Sulfonated [8,9] benzophenoxazine dyes and the use...

Details Sulfonated [8,9] benzophenoxazine dyes and the use... Sulfonated [8,9] benzophenoxazine dyes and the use...

2000-05-02

2002-10-15

Raymond, Richard L. (Department: 1624)

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

Organic compounds

Chalcogen in the nitrogen containing substituent

C530S391300, C530S391500, C530S402000, C530S409000, C536S023100, C536S025320

Reexamination Certificate

active

06465644

ABSTRACT:

I. FIELD OF THE INVENTION
The present invention relates to sulfonated 3,7-diamino-[8,9]benzophenoxazine dye compounds and uses thereof.
II. BACKGROUND OF THE INVENTION
Fluorescent reagents enable life science research in many fields, including biological, biomedical, genetic, fermentation, aquaculture, agriculture, forensic and environmental applications. Fluorescent probes and stains identify biopolymers and detect particular biological components within and outside cells. A common example is the use of fluorescent-labelled antibodies to detect cell-surface receptors. Another example is the widespread use of gel electrophoresis for characterizing nucleic acids, one limitation of which is the sensitivity of the methods used to detect the nucleic acid bands.
Detection of biological analytes utilizing fluorescent labels eliminates the need for radioactive labels, thereby enhancing safety and diminishing the adverse environmental impact and costs associated with radioactive waste disposal. Examples of methods utilizing fluorescent detection methods include automated DNA sequencing, oligonucleotide probe methods, detection of polymerase-chain-reaction products, immunoassays, and the like. In the life and medical sciences, researchers and technicians often need to detect proteins, antigens, and other ligands on the surface of cells. Receptor based assays utilize labelled molecules, e.g. fluorescent labelled peptides, proteins, and antibodies to detect expressed proteins and other ligands.
Dyes that are generally applicable for staining or labelling biopolymers across a broad range of applications preferably have the following properties: (i) the dye-biopolymer conjugate or complex should produce a very high signal with low background so that small quantities of biopolymers can be sensitively detected in both cell-free and cell-based assays; and (ii) the conjugate or complex should be photostable so that the fluorescent signal may be observed, monitored and recorded without significant photo bleaching. For applications involving cell surface binding of dye-peptide or dye-antibody conjugates to membranes or cell surfaces, especially live cells, the dyes preferably (iii) have good water-solubility to achieve effective conjugate concentration and detection sensitivity and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.
While many dye compounds have found use as nucleic acid stains, most available dyes fluoresce in the green region of the visible spectrum. Green lasers are more expensive than red lasers and give higher background signals in live cell assays due to autofluorescence of cellular components and assay equipment. These higher background signals decrease the sensitivity of the assay. Moreover, many cellular components absorb green light, further reducing the sensitivity of the assay. Thus, sensitive dyes that are photostable, have excitation and emission maxima in the red region of the visible spectrum and that are water-soluble are highly desirable.
III. SUMMARY OF THE INVENTION
The present invention relates to a new class of sulfonated 3,7-diamino-[8,9]benzophenoxazine dyes which are useful, among other things, for labelling substrates for fluorescent detection. In one aspect, compounds of the invention emit light in the red region of the light spectrum, with excitation maxima typically 600 nm or greater. The compounds have excellent solubility in aqueous solutions and can enhance the water-solubility of molecules to which they are attached.
Generally, the present invention provides dye compounds comprising a 3,7-diamino-[8,9]benzophenoxazine structure which contains at least one sulfonate substituent. In one embodiment, the invention includes a dye compound defined by the formula (I):
including any associated counter ions, wherein:
R
1
, R
2
, R
4
, R
6
, R
11
, R
12
, R
13
and R
14
, when taken alone, are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C
1
-C
6
alkyl, C
1
-C
6
aminoalkyl, C
5
-C
14
aryl, C
5
-C
14
aryl substituted with one or more of the same or different W groups, —OR
A
, —SR
A
, —NR
A
R
B
, —CN, —NO
2
, —C(O)R
A
or a reactive linking group;
R
1
when taken together with R
2
is C
5
-C
14
aryleno or C
5
-C
14
aryleno substituted with one or more of the same or different W groups;
R
3
, R
3′
, R
7
and R
7′
, when taken alone, are separately hydrogen, a reactive linking group, an aliphatic cationic chain, C
1
-C
6
alkyl or C
5
-C
14
aryl;
R
3
when taken together with R
3′
is C
2
-C
8
alkyldiyl;
R
7
when taken together with R
7′
is C
2
-C
8
alkyldiyl;
R
11
and R
12
, when taken together, are C
5
-C
14
aryleno or C
5
-C
14
aryleno substituted with one or more of the same or different W groups;
R
12
and R
13
, when taken together, are C
5
-C
14
aryleno or C
5
-C
14
aryleno substituted with one or more of the same or different W groups;
R
13
and R
14
, when taken together, are C
5
-C
14
aryleno or C
5
-C
14
aryleno substituted with one or more of the same or different W groups;
each W is independently hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C
1
-C
6
alkyl, —OR
A
, —SR
A
, —NR
A
R
B
, —CN, —NO
2
or —C(O)R
A
; and
each R
A
and each R
B
is independently hydrogen or C
1
-C
6
alkyl;
with the proviso that at least one of R
1
, R
2
, R
4
, R
6
, R
11
, R
12
, R
13
and R
14
is sulfonate, or R
12
and R
13
taken together are benzo containing at least one sulfonate attached to the benzo ring, or at least one of R
3
, R
3′
, R
7
and R
7′
is C
1
-C
6
alkylsulfonate or C
4
-C
10
arylsulfonate.
The invention also includes a dye compound defined by the formula (II):
wherein R
15
, R
16
, R
17
, and R
18
are separately hydrogen, sulfonate, carboxylate, phosphonate, phosphate, halogen, C
1
-C
6
alkyl, C
5
-C
14
aryl, C
5
-C
14
aryl substituted with one or more of the same or different W groups, —OR
A
, —SR
A
, —NR
A
R
B
, —CN, —NO
2
, —C(O)R
A
or a reactive linking group, wherein R
A
and R
B
are as defined above.
In one embodiment of formula I or formula II, a 5- to 7-member ring whose ring atoms are selected from carbon, nitrogen, oxygen, and sulfur is formed by R
2
and R
3
taken together with the C2-ring atom, C3 ring atom, and 3-nitrogen atom; or by R
3′
and R
4
taken together with the 3-nitrogen atom, C3-ring atom, and C4-ring atom; or by R
6
and R
7′
taken together with the C6-ring atom, C7-ring atom, and 7-nitrogen atom; or by R
7
and R
14
taken together with the 7-nitrogen atom, C7-ring atom, C8-ring atom, and C14-ring atom. The 5- to 7-member ring may optionally include a gem-disubstituted carbon atom. For example, the gem disubstituted carbon atom can be substituted with two C
1
-C
6
alkyl groups which may be the same or different, such as methyl.
In further embodiments with reference to formulas I and II above, R
1
, R
2
, R
4
and R
6
are each hydrogen; or R
3
and R
3′
are each independently C
1
-C
3
alkyl; or R
1
and R
2
together are [1,2]benzeno, [1,2]naphthaleno or [2,3]naphthaleno; or R
11
, R
12
, R
13
and R
14
are each hydrogen; or in formula I, R
11
and R
12
together or R
12
and R
13
together or R
13
and R
14
together are [1,2]benzeno.
In another embodiment, the aliphatic cationic chain is —(CH
2
)
n
—NR
2
, —(CH
2
)
n
—
+
NR
3
, —(CH
2
)
n
—
+
NR
2
—(CH
2
)
n
—NR
2
or —(CH
2
)
n
—
+
NR
2
—(CH
2
)
n
—
+
NR
3
, each n is independently an integer from 2 to 3, and each occurrence of R is independently selected from hydrogen and C
1
-C
6
alkyl.
In certain preferred embodiments, alkylsulfonate is —(CH
2
)
n
—SO
3
H, and n is an integer from 1 to 6, or arylsulfonate is:
wherein n is 0 or 1.
The reactive linking group, when present, is preferably succinimidyl ester, isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, phosphoramidite, maleimide, haloacetyl, or iodoacetamide, although ot

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