DYES HAVING RATIOMETRIC FLUORESCENCE RESPONSE FOR DETECTING METABOLITES

• US 20080311675A1
• Filed: 05/21/2008
• Published: 12/18/2008
• Est. Priority Date: 05/22/2007
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Thomas, Joseph, Cash, Michael T.
• Granted Patent: US 8,772,047 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: A61K 8/498 having 6-membered rings or their condensed derivatives, e.g. coumarin
• CPC Class Codes: C09B 23/105 two >CH- groups
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose

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

1. A fluorophore having the formula:

A-Y

wherein;

A is selected from the group consisting of a coumarin nucleus and an aza-coumarin nucleus;

Y is selected from the group consisting of;

wherein;

s and t are each independently an integer from 1 to 8;

each X₁ and X₂ is independently selected from the group consisting of C, S, and N, wherein at least one of X₁ and X₂ is N, under the proviso that (i) when X₁ is C or S, R₁ is Z, or when X₂ is C or S, R₂ is Z, as Z is defined herein below;

(ii) if both X₁ and X₂ are N at the same time, at least 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 selected from the group consisting of;

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃ is O or NR₆;

each R₃, R₅, R₆ 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;

R₄ is —

(CH₂)_m—

X₄;

wherein m is an integer from 1 to 8; and

X₄ is halogen.

2. The fluorophore of claim 1, wherein A is coumarin and the fluorophore has 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, S, and N, wherein at least one of X₁ and X₂ is N, under the proviso that (i) when X₁ is C or S, R₁ is Z, or when X₂ is C or S, R₂ is Z, as Z is defined herein below;

(ii) if both X₁ and X₂ are N at the same time, at least 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 selected from the group consisting of;

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃ is O or NR₆;

each R₃, R₅, 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—

X₄;

wherein m is an integer from 1 to 8; and

X₄ is halogen.

3. The fluorophore of claim 2, wherein the fluorophore is selected from the group consisting of:

4. A biosensor compound having the formula:

A-Y′

—

B

wherein;

A is selected from the group consisting of a coumarin nucleus and an aza-coumarin nucleus;

Y′

is selected from the group consisting of;

wherein;

s′ and

t′

are each independently an integer from 1 to 8;

each X₁′ and

X₂′

is independently selected from the group consisting of C, S, and N, wherein at least one of X₁′ and

X₂′

is N, under the proviso that (i) when X₁′

is C or S, R₁′

is Z, or when X₂′

is C or S, R₂′

is Z, as Z is defined herein below;

(ii) if both X₁′ and

X₂′

are N at the same time, at least 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 selected from the group consisting of;

—

(CH₂)_nX₃′

C(═

O)—

R₄′

,

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃′

is O or NR₆′

;

each R₃′

, R₅′

, R₆′ 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;

R₄′

is —

(CH₂)_m—

B;

wherein m is an integer from 1 to 8; and

B is a binding member having a binding affinity for a ligand or analyte to be detected; and

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.

5. The biosensor compound of claim 4, wherein A is coumarin and the biosensor compound of formula A-Y′

—

B has 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, S, and N, wherein at least one of X₁′ and

X₂′

is N, under the proviso that (i) when X₁′

is C or S, R₁′

is Z, or when X₂′

is C or S, R₂′

is Z, as Z is defined herein below;

(ii) if both X₁′ and

X₂′

are N at the same time, at least 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 selected from the group consisting of;

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃′

is O or NR₆′

;

each R₃′

, R₅′

, 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′

—

X₄′

;

wherein m′

is an integer from 1 to 8; and

B is a binding member having a binding affinity for a ligand or analyte to be detected.

6. The biosensor compound of claim 5, wherein the biosensor compound is selected from the group consisting of:

7. The biosensor compound of claim 4, wherein the binding member is a binding protein.

8. The biosensor compound of claim 7, wherein the binding protein is a periplasmic binding protein.

9. The biosensor compound of claim 8, wherein the binding protein is selected from the group consisting of a glucose binding protein and a glucose/galactose binding protein (GGBP).

10. The biosensor compound of claim 9, wherein the binding protein is selected from the group consisting of W183C, SM4, and Y10C.

11. The biosensor compound of claim 9, 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.

12. The biosensor compound of claim 5, 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 residues.

13. The biosensor compound of claim 12, wherein the receptor has one or more disulfide bridges.

14. The biosensor compound of claim 13, wherein the receptor is an antibody or an antibody fragment.

15. The biosensor compound of claim 14, wherein the receptor is a natural or artificial monoclonal antibody.

32. A biosensor device comprising the biosensor compound of claim 4.

33. A reagent for determining the presence or amount of one or more analytes in a sample, the reagent comprising a biosensor compound of claim 32.

34. A kit for determining the presence or amount of one or more analytes in a sample, the kit comprising the biosensor device of claim 32.

16. A method for determining the presence or amount of one or more analytes in a sample, the method comprising:

(a) providing a biosensor compound having at least one mutated binding protein with a fluorophore covalently attached thereto through a thiol group of the mutated binding protein, wherein the biosensor compound has the following formula;

A-Y′

—

B

wherein;

A is selected from the group consisting of a coumarin nucleus and an aza-coumarin nucleus;

Y′

is selected from the group consisting of;

wherein;

s′ and

t′

are each independently an integer from 1 to 8;

each X₁′ and

X₂′

is independently selected from the group consisting of C, S, and N, wherein at least one of X₁′ and

X₂′

is N, under the proviso that (i) when X₁′

is C or S, R₁′

is Z, or when X₂′

is C or S, R₂′

is Z, as Z is defined herein below;

(ii) if both X₁′ and

X₂′

are N at the same time, at least 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 selected from the group consisting of;

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃′

is O or NR₆′

;

each R₃′

, R₅′

, R₆′ 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;

R₄′

is —

(CH₂)_m—

B;

wherein m is an integer from 1 to 8; and

B is a binding member having a binding affinity for a ligand or analyte to be detected; and

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;

(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.

17. The method of claim 16, wherein A is coumarin and the biosensor compound of formula A-Y′

—

B has 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, S, and N, wherein at least one of X₁′ and

X₂′

is N, under the proviso that (i) when X₁′

is C or S, R₁′

is Z, or when X₂′

is C or S, R₂′

is Z, as Z is defined herein below;

(ii) if both X₁′ and

X₂′

are N at the same time, at least 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 selected from the group consisting of;

wherein;

n, p, and r are each independently an integer from 1 to 8;

X₃′

is O or NR₆′

;

each R₃′

, R₅′

, 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′

—

X₄′

;

wherein m′

is an integer from 1 to 8; and

B is a binding member having a binding affinity for a ligand or analyte to be detected.

18. The method of claim 17, wherein the biosensor compound is selected from the group consisting of:

19. The method of claim 16, wherein the binding member is a binding protein.

20. The method of claim 19, wherein the binding protein is a periplasmic binding protein.

21. The method of claim 19, wherein the binding protein is selected from the group consisting of a glucose binding protein and a glucose/galactose binding protein (GGBP).

22. The method of claim 21, wherein the binding protein is selected from the group consisting of W183C, SM4, and Y10C.

23. The method of claim 21, 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.

24. The method of claim 16, 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.

25. The method of claim 24, wherein the receptor has one or more disulfide bridges.

26. The method of claim 24, wherein the receptor is an antibody or an antibody fragment.

27. The method of claim 26, wherein the receptor is a natural or artificial monoclonal antibody.

28. The method of claim 16, 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.

29. The method of claim 16, 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.

30. The method of claim 16, 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.

31. The method of claim 16, wherein the one or more analytes is selected from the group consisting of glucose, a fatty acid, and lactate.