Anthraquinone and its derivatives

Details Anthraquinone and its derivatives Anthraquinone and its derivatives

2001-02-20

2002-10-22

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S004000, C514S644000, C552S236000, C552S237000, C552S240000

Reexamination Certificate

active

06468753

ABSTRACT:

This invention relates to an anthraquinone and its derivatives, in particular, although not exclusively, including its applications in a range of fluorescence detection technologies.
There are a number of DNA-binding fluorochromes available which cover the UV and visible region of the spectrum. Recently, very bright DNA-intercalating cyanine fluorochromes, based upon modified dimers of thiazole orange, have become commercially available. These cyanine dyes do not share the cell permeant properties of other DNA specific UV-activated fluorochromes. Furthermore, the commonly used DNA-interactive fluorochromes have fluorescent signatures which overlap those of other fluorochromes, activated in the spectral range of visible light, which are used as molecular tags to probe aspects of cell biology or biological structures. Examples of currently known cyanine dyes are disclosed in U.S. Pat. No. 5410030 and U.S. Pat. No. 5436134.
The present invention seeks to develop cell permeant DNA-interactive agents which may provide a fluorescence signature extending in to the infra red region of the spectrum. Such an agent could, for example, be optimally excited by red-line emitting lasers in multi-laser/multi-fluorochrome applications for both fixed specimens and viable cells.
Thus, in accordance with a first aspect of the present invention, there is provided a compound of the following formula (I):
wherein each of X
1
and X
2
are independently NH—A—NR
1
R
2
, and wherein A is a C
2-8
alkylene group and R
1
and R
2
are independently selected from hydrogen, C
1-4
alkyl, C
2-4
hydroxyalkyl and C
2-4
aminoalkyl, or R
1
and R
2
together form a C
2-6
alkylene group which with the nitrogen atom to which R
1
and R
2
are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid.
The term “alkylene” here is used to mean an alkyl chain.
In a preferred embodiment, when R
1
and R
2
form a heterocyclic ring, the ring has 3 to 7 carbon atoms therein. Preferably, both X
1
and X
2
are both NH(CH
2
)
2
NR
1
R
2
. In particular, it is preferred that R
1
and R
2
are both C
1-4
alkyl groups, preferably methyl groups.
According to a second aspect of the present invention, there is provided a compound of the following formula (II):
In one embodiment, compound (II) may be in the form of its N-oxide derivative.
The compound of the general formula (I) and, in particular the specific compound (II) may be used as, for example, a DNA dye and may be a pure synthetic compound which is soluble in biologically compatible solvents including water. Compound (II) has a high infinity for DNA (the DNA binding constant is approximately 10e7 M-1) and has the capacity to enter living cells rapidly.
The absorbance spectrum for compound (II) shows Ex
&lgr;max
near 647 nm and produces a fluorescence spectrum extending from 665 nm out to beyond 780 nm wavelengths (Em
&lgr;max
is about 677.5 nm).
According to a further aspect of the present invention, there is provided a method of preparing a compound of the following formula (I):
wherein each of X
1
and X
2
are independently NH—A—NR
1
R
2
, and wherein A is a C
2-8
alkylene group and R
1
and R
2
are independently selected from hydrogen, C
1-4
alkyl, C
2-4
hydroxyalkyl and C
2-4
aminoalkyl, or R
1
and R
2
together form a C
2-6
alkylene group which with the nitrogen atom to which R
1
and R
2
are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid,
the method comprising the step of reacting a compound of the following formula (III)
with NH
2
—A—NR
1
R
2
, wherein A, R
1
and R
2
are as defined above.
The method preferably further comprises the step of treating the resultant compound with an acid, preferably concentrated sulphuric acid. In addition, in a preferred embodiment, the method may further comprise subsequent treatment with sodium chlorate and/or sodium hydrogen sulphite.
Modelling has demonstrated that the compounds of the present invention can form stable, intercalated complexes with DNA. Thus, according to a further aspect of the present invention, there is provided a fluorescent complex comprising a nucleic acid and a compound of the following formula (I):
wherein each of X
1
and X
2
are independently NH—A—NR
1
R
2
, and wherein A is a C
2-8
alkylene group and R
1
and R
2
are independently selected from hydrogen, C
1-4
alkyl, C
2-4
hydroxyalkyl and C
2-4
aminoalkyl, or R
1
and R
2
together form a C
2-6
alkylene group which with the nitrogen atom to which R
1
and R
2
are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid.
The nucleic acid is preferably DNA. It has been found that the DNA may be present in a living cell. The compounds of the present invention may stain fixed human chromosomes. As the DNA:Compound molar ratio increases there is a bathochromic shift in the compound plus DNA solution spectrum. At high DNA:Compound ratios, attainable within living cells, the spectral shift contributes to an already significant separation of the compound-DNA emission spectrum from that of an example of a red-fluorescing compound Cy 5.
According to a further aspect of the present invention, there is provided a method of analysing a cell or biological material containing one or more nucleic acids, comprising the steps of:
a) preparing a biologically compatible solution containing a compound of the formula (I):
wherein each of X
1
and X
2
are independently NH—A—NR
1
R
2
, and wherein A is a C
2-8
alkylene group and R
1
and R
2
are independently selected from hydrogen, C
1-4
alkyl, C
2-4
hydroxyalkyl and C
2-4
aminoalkyl, or R
1
and R
1
together form C
2-6
alkylene group which with the nitrogen atom to which R
1
and R
2
are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid;
b) treating the cell or biological material with the biologically compatible solution;
c) exciting the compound (I) in the treated cell or biological material with a light source; and
d) detecting the emitted fluorescence signal.
The compound of formula (I) may be present in its free state or be complexed to other molecule(s), for example either by covalent or non-covalent attachment.
The light source preferably provides wavelength(s) in the spectral region of the wavelength(s) of maximum absorption of compound (I).
It has been found that the fluorescence signature of the compounds of the present invention extends to the infra red region of the spectrum. The compound of the present invention may be present in the cell or biological material in combination with one or more other fluorochromes or light-emitting compounds. The other fluorochromes may emit in the UV or visible region of the spectrum. Thus, the compounds of the present invention lend themselves to multi-parameter analysis with other fluorochromes with spectra which overlap with those of the commonly used visible-region DNA probes.
The one or more other compounds may be used, for example, to detect Annexin V and is preferably used in combination with the N-oxide derivative of compound (I). Flow cytometric analysis, for example with the instrument in dual laser mode, may be used. The invention thus may provide a way of discriminating intact viable cells from those undergoing the various stages of cell death.
Thus, the compounds of the present invention provide far red/infra red fluorescent permeant DNA dyes suitable for cellular DNA analysis where intact cells may be required, for example the detection of molecules either on the cell surface (e.g. a receptor molecule or marker f

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