Compounds for fluorescence labeling

Details Compounds for fluorescence labeling Compounds for fluorescence labeling

2002-07-17

2004-09-21

Dentz, Bernard (Department: 1625)

Organic compounds -- part of the class 532-570 series

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C546S014000, C546S079000, C546S018000

Reexamination Certificate

active

06794509

ABSTRACT:

TECHNICAL FIELD
The present invention relates to highly sensitive fluorescence-labeling agents, which are used for DNA sequencing or measurement of physiologically active substance or the like based on fluorescence immunoassay, or used as a fluorescence contrast medium that is administered into a blood vessel to visualize information in vivo and the like. The invention also relates to synthetic intermediates for said agents.
BACKGROUND ART
For DNA sequencing and measurement of physiologically active substance or the like based on fluorescence immunoassay, methods have conventionally been used which comprise the step of labeling a target substance with a radioisotope. However, these methods have problems from viewpoints of safety, storability of agents or the like, and therefore, various methods for labeling a target substance with a fluorescent dye have been studied as alternatives to the above methods. Performances required for fluorescence-labeling agents include, for example, (1) agents should have a high fluorescence quantum yield, (2) should have a high molecular extinction coefficient, (3) should be water-soluble and should not aggregate in an aqueous solvent to cause self-quenching, (4) should be hardly hydrolyzed, (5) should hardly cause photodegradation, (6) should be hardly influenced by background fluorescence, and (7) should be introduced with a reactive substituent that produces a covalent bond with a target substance.
Fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate, which have been known as fluorescence-labeling agents since old days, have a high fluorescence quantum yield. However, they have a low molecular extinction coefficient. In addition, they suffer from a problem that they are susceptible to background fluorescence of a membrane used for blotting since their excitation and emission wavelengths are within the range of 500 nm to 600 nm.
As dyes having a high molecular extinction coefficient, for example, cyanine dyes disclosed in U.S. Pat. No. 5,486,616, Japanese Patent Unexamined Publication (Kokai) Nos. 2-191674, 5-287209, 5-287266, 8-47400, 9-127115, 7-145148 and 6-222059, and polymethine dyes such as oxonol barbiturate disclosed in Journal of Fluorescence, 5, p.231 (1995) have been known. However, these dyes generally have a problem of a low fluorescence quantum yield. Further, these dyes also have a problem that they are hardly soluble in water and that they are hydrolyzed even if they are dissolved in water. In addition, they also suffer from a problem that they form aggregates in an aqueous medium due to their strong intermolecular interaction among dye molecules and thus self-quenching of fluorescence is often observed.
The cyanine dyes disclosed in Japanese Patent Unexamined Publication No. 2-191674 and other publications are superior dyes with water-solubility and suppressed formation of aggregates based on introduction of sulfonate group into a relatively stable chromophore. However, the dyes do not have satisfactorily high fluorescence quantum yield, and they suffers from a problem that synthesis of the dyes becomes difficult due to the introduction of sulfonate group. Under the circumstances, development of fluorescent dyes has been desired which have high water-solubility and stability and are free from self-quenching of fluorescence due to aggregation, as well as characteristic of strong fluorescence.
As other dye backbone structure with strong fluorescence, azaindolenine cyanine dyes disclosed in British Patent No. 870,753 have been known. However, the patent publication is silent about characteristics essential for fluorescence-labeling agents such as water-solubility, aggregation property and stability of aqueous solutions. Furthermore, no example is given as to introduction of a reactive substituent that produces a covalent bond with a target substance. Therefore, their suitability as fluorescence-labeling agents remains completely unknown. Further, Japanese Patent Unexamined Publication Nos. 4-358143, 3-195668, 1-280750 and European Patent Laid-open EP841958 disclose applications of the azaindolenine cyanines for photographic purpose. However, the aforementioned applications utilize absorption characteristics of the azaindolenine cyanines, and do not consider and actively utilize their luminescence characteristics.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a compound that is useful as a highly sensitive fluorescence-labeling agent used for DNA sequencing or measurement of physiologically active substances or the like based on fluorescence immunoassay, or used as a fluorescence contrast medium that is administered into a blood vessel to visualize information in vivo. Another object of the present invention is to provide a compound useful as synthetic intermediates for preparation of the aforementioned compounds. The inventors of the present invention conducted various studies to achieve the aforementioned objects, and as a result, they successfully achieved the aforementioned objects by providing the following compounds.
The present invention thus provides compounds represented by the following general formula (I) or salts thereof:
wherein, V
1
, V
2
, V
3
, V
4
and V
5
each independently represent a hydrogen atom or a group selected from the group consisting of a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic group, cyano group, a hydroxy group, nitro group, carboxyl group, an alkoxy group, an aryloxy group, a silyloxy group, a heterocyclyloxy group, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxy group, an aryloxycarbonyloxy group, an amino group (including an anilino group), an acylamino group, an aminocarbonylamino group, an alkoxycarbonylamino group, an aryloxycarbonylamino group, a sulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylamino group, a mercapto group, an alkylthio group, an arylthio group, a heterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, an arylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, an acyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, a carbamoyl group, a phosphono group, a phosphonato group and a group that can form a covalent bond with a compound to be labeled (each of said group may be substituted), provided that V
1
, V
2
, V
3
, V
4
and V
5
do not simultaneously represent a hydrogen atom and provided that V
1
and V
2
, V
2
and V
3
, and V
4
and V
5
may independently bind to each other to form a saturated or unsaturated ring that may be substituted; R
1
represents a hydrogen atom or a group selected from the group consisting of an alkyl group, an aryl group and a heterocyclic group (each of said group may be substituted); R
3
and R
4
represent an alkyl group that may be substituted, and R
3
and R
4
may bind to each other to form a ring that may be substituted; Q represents a group of atoms required to form a cyanine dye chromophore, a melocyanine dye chromophore or a stilyl dye chromophore; and m and n represent 0 or 1, provided that m+n is 1.
The present invention also provides compounds represented by the following general formula (II):
wherein, V
1
, V
2
, V
3
, V
4
, V
5
, V
6
, V
7
, V
8
, V
9
and V
10
each independently represent a hydrogen atom or a group selected from the group consisting of a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic group, cyano group, hydroxy group, nitro group, carboxyl group, an alkoxy group, an aryloxy group, a silyloxy group, a heterocyclyloxy group, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxy group, an aryloxycarbonyloxy group, an amino group (including an anilino group), an acylamino group, an aminocarbonylamino group, an alkoxycarbonylamino group, an aryloxycarbonylamino group, a sulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylamino group, a mercapto group, an alkylthio group, an arylthio group, a heterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, an arylsulfinyl group, an alky

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