OPTICALLY-DETECTABLE ENZYME SUBSTRATES AND THEIR METHOD OF USE

• US 20120040380A1
• Filed: 06/22/2011
• Published: 02/16/2012
• Est. Priority Date: 01/21/2004
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: CORRY, SCHUYLER BOON, DOWNEY, WILLIAM LOUIS, FILANOSKI, BRIAN, GEE, KYLE RICHARD, GREENFIELD, I. LAWRENCE, HIRSCH, JAMES DAVID, JOHNSON, IAIN, RUKAVISHNIKOV, ALEKSEY
• Granted Patent: US 8,318,450 B2
• Time in Patent Office: Days
• US Class Current: 435/7.94
• CPC Class Codes: C07D 499/00 Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula: , e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• CPC Class Codes: C07D 501/00 Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula: , e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• CPC Class Codes: C07D 501/58 with a nitrogen atom, which is a member of a hetero ring, attached in position 7
• CPC Class Codes: C07D 501/60 with a double bond between positions 3 and 4
• CPC Class Codes: C07D 503/00 Heterocyclic compounds containing 4-oxa-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula: , e.g. oxapenicillins, clavulanic acid derivatives; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 5/022 without C-boron linkages
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 2334/00 O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
• CPC Class Codes: G01N 2333/986 acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The present invention relates to compounds that are substrates for an enzyme, and upon reaction with the enzyme provide a detectable response, such as an optically detectable response. In particular, the compounds have utility in detecting the presence of a β-lactamase in a sample. In addition to the compounds, methods are disclosed for analyzing a sample for the presence of a β-lactmase, for example, as an indicator of expression of a nucleic acid sequence including a sequence coding for a β-lactmase. Kits are disclosed that include the disclosed compounds and additional components, for example, cells, antibodies, a β-lactmase or instructions for using the components in an assay.

Claims

1-37. -37. (canceled)

38. A compound having a structure of one of the following formulae:

39. The compound of claim 38, wherein the W dye moiety is a dye having a structure of one of the following formulae:

40. The compound of claim 38, wherein W″

is not a quencher of the fluorescence emitted by the dye moiety.

41. The compound of claim 38, wherein A is S or SO.

42. The compound of claim 38, wherein W″

is (OH).

43. The compound of claim 38, wherein the W dye moiety is a borapolyazaindacene.

44. The compound of claim 38, wherein the compound has a structure of the formula:

45. A compound having a structure of the formula:

46. A method for determining the presence or absence of β

-lactamase enzyme in a sample, the method comprising;

a) contacting the sample with a β

-lactamase substrate to form a contacted sample;

b) incubating the contacted sample for a sufficient amount of time for the β

-lactamase enzyme to cleave the β

-lactamase substrate to form an incubated sample;

c) illuminating the incubated sample with an appropriate wavelength; and

d) observing the illuminated sample whereby the presence or absence of β

-lactamase enzyme in the sample is determined.

47. The method of claim 46, wherein the β

-lactamase substrate has a structure of one of the following formulae;

48. The method of claim 46, wherein the β

-lactamase substrate has a structure of the formula;

49. The method of claim 46, wherein the β

-lactamase substrate has a structure of the formula;

50. A method of localizing a fluorescent dye product in an environment comprising an aqueous solution and a β

-lactamase, the method comprising;

a) contacting the environment with a non-fluorescent compound comprising a dye moiety and a β

-lactam moiety;

b) incubating the product of step a) for a sufficient amount of time for the β

-lactamase to cleave the dye moiety from the β

-lactam moiety, thereby producing a fluorescent dye product which is insoluble in the aqueous solution, and thereby localizing the fluorescent dye product in the environment.

51. The method of claim 50 wherein the non-fluorescent compound has a structure of one of the following formulae:

52. The method of claim 51 wherein the non-fluorescent compound has a structure of the formula:

53. The method of claim 50, wherein the non-fluorescent compound has a structure of the formula:

54. The method of claim 50, wherein the environment is a member selected from a biological cell and a cell-free environment.

55. The method of claim 50, wherein the biological cell environment is an interior of a cell, an interior of a cell organelle, and an exterior of a cell.

56. A method for detecting the presence or absence of a target antigen in a sample, the method comprising:

a) contacting the sample with an antibody to form a contacted sample, wherein the antibody binds either directly or indirectly to the target antigen, and wherein the antibody comprises a β

-lactamase enzyme;

b) adding a non-fluorescent (β

-lactamase substrate to the incubated sample to form an added sample;

c) incubating the added sample for a sufficient amount of time for the β

-lactamase enzyme to cleave the non-fluorescent β

-lactamase substrate to form a fluorescent dye moiety;

d) illuminating the fluorescent dye moiety with an appropriate wavelength to form an illuminated sample; and

e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.

57. The method of claim 56, wherein the nonfluorescent β

-lactamase substrate has a structure of one of the following formulae;

58. The method of claim 56, wherein the nonfluorescent β

-lactamase substrate has a structure of the formula;

59. A kit for determining the presence or absence of β

-lactamase in a sample, the kit comprising;

a) a compound having a structure of one of the following formulae;

60. The kit of claim 59, further comprising at least one of a reaction buffer, a β

-lactamase, a antibody conjugated to a β

-lactamase enzyme, a nucleic acid sequence coding for a β

-lactamase, and a cell comprising a nucleic acid sequence coding for a β

-lactamase.

61. A kit for determining the presence or absence of β

-lactamase in a sample, the kit comprising;

a) a compound having a structure of the formula;

62. The kit of claim 61, further comprising at least one of a reaction buffer, a β

-lactamase, a antibody conjugated to a β

-lactamase enzyme, a nucleic acid sequence coding for a β

-lactamase, and a cell comprising a nucleic acid sequence coding for a β

-lactamase.