# US 8,772,047 B2 · Dyes having ratiometric fluorescence response for detecting metabolites

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- Number: US 8,772,047 B2
- Document ID: US8772047B2
- Status: Granted
- Kind: B2
- Application number: 12/124,553
- Filed: 2008-05-21
- Published: 2008-12-18
- Granted: 2014-07-08
- Earliest claimed: 2007-05-22
- Assignees: Becton, Dickinson and Company
- Applicants: Joseph Thomas; Michael T. Cash
- Inventors: Joseph Thomas; Michael T. Cash
- Web page: https://patentgrep.com/patent/US8772047B2

## CPC

- G01N 33/582: {with fluorescent label}
- C09B 23/105: {two >CH- groups}
- C09B 57/02: Coumarine dyes
- G01N 33/533: with fluorescent label
- G01N 33/66: involving blood sugars, e.g. galactose
- A61K 8/498: {having 6-membered rings or their condensed derivatives, e.g. coumarin}
- C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- 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)
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

The presently disclosed subject matter provides thiol-reactive, environmentally sensitive fluorescent dyes, or fluorophores, which have an emission wavelength in the visible spectral region. When conjugated with a binding protein, the fluorophores exhibit a ratiometric response to one or more ligands or target analytes. The presently disclosed fluorophore-binding protein conjugates can be used to detect the presence of or amount of physiologically-important metabolites, such as glucose, fatty acids, and lactate, in biological samples.

## Claims

### Claim 1

A fluorophore, wherein the fluorophore is selected from the group consisting of:

### Claim 2

A biosensor compound having the following formula:
wherein, B is a binding member having a binding affinity for a ligand or analyte to be detected
wherein the biosensor compound exhibits a detectable change in a fluorescence property as a result of binding to the ligand or analyte or as a result of a change in concentration of the ligand or analyte in a sample under test.

### Claim 3 (depends on 2)

The biosensor compound of claim 2, wherein the binding member is a binding protein.

### Claim 4 (depends on 3)

The biosensor compound of claim 3, wherein the binding protein is a periplasmic binding protein.

### Claim 5 (depends on 4)

The biosensor compound of claim 4, wherein the binding protein is selected from the group consisting of a glucose binding protein and a glucose/galactose binding protein (GGBP).

### Claim 6 (depends on 5)

The biosensor compound of claim 5, wherein the binding protein is selected from the group consisting of W183C, SM4, and Y10C.

### Claim 7 (depends on 5)

The biosensor compound of claim 5, wherein the glucose/galactose binding protein has at least one amino acid substitution, and wherein the at least one amino acid substitution is selected from the group consisting of:
(a) a cysteine at position 11;
(b) a cysteine at position 14;
(c) a cysteine at position 19;
(d) a cysteine at position 43;
(e) a cysteine at position 74;
(f) a cysteine at position 107;
(g) a cysteine at position 110;
(h) a cysteine at position 112;
(i) a cysteine at position 113;
(j) a cysteine at position 137;
(k) a cysteine at position 149;
(l) a cysteine at position 213;
(m) a cysteine at position 216;
(n) a cysteine at position 238;
(o) a cysteine at position 287;
(p) a cysteine at position 292;
(q) a cysteine at position 112 and a serine at position 238;
(r) a cysteine at position 149 and a serine at position 238;
(s) a cysteine at position 152 and a serine at position 213;
(t) a cysteine at position 213 and a cysteine at position 238;
(u) a cysteine at position 149 and an arginine at position 213;
(v) a cysteine at position 149 and a serine at position 213 and a serine at position 238; and
(x) a cysteine at position 149 and an arginine at position 213 and a serine at position 238.

### Claim 8 (depends on 2)

The biosensor compound of claim 2, wherein the binding member is at least one fragment of a receptor, wherein the receptor is a protein in nature and is capable of binding to a suitable ligand via an active site, and wherein at least one amino acid residue of the fragment is located in a proximity of the active site and is a cysteine residue or is substituted with a cysteine residue.

### Claim 9 (depends on 8)

The biosensor compound of claim 8, wherein the receptor has one or more disulfide bridges.

### Claim 10 (depends on 9)

The biosensor compound of claim 9, wherein the receptor is an antibody or an antibody fragment.

### Claim 11 (depends on 10)

The biosensor compound of claim 10, wherein the receptor is a natural or artificial monoclonal antibody.

### Claim 12 (depends on 2)

A method for determining the presence or amount of one or more analytes in a sample, the method comprising:
(a) providing a biosensor compound of claim 2 having at least one mutated binding protein with a fluorophore covalently attached thereto through a thiol group of the mutated binding protein
(b) contacting the biosensor compound with a sample suspected of containing one or more analytes to bind the one or more analytes, if present, to the binding protein;
(c) irradiating the sample suspected of containing one or more analytes with electromagnetic radiation to induce the fluorophore to fluoresce; and
(d) detecting a fluorescence property to determine the presence or amount of one or more analytes in the sample.

### Claim 13 (depends on 12)

The method of claim 12, wherein the binding member is a binding protein.

### Claim 14 (depends on 13)

The method of claim 13, wherein the binding protein is a periplasmic binding protein.

### Claim 15 (depends on 13)

The method of claim 13, wherein the binding protein is selected from the group consisting of a glucose binding protein and a glucose/galactose binding protein (GGBP).

### Claim 16 (depends on 15)

The method of claim 15, wherein the binding protein is selected from the group consisting of W183C, SM4, and Y10C.

### Claim 17 (depends on 15)

The method of claim 15, wherein the glucose/galactose binding protein has at least one amino acid substitution, and wherein the at least one amino acid substitution is selected from the group consisting of:
(a) a cysteine at position 11;
(b) a cysteine at position 14;
(c) a cysteine at position 19;
(d) a cysteine at position 43;
(e) a cysteine at position 74;
(f) a cysteine at position 107;
(g) a cysteine at position 110;
(h) a cysteine at position 112;
(i) a cysteine at position 113;
(j) a cysteine at position 137;
(k) a cysteine at position 149;
(l) a cysteine at position 213;
(m) a cysteine at position 216;
(n) a cysteine at position 238;
(o) a cysteine at position 287;
(p) a cysteine at position 292;
(q) a cysteine at position 112 and a serine at position 238;
(r) a cysteine at position 149 and a serine at position 238;
(s) a cysteine at position 152 and a serine at position 213;
(t) a cysteine at position 213 and a cysteine at position 238;
(u) a cysteine at position 149 and an arginine at position 213;
(v) a cysteine at position 149 and a serine at position 213 and a serine at position 238; and
(x) a cysteine at position 149 and an arginine at position 213 and a serine at position 238.

### Claim 18 (depends on 12)

The method of claim 12, wherein the binding member is at least one fragment of a receptor, wherein the receptor is a protein in nature and is capable of binding to a suitable ligand via an active site, and wherein at least one amino acid residue of the fragment is located in a proximity of the active site and is a cysteine residue or is substituted with a cysteine residue.

### Claim 19 (depends on 18)

The method of claim 18, wherein the receptor has one or more disulfide bridges.

### Claim 20 (depends on 18)

The method of claim 18, wherein the receptor is an antibody or an antibody fragment.

### Claim 21 (depends on 19)

The method of claim 19, wherein the receptor is a natural or artificial monoclonal antibody.

### Claim 22 (depends on 12)

The method of claim 12, further comprising:
(a) measuring a fluorescence intensity at a first emission wavelength before contacting the biosensor compound with a sample suspected of containing one or more analytes;
(b) measuring a fluorescence intensity at a second emission wavelength after contacting the biosensor compound with a sample suspected of containing one or more analytes; and
(c) determining the ratio of the second emission wavelength to the first emission wavelength to determine the presence or amount of one or more analytes in the sample.

### Claim 23 (depends on 12)

The method of claim 12, further comprising continuously:
(a) contacting the binding protein with the sample suspected of containing one or more analytes;
(b) irradiating the sample with electromagnetic radiation; and
(c) detecting the fluorescence property.

### Claim 24 (depends on 12)

The method of claim 12, wherein the mutated binding protein undergoes a conformation change as a result of changes in analyte concentration of the sample suspected of containing one or more analytes and wherein the method detects changes in the fluorescence property as a result of changes in the analyte concentration.

### Claim 25 (depends on 12)

The method of claim 12, wherein the one or more analytes is selected from the group consisting of glucose, a fatty acid, and lactate.

### Claim 26 (depends on 2)

A biosensor device comprising the biosensor compound of claim 2.

### Claim 27 (depends on 2)

A reagent for determining the presence or amount of one or more analytes in a sample, the reagent comprising a biosensor compound of claim 2.

### Claim 28 (depends on 26)

A kit for determining the presence or amount of one or more analytes in a sample, the kit comprising the biosensor device of claim 26.

### Claim 29

A biosensor compound having the following formula:
wherein:
q′ is an integer from 1 to 8;
each X1′ and X2′ is independently selected from the group consisting of C and N, wherein at least one of X1′ and X2′ is N, under the proviso that (i) when X1′ is C, R1′ is Z, or when X2′ is C, R2′ is Z, as Z is defined herein below; (ii) if both X1′ and X2′ are N at the same time, one of R1′ and R2′ is absent; and (iii) when X1′ is N, R1′ when present is Z″, or when X2′ is N, R2′ when present is Z″, wherein Z″ is
—(CH2)n′X3′C(═O)—R4′,
wherein:
n′ is an integer from 1 to 8;
X3′ is O or NR6′;
each R3′, R6′, R7′, R8′, and Z is independently selected from the group consisting of H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl, alkylaminoalkyl, and alkoxycarbonyl; or
R7′ and R8′ together represent a C2 to C10 alkyl, C2 to C10 substituted alkyl, or C2 to C10 alkylene;
R4′ is —(CH2)m—B;
wherein m′ is an integer from 1 to 8; and
B is SM4, wherein SM4 is a mutated glucose/galactose binding protein (GGBP) having the following substitutions: N39I, G82E, Q83K, N84D, Q175E, Q177H, L178M, W183C, N259E and N260S.

### Claim 30 (depends on 29)

The biosensor compound of claim 29, wherein the biosensor compound is selected from the group consisting of: