund Kevin P
• Sergueev Dmitri
• Gall Alexander

Classifications

• C07D491/052 Primary

  the oxygen-containing ring being six-membered · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C07D491/02 Primary

  in which the condensed system contains two hetero rings · CPC title

• G01N33/533

  with fluorescent label · CPC title