IP Library › Granted Patent US 12,359,068
Granted Patent B2
US 12,359,068 · App. 18/435,635 · Granted Jul 15, 2025

Rhodamine fluorescent compounds and production methods thereof

Inventors: Ada Tong (San Diego, CA); Megha Cila (San Diego, CA); Ronald Graham (Carlsbad, CA)
Assignee: Singular Genomics Systems, Inc.
C09B11/24A61K49/0041A61K49/0054C07H19/20C12Q1/6869
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Quick Facts
Patent No.
US 12,359,068
App. No.
18/435,635
Granted
Jul 15, 2025
Kind
B2
Abstract

Disclosed herein, inter alia, are fluorescent compounds and methods of making and using thereof.

Claims (71)

1. A method of making a compound of formula I,

the method comprising mixing compound A and compound B together in a reaction vessel at a first temperature to form a dye-intermediate, and adding oleum to said reaction vessel at a second temperature, wherein

compound A has the formula:

and

compound B has the formula:

R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 2 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 5 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

wherein R 1 , R 2 , R 5 , or R 6 is hydrogen;

R 1 and R 2 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 6 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 2 and R 4 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 1 and R 3 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 6 and R 8 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 7 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 3 , R 4 , R 7 , R 8 , R 11 , and R 12 are independently hydrogen, —PO 3 H, —PO 4 H, —SO 2 NH 2 , —SO 3 H, —SO 4 H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; wherein R 3 , R 4 , R 7 , or R 8 is —SO 3 H; and

R 9 is substituted or unsubstituted aryl.

2. The method of claim 1 , wherein said dye-intermediate is not isolated or removed from said reaction vessel prior to adding oleum.

3. The method of claim 1 , wherein said reaction vessel comprises a sulfonic acid.

4. The method of claim 1 , wherein said reaction vessel comprises difluoroacetic acid, dichloroacetic acid, ethane sulfonic acid, benzene sulfonic acid, toluene sulfonic acid, methanesulfonic acid, dichlorobenzene, trifluoroacetic acid, or polyphosphoric acid.

5. The method of claim 1 , wherein said reaction vessel comprises methanesulfonic acid.

6. The method of claim 1 , wherein said reaction vessel comprises a Lewis acid, wherein said Lewis acid is selected from aluminum chloride (AlCl 3 ), boron trifluoride (BF 3 ), iron(III) chloride (FeCl 3 ), magnesium chloride (MgCl 2 ), zinc chloride (ZnCl 2 ), chromic acid (H 2 CrO 4 ), stannic chloride (SnCl 4 ), stannous chloride (SnCl 2 ), titanium(IV) chloride (TiCl 4 ), and bromotrifluoromethane (CF 3 Br).

7. The method of claim 1 , wherein said oleum is 20% fuming sulfuric acid.

8. The method of claim 1 , wherein said first temperature is about 80° C. to about 130° C., and said second temperature is about 10° C. to about 30° C.

9. The method of claim 1 , wherein said first temperature is about 95° C. to about 120° C., and said second temperature is about 15° C. to about 25° C.

10. The method of claim 1 , wherein said first temperature is about 100° C., and said second temperature is about 20° C.

11. The method of claim 1 , wherein compound A has the formula:

12. The method of claim 1 , wherein compound A has the formula:

13. The method of claim 1 , wherein compound A has the formula:

wherein

R 10 is independently hydrogen, halogen, —CCl 3 , —CBr 3 , —CF 3 , —CI 3 , —CHCl 2 , —CHBr 2 , —CHF 2 , —CHI 2 , —CH 2 Cl, —CH 2 Br, —CH 2 F, —CH 2 I, —CN, —OH, —NH 2 , —COOH, —CONH 2 , —NO 2 , —SH, —SO 3 H, —SO 4 H, —SO 2 NH 2 , —NHNH 2 , —ONH 2 , —NHC(O)NHNH 2 , —NHC(O)NH 2 , —NHSO 2 H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl 3 , —OCF 3 , —OCBr 3 , —OCI 3 , —OCHCl 2 , —OCHBr 2 , —OCHI 2 , —OCHF 2 , —OCH 2 Cl, —OCH 2 Br, —OCH 2 I, —OCH 2 F, —N 3 , —SF 5 , —SO 2 Cl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and

z10 is an integer from 0 to 5.

14. The method of claim 1 , wherein compound B has the formula:

15. The method of claim 1 , wherein compound B has the formula:

16. The method of claim 1 , wherein R 9 is:

17. The method of claim 1 , wherein the compound of formula I has the formula:

18. A compound made according to claim 1 , having the formula:

wherein

R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 2 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 5 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

wherein R 1 , R 2 , R 5 , or R 6 is hydrogen;

R 1 and R 2 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 6 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 2 and R 4 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 1 and R 3 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 6 and R 8 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 7 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 3 , R 4 , R 7 , R 8 , R 11 , and R 12 are independently hydrogen, —PO 3 H, —PO 4 H, —SO 2 NH 2 , —SO 3 H, —SO 4 H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; wherein R 3 , R 4 , R 7 , or R 8 is —SO 3 H; and

R 9 is substituted or unsubstituted aryl.

19. The compound of claim 18 , having the formula:

20. A nucleotide comprising a monovalent compound having the formula:

wherein

R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 2 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 5 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R 6 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

wherein R 1 , R 2 , R 5 , or R 6 is hydrogen;

R 1 and R 2 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 6 substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 2 and R 4 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 1 and R 3 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 6 and R 8 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 5 and R 7 substituents may optionally be joined to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl;

R 3 , R 4 , R 7 , R 8 , R 11 , and R 12 are independently hydrogen, —PO 3 H, —PO 4 H, —SO 2 NH 2 , —SO 3 H, —SO 4 H, substituted or unsubstituted alkyl, or substituted or unsubstituted heteroalkyl; wherein R 3 , R 4 , R 7 , or R 8 is —SO 3 H;

R 10 is hydrogen, halogen, —COOH, —CONH 2 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;

L 1 is a covalent linker;

z10 is an integer from 1 to 4; and

R 13 is a monovalent nucleotide.

Assignments (2)
SECURITY INTEREST Recorded Mar 7, 2025
From: SINGULAR GENOMICS SYSTEMS, INC.
To: FIRST-CITIZENS BANK & TRUST COMPANY
Reel/Frame 070440/0465 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 20, 2024
From: CILA, MEGHA; GRAHAM, RONALD; TONG, ADA
To: SINGULAR GENOMICS SYSTEMS, INC.
Reel/Frame 067469/0826 →
Continuity (2)
Provisional Application 63444848 · Feb 10, 2023
Related Publication 20240294764A1 · Sep 5, 2024
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