Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-02-15
2004-12-07
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S024000, C435S004000, C435S006120, C435S968000, C435S007720
Reexamination Certificate
active
06828116
ABSTRACT:
The present invention relates to the field of fluorescence resonance energy transfer. In particular, the invention relates to fluorogenic assays which include a novel class of non-fluorescent quenching dyes, and to novel quenching dye compounds thereof.
Fluorescence resonance energy transfer (FRET) occurs between the electronic excited states of two fluorophores when they are in sufficient proximity to each other, in which the excited-state energy of the donor fluorophore is transferred to the acceptor fluorophore. The result is a decrease in the lifetime and a quenching of fluorescence of the donor species and a concomitant increase in the fluorescence intensity of the acceptor species. In one application of this principle, a fluorescent moiety is caused to be in close proximity to a quencher molecule. In this configuration, the energy from the excited donor fluorophore is transferred to the quencher and dissipated as heat rather than fluorescence energy.
The use of fluorescence resonance energy transfer (FRET) labels in biological systems is well known. The principle has been used in the detection of binding events or cleavage reactions in assays which employ fluorescence resonance energy transfer. In the case of peptide cleavage reactions, a fluorescent donor molecule and fluorescent acceptor molecule are attached to a peptide substrate on either side of the peptide bond to be cleaved and at such a distance that non-radiative energy transfer between the donor and the acceptor species takes place. For example, EPA 428000 discloses a novel fluorogenic peptide substrate involving a fluorescent donor molecule and a quenching acceptor molecule attached thereto. The labelled substrate can be used in the detection and assay of a viral protease enzyme, whereby, if there is enzyme present in a test sample, the substrate is cleaved and the iikonor and acceptor species are thereby separated. The resultant fluorescent emission of the donor species can be measured. Suitable fluorescent donors include fluorescein derivatives, coumarins and 5-((2-aminoethyl)amino)-naphthalene-1-sulphonic acid (EDANS). Suitable quenching acceptors include 2,4-dinitrophenyl (DNP) and 4-(4-dimethylaminophenyl)azobenzoic acid (DABCYL).
Fluorescence energy transfer has also been used in the study of nucleic acid hybridisation. For example, Tyagi and Kramer (Nature Biotechnology, 14, 303-8, (1996)) disclose homogeneous hybridisation assays which utilise fluorescent labelled probes. The hair-pin probes comprise a single-stranded nucleic acid sequence that is complementary to the target nucleic acid, together with a stem sequence formed from two complementary arms which flank the probe sequence. A fluorophore (EDANS) is attached to one arm and the non-fluorescent quencher moiety (DABCYL) is attached to the complementary arm. In the absence of target, the stem keeps the fluorescent and quenching groups in close proximity causing the fluorescence of the fluorophore to be quenched. When the probe is allowed to bind to a nucleic acid target, it undergoes a conformational change, forming a more stable hybrid with the target and forcing the arm sequences (and the fluorophore and quencher) to move apart. The fluorophore will then emit fluorescence when excited by light of a suitable wavelength.
The success of the fluorescence resonance energy transfer approach is dependent upon the choice of the appropriate donor/acceptor pair. If energy transfer between the donor and acceptor can be optimised, residual fluorescence is minimised when the donor/quencher pair are in close proximity and a large change in signal can be obtained when they are separated. There is an increasing trend towards assay miniaturisation and in high throughput screening assays and, as a result, it is beneficial to use fluorophores with high extinction coefficients in order to achieve the sensitivity levels required. A further problem associated with such assays is due to colour quenching caused by the presence in the assay medium of coloured samples which tend to absorb strongly in the 350-450 nm region of the spectrum.
The present invention provides a non-fluorescent cyanine acceptor dye that can be used as one component of a fluorescent donor/acceptor pair for assays involving the detection of binding and/or cleavage events in reactions involving biological molecules. The fluorescent donor dye possesses a high extinction coefficient, thereby enabling the detection of low levels of the fluorophore. Moreover, the fluorescent dye pair have excitation and emission wavelengths in a range which is substantially free from auto-fluorescence associated with biological samples and from quenching due to coloured samples. Additionally, the dyes are relatively pH insensitive and they possess a high degree of spectral overlap, allowing efficient energy transfer.
Accordingly, the present invention relates to a compound of formula (1):
wherein the linker group Q contains at least one double bond and forms a conjugated system with the rings containing X and Y;
groups R
3
, R
4
, R
5
and R
6
are attached to the rings containing X and Y, or optionally, are attached to atoms of the Z
1
and Z
2
ring structures;
Z
1
and Z
2
each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms;
X and Y are the same or different and are selected from bis-C
1
-C
4
alkyl- and C
4
-C
5
spiro alkyl-substituted carbon, oxygen, sulphur, selenium, —CH═CH— and N—W wherein N is nitrogen and W is selected from hydrogen, a group —(CH
2
)
m
R
8
where m is an integer from 1 to 26 and R
8
is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary ammonium, nitro, primary amide, substituted amide., and groups reactive with amino, hydroxyl, carbonyl, carboxyl, phosphoryl, and sulphydryl groups;
at least one of groups R
1
, R
2
, R
3
, R
4
, R
5
, R
6
and R
7
is a target bonding group;
any remaining groups R
3
, R
4
, R
5
, R
6
and R
7
groups are independently selected from the group consisting of hydrogen, C
1
-C
4
alkyl, OR
9
, COOR
9
, nitro, amino, acylamino, quaternary ammonium, phosphate, suiphonate and sulphate, where R
9
is selected from H and C
1
-C
4
alkyl;
any remaining R
1
and R
2
are selected from C
1
-C
10
alkyl which may be unsubstituted or substituted with phenyl the phenyl being optionally substituted by up to two substituents selected from carboxyl, sulphonate and nitro groups;
characterised in that at least one of the groups R
1
, R
2
, R
3
, R
4
, R
6
, R
6
and R
7
comprises a substituent which reduces the fluorescence emission of said dye such that it is essentially non-fluorescent;
provided that the linker group Q is not a squaraine ring system.
Preferably the linker group Q contains 1, 2 or 3 double bonds in conjugation with the rings containing X and Y.
Preferably Q is the group:
wherein the groups R
10
are selected from hydrogen and C
1
-C
4
alkyl which may be unsubstituted or substituted with phenyl, or two or more of R
10
together with the group:
form a hydrocarbon ring system substituted with R
7
and which may optionally contain a heteroatom selected from —O—, —S—, or >NR
7
, wherein R
7
is hereinbefore defined; and
n=1, 2 or 3.
Suitably, the non-fluorescent cyanine dye for use in the present invention is a compound having the formula (2):
wherein groups R
3
, R
4
, R
5
and R
6
are attached to the rings containing X and Y or, optionally, are attached to atoms of the Z
1
and Z
2
ring structures and n is an integer from 1-3;
Z
1
and Z
2
each represent a bond or the atoms necessary to complete one or two fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms;
X and Y are the same or different and are selected from bis-C
1
-C
4
alkyl-and C
4
-C
5
spiro alkyl-subst
Birch Martyn N.
Bosworth Nigel
Hamilton Alan L.
Hatcher Malcolm J.
Scott Brian
Amersham Biosciences UK Limited
Leary Louise N.
Ronning, Jr. Royal N.
Ryan Stephen G.
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