Extended rhodamine compounds useful as fluorescent labels

Details Extended rhodamine compounds useful as fluorescent labels Extended rhodamine compounds useful as fluorescent labels

1999-06-03

2001-06-19

Rotman, Alan L. (Department: 1612)

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

Organic compounds

Chalcogen in the nitrogen containing substituent

C544S180000, C544S336000, C544S359000, C546S028000, C546S036000, C546S041000, C546S071000, C546S073000, C546S075000, C546S101000, C546S104000, C546S108000, C546S111000, C548S131000, C548S206000, C548S240000, C548S250000, C548S311400, C548S311700

Reexamination Certificate

active

06248884

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to fluorescent dye compounds. More specifically, this invention relates to extended rhodamine dyes useful as fluorescent labeling reagents.
BACKGROUND
The non-radioactive detection of biological analytes utilizing fluorescent labels is an important technology in modern molecular biology. By eliminating the need for radioactive labels, safety is enhanced and the environmental impact and costs associated with reagent disposal is greatly reduced. Examples of methods utilizing such non-radioactive fluorescent detection include automated DNA sequencing, oligonucleotide hybridization methods, detection of polymerase-chain-reaction products, immunoassays, and the like.
In many applications it is advantageous to employ multiple spectrally distinguishable fluorescent labels in order to achieve independent detection of a plurality of spatially overlapping analytes, i.e., multiplex fluorescent detection. Examples of methods utilizing multiplex fluorescent detection include single-tube multiplex DNA probe assays and multi-color automated DNA sequencing. In the case of multiplex DNA probe assays, by employing multiplex fluorescent detection, the number of reaction tubes may be reduced thereby simplifying experimental protocols and facilitating the production of application-specific reagent kits. In the case of multi-color automated DNA sequencing, multiplex fluorescent detection allows for the analysis of multiple nucleotide bases in a single electrophoresis lane thereby increasing throughput over single-color methods and reducing uncertainties associated with inter-lane electrophoretic mobility variations.
Assembling a set of multiple spectrally distinguishable fluorescent labels useful for multiplex fluorescent detection is problematic. Multiplex fluorescent detection imposes at least six severe constraints on the selection of component fluorescent labels, particularly for applications requiring a single excitation light source, an electrophoretic separation, and/or treatment with enzymes, e.g., automated DNA sequencing. First, it is difficult to find a set of structurally similar dyes whose emission spectra are spectrally resolved, since the typical emission band half-width for organic fluorescent dyes is about 40-80 nanometers (nm). Second, even if dyes with non-overlapping emission spectra are identified, the set may still not be suitable if the respective fluorescent quantum efficiencies are too low. Third, when several fluorescent dyes are used concurrently, simultaneous excitation becomes difficult because the absorption bands of the dyes are usually widely separated. Fourth, the charge, molecular size, and conformation of the dyes must not adversely affect the electrophoretic mobilities of the analyte. Fifth, the fluorescent dyes must be compatible with the chemistry used to create or manipulate the analyte, e.g., DNA synthesis solvents and reagents, buffers, polymerase enzymes, ligase enzymes, and the like. Sixth, the dye must have sufficient photostability to withstand laser excitation.
Currently available multiplex dye sets suitable for use in four-color automated DNA sequencing applications require blue or blue-green laser light to adequately excite fluorescence emissions from all of the dyes making up the set, e.g., argon-ion lasers. Use of such lasers in commercial automated DNA sequencing systems is disadvantageous because of their high cost and limited lifetime.
Thus, there exists a need for fluorescent dye compounds which satisfy the above constraints and are excitable by laser light having a wavelength above about 630 nm.
SUMMARY
The present invention is directed towards our discovery of a class of extended rhodamine dye compounds suitable for the creation of sets of spectrally-resolvable fluorescent labels useful for multiplex fluorescent detection. The subject dye compounds are particularly well suited for use in automated fluorescence-based DNA sequencing systems using an excitation light source having a wavelength greater than about 630 nm, e.g., a helium-neon gas laser or a solid state diode laser.
In a first aspect, the invention comprises an extended rhodamine compound having the structure
wherein the composition of moieties R
1
through R
11
, R
13
, and Y
1
through Y
4
are as follows. Taken alone, R
1
through R
7
, R
9
through R
11
, and R
13
is each independently selected from the group consisting of —H, alkyl, alkyl independently substituted with one or more Z
1
, heteroalkyl, heteroalkyl independently substituted with one or more Z
1
, aryl, aryl independently substituted with one or more Z
1
, heteroaryl, heteroaryl independently substituted with one or more Z
1
, arylalkyl, arylalkyl independently substituted with one or more Z
1
, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z
1
, halogen, —OS(O)
2
OR, —S(O)
2
OR, —S(O)
2
R, —S(O)
2
NR, —S(O)R, —OP(O)O
2
RR, —P(O)O
2
RR, —C(O)OR, —NRR, —NRRR, —NC(O)R, —C(O)R, —C(O)NRR,—CN, and —OR. As used here, and throughout this Summary section, each R may be independently —H, allyl, heteroalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl or linking group, and each Z
1
may be independently any one of —R, halogen, —OS(O)
2
OR, —S(O)
2
OR, —S(O)
2
R, —S(O)
2
NR, —S(O)R, —OP(O)O
2
RR, —P(O)O
2
RR,—C(O)OR, —NRR, —NRRR, —NC(O)R, —C(O)R, —C(O)NRR,—CN, —O or —OR.
R
8
is selected from the group consisting of —H, alkyl, alkyl in dependently substituted with one or more Z
1
, heteroalkyl, heteroalkyl independently substituted with one or more Z
1
, aryl, aryl independently substituted with one or more Z
1
, heteroaryl, heteroaryl independently substituted with one or more Z
1
, arylalkyl, arylalkyl independently substituted with one or more Z
1
, heteroarylalkyl, heteroarylalkyl independently substituted with one or more Z
1
.
Taken alone, nitrogen substituents Y
1
through Y
4
are each independently selected from the group consisting of —H, alkyl, alkyl independently substituted with one or more Z
1
, heteroalkyl, heteroalkyl independently substituted with one or more Z
1
, aryl, aryl independently substituted with one or more Z
1
, heteroaryl, heteroaryl independently substituted with one or more Z
1
, arylalkyl, arylalkyl independently substituted with one or more Z
1
, heteroarylalkyl, and heteroarylalkyl independently substituted with one or more Z
1
.
Alternatively, rather than being taken alone, substituents R
1
through R
7
, R
9
through R
11
, R
13
, Y
1
through Y
4
can be taken together in various selected combinations. In particular, R
1
may be taken together with R
2
, Y
1
or Y
2
, R
2
may be taken together with R
1
, R
3
may be taken together with R
4
, R
4
may be taken together with R
3
, Y
3
or Y
4
, R
5
may be taken together with R
6
, Y
3
or Y
4
, R
6
may be taken together with R
5
, R
7
, Y
3
or Y
4
, R
7
may be taken together with R
6
, R
9
may be taken together with R
10
, R
10
may be taken together with R
9
or R
11
, R
11
may be taken together with R
10
, Y
1
or Y
2
, R
13
may be taken together with Y
1
or Y
2
, Y
1
may be taken together with R
1
, R
11
, or Y
2
, Y
2
may be taken together with R
1
, R
11
, or Y
1
, Y
3
may be taken together with R
4
, R
5
, R
6
, R
13
, or Y
4
, or Y
4
may be taken together with R
4
, R
5
, R
6
, R
13
, or Y
3
.
In a second aspect, the invention comprises intermediate compounds useful for the synthesis of the extended rhodamine compounds of the first aspect, such intermediates having the structure
wherein the composition of moieties R
1
, R
2
, R
9
through R
12
, Y
1
and Y
2
are as follows. Taken alone, R
1
, R
2
, R
9
, R
10
and R
11
is each independently selected from the group consisting of —H, alkyl, alkyl independently substituted with one or more Z
1
, heteroalkyl, heteroalkyl independently substituted with one or more Z
1
, aryl, aryl independently substituted with one or more Z
1
, heteroaryl, heteroaryl independently substituted with one or more Z
1
, arylalkyl, arylalkyl independently substituted with one or more Z
1
, heteroarylalkyl, h

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