# US 2014/0193926 A1 · Pyrenyloxysulfonic acid fluorescent agents

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- Number: US 2014/0193926 A1
- Document ID: US20140193926A1
- Status: Published application
- Kind: A1
- Application number: 14/080,559
- Filed: 2013-11-14
- Published: 2014-07-10
- Earliest claimed: 2003-12-09
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Richard HAUGLAND; Wai-Yee Leung; Jixiang Liu
- Web page: https://patentgrep.com/patent/US20140193926A1

## CPC

- G01N 33/536: with immune complex formed in liquid phase
- C07C 309/38: formed by at least three rings
- C07D 207/16: Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D 207/46: with hetero atoms directly attached to the ring nitrogen atom
- C07D 211/56: Nitrogen atoms (nitro radicals C07D211/38)
- C07D 211/60: Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D 211/62: attached in position 4
- C07D 401/12: linked by a chain containing hetero atoms as chain links
- C07H 17/08: Hetero rings containing eight or more ring members, e.g. erythromycins
- C07H 19/04: Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07K 14/77: Ovalbumin
- C07K 16/18: against material from animals or humans
- C09B 57/001: {Pyrene dyes}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

The invention provides a novel class of reactive fluorescent agents that are based on a pyrene sulfonic acid nucleus. The agents are readily incorporated into conjugates with other species by reacting the reactive group with a group of complementary reactivity on the other species of the conjugate. Also provided are methods of using the compounds of the invention to detect and/or quantify an analyte in a sample. In an exemplary embodiment, the invention provides multi-color assays incorporating the compounds of the invention.

## Claims

### Claim 1

A compound having the formula:
wherein
R and R1′ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl moieties;
X and X1 are independently selected from O, NH or S;
s and t are independently selected from the integers from 0 to 3, with the proviso that at least one of s and t is at least 1;
R2 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted heterocycloalkyl;
R3 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl,
wherein at least one of R2 and R3 is substituted with a member selected from a reactive group, a moiety comprising a reactive group and a component of a conjugate, and
R2 and R3, together with the nitrogen to which they are bound are optionally joined to form a ring which is substituted with a member selected from a reactive group, a moiety comprising a reactive group and a component of a conjugate,
with the proviso that, when R2 is H, R3 is a cyclic structure substituted with a member selected from a reactive group, a moiety comprising a reactive group and a component of a conjugate.

### Claim 2 (depends on 1)

The compound according to claim 1, wherein X and X1 are 0; R1 and R1′ are H; and R2 and R3 are members independently selected from substituted or unsubstituted alkyl, with the proviso that at least one of R2 and R3 are substituted with a member selected from a reactive group, a moiety substituted with a reactive group and a bond to a component of a conjugate.

### Claim 3 (depends on 1)

The compound according to claim 1, wherein X and X1 are O; R1 and R1′ are H; and R2 and R3, together with the nitrogen to which they are bonded are joined to form a ring substituted with a member selected from a reactive group, a moiety substituted with a reactive group and a bond to a component of a conjugate.

### Claim 4 (depends on 1)

The compound according to claim 1, wherein X and X1 are O; R1 and R1′ are H; R2 is H and R3 is a cyclic structure substituted with a member selected from a reactive group, a moiety substituted with a reactive group and a bond to a component of a conjugate.

### Claim 5 (depends on 1)

The compound according to claim 1, wherein s is 1; and t is 2.

### Claim 6 (depends on 1)

The compound according to claim 1, having the formula:
wherein
R4, R5, R6 and R7 are members independently selected from hydrogen, halogen, substituted or unsubstituted C1-C18 alkyl, substituted or unsubstituted C1-C18 alkoxy, substituted or unsubstituted C1-C18 alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, sulfo, nitro, cyano, carboxyl, substituted or unsubstituted C1-C18 carbamoyl, amino, a reactive group, hydroxyl and a bond to a component of a conjugate; and
n is 1 or 2.

### Claim 7 (depends on 6)

The compound according to claim 6, wherein at least one of R4, R5, R6 and R7 is a carboxyl moiety or an active ester thereof.

### Claim 8

(canceled)

### Claim 9 (depends on 1)

The compound according to claim 1, wherein said reactive group is a member selected from an acrylamide, an activated ester of a carboxylic acid, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a carboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, a hydrazine a hydrazide, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a sulfonyl halide, a thiol group, and a photoactivatable group.

### Claim 10

(canceled)

### Claim 11

A fluorescent labeled conjugate comprising:
a component which is a member selected from an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus and combinations thereof covalently bonded to a first fluorescent moiety having the formula:
wherein
R and R1′ are members independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl moieties;
X and X1 are independently selected from O, NH or S;
s and t are independently selected from the integers from 0 to 3;
R2 is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl;
R3 is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl,
wherein at least one of R2 and R3 is substituted with a member selected from a reactive group, a moiety comprising a reactive group, and a component of a conjugate, and
R2 and R3, together with the nitrogen to which they are bound are optionally joined to form a ring which is substituted with a member selected from a reactive group, a moiety comprising a reactive group and a component of a conjugate,
with the proviso that, when R2 is H, R3 is a cyclic structure substituted with a member selected from a reactive group, a moiety comprising a reactive group, and said component of said conjugate; and
with the proviso that at least one member selected from R2 and R3 comprises at least one moiety derived from said reactive group by its reaction with a reactive moiety of said component.

### Claim 12 (depends on 11)

A composition comprising:
(a) a first conjugate according to claim 11; and
(b) a second conjugate, comprising a component covalently bonded to a second fluorophore having a structure different from said first fluorophore.

### Claim at position 13

13-18. (canceled)

### Claim 19 (depends on 11)

A method for detecting an analyte in a sample, said method comprising:
(a) contacting said sample with a conjugate according to claim 11 wherein said component is a binding partner for said analyte;
(b) incubating said conjugate with said sample for a sufficient amount of time for said analyte and said component to interact, thereby forming a fluorescent analyte; and
(c) illuminating said fluorescent analyte with an appropriate wavelength whereby the presence of said analyte is determined in said sample.

### Claim 20 (depends on 19)

The method according to claim 19, wherein further comprising, between steps (b) and (c):
(d) separating said fluorescent analyte from said sample.

### Claim 21 (depends on 12)

A method for the detecting a first analyte and a second analyte in a sample, said method comprising:
(a) incubating said sample with a composition according to claim 12, wherein said first component is a binding partner for said first analyte and said second component is a binding partner for said second analyte, for a time sufficient for said first analyte to interact with said first conjugate and said second analyte to interact with said second conjugate, thereby forming a fluorescent first analyte and a fluorescent second analyte, respectively;
(b) illuminating said first fluorescent analyte with an appropriate wavelength whereby the presence of said first analyte is detected in said sample; and
(c) illuminating said second fluorescent analyte with an appropriate wavelength whereby the presence of said second analyte is detected in said sample.

### Claim 22

(canceled)

### Claim 23 (depends on 1)

A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 1.

### Claim at position 19

24-25. (canceled)

### Claim 26

A compound having structure VI:
wherein
designates a linker arm covalently attached to a reactive group;
the reactive group is an activated ester of a carboxylic acid comprising an aryloxy group substituted one or more times by nitro, fluoro, chloro, cyano, trifluoromethyl, or a combination thereof; and
s and t are independently selected from the integers from 0 to 3, with the proviso that at least one of s and t is at least 1.

### Claim 27 (depends on 26)

The compound according to claim 26, wherein the linker arm comprises a single covalent bond or a series of stable covalent bonds incorporating 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, S and P.

### Claim 28 (depends on 26)

The compound according to claim 26, wherein the linker arm comprises a cleavable group.

### Claim 29 (depends on 26)

The compound according to claim 26, wherein the linker arm comprises —C(O)NH—, —C(O)O—, —NH—, —S—, or —O—.

### Claim 30 (depends on 26)

The compound according to claim 26, wherein s is 1, and t is 2.

### Claim 31 (depends on 26)

A fluorescently labeled conjugate resulting from covalent attachment of the compound of claim 26 and a component which is a member selected from an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus and a combination thereof.

### Claim 32 (depends on 26)

A kit for the detection of an analyte in a sample, wherein said kit comprises a compound according to claim 26.

### Claim 33

A kit for the detection of an analyte in a sample, wherein said kit comprises a compound having structure VI:
wherein
s is 1;
t is 2; and
is a linker arm and the linker arm is covalently attached to a reactive group having the structure: