Fluorescent dye

Details Fluorescent dye Fluorescent dye

2002-02-28

2004-03-16

Powers, Fiona T. (Department: 1626)

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

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

C548S120000, C548S151000, C548S218000, C548S302100, C548S433000, C008S648000, C435S006120, C436S094000, C436S172000, C436S800000

Reexamination Certificate

active

06706879

ABSTRACT:

BACKGROUND OF THE INVENTION
Fluorescent dyes are widely used for biological, biochemical or chemical applications in which a highly sensitive detection reagent is desirable. Molecules labeled with sensitive dye reagents enable the researcher to determine the presence, quantity or location of such molecules by monitoring their fluorescence. The quenching and energy transfer properties of fluorescent dyes also render the dyes useful for a variety of methods permitting investigators to monitor the in vivo and in vitro interactions between labeled molecules (e.g., protein:protein, protein:nucleic acid, nucleic acid:nucleic acid, and protein: or nucleic acid: interactions with drugs, drug candidates or other chemical entities). Fluorescent dyes are also useful for non-biological applications, such as photographic media.
In comparison to radiolabels frequently used for similar purposes, fluorescent dyes have the advantages of being safe to handle and dispose of and relatively stable. In addition, fluorescent dyes are detectable in real time, and differing fluorescence excitation and emission spectra can be exploited to permit differential detection of two, three or more different labeled species in a given mixture.
There is a wide variety of fluorescent dyes known and used in various fields. One class, the cyanine dyes, is very frequently used in biological and biochemical applications. Cyanine dyes are generally characterized by the presence of a pair of nitrogen-containing heterocycles (“terminal heterocycles”) connected by a polymethine bridge over which bond resonance occurs. Most cyanine dyes exhibit high visible absorbance and reasonable resistance to photodegradation. The general structure of cyanine dyes is given by the following formula:
In the above formula, X and Y are typically heteroatoms (e.g., O, S, N) or disubstituted carbon atoms (e.g., C—(CH
3
)
2
). Those dyes wherein n=0 are typically referred to as “cyanine” dyes. Where n=1 the dyes are termed “carbocyanine” dyes, while where n=2 the dyes are “dicarbocyanine” dyes and if n=3 the dyes are “tricarbocyanine” dyes, and so on. This class of dyes is generically referred to as “cyanine” dyes regardless of the specific number of methine groups between the ring systems.
The substituents R
1
and R
2
are typically saturated or unsaturated alkyl groups that are optionally further substituted by a wide variety of other functional groups. Other positions on the terminal heterocycles may be substituted with various organic functional groups, as well as additional fused or unfused rings that may themselves be additionally substituted.
There is a need in the art for additional fluorescent dyes. In particular, there is a need for dyes with fluorescence characteristics that permit distinct detection in multiple labeling assays, both in vitro and in vivo. Dye characteristics that can be altered to advantage include, for example, fluorescence excitation and emission spectra, fluorescence efficiency and quantum yield, fluorescence intensity, and characteristics such as solubility, chemical stability and compatibility with given assay conditions (e.g., in vitro, in vivo, aqueous, non-aqueous, etc.).
Fluorescence characteristics of the cyanine dyes can be altered, for example, by changing the aromatic nature of, or substituents on, the terminal heterocycles, or by changing the number of methine groups between the aromatic moieties. Generally, the longer the polymethine bridge, the higher the wavelengths of excitation and emission (i.e., longer polymethine bridges tend to shift excitation and emission spectra to the red, a so-called “red shift”). However, in general, the stability of the dye and the fluorescence efficiency decreases with increasing polymethine bridge length. It is desirable to alter fluorescence characteristics without dramatically increasing the size of the polymethine bridge.
SUMMARY OF THE INVENTION
The invention relates to novel fluorescent cyanine dyes and to molecules labeled with them. The dyes according to the invention are water soluble and can be used in any application normally requiring water soluble fluorescent dyes. The invention encompasses compositions comprising the novel fluorescent cyanine dyes disclosed herein, as well as nucleosides, nucleotides, polynucleotides, or other molecules or biomolecules labeled with such dyes.
In one aspect, fluorescent cyanine dyes according to the invention have a hetero cyclic structure integrated into the characteristic polymethine bridge between the terminal heterocycles that are characteristic of cyanine dyes. Dyes according to this aspect of the invention have longer emission wavelengths than dyes having similar terminal heterocycles but lacking the heterocycle integrated into the polymethine bridge.
In other aspects, the fluorescent cyanine dyes according to the invention have novel arrangements of functional groups for linkage to molecules of interest, novel combinations or arrangements of terminal heterocycles and/or structures providing structural rigidity. Each of the dyes according to these aspect of the invention has different fluorescence characteristics, making them useful for multiparameter assays and/or assays based on energy transfer.
The invention encompasses a fluorescent cyanine dye having the formula:
T
1
(—CH═)
n1
A(—CH═)
n2
T
2
wherein: n≧1 and n
1
is the same as or different from n
2
; A comprises the formula:
wherein: X
1
and Y
1
are selected from the group consisting of C(CH
3
)
2
, CH═CH, O, N, S, Se and Te and either X
1
or Y
1
is N; X
2
and Y
2
are selected from the group consisting of C(CH
3
)
2
, CH═CH, O, N, S, Se and Te and either X
2
or Y
2
is N; or A comprises the formula:
wherein: Z
1
and Y
1
are selected from the group consisting of C(CH
3
)
2
, CH═CH, O, N, S, Se and Te and either Z
1
or Y
1
is N; Z
2
and Y
2
are selected from the group consisting of C(CH
3
)
2
, CH═CH, O, N, S, Se and Te and either Z
2
or Y
2
is N; and wherein a and b are 0 or 1, and a+b=1; and where X, Y or Z is N, R
2
and R
3
are substituents on N and are the same or different and are selected from the group consisting of H, methyl, ethyl, C(CH
3
)
2
and (CH
2
)
q
V, wherein q is an integer from 1 to 25 and V is a reactive group or H; and wherein T1 and T2 are the same or different and have the formula:
wherein: Q is selected from the group consisting of O, S, CH
2
, (CH═CH) and C(CH
3
)
2
; R
1
and R
4
are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH
2
)
q
V, wherein q is an integer from 1 to 25 and V is a reactive group or H; each of W
1-8
is the same or different and may be H or a hydrophilic moiety; at least one occurrence of W is a hydrophilic moiety; and wherein at least one of R
1
-R
4
has a reactive group.
In one embodiment, one or both of Y
1
and Y
2
are N. It is preferred that in the dye of this embodiment, one or both of X
1
and X
2
are S. It is also preferred in this embodiment that one or both of X
1
and X
2
are O. It is also preferred in this embodiment that one or both of X
1
and X
2
are CH
2
. It is also preferred in this embodiment that one or both of X
1
and X
2
are (CH═CH). It is also preferred in this embodiment that one or both of Y
1
and Y
2
are S.
In another embodiment, Z
1
and Y
2
are S.
In another embodiment, Y
1
and Z
2
are S.
In another embodiment, Q is CH
2
.
In another embodiment, Q is C(CH
3
)
2
.
The invention further encompasses a composition comprising a dye as described above.
The invention further encompasses a fluorescent cyanine dye having the formula:
wherein: n≧1; Q is selected from the group consisting of O, S, CH
2
, (CH═CH) and C(CH
3
)
2
; R
1
-R
4
are the same or different and are selected from the group consisting of H, methyl, ethyl and (CH
2
)
q
V, wherein q is an integer from 1 to 25 and V is a reactive group or H, and at least one of R1-R4 has a reactive group; each of W
1-8
is the same or different and may be H or a hydrophilic

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