WO2009014513A1

WO2009014513A1 - Novel dyes for the detection or quantification of desirable target molecules

Info

Publication number: WO2009014513A1
Application number: PCT/US2007/016581
Authority: WO
Inventors: Praveen Pande; Maciej Szczepanik; Yiu-Jun Xiang
Original assignee: Enzo Life Sciences, Inc.
Priority date: 2007-07-24
Filing date: 2007-07-24
Publication date: 2009-01-29

Abstract

The present invention provides dyes, reactive dyes and labeled reagents that may be used in the detection or quantification of desirable target molecules, such as proteins and nucleic acids. Dyes are provided that may be used free in solution where the binding of the dye to the target molecule provides signal generation. Dyes are also provided that comprise reactive groups that may be used to attach the dyes to probes that will bind to desirable target molecules. The novel dyes of the present invention have been modified to provide beneficial properties.

Description

NOVEL DYES FOR THE DETECTION OR QUANTIFICATION OF DESIRABLE TARGET MOLECULES

FIELD OF THE INVENTION

This invention relates to field of labeling compositions, reagents and processes that are useful in applications related to the detection, quantification and localization of target molecules of interest that include nucleic acids and proteins.

All patents, patent applications, patent publications, scientific articles and the like, cited or identified in this application are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Patent Application Serial No. 1 1 /137,771 , filed May 24, 2005, published as US-2006-0269926-A1 on November 30, 2006, and it is also a continuation-in-part of U.S. Patent Application Serial No. 1 1 /177,923, filed on July 7, 2005, published as US-2006-0269931 -A1 on November 30, 2006. This application claims priority to both aforementioned applications.

BACKGROUND OF THE INVENTION

There are a variety of properties that might be desirable for dyes that are intended for use as markers for detection of proteins or nucleic acid hybridization. These can include the ability to bind to a protein, lipid or nucleic acid, the capability of incorporation into nucleic acids by enzymatic means when attached to a nucleotide, a lack of steric hindrance that could potentially interfere with hybridization, water solubility, lack of aggregation, ability to intercalate into double- stranded nucleic acids and the presence of a reactive group that allows attachment of the dye to a nucleotide or other desirable target. Suitable dyes could have many of these properties but do not need to have them all. For instance, the ability to intercalate may allow detection of hybridization events in the presence of unhybridized probes or it may provide increased hybridization stabilization. Examples of these applications are disclosed in European Patent Application EP 0 231 495, U.S. Patent No. 5,994,056 and U.S. Patent No. 6,174,670, all of which are incorporated by reference. Similarly, the ability to be incorporated by an enzyme is a useful property when carrying out enzymatic labeling of nucleic acids. Labels that are inhibitory towards incorporation can still be used in some methods where nucleic acids are chemically synthesized rather than using enzymatic means. Also, nucleotides with reactive groups such as allyl-amine may be incorporated enzymatically into nucleic acids and then in a second step they are post- synthetically modified by attachment of dyes. Steric hindrance may be compensated to some degree by the nature of the linker joining the dye to a nucleotide with regard to both the length and the constituents of the linker. For a discussion of this last point, see U.S. Patent Application Publication No. 2003/0225247, hereby incorporated by reference.

The particular spectral characteristics of dyes are also important qualities. Although broad-spectrum white light can be used as a source of excitation, lasers with defined set wavelengths are most commonly employed. As such, dyes that would find most immediate use would have excitation wavelengths that can make use of such laser emissions. Emission wavelengths are of a more flexible nature since filters can be used to isolate a particular part of the spectrum. However, it should be noted that there are a number of machines used for detection of labeled nucleic acids that have been designed with dyes that are commonly used. For instance, there are a number of slide scanners that have been optimized for detection of nucleic acids labeled with the Cy3 and Cy5 dyes described by Waggoner et al. in U.S. Patent No. 5,268,486 (incorporated herein by reference). On the other hand, the availability of dyes that have useful properties but have wavelengths that are not commonly used can prove to be an incentive to adopt lasers with compatible wavelengths. A set of dyes with well separated emission spectra may find use where more than one fluorophor is to be used at the same time. Well known applications in this are immunostaining for various proteins in cells, in situ hybridization for multiple targets, non-radioactive sequencing, nucleic acid array analysis, protein array analysis, as well as non-specific cellular staining with dyes having general affinities for proteins or lipids. On the other hand, overlapping spectral characteristics also have applications; for instance, emission by one fluorophor may be used to excite a second fluorophor through energy transfer when distances are sufficiently close.

Among the dyes that have been most widely used as markers for proteins and nucleic acid labeling are members of the xanthene, coumarin, cyanine and asymmetric cyanine dye families. Xanthene dyes are among the earliest dyes used for biological staining, where fluorescein was used to work out many of the techniques for labeling proteins and nucleic acids. The basic structure of fluorescein molecules can be depicted as:

Related xanthene compounds that have also been used as labels include rhodols and rhodamines. Their basic structure is as follows:

The R group attached to the central structure is typically a substituted phenyl group although as described in U.S. Patent Application Publication No. 2003/0225247 (hereby incorporated by reference), aphenylic versions are also suitable as dyes.

Another family of dyes that have enjoyed widespread use is based upon derivatives of coumarin. The basic structure of coumarin is as follows:

Typically, coumarin derivatives will be dyes when R is an OH or an amine group. Useful compounds have also been made where R is further modified such that an enzymatic cleavage event converts the R group into an OH or amine group. Thus this proto-dye or dye precursor can be used as marker for the presence of an enzyme that is capable of converting a coumarin compound into a fluorescent dye. Discussions of such methods are disclosed in U.S. Patent No. 5,696,157 and U.S. Patent No. 5,830,912, both of which are incorporated by reference.

As described above, a large number of useful dyes are based upon cyanine dyes. The basic structure of cyanine dyes is as follows

As will be discussed later, major factors in the particular spectral qualities of these dyes is dependent upon the number "n", the nature of "X" and "Y " and functional groups that extend the aromaticity of the dyes.

Other compounds that were functionally considered to be cyanine-type dyes (see U.S. Patent No. 5,268,486 hereby incorporated by reference) are the merocyanine and styryl dyes whose structures are:

and

There are a variety of atoms that have been used in the X and Y positions. These have included carbon, sulfur, oxygen, nitrogen and selenium. When X or Y is a carbon, this portion of the dye is an indolinium moiety. When X or Y is substituted by sulfur, oxygen or nitrogen this portion is respectively described as a benzothiazolium, benzoxazolium or a benzimidazolium moiety.

Another version of styryl dyes can have picoline or quinoline moieties instead of the benzazolium group, thereby having the structures:

and

Asymmetric cyanine dyes contain one portion that is essentially the benzazolium portion of the cyanine dye family but connected to this portion by the methine bridge is a different aromatic compound. Their structure is as follows:

The aromatic moiety can be a six membered aromatic or heteroaromatic ring

Improvements to these dyes have been carried out by substitution of various groups onto the basic structure, i.e. on the carbons and nitrogens of the preceding structures or where H or R groups are featured. Additionally, other rings may be fused to various parts of the rings in the structures above, thereby generating more complex structures. These modifications have led to shifts in the excitation and emission characteristics of the dyes that allow a large number of dyes with same basic structure but having different spectral characteristics, i.e. modifications can be made in their structure that can alter the particular wavelengths where these compounds will absorb and fluoresce light. As described above, the cyanine dyes can have a general structure comprising two benzazolium-based rings connected by a series of conjugated double bonds. The dyes are classified by the number (n) of central double bonds connecting the two ring structures; monocarbocyanine or trimethinecarbocyanine when n = 1 ; dicarbocyanine or pentamethinecarbocyanine when n = 2; and tricarbocyanine or heptamethinecarbocyanine when n = 3. The spectral characteristics of the cyanine dyes have been observed to follow specific empirical rules. For example, each additional conjugated double bond between the rings usually raise the absorption and emission maximum about 100 nm. Thus, when a compound with n = 1 has a maximum absorption of approximately 550 nm, equivalent compounds with n = 2 and n = 3 can have maximum absorptions of 650 nm and 7-50 nm respectively. Addition of aromatic groups to the sides of the molecules has lesser effects and may shift the absorption by 15 nm to a longer wavelength. The groups comprising the indolenine ring can also contribute to the absorption and emission characteristics. Using the values obtained with gem- dimethyl group as a reference point, oxygen substituted in the ring for the gem- dimethyl group can decrease the absorption and emission maxima by approximately 50 nm. In contrast, substitution of sulfur can increase the absorption and emission maxima by about 25 nm. R groups on the aromatic rings such as alkyl, alkyl- sulfonate and alkyl-carboxylate usually have little effect on the absorption and emission maxima of the cyanine dyes (U.S. Patent No. 6,1 10,630, hereby incorporated by reference).

As described above, alteration of spectral qualities is only one useful modification that can be made to a dye. In another instance, modification of a dye by a sulfonate group may increase the stability of many dyes and thereby resist "bleaching" after illumination. Modification of dyes by sulfonation was later applied in the modification of cyanine dyes with reactive groups (US patent serial No. 5,569,766 hereby incorporated by reference), where it was reported that the sulfonation decreases aggregation of labeled materials. It was further applied to xanthenes, coumarins and the non-benzazolium portion of asymmetric cyanine dyes (U.S. Patent No. 5,436,134, U.S. Patent No. 6,130,101 and U.S. Patent No. 5,696,157, all of which are hereby incorporated by reference). Modifications of dyes haves also been made to increase their affinity or selectivity towards binding to nucleic acids (European Patent No. EP 0 231 495, U.S. Patent Application Publication No. 2003/0225247 and U.S. Patent No. 5,658,751 , all of which are incorporated by reference).

In many cases, the utility of these dyes has been achieved by synthesis of compounds with a reactive group that allows attachment of the dye to a target molecule. For instance, although cyanine dyes in themselves had been known for many years, it was only when derivatives were described with reactive groups (U.S. Patent No. 5,268,486 hereby incorporated by reference) that they found widespread use in labeling proteins and nucleic acids. Their versatility was then increased by disclosure of other groups that could be used to attach cyanine dyes to suitable partners (U.S. Patent No. 6,1 14,350 and U.S. Patent Application Publication No. 2003/0225247, both of which are hereby incorporated by reference). An exemplarary list of electrophilic groups and corresponding nucleophilic groups that can be used for these purposes are given in Table 1 of U.S. Patent No. 6,348,596 (hereby incorporated by reference).

A variety of linker arms may be used to attach dyes to targets. Commonly used constituents for linkers are chains that contain varying amounts of carbon, nitrogen, oxygen and sulfur. Examples of linkers using some of these combinations are given in U.S. Patent No. 4,707,440, hereby incorporated by reference. Bonds joining together the constituents can be simple carbon-carbon bonds or they may be acyl bonds (U.S. Patent No. 5,047,519), sulfonamide moieties (U.S. Patent No. 6,448,008 B1 ) and polar groups (U.S. Patent Application Publication No. 2003/0225247) all of which are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides a dye compound having the formula (A)

or (B)

wherein X and Y independently comprise CR₃₀R₃₁, NR₃₀, O, S or Se, wherein R₃₀ and R₃₁ independently comprise hydrogen, a halogen, an amino group, an alkγl group wherein said alkyl group is saturated or unsaturated linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated branched or linear, substituted or unsubstituted, or when taken together R₃₀ and R₃₁ comprise a 5 or 6 membered ring; wherein m and n are each independently integers from 0 to 5; wherein A is a linker connecting an aromatic moiety to the methine bridge wherein said linker comprises one or more atoms selected from C, O, S. P, N and combinations thereof, wherein said linker may be substituted or unsubstituted, saturated or unsaturated, branched or linear; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₃₀ or R₃₁ comprises Q, wherein Q comprises a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Q is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof and wherein when Q is a sulfonamide, Q does not have a terminal reactive group or a linker arm joining the dye to a target molecule; wherein R₁₆, R₁₇,

R19/ R20' "21 < "22» "23» R24' °2sι °2β> "27» °28 ' "29' "33' R₃₃' R₃₄' R₃₅' R₃₆/ R₃₇ ^ano< the remaining R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ , R₁₅, R₃₀ and R₃₁ independently comprise hydrogen, Z, a halogen, an amino group, an aryl group wherein said aryl group is substituted or unsubstituted, an alkyl group wherein said alkγl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₂ and R₃, R₃ and R₄, R₄ and R₅, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, R₁ and R₁₆, R₆ and R₂₃, R₃₃ and R₃₄, R₃₄ and R₃₅, R₃₆ and R₃₇ independently comprise a five or six membered ring; wherein Z comprises a carboxyl group (CO₂ ), a carbonate ester (COER₃₃), a sulfonate (SO₃ ), a sulfonate ester (SO₂ER₃₃), a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ^"ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂"), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Z is attached directly or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm is saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof; and wherein any of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₃₀ or R₃₁ may further comprise a heteroatom containing side chain, wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (-OR₃₈), a thioether linkage (-SR₃₈), or an amine linkage (-NR₃₈R₃₉ or - N ⁺ R₃₈R₃₉R₄₀), and wherein R₃₈, R₃₉ and R₄₀ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₈ and R₃₉ and R₃₉ and R₄₀ independently comprise a five or six membered ring, and wherein any of R₃₈, R₃₉ or R₄₀ may further comprise said heteroatom containing side chain;

The present invention also provides a process for detecting the presence or quantity of a target comprising the steps of a) providing i) a sample in which the presence or quantity of a target is unknown or sought to be detected, and ii) a composition comprising a first portion and a second portion wherein said first portion comprises a dye and said second portion comprises a target specific moiety, said dye having the formula:

(A)

or (B)

wherein X and Y independently comprise CR₃₀R₃₁, NR₃₀, O, S or Se, wherein R₃₀ and R₃₁ independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated linear or branched, substituted or unsubstituted, an arγl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated branched or linear, substituted or unsubstituted, or when taken together R₃₀ and R₃₁ comprise a 5 or 6 membered ring; wherein m and n are each independently integers from 0 to 5; wherein A is a linker connecting an aromatic moiety to the methine bridge wherein said linker comprises one or more atoms selected from C, O, S. P, N and combinations thereof, wherein said linker may be substituted or unsubstituted, saturated or unsaturated, branched or linear; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, ^Ri₅' R₃₀ ^or R₃₁ comprises Q, wherein Q comprises a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (PO₄=), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼ ), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ^"), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Q is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof and wherein when Q is a sulfonamide, Q does not have a terminal reactive group or a linker arm joining the dye to a target molecule; wherein R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R21 , R22' R23' ^24» ^25» R26' ^27» R_∑β ' ^₂₉» R₃₃' R₃₃' ^R ₃₄' R₃₅' R₃₆' R₃₇ and the remaining R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, Ri₃'Ri_{4 >} Ri₅' R₃₀ ^and R₃i independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₂ and R₃, R₃ and R₄, R₄ and R₅, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, R₁ and R₁₆, R₆ and R₂₃, R₃₃ and R₃₄, R₃₄ and R₃₅, R₃₆ and R₃₇ independently comprise a five or six membered ring; wherein Z comprises a carboxyl group (CO₂ ), a carbonate ester (COER₃₃), a sulfonate (SO₃ ), a sulfonate ester (SO₂ER₃₃), a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₆), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (P0₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼ ), a thiophosphate monoester (PSO₂ ^"ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Z is attached directly or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm is saturated or unsaturated, linear or branched, substituted or uπsubstituted, or any combinations thereof; and wherein any of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₃₀ or R₃₁ may further comprise a heteroatom containing side chain, wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (-OR₃₈), a thioether linkage (-SR₃₈), or an amine linkage (-NR₃₈R₃₉ or - N⁺R₃₈R₃₉R₄₀), and wherein R₃₈, R₃₉ and R₄₀ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₈ and R₃₉ and R₃₉ and R₄₀ independently comprise a five or six membered ring, and wherein any of R₃₈, R₃₉ Or R₄₀ may further comprise said heteroatom containing side chain; and wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, Ri₃/ Ri₄, Ri₅/ R₃₀ or R₃₁ is linked to said second portion.

Other steps of this process include b) allowing any targets present in said sample i) to bind with said target specific moiety comprising said composition ii); and c) quantifying the amount of said composition ii) bound to any of said target in the sample, thereby detecting the presence or quantity of said target. DESCRIPTION OF THE INVENTION

This invention provides Near Infrared Dyes (NIRD) and their reactive esters that may be used for in-vivo or in-vitro detection of cellular processes in animal models. The series of dyes of the present invention have been modified by the addition of groups to provide, hydrophilic, hydrophobic and lipophilic properties.

The present dye series is based on cyanine chromophore which is known to absorb far-red (600-700 nm) and near-infrared (700-1200 nm) light. This makes these cyanine dyes less susceptible to interference from auto fluorescence of biomolecules and hence increasing the signal-to-noise ratio. Additionally, near- infrared dyes can be easily excited with inexpensive diode lasers such as Cd-Se (exciting between 780 nm-830 nm). In general, near infrared detection is more sensitive than visible fluorescence and equal to or more sensitive than chemiluminescent detection. Most of the near infrared dyes that are currently available in the market are poly-sulfonated, thus making them excellent water soluble dyes. Examples include, Alexa" 750 and Alexa^* 790 (Invitrogen, U.S. Patent No. 6,974,873 B1 and U.S. Patent No. 6,977,305 B1 ), Cy5.5 and Cy7(GE Healthcare, U.S. Patent No. 5,268,486 and U.S. Patent No. 5,569,766), IRDye^* 800CW and IRDye" 700DX (both from Li-Cor Biosciences, U.S. Patent No. 6,593,148 B1 and U.S. Patent No. 6,995,274 B1 ). However, this property of these dyes makes them impervious to cell membrane, thus limiting their use in live cell imaging. Furthermore, the choices of near infrared dyes are currently limited in the market. Hence, developing new and better near-infrared dyes, in particular dyes with cell permeable characteristics still remains a challenge and demand.

This invention relates to modified cyanine dyes which absorbs in near infrared spectrum. For these dyes an ethylene unit is replaced by a cyclic conjugated ring, in the long poly methine chain. This improves the stability of these dyes towards oxidation and photobleaching. See, for example, U.S. Patent No. 6,593,148 B1 , issued on July 15, 2003, the contents of which are hereby incorporated by reference. The present invention provides a dye having the following formulas:

wherein X and Y independently comprise CR₃₀R₃₁, NR₃₀, O, S or Se, wherein R₃₀ and R₃₁ independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxγ group is saturated or unsaturated branched or linear, substituted or unsubstituted, or when taken together R₃₀ and R₃₁ comprise a 5 or 6 membered ring; one of R₃₀ and R₃₁ can be - L-R_x, where L is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1 -25 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or a phosphorous- oxygen, phosphorous-sulfur, nitrogen-nitrogen, nitrogen-oxygen or nitrogen- platinum bonds; or aromatic or heteroaromatic bonds; R_x is a reactive group; wherein m and n are each independently integers from 0 to 5; A is H, Cl, S, O, N, CN, -C C, -CH = CH- or is connected by the following ring:

leading to the dye having the formulas:

wherein A is O, S, -CdC₁ -CH = CH- or NR₃₂ wherein R₃₂ is H, alkyl, phenyl, benzyl, or substituted benzyl, wherein said alkyl or phenyl or benzyl groups are saturated or unsaturated, linear or branched, substituted or unsubstituted, an alkoxγ group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, ; R₁₁-R₁₅ are each independently H, alkyl, substituted alkyl, cyano, isocyanate, isothiocyanate, halo, carboxy, amino, phosphate, thiophosphate, phosphonate, thiophosphonate, amide, maleimide or SO₃ ^"Cat⁺, wherein Cat⁺ is a cation;

R₁ and R₆ are each independently alkyl, benzyl, substituted benzyl, (CH₂)_qR_a or (CH₂)_qR_b; wherein at least one of R₁ and R₆ is (CH₂)_qR_b and wherein q is an integer from 1 to 25, and R_a and R_b are functional groups that can directly react with a carboxyl, hydroxyl, amino, or a thiol group. Furthermore, R₁ and R₆ can be a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof. In a preferred embodiment, one of R₁ and R₆ is (CH₂)_qR_a and the other is (CH₂)_qR_b. Particularly preferred R₆ and R_b groups include but are not limited to hydrogen, alkyl, hydroxyl, sulfonate, mercapto, carboxyl, amino, haloalkyl, phosphoramidityl, N-hydroxy succinimidyl ester, sulfo N-hydroxy succinimidyl ester, isocyanate, isothiocyanate, haloacetamide, maleimide, acid halides, benzotriazole, imidazole and chlorotriazole, monochlorotriazine, dichlorotriazine, 4,6,-dichloro-1 ,3,5- triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, sulfonyl halide, imido ester, hydrazine, azidonitrophenol, azide, 3- (2-pyridyl dithio)-proprionamide, glyoxal or aldehyde group, and any combination thereof; wherein at least one of R₂, R₃, R₄, R₅, R₇, R₈, Rg or R₁₀ comprises Q, wherein Q comprises a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (PO₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼ ), a thiophosphate monoester (PSO₂TR₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Q is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof and wherein when Q is a sulfonamide, Q does not have a terminal reactive group or a linker arm joining the dye to a target molecule; wherein R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂₈ and R₂₉ independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxγ group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted; wherein R₃₃, R₃₃, R₃₄, R₃₅, R₃₆, R₃₇ and the remaining R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃,R₁₄ and R₁₅ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkγl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₂ and R₃, R₃ and R₄, R₄ and R₅, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, R₁ and R₁₆, R₆ and R₂₃, R₃₃ and R₃₄, R₃₄ and R₃₅, R₃₆ and R₃₇ independently comprise a five or six membered ring; wherein Z is attached directly or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm is saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof; and wherein any of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, or R₁₅, may further comprise a heteroatom containing side chain, wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (- OR₃₈), a thioether linkage (-SR₃₈), or an amine linkage (-NR₃₈R₃₉ or -N⁺R₃₈R₃₉R₄₀), and wherein R₃₈, R₃₉ and R₄₀ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkγl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₈ and R₃₉ and R₃₉ and R₄₀ independently comprise a five or six membered ring, and wherein any of R₃₈, R₃₉ Or R₄₀ may further comprise said heteroatom containing side chain; further, R₃₃ and R₃₄ independently comprise hydrogen, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, unsubstituted or substituted with sulfonate or NR₄₁R₄₂ or N⁺ R₄₁R₄₂R₄₃, wherein R₄₁, R₄₂ and R₄₃ are independently comprise hydrogen, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, unsubstituted or substituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₃ and R₃₄ comprise a five or six membered ring.

In the present invention, sulfonates are considered to be any group with the formula SO₃ ^' including both sulfonic acid as well as various sulfonate salts. The addition of a sulfonate group provides a charged moiety that can increase solubility, inhibit bleaching and reduce aggregation. The addition of phosphonate (PO₃ ^"), phosphate (0-PO₃ -) moieties or their derivatives may also provide such qualities.

Transformation of the foregoing charged species into esters may convert a charged group into a polar group. Derivatives that may find use with the present invention can include thioanalogues such as thiophosphates, thiophosphonates and thioesters. Other derivatives that may find use can include phosphoramides and phosphonamides.

In the present invention, sulfones are considered to be any groups that have the formula C-SO₂-C where carbon atoms are attached to the intervening sulfur atom. One of the carbon atoms may be part of a ring structure of the dye or it may part of an intervening alkyl group connecting the sulfone to the dye. When one of the carbons of a sulfone is replaced by a nitrogen atom the group is a sulfonamide. The presence of the polar groups may help nucleotide incorporation since dyes with polar groups will be less negatively charged than their ionized equivalents and thus be less repelled by the negatively charged phosphate backbone of a nucleic acid template. The sulfone or sulfonamide group can be modified as desired by linkage to other moieties to add desirable properties. It is also understood that the degree of charge or polarity can be determined by the user by the addition of appropriate combinations of charged and polar groups to a dye.

In the present invention, sulfoxides (SOR₃₃), sulfones (SO₂CR₃₃R₃₄R₃₅) and sulfonamides (SO₂NR₃₃R₃₄) are respectively defined as having the structures:

In the present invention, phosphates (PO₄ ), their monoesters (PO₃ E R₃₃), diesters (PO₂ER₃₃ER₃₄), are respectively defined as having the structures:

E is an oxygen in the monoester and diester and

when E is a sulfur. In the present invention, phosphonates (PO₃ ), their esters (PO₂ ^"ER₃₃ and

POER₃₃ER₃₄) are respectively defined as having the structures:

when E is ah oxygen in the monoester and diester and

O O

I l I l

-^R33 ^^R33

-p-

O S

R34 when E is a sulfur.

In the present invention, thiophosphates (PSO₃"), their esters (PSO₂-ER₃₃ and PSOER₃₃ER₃₄) are respectively defined as having the structures:

S S S 0 — p Il — Θ 0 0 — p Il — 0 /^R» 0 — p Il — 0 -^R" θ 'θ

O O O

R₃4

when E is an oxygen in the monoester and diester and

<33 R33

-O P- -O P S

S

R34 when E is a sulfur.

In the present invention, thiophosphonates (PSO₂ ⁼ ), their esters (PSO ER₃₃ and PSER₃₃ER₃₄) are respectively defined as having the structures:

when E is an oxygen in the monoester and diester and

when E is a sulfur

In the present invention, sulfonates (SO₃ ^"), their esters (SO₂ER₃₃) are respectively defined as having the structures:

when E is an oxygen or sulfur in the ester linkage.

In the present invention, phosphonamides (PONR₃₃R₃₄NR₃₆R₃₇), phosphoramides (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇) and phosphoramidites (PO₂R₃₆NR₃₃R₃₄) are respectively defined as having the structures:

and their thioanalogues (PSNR₃₃R₃₄NR₃₆R₃₇), (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇) and (POSR₃₆NR₃₃R₃₄) having respectively the structures:

It is also understood that when a dye comprises anionic group, there will also be a cationic counterion present. Any cation may serve this purpose as long as it doesn't interfere with the use of the dye. Examples of cations that may serve as counterions can include but not be limited to hydrogen, sodium, potassium, lithium, calcium, cesium, ammonium, alkyl ammonium, alkoxy ammonium and pyridinium. It is also understood that when a dye comprises a cationic group, there will also be an anionic counterion present. Any anion may serve this purpose as long as it doesn't interfere with the use of the dye. Examples of anions that may serve as counterions can include but not be limited to halides such as a bromide, chloride, fluoride and iodide. Other examples can include but not be limited to perchlorate (CIO₄ ^"), sulfate (SO₄ ⁼), sulfonate, alkane sulfonate, aryl sulfonate, phosphate, tosylate, mesylate and tetrafluoroborate moieties. In some cases the counterion or counterions are provided by the dye being a salt where they exist as separate ionic species. In other cases, the counterion or counterions may be present as part of the compound (sometimes called inner salts). It is understood that there may also be a combination of ions that are provided by the compound and salts. With regard to acid moieties that are shown in forms such as COOH it is also understood that these compounds may be found in ionized forms such as COO^".

Alkyl, aryl or alkoxy R groups may be substituted or unsubstituted. Examples of substitutions can include but not be limited to one or more fluorine, chlorine, bromine, iodine, hydroxy, carboxy, carbonyl, amino, cyano, nitro or azido groups as well as other alkyl or alkoxy groups. The length of the alkoxy groups may be as desired. For instance, they may independently comprise from 1 to 25 carbons in length. They may be shorter as well, for instance they may be only 1 to 6 carbons in length in a dye molecule of the present invention.

The polar groups, charged groups and other substituent may be connected to the dye directly or they may be connected by a linker arm comprising carbon, nitrogen, sulfur, oxygen or any combination thereof. The linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted as well as any combination of the foregoing.

Previously, cyanine dyes have been disclosed that comprise an SO₃ group (U.S. Patent No. 5,268,486, U.S. Patent No. 5,486,616, U.S. Patent No. 5,569,766, U.S. Patent No. 6,027,709, U.S. Patent No. 6,593,148, U.S. Patent No. 6,995,274, U.S. Patent No. 6,974,873 and U.S. Patent No. 6,977,305) but the use of sulfone (SO₂) groups to modify the properties of cyanine dyes has not been disclosed. The addition of a sulfonamide group to a cyanine dye has been previously disclosed but only in the context of being part of a linker arm (U.S. Patent No. 6,448,008) thereby being part of the connection between the dye and a terminal reactive group. Cyanine dyes lacking reactive groups, or cyanine dyes with sulfonamide groups in moieties other than the linker arm with a combination of ring substitution in the methine bridge which is further substituted with other groups have not been disclosed.

Complex ring structures

As described above some of the R groups may be joined together to form one or more fused 5 or 6 membered ring structures. It is understood that the complex rings that are formed by closure of R groups may be further substituted with any of the R groups described previously. Examples of complex rings that may be formed for the benzazolium portion of cyanine and asymmetric cyanine dyes can comprise but not be limited to:

If desired, a variation of the preceding dyes can be the substitution of an azabenzazolium instead of a benzazolium moiety in the cyanine, asymmetric cyanine and styrene dyes; i.e. a nitrogen replaces the carbon in the positions where R², R³, R⁴, R⁵, R⁷, R⁸, R⁹ or R¹⁰ are connected to the benzazolium moiety of cyanine dyes or to the R², R³, R⁴ or R⁵ positions of the asymmetric cyanine and styrene dyes disclosed previously. Methods for the synthesis and use of an azabenzazolium based dyes are disclosed in U.S. Patent No. 6,664,047 B1 , hereby incorporated by reference. As such these moieties would have the structures:

Examples of rings and complex rings that may comprise the non-benzazolium portion of an asymmetric cyanine dye can comprise but not be limited to:

Reactive Groups and Targets

In another aspect of the present invention, one of the R groups is a reactive group thereby allowing the dyes of the present invention to be attached to a useful target molecule. Examples of reactive groups that may find use in the present invention can include but not be limited to a nucleophilic reactive group, an electrophilic reactive group, a terminal alkene, a terminal alkγne, a platinum coordinate group or an alkylating agent.

There are a number of different electrophilic reactive groups that may find use with the present invention; examples can include but not be limited to isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,-dichloro- 1 ,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)-propionamide, glγoxal and aldehyde groups. Nucleophilic reactive groups can include but not be limited to reactive thiol, amine and hydroxγl groups. For purposes of synthesis of dyes, reactive thiol, amine or hydroxyl groups can be protected during various synthetic steps and the reactive groups generated after removal of the protective group. Use of a terminal alkene or alkyne groups for attachment of markers has been previously described in U.S. Patent Application Serial No. 2003/0225247, hereby incorporated by reference. The use of platinum coordinate groups for attachment of other dyes has been previously disclosed in U.S. Patent No. 5,580,990 and the use of alkyl groups has been previously described in U.S. Patent No. 6,593,465 B1 , both of which patents are hereby incorporated by reference.

Examples of useful target molecules can include but not be limited to a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, liposomes, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, natural polymers, synthetic polymers, plastic particle, plastic surface, silicaceous particle, silicaceous surface, organic molecule, dyes and derivatives thereof.

The nucleoside, nucleotide, oligonucleotide, or polynucleotide can comprise one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues and any combination thereof.

As described above, the dyes of the present invention may have dyes as targets thereby creating composite dyes. By joining the dyes of the present invention to another dye, unique properties may be enjoyed that are not present in either dye alone. For instance, if one of the dyes of the present invention is joined to another dye such that it creates an extended conjugation system, the spectral characteristics of the dye may be different than either dye component. Another example of this method is where the conjugation systems do not overlap but the proximity allows an internal energy transfer to take place thereby extending the Stokes shift. For an example of this, see U.S. Patent No. 5,401 ,847, U.S. Patent No. 6,008,373 B1 and U.S. Patent No. 5,800,996, all three of which patents are hereby incorporated by reference. Other properties may also be enhance by this joining, for example, it has been previously described that the joining together of two ethidium bromide molecules generates a dye that has enhanced binding to nucleic acids (U.S. Patent Application Publication No. 2003/0225247, hereby incorporated by reference). Other composite dyes have been described that simultaneously enjoy both properties, i.e. enhanced binding and energy transfer (U.S. Patent No. 5,646,264, hereby incorporated by reference). Furthermore, these composites dyes are not limited to binary constructs of only two dyes, but may comprise oligomeric or polymeric dyes. These composite dyes may be comprised of the same dye or different dyes may be joined together depending upon the properties desired.

Utility may also be achieved by attaching a dye of the present invention to a target specific moiety. Thus, binding between the target specific moiety and its corresponding target may be monitored by essentially determining the presence or amount of dye that is bound to the target. Well-known examples of such assays are hybridizations between complementary nucleic acids as well as binding that take place between antibodies and their corresponding antigens. Other binding pairs that may be of interest can include but not be limited to ligand/ receptor, hormone/hormone receptor, carbohydrate/lectin and enzyme/substrate. Assays may be carried out where one component is fixed to a solid support and a corresponding partner is in solution. By binding to the component fixed to the support, the partner now becomes attached to the support as well. A well-known example of this method is the microarray assays where labeled analytes become bound to discrete sites on the microarray. Homogeneous probe dependent assays are also well known in the art and may take advantage of the present invention. Examples of such methods are energy transfer between adjacent probes (U.S. Patent No. 4,868,103), the Taqman exonuclease assay (U.S. Patent No. 5,538,848 and U.S. Patent No. 5,210,015), Molecular Beacons (U.S. Patent No. 5,1 18,801 and U.S. Patent No. 5,925,517) and various real time assays (U.S. Patent Application Serial No. 10/096,076), all of which are incorporated by reference.

Antibodies labeled with dyes of the present invention may be used in various formats. For example, an antibody with one of the dyes of the present invention may be used in an immunofluorescent plate assay or in situ analysis of the cellular location and quantity of various antigenic targets. Antibodies labeled with dyes may also be used free in solution in cell counting or cell sorting methods that use a flow cytometer or for in-vitro and tn-vivo imaging of animal models. The presence or absence of a signal may then be used to indicate the presence or absence of the target itself. An example of this is a test where it is sufficient to know whether a particular pathogen is present in a clinical specimen. On the other hand, quantitative assays may also be carried out where it is not so much the intention of evaluating if a target is present but rather the particular amount of target that is present. An example of this is the previously cited microarray assay where the particular rise or fall in the amount of particular mRNA , species may be of interest.

In another embodiment of the present invention, dyes that have been disclosed above as well as dyes described previous literature may be attached to a carrier with a more general affinity. Dyes may be attached to intercalators that in themselves do not provide signal generation but by virtue of their binding may bring a dye in proximity to a nucleic acid. A further example is attachment of dyes to SDS molecules thereby allowing dyes to be brought into proximity to proteins. Thus this embodiment describes the adaptation of a dye or dyes that lack affinity to a general class of molecules may be adapted by linking them to non-dye molecules or macromolecules that can convey such properties.

Various applications may enjoy the benefits of binding the dyes of the present invention to appropriate targets. As described above, staining of macromolecules in a gel is a methodology that has a long history of use. More recent applications that also may find use are real time detection of amplification (U.S. Patent No. 5,994,056, U.S. Patent No. 6,174,670 and U.S. Patent Application Serial No. 10/096,076, all of which are hereby incorporated by reference), and binding of nucleic acids to microarrays. In situ assays may also find use where the binding of dyes of the present invention is used to identify the location or quantity of appropriate targets.

Selected embodiments of the dyes of this invention include but are not limited to following: 

NIRD-101-NHS Ester:

NIRD- 102-NHS Ester:

NIRD-105-NHS Ester:

NIRD-1O6-NHS Ester:

NIRD-107-NHS Ester:

NIRD-109-NHS Ester:

NIRD-110-NHS Ester:

NIRD-120-NHS Ester:

NIRD-130-NHS Ester:

NIRD-140-NHS Ester:

NIRD-150-NHS Ester:

NIRD- 160-NHS Ester:

NIRD-170-N HS Ester:

NIRD- 180-NHS Ester:

NIRD-190-NHS Ester:

NIRD-200-NHS Ester:

NIRD-210-NHS Ester:

NIRD-220-NHS Ester:

NIRD-230-NHS Ester:

NIRD-240-NHS Ester:

NIRD-250-NHS Ester:

NIRD-260-NHS Ester:

NIRD-280-NHS Ester:

NIRD-290-NHS Ester:

NIRD-300-NHS Ester:

NIRD-310-NHS Ester:

NIRD-320-isothiocyanate:

NIRD-330-isothiocyanate:

NIRD-340-NHS Ester:

The examples which follow are set forth to illustrate various aspects of the present invention but are not intended in any way to limit its scope as more particularly set forth and defined in the claims that follow thereafter. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

Synthesis of NIRD- 101 a) Preparation of 2,3,3-trimethyl-3//-indole-5-sulfonyl chloride (Compound 1 )

Chlorosulfonic acid (37 ml, 314.0 mmol) was cooled in an ice bath and 2,3,3-trimethylindolenine (10.0 g, 62.8 mmol) was carefully added drop wise over a period of 15 minutes. The combined mixture was continued to stir in the ice bath until all fumes subsided and then heated at -1 10 ⁰C for 18 hours. The mixture after cooling was added very slowly to ca. 400 ml ice/water mix. The brownish turbid aqueous solution was then extracted with chloroform (400 ml). The organic layer was washed with water (2x, 500 ml), washed with brine (1 x, 500 ml), dried and evaporated to yield 6.77 g of Compound 1 as a brownish/orange solid, R, = 0.35 (30% ethyl acetate in hexane). The structure of this compound is given below:

b) Preparation of Λ/-(2-(dimethylamino)ethyl)-2,3,3-trimethyl-3#-indole-5- sulfonamide (Compound 2)

To a solution of Compound 1 (7.79 g, 30.23 mmol) in chloroform (200 mL), a mixture of triethyl amine (8.4 mL, 60.46 mmol) and /V,/V-dimethylethylene diamine (5 mL, 45.34 mmol) in 20 mL THF was added drop wise. The combined mixture was stirred at room temperature for 1 hour during which time TLC showed complete disappearance of Compound 1. The reaction mixture was transferred to a 500 mL separatory funnel and it was washed with water (2x, 200 mL), brine (1 x, 200 mL), dried (sodium sulfate) and evaporated to provide compound 2 as reddish brown sticky product {8.35 g, 89% yield). It was used further in the synthesis without any purification. The structure of this compound is given below:

c) Preparation of 1 ,2,3,3-tetramethyl-5-(N-(2-(trimethyammonium) ethyl)sulfamoyl)-3/V-indolium ditosylate (Compound 3)

To a solution of compound 2 (8.35 g, 26.9 mmol) in acetonitrile (50 mL), methyl p-toluene sulfonate (16.3 mL, 107.9 mmol) was added. The combined mixture was refluxed for 22 hours and after cooling was added to 350 mL ethyl acetate. A sticky pink-colored solid was obtained. Solvents were decanted and more ethyl acetate was added to the sticky residue and it was vigorously stirred. This process was repeated until a free-flowing solid was obtained. The solid was then collected by centrifugation, washed with ethyl acetate and dried under vacuum to yield 20.31 g of Compound 3, whose structure is given below:

d) Preparation of 1 -( -carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (Compound 4)

A mixture of potassium salt of indoleninium sulfonate (8.0 g, 28.8 mmol) and 6-bromohexanoic acid (7.32 g, 37.5 mmol, ALDRICH) were heated in a pressure tube at 120-125 ⁰C for 16 hours. The resulting mass was dissolved in boiling DMF (20 mL) and this solution was drop wise added to 200 mL ethyl acetate. The pink solid that precipitated was collected by centrifugation, washed with ethyl acetate and dried under vacuum to yield 21 .6 g (89%) of Compound 4 with the structure:

e) Preparation of NIRD-101 -Cl

A mixture of Compound 3 (0.7 g, 1 .03 mmol) and Λ/-[(3-(Anilinomethylene)- 2-chloro-1 -cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.41 g, 1.13 mmol, ALDRICH) in acetic acid (5 ml) and acetic anhydride (5 mL) was heated and refluxed. The reaction progress was monitored by increase in absorbance at 650 nm (using Nanodrop machine, 3 μL reaction mixture diluted to 5 ml_ ethanol). The reaction mixture (hot) was then added to a mixture of ethyl ether (20 ml) and ethyl acetate (20 mL) in 2-50 mL Falcon tubes. The precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (2x, 30 mL) and dried under Argon. To this solid, a solution of 1 -( -carboxypentynyl)-2,3,3- trimethylindoleninium-5-sulfonate (0.73 g, 1 .55 mmol) in acetic anhydride (6 mL) and pyridine (6 mL) was added. The combined mixture was stirred in the dark at room temperature for 20 hours. Reaction mixture was then added to 10-fold ethyl acetate and precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (4 x 40 mL) and dried under vacuum to provide NIRD-101 -CI (1 .38 g). Abs (max, in PBS) = 783 nm; Em = 802 nm. This dye was used further in the synthesis without any purification. The structure of NIRD-101 -CI is given below:

f) Preparation of NIRD-101 -COOH

To a solution of NIRD-101 -CI (0.6 g, 0.72 mmol) in DMF (3 mL), a solution of sodium phenoxide (0.1 9 g, 1 .1 mmol, ALDRICH) in DMF (3.5 mL) was added. The combined mixture was stirred at room temperature for 3 hours and then slowly added to vigorously stirred ethyl acetate (2x, 40 mL). Precipitated brown solid was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to provide NIRD-101 -COOH (0.54 g). Abs (max, in methanol) = 780 nm. The structure of NIRD-101 -COOH is given below:

Example 2

Synthesis of MRD-105 a) Preparation of 3-(5-carboxypentyl)-1 ,1 ,2-trimethyl-1 AΛbenzo[e]indolium bromide (Compound 5)

To a solution of 2,3,3-trimethylbenzo[e]indolenine (5.0 g, 23.9 mmol, ALFA AESAR) in acetonitrile (50 mL), 6-bromohexanoic acid (14.0 g, 71 .8 mmol, ALDRICH) was added. The combined mixture was refluxed for 18 hours, cooled and slowly added to well-stirred ethyl acetate (400 mL). Precipitated solid (light blue-gray) was collected by centrifugation, washed with ethyl acetate and dried under vacuum to yield 5.3 g (55%) of Compound 5 with the structure:

b) Preparation of NIRD-105-Cl

A mixture of Compound 3 (0.7 g, 1.03 mmol) and Λ/-[(3-(Anilinomethylene)- 2-chloro-1 -cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.41 g, 1 .13 mmol, ALDRICH) in acetic acid (5 ml) and acetic anhydride (5 mL) was heated and refluxed for 2 hours. The reaction mixture (hot) was then added to a mixture of ethyl ether (20 ml) and ethyl acetate (20 mL) in 2-50 mL Falcon tubes. The precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (2x, 30 mL) and dried under Argon. To this solid, a solution of Compound 5 (0.63 g, 1 .55 mmol) in acetic anhydride (6 mL) and pyridine (6 mL) was added. The combined mixture was stirred in the dark at room temperature for 20 hours. Reaction mixture was then added to ethyl acetate (150 mL) and precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (4 x 40 mL) and dried under vacuum to provide NIRD-105-Cl (0.92 g); Abs (max, in PBS) = 782 nm. This dye was used further in the synthesis without any purification. The structure of NIRD-105-Cl is given below:

c) Preparation of NIRD-105-COOH

To a solution of NIRD-105-Cl (0.31 g, 0.25 mmol) in DMF (2.5 mL), a solution of benzenethiol, sodium salt (0.07 g, 0.5 mmol, ALDRICH) in DMF (1 .5 mL) was added. The combined mixture was stirred at room temperature for 1 hour and then slowly added to well-stirred ethyl acetate (40 ml_). Precipitated dark solid was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to provide crude NIRD-105-COOH (0.25 g). This dye was purified on Biotage (Silica column, 25 + M) using an isocratic system of methanol:water:acetic acid = 85:10:5. Appropriate fractions were combined, evaporated to dryness, and then dissolved in ~5 mL methanol. It was then drop wise added to 40 mL ethyl acetate and precipitated dark colored dye was collected by centrifugation, washed with ethyl acetate and dried to yield 48 mg of NIRD- 105-COOH; Abs (max, in methanol) = 825 nm. The structure of NIRD-105-COOH is given below:

Example 3

Synthesis of NIRD -106 a) Preparation of Compound 6

Chlorosulfonic acid (1 5.9 ml, 239.0 mmol) was cooled in an ice bath and 2,3,3-trimethylbenzo[e]indolenine (10.0 g, 47.8 mmol) was carefully added drop wise over a period of 15 minutes. The combined mixture was continued to stir in the ice bath until all fumes subsided and then heated at ~1 10 ⁰C for 19 hours. The mixture after cooling was added very slowly to ca. 300 ml ice/water mix. The aqueous solution was then extracted with chloroform (400 ml). The organic layer was washed with water (2x, 500 ml), washed with brine {1 x, 500 ml), dried and evaporated to yield 6.48 g of Compound 6 as a brownish/green solid, R_f = 0.1 1 (30% ethyl acetate in hexane). The structure of this compound is given below:

b) Preparation of Compound 7

To a solution of Compound 6 (3.0 g, 10.0 mmol) in chloroform (30 ml_), a mixture of triethyl amine (2.8 ml_, 20.0 mmol) and Λ/,/V-dimethylethylene- diamine (1.65 ml_, 1 5.0 mmol) in 3 mL THF was added drop wise. The combined mixture was stirred at room temperature for 18 hours during which time TLC showed complete disappearance of Compound 6. The reaction mixture was transferred to a 500 mL separatory funnel and it was washed with water (2x, 200 mL), brine (1 x, 200 mL), dried (sodium sulfate) and evaporated to provide Compound 7 as orange sticky product (2.49 g, 69% yield). It was used further in the synthesis without any purification. The structure of this compound is given below:

c) Preparation of Compound 8

To a solution of Compound 7 (2.49 g, 6.92 mmol) in acetonitrile {30 mL), methyl p-toluene sulfonate (5.2 mL, 34.6 mmol) was added. The combined mixture was refluxed for 22 hours and after cooling was added to 350 mL ethyl acetate. Precipitated solid (dark grey) was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to yield 4.75 g (94%) of Compound 8, whose structure is given below:

d) Preparation of NI RD- 106-Cl

This procedure was carried out as described previously in step (b) of Example 2, using Compound 8 (0.73 g, 1 .0 mmol), Λ/-[(3-(Anilinomethylene)-2-chloro-1 - cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.4 g, 1.1 mmol, ALDRICH) acetic acid (4 ml), acetic anhydride (10 mL), Compound 5 (0.6 g, 1 .5 mmol from step (a) of Example 2) and pyridine (6 mL). The dye was obtained as a black solid (0.25 g), whose structure is given below:

Example 4

Synthesis of NIRD- 109

Preparation of NIRD-109-C00H

To a solution of NIRD-101 -CI (0.1 g, 0.12 mmol) in DMF (0.5 mL), a solution of MM/V'-trimethylethylenediamine (0.03 g, 0.3 mmol, ALDRICH) in DMF (3.5 mL) was added. The combined mixture was stirred at room temperature for 4 hours and then slowly added to vigorously stirred ethyl acetate (2x, 40 mL). Precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to provide NIRD-109-COOH (84 mg). Abs (max, in PBS) = 680 nm. The structure of NIRD-109-COOH is given below:

Example 5

Synthesis of NIRD-120 a) Preparation of Λ/,Λ/,2,3,3-pentamethyl-3#-indole-5-sulfonamide (Compound 9)

To a warm solution of Compound 1 (6.77 g, 26.26 mmol) in chloroform (100 mL), a mixture of triethyl amine (5 mL, 34.14 mmol) and THF solution of N,N- dimethylamine (17 mL of 2M solution) was added. The combined mixture was stirred at room temperature for 30 minutes during which time TLC showed complete disappearance of Compound 1. The reaction mixture was washed with water (2x, 150 mL), brine (1 x, 150 mL), dried (sodium sulfate) and evaporated to provide a reddish brown viscous liquid. This residue was dissolved in ~5 mL ethyl acetate (with slight heating) and added slowly to 40 mL hexane (in 2-Falcon tubes). A sticky brownish/orange solid separated which was vigorously vortexed until a free floating solid was obtained. Solid was then collected by centrifugation, washed with hexane (2x, 40 mL) and dried to provide Compound 9 (5.44 g, yield = 78%), R, = 0.14 (50% ethyl acetate in hexane). The structure of this compound is given below:

b) Preparation of δ-fN.N-dimethylsulfamoyD-i ^^^-tetramethyl-SH-indolium tosylate (Compound 10)

To a solution of Compound 9 (12 g, 26.5 mmol) in acetonitrile (80 mL), methyl p-toluene sulfonate (6.0 mL, 39.8 mmol) was added. The combined mixture was refluxed for 22 hours and after cooling was added to 800 mL ethyl acetate. A sticky pink-colored solid was obtained. Solvents were decanted and more ethyl acetate was added to the sticky residue and it was vigorously stirred. This process was repeated until a free-flowing solid was obtained. The solid was then collected by centrifugation, washed with ethyl acetate and dried under vacuum to yield 18 g of Compound 10, whose structure is given below:

c) Preparation of NIRD-120-Cl

A mixture of Compound 10 (1 .0 g, 2.2 mmol) and Λ/-[(3-(Anilinomethylene)- 2-chloro-1 -cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.87 g, 2.42 mmol, ALDRICH) in acetic acid (5 ml) and acetic anhydride (5 mL) was heated and refluxed. The reaction progress was monitored by increase in absorbance at 650 nm (using Nanodrop machine, 3 μL reaction mixture diluted to 5 mL ethanol). The reaction mixture (hot) was then added to a mixture of ethyl ether (20 mL) and ethyl acetate (20 mL) in 2-50 mL Falcon tubes. The precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (2X, 30 mL) and dried under Argon. To this solid, a suspension of 1 -{ -carboxypentynyl)-2,3,3- trimethylindoleninium-5-sulfonate (1 .6 g, 3.3 mmol) in acetic anhydride (12 mL) and pyridine (12 mL) was added. The combined mixture was stirred in the dark at room temperature for 20 hours. Reaction mixture was then added to 10-fold ethyl acetate and precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to provide NIRD-120-CI (1 .99 g). Abs (max, in methanol) = 784 nm. This dye was used further in the synthesis without any purification. The structure of NIRD-120-CI is given below:

d) Preparation of NIRD-120-COOH

To a solution of NIRD-120-CI (0.5 g, 0.65 mmol) in DMF (3.5 mL), a solution of benzenethiol, sodium salt (0.17 g, 1 .3 mmol, ALDRICH) in DMF (1 .5 mL) was added. The combined mixture was stirred at room temperature for 3 hours and then slowly added to vigorously stirred ethyl acetate (40 mL). Precipitated dark brown solid was collected by centrifugation, washed with ethyl acetate (4x, 40 mL) and dried under vacuum to provide crude NIRD-120-COOH (0.46 g). This dye was purified on Biotage (Silica column, 25 + M) using a 2-step gradient; (a) methanol (0 to 30%) in chloroform and (b) methanol (0 to 30%) in acetonitrile. For 2^nd gradient, the column was first washed with acetonitrile and then eluted with methanol gradient. Appropriate fractions were combined, evaporated to dryness, and then dissolved in ~5 ml_ DMF. It was then drop wise added to 40 mL ethyl acetate and precipitated dark green dye was collected by centrifugation, washed with ethyl acetate and dried to yield 57 mg of NIRD-120-COOH. Abs (max, in methanol) = 800 nm, Em (max, in methanol) = 819 nm. The structure of NIRD- 120-COOH is given below:

e) Preparation of NIRD- 120-NHS ester

To a solution of NIRD-1 20-COOH (55 mg, 65.2 //mol) in 1 mL DMF, 2- succinimido-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (22 mg, 71 .6 //mol) and /V,Λ/-diisopropyl ethylamine (24 μl, 136.9 //mol) were added. Combined mixture was stirred at room temperature for 30 mins and then added drop wise to 15 mL ethyl acetate. Precipitated dark green dye was collected by centrifugation, washed with ethyl acetate (3x, 30 mL) and dried to provide 54 mg of NIRD-120-NHS ester. Abs (max, in methanol) = 800 nm, Em (max, in methanol) = 819 nm. The structure of this compound is given below:

Example 6

Synthesis of NIRD- 130 a) Preparation of 4-(5-(Λ/,Λ/-dimethylsulfamoyl)-2,3,3-trimethyl-3/-/-indolium-1 - yl)butane-1 -sulfonate (Compound 1 1 )

A mixture of Compound 9 (5.0 g, 18.7 mmol) from step (a) of Example 3 and 1 -4-butane sultone (5.1 g, 37.5 mmol) was heated in a pressure tube at 160 ⁰C for 18 hours. The mixture was allowed to cool, the resulting mass was dissolved in 10 ml_ of hot DMF and then added to 150 mL of vigorously stirred ethyl acetate. A sticky solid that precipitated was triturated with ethyl acetate until a free floating dark pink solid separated. The solid was collected by centrifugation, further washed with ethyl acetate and dried under vacuum to yield 6.5 g (86%) of Compound 1 1 with the structure:

b) Preparation of NIRD-130-Cl

A mixture of Compound 1 1 (0.5 g, 1 .24 mmol) and ΛM(3-(Anilinomethylene)- 2-chloro-1 -cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.51 g, 1.43 mmol, ALDRICH) in acetic acid (2.5 mL) and acetic anhydride (2.5 mL) was heated and refluxed. The reaction progress was monitored by increase in absorbance at 658 nm (using Nanodrop machine, 3 μL reaction mixture diluted to 5 mL ethanol). The reaction mixture (hot) was then added to ethyl acetate (40 mL) and precipitated dark colored solid was collected by centrifugation. It was then washed with ethyl acetate (3x, 30 mL) and dried under Argon. To this solid, a suspension of 1 -( -carboxypentynyl)-2,3,3-trimethylindoleninium-5-sulfonate (0.7 g, 1 .5 mmol) in acetic anhydride (6 mL) and pyridine (6 mL) was added. The combined mixture was stirred in the dark at room temperature for 23 hours. Reaction mixture was then added to 10-fold ethyl acetate and precipitated dark colored solid was collected by centrifugation, washed with ethyl acetate (4 x 40 mL) and dried under vacuum to provide NIRD-130-Cl (1 .13 g); Abs (max, in methanol) = 783 nm. This dye was used further in the synthesis without any purification. The structure of NIRD-130-CI is given below:

c) Preparation of NIRD-130-COOH

This procedure was carried out as described previously in step (d) of Example 3, using NIRD-130-CI (1 .05 g, 1 .18 mmol), benzenethiol, sodium salt (0.35 g, 2.36 mmol, ALDRICH) and DMF (10 mL). The Dye was obtained as a brown solid (1 .0 g). This crude dye (ca. 215 mg) was purified on Biotage (Silica column, 25 + M) using a gradient of methanol (0 to 50%) in acetonitrile. Appropriate fractions were combined, evaporated to dryness, and then dissolved in -5 mL DMF. It was then drop wise added to 40 mL ethyl acetate and precipitated dark colored dye was collected by centrifugation, washed with ethyl acetate and dried to yield 43 mg of NIRD-130-COOH. Abs (max, in methanol) = 804 nm, Em (max, in methanol) = 825 nm. The structure of NIRD-130-COOH is given below:

d) Preparation of NIRD-130-NHS ester

To a solution of NIRD-1 30-COOH (43 mg, 44.5 μmol) in 1 mL DMF, 2- succinimido-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (1 5 mg, 49 //mol) and Λ/,Λ/-diisopropyl ethylamine (16 μl, 93.5 //mol) were added. Combined mixture was stirred at room temperature for 1 hour and then added drop wise to 10 mL ethyl acetate. Precipitated dark green dye was collected by centrifugation, washed with ethyl acetate (3 X 30 mL) and dried to provide 35 mg of NIRD-130-NHS ester. Abs (max, in methanol) = 804 nm, Em (max, in methanol) = 825 nm. The structure of this compound is given below:

Example 7

Synthesis of NIRD- 140 a) Preparation of NIRD- 140-Cl

A solution of Compound 10 (0.2 g, 0.44 mmol) and Λ/-[(3-

(Anilinomethylene)-2-chloro-1 -cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.09 g, 0.22 mmol, ALDRICH) in a mixture of n-butanol (3.5 mL) and toluene (1 .5 mL) was heated to reflux for 4 hours. Upon cooling the reaction mixture was added to vigorously stirred diethyl ether (40 mL) and precipitated dye was collected by centrifugation, washed with ether (3 x 35 mL) and dried under vacuum to provide NIRD-140-CI (0.5 g); Abs (max, in methanol) = 775 nm; Em (max, in methanol) = 799 nm. The structure of this dye is given below:

b) Preparation of NIRD-140-COOH

To a solution of NIRD-140-Cl (0.35 g, 0.4 mmol) in DMF (3.5 mL), a solution of 4-mercaptobenzoic acid (0.12 g, 0.8 mmol, ALDRICH) in DMF (1 .5 mL) was added. The combined mixture was stirred at room temperature for 4 hours and then slowly added to vigorously stirred diethyl ether (40 mL). Precipitated sticky dye was collected by centrifugation, washed with ether (4x, 40 mL) and dried under vacuum to provide crude NIRD-140-COOH (0.28 g). This dye was purified on Biotage (Silica column, 25 + M) using a gradient of methanol (0 to 100% over 16 column volume) in acetonitrile. Appropriate fractions were combined, evaporated to dryness, and then dissolved in -4 mL DMF. It was then drop wise added to 40 mL diethyl ether and precipitated dark green dye was collected by centrifugation, washed with diethyl ether and dried to yield 64 mg of NIRD-140- COOH. Abs (max, in methanol) = 796 nm, Em (max, in methanol) = 812 nm. The structure of NIRD-140-COOH is given below:

c) Preparation of NIRD-140-NHS ester

To a solution of NIRD-1 20-COOH (62 mg, 63 //mol) in 1.5 mL DMF, 2- succinimido-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (21 mg, 69 //mol) and Λ/,/V-diisopropyl ethylamine (23 //L, 130 //mol) were added. Combined mixture was stirred at room temperature for 30 mins and then added drop wise to 15 mL diethyl ether. Precipitated dark green dye was collected by centrifugation, washed with diethyl ether (3x, 15 mL) and dried to provide 59 mg of NIRD-120-NHS ester. Abs (max, in methanol) = 796 nm, Em (max, in methanol) = 812 nm. The structure of this dye is given below:

Example 8

Synthesis of MRD-150

Preparation of NIRD-150-CI

This procedure was carried out as described previously in step (a) of Example 5, using Compound 3 (1 .0 g, 1 .46 mmol), Λ/-[(3-(Anilinomethylene)-2-chloro-1 - cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.26 g, 0.73 mmol, ALDRICH) and a mixture of n-butanol (3.5 ml_) and toluene (1 .5 mL). The crude dye was obtained as a dark colored solid (1 .2 g). The structure of this dye is given below:

Example 9

Synthesis of NlRD-160 a) Preparation of 1 -benzyl-5(Λ/,Λ/-dimethylsulfamoyl)-2,3,3-trimethyl-3//- indolium bromide (Compound 1 2)

To a solution of Compound 9 (2.2 g, 8.3 mmol) in acetonitrile (8 ml_), benzyl bromide (3.0 rτ»L, 25.2 mmol, ALDRICH) was added. The combined mixture was refluxed for 22 hours and after cooling was added to 100 ml_ diethyl ether. A dark purple solid was obtained which was collected by centrifugation, washed with diethyl ether (2x, 40 mL) and dried under vacuum to yield 3.4 g of Compound 12, whose structure is given below:

b) Preparation of NIRD-160-Cl

This procedure was carried out as described previously in step (a) of Example 5, using Compound 12 (0.8 g, 1 .83 mmol), Compound 4 (0.86 g, 1 .83 mmol), N- [(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (0.66 g, 1 .83 mmol, ALDRICH), and a mixture of n-butanol (3.5 mL) and toluene (1 .5 mL). The crude dye was obtained as a dark colored solid (1 .6 g). The structure of this dye is given below:

Example 10 Synthesis of NIRD-170 a) Preparation of NIRD-170-Cl

This procedure was carried out as described previously in step (a) of Example 5, using Compound 12 (0.8 g, 1 .83 mmol), Λ/-[(3-(Anilinomethylene)-2-chloro-1 - cyclohexen-1 -yl)methylene]aniline monohydrochloride (0.33 g, 0.92 mmol, ALDRICH) and a mixture of n-butanol (21 mL) and toluene (9 mL). The crude dye was obtained as a dark colored solid (0.7 g). Abs (max, in methanol) = 804 nm. The structure of this dye is given below:

b) Preparation of NIRD-170-COOH

This procedure was carried out as described previously in step (b) of Example 5, using NIRD-170-Cl (0.7 g, 0.75 mmol), 4-mercaptobenzoic acid (0.23 g, 1 .5 mmol, ALDRICH) and DMF (10 mL). The crude dye was obtained as a dark colored solid (0.7 g) and was purified (ca. 300 mg) on Biotage (Silica column, 25 + M) using a gradient of methanol (0 to 100% over 16 column volume) in acetonitrile. Appropriate fractions were combined, evaporated to dryness, and then dissolved in ~1 mL DMF. It was then drop wise added to 10 mL diethyl ether and precipitated dark green dye was collected by centrifugation, washed with diethyl ether and dried to yield 26 mg of NIRD-170-COOH. Abs (max, in methanol) = 804 nm, Em (max, in methanol) = 817 nm. The structure of NIRD-170-COOH is given below:

c) Preparation of NIRD-170-NHS ester

This procedure was carried out as described previously in step (d) of Example 6, using NIRD-170-COOH (26 mg, 24.8 μmol), 2-succinimido-1 ,1 ,3,3- tetramethyluronium tetrafluoroborate (9.0 mg, 27.3 //mol), Λ/,/V-diisopropyl ethylamine (9 μl, 52.1 //mol) and DMF (0.5 ml_). The dye was obtained as a dark colored solid (25 mg). Abs (max, in methanol) = 804 nm, Em (max, in methanol) = 817 nm. The structure of this dye is given below:

Many obvious variations will be suggested to those of ordinary skill in the art in light of the above detailed descriptions of the present invention. All such obvious variations are fully contemplated and are embraced by the scope and spirit of the present invention as set forth in the claims that now follow.

Claims

WHAT IS CLAIMED IS:

1 . A dye compound having the formula (A)

or (B)

wherein X and Y independently comprise CR₃₀R₃₁, NR₃₀, O, S or Se, wherein R₃₀ and R₃₁ independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated branched or linear, substituted or unsubstituted, or when taken together R₃₀ and R₃₁ comprise a 5 or 6 membered ring; wherein m and n are each independently integers from 0 to 5; wherein A is a linker connecting an aromatic moiety to the methine bridge wherein said linker comprises one or more atoms selected from C, O, S. P, N and combinations thereof, wherein said linker may be substituted or unsubstituted, saturated or unsaturated, branched or linear; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, Ri₅' R₃₀ ^or R₃₁ comprises Q, wherein Q comprises a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (P0₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃"), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Q is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof and wherein when Q is a sulfonamide, Q does not have a terminal reactive group or a linker arm joining the dye to a target molecule; wherein R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂₈ , R₂g, R₃₃, R₃₃' ^₃₄» ^₃₅» ^₃₆' R₃₇ ^{an0< tne} remaining R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, Rn, R^ R₁₃,R₁₄ , Ri₅, R₃₀ ^{and R} ₃i independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an arγl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₂ and R₃, R₃ and R₄, R₄ and R₅, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, R₁ and R₁₆, R₆ and R₂₃, R₃₃ and R₃₄, R₃₄ and R₃₅, R₃₆ and R₃₇ independently comprise a five or six membered ring; wherein Z comprises a carboxyl group (CO₂ ), a carbonate ester (COER₃₃), a sulfonate (SO₃ ), a sulfonate ester (SO₂ER₃₃), a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (P0₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Z is attached directly or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm is saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof; and wherein any of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, Ri₅/ R₃₀ ^or R₃₁ ^maY further comprise a heteroatom containing side chain, wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (-OR₃₈), a thioether linkage (-SR₃₈), or an amine linkage (-NR₃₈R₃₉ or - N⁺R₃₈R_SgFUo)' ^and wherein R₃₈, R₃₉ and R₄₀ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₈ and R₃₉ and R₃₉ and R₄₀ independently comprise a five or six membered ring, and wherein any of R₃₈, R₃₉ Or R₄₀ may further comprise said heteroatom containing side chain;

2. The dye compound of claim 1 , wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇» R₈» R₉' Rio/ Ri₃' Ri4' Ri_S' R₃₀ ^or R₃₁ comprises a reactive group Rx, wherein said reactive group Rx comprises a nucleophilic reactive group, an electrophilic group, terminal alkene, a terminal alkyne, a coordinate group or an alkylating agent.

3. The compound dye of claim 2, wherein said nucleophilic reactive group comprises a thiol, amine or hydroxyl group.

4. The compound dye of claim 2, wherein said electrophilic reactive group comprises an isocyanate, isothiocyanate, monochlorotriazine, dichlorotriazine, 4,6,- dichloro-1 ,3,5-triazines, mono- or di-halogen substituted pyridine, mono- or di- halogen substituted diazine, maleimide, haloacetamide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, imidazole, benzotriazole, hydrazine, azidonitrophenol, azide, 3-(2-pyridyl dithio)- propionamide, glyoxal or aldehyde group.

5. The dye compound of claim 2, having the structure

6. The dye compound of claim 2, having the structure

7. The dye compound of claim 2, having the structure

8. The dye compound of claim 2, having the structure

9. The dye compound of claim 2, having the structure

10. The dye compound of claim 2, having the structure

1 1 . The dye compound of claim 2, having the structure

12. The dye compound of claim 2, having the structure

13. The dye compound of claim 2, having the structure

14. The dye compound of claim 2, having the structure

15. The dye compound of claim 2, having the structure

16. The dye compound of claim 2, having the structure

17. The dye compound of claim 2, having the structure

1 8. The dye compound of claim 2, having the structure

19. The dye compound of claim 2, having the structure

20. The dye compound of claim 2, having the structure

21 . The dye compound of claim 2, having the structure

22. The dye compound of claim 2, having the structure

23. The dye compound of claim 2, having the structure

24. The dye compound of claim 2, having the structure

25. The dye compound of claim 2, having the structure

26. The dye compound of claim 2, having the structure

27. The dye compound of claim 2, having the structure

28. The dye compound of claim 2, having the structure

29. The dye compound of claim 2, having the structure

30. The dye compound of claim 2, having the structure

31 . The dye compound of claim 2, having the structure

32. The dye compound of claim 2, having the structure

33. The dye compound of claim 2, having the structure

34. The dye compound of claim 2, having the structure

35. The dye compound of claim 1 , wherein said dye compound is linked to a target molecule.

36. The dye compound of claim 35, wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, locked nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, Iγmphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenrn, carbohydrate, oligosaccharide, polysaccharide, lipid, liposome, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, siliceous particle, siliceous surface, organic molecule, dye or a derivative thereof.

37. The dye compound of claim 36, wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, dideoxyribonucleoside moieties, dideoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.

38. A method for detecting the presence or quantity of a target comprising the steps of: a) providing i) a sample in which the presence or quantity of a target is unknown or sought to be detected, and ii) a composition comprising a first portion and a second portion wherein said first portion comprises a dye and said second portion comprises a target specific moiety, said dye having the formula:

(A)

or (B)

wherein X and Y independently comprise CR₃₀R₃₁ , NR₃₀, O, S or Se, wherein R₃₀ and R₃₁ independently comprise hydrogen, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated branched or linear, substituted or unsubstituted, or when taken together R₃₀ and R₃₁ comprise a 5 or 6 membered ring; wherein m and n are each independently integers from 0 to 5; wherein A is a linker connecting an aromatic moiety to the methine bridge wherein said linker comprises one or more atoms selected from C, O, S. P, N and combinations thereof, wherein said linker may be substituted or unsubstituted, saturated or unsaturated, branched or linear; wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, Ri₅' R₃₀ ^or R₃₁ comprises Q, wherein Q comprises a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (P0₄ ⁼), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼ ), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Q is attached directly, or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm may be saturated or unsaturated, linear or branched, substituted or unsubstituted and any combinations thereof and wherein when Q is a sulfonamide, Q does not have a terminal reactive group or a linker arm joining the dye to a target molecule; wherein R₁₆, R₁₇, R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂β » ^R ₂₉' ^R ₃₃» R₃₃' ^_3Λ> R₃₅' ^R ₃₆' ^R ₃₇ and the remaining R₂, R₃, R₄, R₅, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, Ri₃'Ri_{4 >} Ri₅' ^R ₃o ^ar|d ^R ₃i independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₂ and R₃, R₃ and R₄, R₄ and R₅, R₇ and R₈, R₈ and R₉, R₉ and R₁₀, R₁ and R₁₆, R₆ and R₂₃, R₃₃ and R₃₄, R₃₄ and R₃₅, R₃₆ and R₃₇ independently comprise a five or six membered ring; wherein Z comprises a carboxyl group (CO₂ ^"), a carbonate ester (COER₃₃), a sulfonate (SO₃ ), a sulfonate ester (SO₂ER₃₃), a sulfoxide (SOR₃₃), a sulfone (SO₂CR₃₃R₃₄R₃₅), a sulfonamide (SO₂NR₃₃R₃₄), a phosphate (P0₄ ⁼ ), a phosphate monoester (PO₃ ER₃₃), a phosphate diester (PO₂ER₃₃ER₃₄), a phosphonate monoester (PO₂ ER₃₃), a phosphonate diester (POER₃₃ER₃₄), a thiophosphate (PSO₃ ⁼ ), a thiophosphate monoester (PSO₂ ER₃₃), a thiophosphate diester (PSOER₃₃ER₃₄), a thiophosphonate (PSO₂ ⁼ ), a thiophosphonate monoester (PSO ER₃₃), a thiophosphonate diester (PSER₃₃ER₃₄), a phosphonamide (PONR₃₃R₃₄NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₆R₃₇), a phosphoramide (PONR₃₃R₃₄NR₃₅NR₃₆R₃₇), its thioanalogue (PSNR₃₃R₃₄NR₃₅NR₃₆R₃₇), a phosphoramidite (PO₂R₃₆NR₃₃R₃₄), or its thioanalogue (POSR₃₆NR₃₃R₃₄), wherein any of E independently comprises O or S; wherein Z is attached directly or indirectly through a linker arm comprising carbon, sulfur, oxygen, nitrogen, and any combinations thereof and wherein said linker arm is saturated or unsaturated, linear or branched, substituted or unsubstituted, or any combinations thereof; and wherein any of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, Ri₅' R₃₀ ^or R₃₁ ^maY further comprise a heteroatom containing side chain, wherein said side chain is joined to the R group by a linkage which comprises an ether linkage (-OR₃₈), a thioether linkage (-SR₃₈), or an amine linkage (-NR₃₈R₃₉ or - N ⁺ R₃₈R₃₉R₄₀), and wherein R₃₈, R₃₉ and R₄₀ independently comprise hydrogen, Z, a halogen, an amino group, an alkyl group wherein said alkyl group is saturated or unsaturated, linear or branched, substituted or unsubstituted, an aryl group wherein said aryl group is substituted or unsubstituted, an alkoxy group wherein said alkoxy group is saturated or unsaturated, branched or linear, substituted or unsubstituted, or when taken together R₃₈ and R₃₉ and R₃₉ and R₄₀ independently comprise a five or six membered ring, and wherein any of R₃₈, R₃₉ or R₄₀ may further comprise said heteroatom containing side chain; and wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ , R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, R₃₀ or R₃₁ is linked to said second portion; b) allowing any targets present in said sample i) to bind with said target specific moiety comprising said composition ii); and c) quantifying the amount of said composition ii) bound to any of said target in the sample, thereby detecting the presence or quantity of said target.

39. The process of claim 38, wherein said target molecule comprises a nucleoside, nucleotide, oligonucleotide, polynucleotide, peptide nucleic acid, locked nucleic acid, protein, peptide, enzyme, antigen, antibody, hormone, hormone receptor, cellular receptor, lymphokine, cytokine, hapten, lectin, avidin, strepavidin, digoxygenin, carbohydrate, oligosaccharide, polysaccharide, lipid, liposome, glycolipid, viral particle, viral component, bacterial cell, bacterial component, eucaryotic cell, eukaryotic cell component, natural drug, synthetic drug, glass particle, glass surface, plastic particle, plastic surface, siliceous particle, siliceous surface, organic molecule, dye or a derivative thereof.

40. The process of claim 39, wherein said nucleoside, nucleotide, oligonucleotide, or polynucleotide comprises one or more ribonucleoside moieties, ribonucleotide moieties, deoxyribonucleoside moieties, deoxyribonucleotide moieties, dideoxyribonucleoside moieties, dideoxyribonucleotide moieties, modified ribonucleosides, modified ribonucleotides, modified deoxyribonucleosides, modified deoxyribonucleotides, ribonucleotide analogues, deoxyribonucleotide analogues or any combination thereof.

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