# US 8,772,048 B2 · Fluorogenic compounds converted to fluorophores by photochemical or chemical means and their use in biological systems

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- Number: US 8,772,048 B2
- Document ID: US8772048B2
- Status: Granted
- Kind: B2
- Application number: 13/409,818
- Filed: 2012-03-01
- Published: 2012-07-26
- Granted: 2014-07-08
- Earliest claimed: 2008-05-23
- Assignees: Kent State University; Leland Stanford Junior University
- Applicants: Robert J. Twieg; William E. Moerner; Samuel J. Lord; Na Liu; Reichel Samuel
- Inventors: Robert J. Twieg; William E. Moerner; Samuel J. Lord; Na Liu; Reichel Samuel
- Web page: https://patentgrep.com/patent/US8772048B2

## CPC

- C07D 307/30: with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D 213/42: having hetero atoms attached to the substituent nitrogen atom (nitro radicals C07D213/26)
- C07D 249/16: condensed with carbocyclic rings or ring systems
- C07D 265/38: [b, e]-condensed with two six-membered rings
- C07D 271/12: condensed with carbocyclic rings or ring systems
- C07D 309/34: having three or more double bonds between ring members or between ring members and non-ring members
- Y10S 436/80: Fluorescent dyes, e.g. rhodamine
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Fluorophores derived from photoactivatable azide-pi-acceptor fluorogens or from a thermal reaction of an azide-pi-acceptor fluorogen with an alkene or alkyne are disclosed. Fluorophores derived from a thermal reaction of an alkyne-pi-acceptor fluorogen with an azide are also disclosed. The fluorophores can readily be activated by light and can be used to label a biomolecule and imaged on a single-molecule level in living cells.

## Claims

### Claim 1

A label comprising:
a fluorophore covalently linked to a biomolecule, wherein said biomolecule comprises a DNA, RNA, monosaccharide, polysaccharide, nucleotide (ATP, GTP, cAMP), lipid, peptide, or a protein including enzyme and other structural proteins, or any combination thereof;
wherein said fluorophore is derived from a photoactivatable fluorogen, said photoactivatable fluorogen comprising an azide conjugated covalently to an acceptor group through a pi-conjugated system (pi-bridge);
wherein said fluorophore is derived from said photoactivatable fluorogen by a photochemical conversion whereby said azide group is converted to an amine donor group and wherein said fluorophore comprises said amine donor group conjugated covalently to said acceptor group through said pi-bridge so that an electron from the amine donor can be delocalized to said acceptor group through said pi-bridge;
wherein said pi-bridge of said fluorogen is a conjugated system and is derived from one or more contiguous alkenes each, independently, containing from 2 to about 12 carbon atoms, or wherein said one or more contiguous alkenes is a replaced —(CH═N)— unit, or wherein said one or more contiguous alkenes are substituted with an aliphatic group containing from 1 to about 18 carbon atoms;
or is derived from one or more alkynes each, independently, containing from 2 to about 12 carbon atoms;
or is derived from one or more aromatic or heterocyclic aromatic or mono or polysubstituted aromatic or heterocyclic wherein each, independently, aromatic group has from 3 to about 26 total ring atoms, or each, independently, heterocyclic aromatic group has from 3 to 26 total ring atoms and from 1 to 3 nitrogen, oxygen, sulfur, phosphorus, or selenium atoms, and wherein each substituent, independently, is a) one or more alkyl groups having a total of from 1 to about 18 carbon atoms, or b) is one or more alkene groups having a total of from 2 to about 4 carbon atoms, or c) one or more alkyne groups having a total of from 2 to 10 carbon atoms; or d) is an unsubstituted heterocyclic group having a total of from 3 to about 26 carbon atoms, and, independently, from 1 to 3 nitrogen, oxygen, sulfur, phosphorus, or selenium atoms; or e) wherein said substituent is one or more halides; or f) wherein said substituent can be connected to 2 or more of said aromatic groups; or
any combination of the preceding;
wherein said acceptor group of said fluorophore is
pyridinium wherein R1 is not H and X— is a counterion, or
where R3, R4, R5, and R6, each, independently, can be the same as R1 or R2, except that R5 and R6 can be combined as a cycloaliphatic group; or
or any combination thereof, and
wherein said R1 and said R2, independently, comprises H; or, an alkyl group having from 1 to about 18 carbons; or an aryl group having from 4 to about 14 carbon atoms; or an aromatic heterocyclic group containing one or more of oxygen, nitrogen, or sulfur atoms and from 4 to about 14 carbon atoms, or any combination thereof; or an alkene or an alkyne group each, independently, containing from 2 to about 18 carbon atoms; or wherein said alkyl, alkene, alkyne, or said aromatic heterocyclic group, aryl group, each, independently and optionally, contain a functional group comprising an ether, an amine, a cyano, an alcohol, a carboxylic acid, or a sulfonic acid, or any combination thereof;
wherein said pi-bridge of said fluorophore is the same as said pi-bridge of said fluorogen, and wherein said acceptor group of said fluorophore is the same as said acceptor group of said fluorogen; and
wherein said fluorophore is excitable at a wavelength of greater than about 440 nanometers.

### Claim 2 (depends on 1)

A label comprising the conjugated covalently linked fluorophore-biomolecule according to claim 1, wherein with respect to said pi group said one or more aromatic or heterocyclic aromatic or mono or polysubstituted aromatic or heterocyclic aromatic groups each, independently, contains from about 5 to about 14 total ring atoms, wherein said one or more substituent alkyl groups each, independently, contains from 1 to 8 carbon atoms, and wherein said heterocyclic aromatic group ring contains one or more of only said nitrogen, oxygen, or sulfur atoms therein.

### Claim 3 (depends on 2)

A label comprising the conjugated covalently linked fluorophore-biomolecule according to claim 2, wherein said one or more electron accepting groups is bonded to one or more alkenes, independently, having from 2 to 20 carbon atoms or bonded to at least one other electron accepting group through one or more alkenes, or one or more heterocyclic aromatic rings containing one or more nitrogen atoms and/or one or more oxygen atoms; or any combination thereof, and
wherein said azide group is derived from an azide having the formula R-N3 where R comprises an aromatic having one or more rings, or a substituted aromatic wherein said substituted group is an aliphatic having from 1 to about 12 carbon atoms, or a heterocyclic containing a total of 3 to about 12 atoms with one or more atoms being O, S, N, or a halide, or a combination thereof.

### Claim 4 (depends on 1)

A label comprising a conjugated covalently linked fluorophore-biomolecule according to claim 1, wherein said fluorophore is a compound comprising
wherein n is 0, or 1 to about 4, wherein said R1 and said R2, independently, comprises H; or, an alkyl group having from 1 to about 18 carbons; or an aryl group having from 4 to about 14 carbon atoms; or an aromatic heterocyclic group containing one or more of oxygen, nitrogen, or sulfur atoms and from 4 to about 14 carbon atoms, or any combination thereof; or an alkene or an alkyne group each, independently, containing from 2 to about 18 carbon atoms; or wherein said alkyl, alkene, alkyne, or said aromatic heterocyclic group, aryl group, each, independently and optionally, contain a functional group comprising an ether, an amine, a cyano, an alcohol, a carboxylic acid, or a sulfonic acid, or any combination thereof,
wherein said amine end group comprises —NH2, H-alkyl-N—, dialkyl-N—, H-phenyl-N—, diphenyl-N—, wherein said alkyl group, independently, contains from 1 to about 15 carbon atoms and can be linear, branched, or functionalized, wherein said phenyl group, independently, can be substituted and contain from 1 to about 15 carbon atoms, or wherein an organic heterocyclic group having a total of from 3 to about 12 atoms can be substituted on said nitrogen atom and optionally include one or more O, S, or N atoms, or wherein said nitrogen atom is part of a ring.

### Claim 5 (depends on 2)

A label comprising a conjugated covalently linked fluorophore-biomolecule according to claim 2, wherein said fluorophore is a compound comprising
wherein n is 0, or 1 to about 4, wherein said R1 and said R2, independently, comprises H; or, an alkyl group having from 1 to about 18 carbons; or an aryl group having from 4 to about 14 carbon atoms; or an aromatic heterocyclic group containing one or more of oxygen, nitrogen, or sulfur atoms and from 4 to about 14 carbon atoms, or any combination thereof; or an alkene or an alkyne group each, independently, containing from 2 to about 18 carbon atoms; or wherein said alkyl, alkene, alkyne, or said aromatic heterocyclic group, aryl group, each, independently and optionally, contain a functional group comprising an ether, an amine, a cyano, an alcohol, a carboxylic acid, or a sulfonic acid, or any combination thereof,
wherein said amine end group comprises —NH2, H-alkyl-N—, dialkyl-N—, H-phenyl-N—, diphenyl-N—, wherein said alkyl group, independently, contains from 1 to about 15 carbon atoms and can be linear, branched, or functionalized, wherein said phenyl group, independently, can be substituted and contain from 1 to about 15 carbon atoms, or wherein an organic heterocyclic group having a total of from 3 to about 12 atoms can be substituted on said nitrogen atom and optionally include one or more O, S, or N atoms, or wherein said nitrogen atom is part of a ring.

### Claim 6 (depends on 3)

A label comprising a conjugated covalently linked fluorophore-biomolecule according to claim 3, wherein said fluorophore is a compound comprising
wherein n is 0, or 1 to about 4, wherein said R1 and said R2, independently, comprises H; or, an alkyl group having from 1 to about 18 carbons; or an aryl group having from 4 to about 14 carbon atoms; or an aromatic heterocyclic group containing one or more of oxygen, nitrogen, or sulfur atoms and from 4 to about 14 carbon atoms, or any combination thereof; or an alkene or an alkyne group each, independently, containing from 2 to about 18 carbon atoms; or wherein said alkyl, alkene, alkyne, or said aromatic heterocyclic group, aryl group, each, independently and optionally, contain a functional group comprising an ether, an amine, a cyano, an alcohol, a carboxylic acid, or a sulfonic acid, or any combination thereof,
wherein said amine end group comprises —NH2, H-alkyl-N—, dialkyl-N—, H-phenyl-N—, diphenyl-N—, wherein said alkyl group, independently, contains from 1 to about 15 carbon atoms and can be linear, branched, or functionalized, wherein said phenyl group, independently, can be substituted and contain from 1 to about 15 carbon atoms, or wherein an organic heterocyclic group having a total of from 3 to about 12 atoms can be substituted on said nitrogen atom and optionally include one or more O, S, or N atoms, or wherein said nitrogen atom is part of a ring.

### Claim 7 (depends on 1)

A visualizable label comprising the composition of claim 1.

### Claim 8 (depends on 2)

A visualizable label comprising the composition of claim 2.

### Claim 9 (depends on 3)

A visualizable label comprising the composition of claim 3.

### Claim 10 (depends on 4)

A visualizable label comprising the composition of claim 4.

### Claim 11 (depends on 5)

A visualizable label comprising the composition of claim 5.

### Claim 12 (depends on 6)

A visualizable label comprising the composition of claim 6.