# US 8,465,985 B2 · Fluorescent probe

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- Number: US 8,465,985 B2
- Document ID: US8465985B2
- Status: Granted
- Kind: B2
- Application number: 12/527,898
- Filed: 2008-02-29
- Published: 2011-05-19
- Granted: 2013-06-18
- Earliest claimed: 2007-03-01
- Assignees: The University of Tokyo
- Applicants: Tetsuo Nagano; Hirotatsu Kojima; Sakiko Aizawa; Kazuki Kiyose
- Inventors: Tetsuo Nagano; Hirotatsu Kojima; Sakiko Aizawa; Kazuki Kiyose
- Web page: https://patentgrep.com/patent/US8465985B2

## CPC

- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C09K 11/06: containing organic luminescent materials
- C09K 2211/1007: Non-condensed systems
- C09K 2211/1029: containing one nitrogen atom as the heteroatom
- G01N 2021/7786: {Fluorescence}
- Y10T 436/141111: Diverse hetero atoms in same or different rings [e.g., alkaloids, opiates, etc.]
- 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)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A fluorescent probe comprising a compound represented as (Fluorophore A)-S-(Fluorophore B) (Fluorophore A and Fluorophore B are fluorophores which emit fluorescence when they are irradiated with an excitation light of a wavelength of 600 to 950 nm, Fluorophore A has a property that it shows change of fluorescence characteristic before and after a specific reaction with an substance to be measured, and S represents a spacer which connects Fluorophore A and Fluorophore B), which compound shows substantial change in efficiency of fluorescence resonance energy transfer between Fluorophore A and Fluorophore B before and after the specific reaction with the objective substance, wherein Fluorophore A is represented by the following formula (AI): which uses and shows an excitation light wavelength/fluorescence wavelength in the near infrared region, lights of which region show superior biological tissue permeability, and enables measurement of an substance to be measured by the ratio method.

## Claims

### Claim 1

A fluorescent probe comprising a compound represented by the following general formula (I):
(Fluorophore A)-S-(Fluorophore B) (I)
wherein Fluorophore A and Fluorophore B are fluorophores which emit fluorescence when they are irradiated with an excitation light of a wavelength of 600 to 950 nm, Fluorophore A has a property that it shows change of fluorescence characteristic before and after a specific reaction with a substance to be measured, and S represents a spacer which connects Fluorophore A and Fluorophore B, which compound shows substantial change in efficiency of fluorescence resonance energy transfer between Fluorophore A and Fluorophore B before and after the specific reaction with the substance to be measured,
wherein Fluorophore A is represented by the following formula (AI):
wherein R1 and R2 independently represent a C1-18 alkyl group which may have a substituent; R3 and R4 independently represent hydrogen atom, or a substituent for detecting the substance to be measured, provided that R3 and R4 do not simultaneously represent hydrogen atom, and R3 and R4 may bind to each other to form a ring structure for detecting the substance to be measured; Y1 and Y2 independently represent —O—, —S—, —Se—, —CH═CH—, —C(R5)(R6)—, or —N(R7)—(wherein R5, R6 and R7 independently represent hydrogen atom, or a C1-6 alkyl group which may have a substituent); n and n′ independently represent 0, 1 or 2; Z1 and Z2 independently represent a nonmetallic atom group required to form a benzo-condensed ring which may have a substituent or a naphtho-condensed ring which may have a substituent; L1, L2, L3, L5, L6 and L7 independently represent a substituted or unsubstituted methine group, L4 represents a substituted methine group, provided that when n or n′ is 2, each two of L1 and L2, or each two of L6 and L7 may be the same or different; M−represents a counter ion in a number required for neutralizing electrical charge, and the combination of R3 and R4 is a combination that provides change in the maximum excitation wavelength of Fluorophore A represented by the formula (AI) before and after detection of the substance to be measured;
Fluorophore B is a tetramethylindodicarbocyanine derivative; and
S is a spacer of a length so that FRET is induced between Fluorophore A and Fluorophore B.

### Claim 2 (depends on 1)

The fluorescent probe according to claim 1, wherein the substance to be measured is a proton, a metal ion, an active oxygen species, an enzymatic reaction substrate, or an enzymatic reaction product.

### Claim 3 (depends on 1)

The fluorescent probe according to claim 1, wherein the fluorescent probe is used for pH measurement, and Fluorophore A is represented by the formula (AI), wherein R1 and R2 independently represent methyl group or 4-sulfobutyl group; one of R3 and R4 is hydrogen atom, and the other is a C1-18 alkyl which may have a substituent; Y1 and Y2 represent —C(CH3)2—; both n and n′ represent 1; both Z1 and Z2 represent an unsubstituted benzo-condensed ring; L1, L2, L3, L5, L6 and L7 represent unsubstituted methine group; and M−represents a counter ion in a number required for neutralizing electrical charge.

### Claim 4 (depends on 1)

The fluorescent probe according to claim 1, wherein the fluorescent probe is used for pH measurement, and Fluorophore A is represented by the formula (AI), wherein R1 and R2 independently represent methyl group or 4-sulfobutyl group; R3 and R4 independently represent a C1-18 alkyl group which may have a substituent, an aryl group which may have a substituent, or —(CH2)m—N(R21)(R22), one of R3 or R4 is —(CH2)m—N(R21)(R22) (R21 and R22 independently represent a C1-6 alkyl group which may have a substituent, or an aryl group which may have a substituent, or R21 or R22 binds to R3 or R4, respectively, to represent a C1-3 alkylene group which may have a substituent, and m represents 1 or 2); Y1 and Y2 represent —C(CH3)2—;both n and n′ represent 1, Z1 and Z2 represent an unsubstituted benzo-condensed ring, and L1, L2, L3, L5, L6 and L7 represent unsubstituted methine group; and M−represents a counter ion in a number required for neutralizing electrical charge.

### Claim 5 (depends on 1)

The fluorescent probe according to claim 1, which is a fluorescent probe for pH measurement and contains the following compound;

### Claim 6 (depends on 1)

A method for fluorometry of a substance to be measured, which comprises (a) contacting a compound represented by the general formula (I) according to claim 1 and a substance to be measured, and (b) measuring fluorescence intensity of the compound represented by the general formula (I) after the contacting of (a) to detect change of FRET efficiency originating in the contacting with the substance to be measured.

### Claim 7 (depends on 5)

A method for fluorometry of a substance to be measured, which comprises (a) contacting the compound according to claim 5 and a substance to be measured, and (b) measuring fluorescence intensity of the compound after the contacting of (a) to detect change of FRET efficiency originating in the contacting with the substance to be measured.