Fluorogenic compounds and uses therefor

• US 6,566,508 B2
• Filed: 03/11/2002
• Issued: 05/20/2003
• Est. Priority Date: 07/07/2000
• Status: Expired due to Fees

• Current Assignee: 3M Innovative Properties Company (3M Company)
• Original Assignee: 3M Innovative Properties Company (3M Company)
• Inventors: Bentsen, James Gregory, Knudson, Orlin Bruce, Lewandowski, Kevin Michael, Mickelson, Christopher Allen
• Primary Examiner(s): Ponda, Kathleen K.
• Application Number:
  US10/094,678
• Publication Number:
  US 20020147317A1
• Time in Patent Office: 435 Days
• Field of Search: 536/4.1, 536/17.2, 536/17.4, 536/18.1, 536/18.4, 530/300, 530/331, 548/100, 548/146, 548/200, 548/202, 548/215, 548/235, 548/300.1, 548/311.7, 548/333.5, 549/29, 549/30, 549/70, 549/72, 549/218, 549/368, 549/402, 435/7.2, 435/7.32, 435/7.33, 435/7.34, 435/7.35, 435/29, 435/31
• US Class Current: 536/4.1
• CPC Class Codes: C07H 17/075 Benzo[b]pyran-2-ones
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 2334/20 Coumarin derivatives
• CPC Class Codes: G01N 2333/924 acting on glycosyl compounds (3.2)

Abstract

Disclosed are novel coumarin based fluorogenic compounds useful in assaying for biological activity. Specifically, these fluorogenic compounds exhibit fluorescence at particular wavelengths when cleaved by target enzymes. Preferred compounds include sugar and peptide derivatives of umbelliferone derivatives bearing a heterocyclic five membered ring at the 3-position. These compounds can be used for rapidly detecting food pathogens and for determining sterilization effectiveness. The compounds may also be used in a form bounded to a polymeric support or to a biomolecule or macromolecule.

Claims

1. An enzyme sensing composite structure comprising:

a support; and

a fluorogenic compound of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, arid a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

wherein the compound is covalently bound to the support through at least one R², R³ or R⁴ by means of one of a bond and a linking group, said linking group comprising functionalities at both ends, the functionality at one end of said linking group being complementary to the functionality of R², R³ or R⁴ and the functionality at the other end being complementary to a functional group on said support.

2. A sensing composite structure of claim 1, wherein the functionalities of the linking moiety independently are an amine, amide, ester, oxirane, olefin, urea, silanol, carbamate, isocyanate, thioisocyanate, sulfonamide, sulfonyl chloride, sulfonic acid, carboxylic acid, carboxyl, chlorotriazine, hydrazine, hydrazide, or aldehyde.

3. The composite structure of claim 1, wherein the linking group is hydrophilic.

4. The composite structure of claim 1, wherein the support is hydrophilic.

5. The composite structure of claim 1, wherein the support is a polymeric material.

6. The composite structure of claim 5, wherein the polymeric material is water-swellable.

7. The composite structure of claim 5, wherein the polymeric material is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinylacetate, polymers and copolymers of cellulose, vinylalkyl and -dialkyl azlactone, and copolymers of one of acrylate esters, methacrylate esters, acrylamides, and methacrylamides with one of acrylic acid and methacrylic acid.

8. A film comprising the composite structure of claim 1.

9. An article comprising the composite structure of claim 1 coated on a substrate.

10. The article of claim 9, wherein the composite structure is coated in a discontinuous pattern on the substrate.

11. The article of claim 10, wherein the pattern is a discernable pattern selected from the group consisting of a bar code, an alphanumeric string, a design, and a machine-readable pattern.

12. The article of claim 8, wherein a plurality of composite structures is coated at a plurality of individual locations on a substrate.

13. A fluorogenic macromolecular conjugate comprising:

a ligand selected from the group consisting of a molecular ligand and a macromolecular ligand; and

a fluorogenic compound of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

wherein the compound is covalently bound to the ligand through at least one R², R³ or R⁴ by means of one of a bond and a linking group, said linking group comprising functionalities at both ends, the functionality at one end of said linking group being complementary to the functionality of R², R³ or R⁴ and the functionality at the other end being complementary to a functional group on said ligand.

14. The conjugate of claim 13, wherein the functionalities of the linking moiety independently are an amine, amide, ester, oxirane, olefin, urea, silanol, carbamate, isocyanate, thioisocyanate, sulfonamide, sulfonyl chloride, sulfonic acid, carboxylic acid, carboxyl, chlorotriazine, hydrazine, hydrazide, or aldehyde.

15. The conjugate of claim 13, wherein the ligand is selected from the group consisting of a protein, a polypeptide, a glycoprotein, a carbohydrate, a steroid, a non-biological organic compound, and a non-biological organic polymer, and combinations thereof.

16. The conjugate of claim 13, wherein the ligand is selected from the group consisting of an antibody, an antigen to an antibody, a hapten, an antibody to a hapten, a hormone, a vitamin, a pharmacological agent, a metabolite of a pharmacological agent, a receptor for at least one of a hormone, a vitamin, a pharmacological agent and a metabolite of a pharmacological agent, and a binding substrate for at least one of a hormone, a vitamin, a pharmacological agent and a metabolite of a pharmacological agent.

32. A method of detecting a biological target molecule in a test sample, the method comprising the steps of:

(a) providing a fluorogenic macromolecular conjugate of claim 13 wherein the ligand is identical to the biological target molecule;

(b) incubating the test sample with a predetermined amount of the fluorogenic macromolecular conjugate and a predetermined amount of a specific binding partner for the target molecule, wherein the target molecule and the fluorogenic macromolecular conjugate compete for binding by the specific binding partner, further wherein the predetermined amount of the fluorogenic macromolecular conjugate is chosen such that a significant fraction of the fluorogenic macromolecular conjugate becomes bound and rendered inaccessible to enzymatic hydrolysis;

(c) adding to the test sample an enzyme that will enzymatically hydrolyze the unbound fluorogenic macromolecular conjugate to form a cleaved fluorogenic compound, whereby the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around λ

₁, is capable of emitting light of a wavelength centered around λ

₂, wherein λ

₂ is at least 10 nm greater than λ

₁, λ

₁ is at least 380 nm and λ

₂ is no more than about 700 nm; and

(d) exciting the cleaved fluorogenic compound with a light of a wavelength range centered around λ

₁ for a time sufficient for the cleaved compound to emit visible light of wavelength λ

₂; and

(e) detecting and analyzing the emitted light.

33. The method of claim 32, wherein the cleaved fluorogenic compound is interrogated with light generated from a diode selected from the group consisting of a light emitting diode and a laser diode.

34. The method of claim 32, wherein the diode is selected from the group consisting of GaN, InGaN, SiC, and ZnSe diodes.

17. A method of detecting bacteria in a test sample, comprising the steps of:

(a) incubating the bacteria in a growth media containing a fluorogenic compound of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

and allowing the diffusion of one or more of a bacterial enzyme and the fluorogenic compound such that the fluorogenic compound is hydrolyzed by the enzyme to form a cleaved fluorogenic compound, whereby the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around X1, is capable of emitting light of a wavelength centered around λ

₂, wherein λ

₂ is at least 10 nm greater than λ

₁, λ

₁ is at least 380 nm, and λ

₂ is no more than about 700 nm; and

(b) exciting the cleaved fluorogenic compound with a light of a wavelength range centered around λ

₁ for a time sufficient for the cleaved compound to emit visible light of wavelength λ

₂ and

(c) detecting and analyzing the emitted light.

18. A method of claim 17, wherein the bacteria are selected from the group consisting of Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Bacillus cereus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus sphaericus, Bacteroides fragilis, Bacteroides intermedium, Candida albicans, Citrobacter freundii, Clostridium perfringens, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumonia, Lactococcus lactis, Mycobacterium fortuitum, Neisseria gonorrhoeae, Organella morganii, Peptostreptococcus anaerobius, Peptococcus magnus, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas pudita, Salmonella typhimurium, Serratia liquefaciens, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus simulans, Streptococcus agalactiae B, Streptococcus anginosus, Streptococcus constellatus, Streptococcus faecalis D, Streptococcus mutans, Streptococcus pyogenes, Streptococcus uberis, and Xanthomonas maltophilia.

19. The method of claim 17, wherein said bacterial enzyme is selected from the group consisting of a peptidase enzyme, an esterase enzyme, and a glycosidase enzyme.

20. The method of claim 17, wherein said test sample is taken from at least one of a food sample, a clinical sample, a cosmetic sample, a beverage sample, a water sample, and a soil sample.

21. The method of claim 17, wherein the cleaved fluorogenic compound is excited with light generated from a diode selected from the group consisting of a light emitting diode and a laser diode.

22. The method of claim 21, wherein the diode is selected from the group consisting of GaN, InGaN, SiC, and ZnSe diodes.

23. The method of claim 17, wherein the test sample is partitioned into a plurality of sub-samples, wherein each sub-sample is incubated separately.

24. The method of claim 23, wherein each sub-sample is incubated with a fluorogenic compound wherein each fluorogenic compound is unique and is cleaved by a specific enzyme to provide a fluorescent cleavage product in each sub-sample that is characteristic of an amount of an enzyme present in the bacteria to be detected in the test sample.

25. A method of determining the effectiveness of a sterilization procedure, comprising the steps of:

(a) placing a test indicator containing an active enzyme in a detectable concentration into a sterilization chamber,

(b) performing the sterilization procedure within the chamber;

(c) introducing a fluorogenic compound of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

and allowing or providing a means for enzyme to diffuse such that the fluorogenic compound is hydrolyzed by the enzyme to form a cleaved compound, whereby the cleaved compound, when exposed to light of a wavelength range centered around λ

₁, is capable of emitting light of a wavelength centered around λ

₂, wherein λ

₂ is at least 10 nm greater than λ

₁, λ

₁ is at least 380 nm and λ

₂ is no more than about 700 nm; and

(d) exciting the cleaved compound with light of a wavelength range centered around λ

₁ for a time sufficient for the cleaved compound to emit visible light of wavelength λ

₂; and

(e) detecting and analyzing the emitted light.

26. The method of claim 25, wherein the enzyme is selected from the group consisting of β

-D-glucosidase, α

-D-glucosidase, alkaline phosphatase, acid phosphatase, butyrate esterase, caprylate esterase lipase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, chymotrypsin, phosphohydrolase, α

-D-galactosidase, β

-D-galactosidase, α

-L-arabinofuranosidase, β

-D-glucuronidase, N-acetyl-β

-glucosaminidase, β

-D-cellobiosidase, alanine aminopeptidase, proline aminopeptidase, tyrosine aminopeptidase, leucine aminopeptidase, phenylalanine aminopeptidase and a fatty acid esterase derived from spore-forming microorganisms.

27. The method of claim 25, wherein the enzyme is associated with a spore selected from Bacillus stearothermophilus and Bacillus subtilus.

28. The method of claim 25, wherein the cleaved compound is excited with light generated from a diode selected from the group consisting of a light emitting diode and a laser diode.

29. The method of claim 25, wherein the diode is selected from the group consisting of GaN, InGaN, SiC, and ZnSe diodes.

30. The method of claim 25, wherein the test indicator containing an active enzyme in a detectable concentration is arrayed in a discontinuous pattern on a support.

31. The method of claim 30, wherein the discontinuous pattern is a discernable pattern selected from the group consisting of a bar code, an alphanumeric string, a design, and a machine-readable pattern.

35. An enzyme sensing element comprising:

(1) one or more fluorogenic compounds of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NW, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_{x, or CHy} where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

(2) a fluid handling architecture structured and adapted to provide mixing of one or more enzyme-containing samples with at least one of the fluorogenic compounds so as to enable an enzymatic reaction wherein a cleaved fluorescent product is formed, such that the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around λ

₁, emits light of a wavelength λ

₂, wherein λ

₂ is at least 10 nm greater than λ

₁, wherein λ

₁ is at least 380 nm and λ

₂ is no greater than about 700 nm.

36. The sensing element of claim 35, wherein the fluid handling architecture is selected from the group consisting of a test card, a microwell array, a capillary array, a microfluidic chip, a sensor disk, an array of sensor disks, and combinations thereof.

37. The sensing element of claim 36, wherein the fluid handling architecture comprises one or more microlocations.

38. The sensing element of claim 35, wherein the fluid handling architecture is configured to absorb a fluid sample containing viable microorganisms and to support the growth of the viable microorganisms in microcolonies.

39. A sensing system for detecting the presence of enzyme activity in a biological sample, the sample optionally having a unique identifier;

the system comprising;

(1) at least one sensing clement comprising;

(a) one or more fluorogenic compounds of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof;

(b) a fluid handling architecture structured and adapted to provide mixing of one or more enzyme-containing samples with at least one of the fluorogenic compounds so as to enable an enzymatic reaction wherein a cleaved fluorescent product is formed, such that the cleaved fluorogenic compound, when exposed to light of a wavelength range centered around λ

₁, emits light of a wavelength λ

₂, wherein λ

₂ is at least 10 nm greater than λ

₁, wherein λ

₁ is at least 380 nm and λ

₂ is no greater than about 700 nm; and

(2) an excitation assembly capable of emitting light of at least 380 nm and no more than about 690 nm.

40. The sensing system of claim 39, wherein the excitation assembly comprises at least one of a solid state light emitting diode and a laser diode.

41. The sensing system of claim 40, wherein the diode is selected from the group consisting of GaN, InGaN, SiC, and ZnSe diodes.

42. The sensing system of claim 39, further comprising

(3) a detection assembly to detect at least one of the intensity and the location of the light emitted from the sensing element.

43. The sensing system of claim 42, further comprising

(4) a processor assembly to analyze the emitted light so as to determine at least one of the concentration, the location and the enumeration of the biomolecules, biomacromolecules or microorganisms and produce processed data.

44. The sensing system of claim 43, wherein the processor assembly includes a relational data base.

45. The sensing system of claim 44, wherein the relational database correlates the processed data with the unique identifier of the biological sample.

46. The sensing system of claim 39, wherein the fluid handling architecture is selected from the group consisting of a test card, a microwell array, a capillary array, a microfluidic chip, a sensor disk, an array of sensor disks, and combinations thereof.

47. The sensing system of claim 46, wherein the fluid handling architecture comprises one or more microlocations.

48. The sensing system of claim 39, wherein the fluid handling architecture is configured to absorb a fluid sample containing viable microorganisms and to support the growth of the viable microorganisms in microcolonies.

49. The sensing system of claim 39, wherein the excitation assembly selectively excites at least one microlocation within a sensing element.

50. The sensing system of claim 49, wherein the excitation assembly includes a raster-scanning means for exciting microlocations on the surface of the sensing element corresponding to growing microcolonies of microorganisms.

51. The sensing system of claim 42, wherein

(1) the excitation assembly comprises at least one solid-state light source that directs light toward the sensing element;

and

(2) the detection assembly comprises

(a) at least one light detector that detects the quantity of light from the sensing element; and

(b) at least one signal converter electrically connected to the light detector to provide at least one output signal in response to the quantity of light from the sensing element.

52. The sensing system of claim 51, wherein the excitation assembly and the detection assembly are colocated in a hand-held housing that is in optical registration with the sensing element.

53. The sensing system of claim 51, wherein the at least one solid state light source is selected from the group consisting of a GaN source, an InGaN source, a SiC source, and a ZnSe source.

54. The sensing system of claim 51, wherein the at least one signal converter comprises at least one analog-to-digital signal converter.

55. The sensing system of claim 47, wherein the output signal is a telemetric output signal.

56. A sensing system for detecting the presence of enzyme activity in a biological sample;

the system comprising;

(1) at least one sensing element comprising;

(a) one or more fluorogenic enzyme substrates comprising a compound of the formula;

wherein;

Q is a enzymatically hydrolyzable group selected from the group consisting of a glycone, a glycosyl phosphate, an ester, and a peptide;

each R² independently is a sterically non-interfering group;

R³ is an electron withdrawing or non-electron withdrawing group;

Z is O or NR⁵, wherein R⁵ is hydrogen or a hydrocarbyl-containing group;

Y and Y¹ independently are O, S, NH_x, or CH_y where x is 0 or 1 and y is 1 or 2, and at least one of Y and Y¹ is O, S, or NH_x; and

each R⁴ independently is selected from the group consisting of hydrogen and carboxyl;

or a salt thereof; and

(b) a fluid handling architecture structured and adapted to provide mixing of one or more of the enzyme-containing samples with at least one of the fluorogenic compounds so as to enable an enzymatic reaction wherein a cleaved fluorescent product is formed;

(2) a device having a means for receiving the at least one sensing element, the device comprising;

(a) a light source;

(b) a first plurality of optical fibers optically coupled to the light source and directing light toward the sensing element from the light source;

(c) a light detector;

(d) a second plurality of optical fibers optically coupled to the light detector and directing light from the sensing element to the detector; and

(e) a signal converter connected to the light detector capable of providing a digital output signal that varies in response to the quantity of light detected by the light detector.

57. The sensing system of claim 56, further comprising an optical reference material, a first reference pathway that directs a portion of the light from the at least one light source toward the optical reference material, and a second reference pathway that directs a portion of the light from the optical reference material toward the at least one light detector.

58. A sterilization efficacy measuring device comprising the sensing system of claim 56.