FIELD OF THE INVENTION

The present invention relates generally to dyes for labelling nucleic acids. More specifically, the present invention relates to intercalating cyanine dyes for the detection and enumeration of nucleic acids.

BACKGROUND OF THE INVENTION

Intercalating dyes which exhibit enhanced fluorescence upon binding to DNA or RNA are a basic tool in molecular and cell biology. In general, intercalating dyes bind noncovalently to DNA through a combination of hydrophobic interactions with the DNA base-pairs and ionic binding to the negatively charged phosphate backbone. The fluorescence of the dye is ideally increased several-fold upon binding to DNA, thereby enabling the detection of small amounts of nucleic acids. Examples of fluorescent πoncovalent DNA binding dyes include ethidium bromide which is commonly used to stain DNA in agarose gels after gel electrophoresis, and propidium iodide and Hoechst 33258 which are used in flow cytometry to determine the

DNA ploidy of cells.

Fluorescent nucleic acid labelling dyes preferably absorb light between about 300 and 900 nm and preferably have a Stokes shift of at least about 10 nm. Dyes that absorb light in the 500 to 900 nm range are preferred because they are spectrally removed from other components that may be present in a biological sample and because they may be used with inexpensive light sources. Fluorescent dyes that have a high extinction coefficient, a high quantum yield, and significantly enhanced fluorescence when bound to a nucleic acid are also preferred. Few new dye chromophores were described until the introduction of

Thiazole Orange as a reticulocyte stain in 1986. Lee, et al., 'Thiazole Orange: A New Dye for Reticulocyte Analysis", Cvtometrv 1986 7, 508-517. Thiazole

Orange is an asymmetric cyanine dye. Although many asymmetric cyanine dyes have been described in the art (e.g., Lincoln, et al., U.S. Patent No.

3,282,932), Thiazole Orange's fluorescence properties when bound to DNA and RNA and its utility for labelling nucleic acids had not been previously identified. Lee, et al., U.S. Patent No. 4,957,870. For example, unlike most asymmetric cyanine dyes, Thiazole Orange exhibits a several thousand-fold enhancement in fluorescence upon binding to DNA.

Since the discovery of Thiazole Orange as a nucleic acid dye, several improvements to Thiazole Orange and its trimethine homologs have been developed to provide dyes with tighter binding to DNA and greater water solubility. Xue, et al. U.S. Patent No. 5,321,130 and Glazer, et al. U.S. Patent

No. 5,312,921. These dyes generally involve a modification to the quinolinium portion of the dye.

A continuing need exists for new and improved dyes for labelling nucleic acids. In particular, a need exists for dyes which exhibit longer wavelengths and significantly enhanced fluorescence when bound to DNA or RNA.

SUMMARY OF THE INVENTION

The present invention relates asymmetric cyanine dyes for non- covalently labelling nucleic acids in which the benzothiazole portion of the dye has been modified to provide improved physical properties to the dye, such as longer wavelengths and improved fluorescence enhancement when bound to

DNA or RNA.

More specifically, the invention relates to cyclized fluorescent cyanine dyes for non-covalently labelling nucleic acids. The cyclized fluorescent cyanine dyes according to the present invention are represented by General Formula I

where: n is 0, 1 or 2; Y may be either S or O;

R, and R ₂ are taken together to form a 5, 6, 7 or 8 membered ring;

R ₃ and R ₄ may each independently be either hydrogen, C, - C ₁₀ alkyl, C, - C ₁₀ alkoxy, or C, - C ₁₀ alkylthio; Rj may be a C, - C _∞ alkyl, preferably substituted with one or more polar substituents which preferably includes one or more positively charged atoms, or a cyclized fluorescent cyanine dye of the

present invention, i.e., where R ₅ is a linker between two cyclized fluorescent cyanine dyes;

R, and R ₇ may each independently be either H or C,., ₀ alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C,^ alkyl or C, - C ₁₀ alkoxy groups;

R ₈ and Rg may each independently be either H or C,., ₀ alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C,^ alkyl or C, - C ₁₀ alkoxy groups; and

R ₁₀ may be either H, C,^ alkyl, C, - C ₁₀ alkoxy or a fused benzene. As used above, alkyl and alkoxy refer to any substituent having a carbon backbone having the specified range of carbon atoms. The carbon backbone may form a straight chain, may be branched or may be cyclic. The alkyl and alkoxy groups may be optionally substituted by a wide variety of substituents including, for example, alcohols, amines, thiols, phosphates, halides, ethers, esters, ketones, aldehydes, carboxylic acids, amides, cydoalkyls, and aromatic rings. The invention also relates to the composition of a cyanine dye according to the present invention non-covalently bound to a nucleic acid sequence, i.e., RNA or DNA, which enables the nucleic acid sequence to be analytically detected.

The invention also relates to a method for detecting nucleic acids in a sample by contacting the nucleic acids with a fluorescent cyanine dye according to the present invention and monitoring the change in fluorescence emission of the dye.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates asymmetric cyanine dyes for non- covalently labelling nucleic acids in which the benzothiazole portion of the dye

has been modified to provide improved physical properties to the dye, such as longer wavelengths and improved fluorescence enhancement when bound to DNA or RNA.

In one embodiment, the present invention relates to cyclized fluorescent cyanine dyes generally represented by General Formula I

where: n is 0, 1 or 2; Y may be either S or O; R, and R ₂ are taken together to form a 5, 6, 7 or 8 membered ring;

R ₃ and R ₄ may each independently be either hydrogen, C, - C ₁₀ alkyl, C, - C ₁₀ alkoxy, or C, - C ₁₀ alkylthio;

Rs may be a C, - _x alkyl, preferably substituted with one or more polar substituents which preferably includes one or more positively charged atoms, or a cyclized fluorescent cyanine dye of the present invention, i.e., where R ₅ is a linker between two cyclized fluorescent cyanine dyes;

R, and R ₇ may each independently be either H or C,. ₁₀ alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6 membered aromatic ring, optionally substituted with C^ alkyl or C, - C ₁₀ alkoxy groups;

R ₈ and R ₉ may each independently be either H or C,. ₁₀ alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 6

membered aromatic ring, optionally substituted with C,., alkyl or C, - C ₁₀ alkoxy groups; and

R ₁₀ may be either H, C _M alkyl, C, - C ₁₀ alkoxy or a fused benzene. As used above, alkyl and alkoxy refer to any substituent having a carbon backbone having the specified range of carbon atoms. The carbon backbone may form a straight chain, may be branched or may be cyclic. The alkyl and alkoxy groups may be optionally substituted by a wide variety of substituents including, for example, alcohols, amines, thiols, phosphates, halides, ethers, esters, ketones, aldehydes, carboxylic adds, amides, cydoalkyls, and aromatic rings.

The cyclized cyanine dyes of the present invention provide the advantage over previous cyanine dyes of having higher absorbance and emission wavelengths. The cyclized cyanine dyes preferably absorb light at a wavelength of at least about 640 nm, more preferably at least about 649 nm and emit fluorescence at a wavelength of at least about 650 nm, more preferably at least about 663 nm. The cyclized cyanine dyes also preferably have a positive Stoke's shift ^{at least about 1 2 πm} - In particular, cyclized cyanine dyes having General Formula I where R and R ₂ are taken together to form a 5, 6, 7 or 8 membered ring have been found to absorb light and fluoresce when bound to a nucleic acid polymer at unexpectedly higher wavelengths than has been previously achieved by cyanine dyes where R, and R ₂ do not form a ring structure.

Fluorescent cyanine dyes having the General Formula I where R and R ₂ are taken together to form a 7 membered ring have also been found to have the greatest Stoke's shift (^Emission'^Abs.)

TABLE 1 : Absorbance and Emission Maxima of Intercalating Dyes in PBS with Excess DNA ( [bp]/[dye] = 100)

COMPOUND & s ^■ ,max _ΠHX E-.

Abs^^ -Absorbance maximum (bounds to DN _A) Em ^S m-Emission maximum (bound to DNA ₎ F.E. -fluorescence enhancement (bounds vs. not _b oun _d to DNA or RNA)

* ^C ompound 5 is taught in U.S. Patent No. 5, ₃ 21,1 ₃₀ to Yue, et al.

Table 1 summarizes the absorbance maximum and fluorescence emission maximum wavelengths (both when bound to DNA) of some exemplary cyclized cyanine dyes of the present invention.

As illustrated in Table 1 , it was found that the addition of a cyclic aliphatic side chain to the basic cyanine dye structure, i.e., formation of a 5-8 membered ring by combining R, and R ₂ , was found to increase the absorbance and fluorescence emission wavelengths of the corresponding acyclic cyanine dye by about 12 nm. For example, as shown with regard to dyes 2 and 5, dye

2 has an Abs _m πiα _o λ _v at 654 nm as compared to 641 nm and an Ems _m ma _n x„ at 667 nm as compared to 655 nm. In addition, dye 4 is the longest wavelength trimethine intercalating dye yet reported.

With regard to n, n may equal 1. Accordingly, the present invention includes cydized cyanine dyes having the General Formula II (i.e. where n = 1 )

where Y, R _{1 t} R ₂ , R ₃ , R _4l R ₅ , Re, R ₇ , R _β , R _g and R ₁₀ are as specified above.

Y may be either S or O. and is most preferably S.

R ₃ and R ₄ may each independently be either hydrogen, C, - C ₁₀ alkyl, C, - C ₁₀ alkoxy, or C, - C ₁₀ alkylthio, and are preferably H. R ₅ may be a C, - C _∞ alkyl. Since DNA and RNA to which the cyclized cyanine dyes bind contain negatively charged phosphate backbones, it is preferred that R ₅ be substituted with one or more polar substituents. It is most preferred that R ₅ include one or more positively charged atoms in the polar

substituenL U.S. Patent No. 5,321,130 to Yue, et al. teaches unsymmetrical cyanine dyes having an aminoalkyi chain containing a backbone of 3-42 carbons and 1-5 positively charged nitrogen atoms. The cationic tail described in U.S. Patent No. 5,321,130 exemplifies one of the positively charged R- _* substituents that may be used in combination with the cydic cyanine dyes of the present invention and is incorporated herein by reference, in addition to the positively charged Re substituents described in U.S. Patent No. 5,321,130, R ₁₂ is also intended to indude aminoalkyi chains induding a positively charged cydic aminoalkyi group having 1-5 positively charged nitrogen atoms. Alternatively, R-j may form part of a linker between two cydized fluorescent cyanine dyes as illustrated by General Formula IV

O

According to this embodiment, Y. R,, Rj, R R ₄ , Re, R ₇ , Re, R* and R ₁₀ are as specified above. It should be noted that the two linked cyanine dyes may be the same or different cyanine dyes. In general, it is preferred that the linked cyanine dyes be the same since different dyes will have different spectral properties.

Re and R ₇ may each independently be either H, C,. ₁₀ alkyl, or are taken together to form a 5 or 6 membered ring, most preferably a 5 or 6 membered aromatic ring, optionally substituted with C _M alkyl or C, - C ₁₀ alkoxy groups. R, and R, may each independently be either H, C,.,-, alkyl, or are taken together to form a 5 or 6 membered ring, most preferably a 5 or 6 membered aromatic ring, optionally substituted with C _1<β alkyl or C, - C ₁₀ alkoxy groups. in general, it is prefe ed either R, and R _τ or R _β and R, are taken together to form a 5 or 6 membered aromatic ring, optionally substituted with

C,^ alkyl or C, - C ₁₀ alkoxy groups. The R, and R ₇ or R ₈ and R ₉ groups that do not form the aromatic ring are preferably H.

R ₁₀ may be either H, C^ alkyl, C, - C ₁₀ alkoxy or a fused benzene.

In a particularly preferred embodiment, the cyclized cyanine dye has the General Formula V where the ring formed by R, and R ₂ includes a positively charged substituent R ₂₇ As discussed herein, inclusion of a positively charged substituent, such as R ₂₇ , to a substituent on the positively charged nitrogen on the benzothiazole ring improves the net fluorescence enhancement of the dye with DNA.

R ₂₇ is a positively charged alkyl substituent which may be attached to any atom used to form the 5, 6, 7 or 8 membered ring. R ₂₇ is more preferably a positively charged aminoalkyi substituent. For example, R ₁₂ can be an aminoalkyi chain containing a backbone of 3-42 carbons and 1-5 positively charged nitrogen atoms as described in U.S. Patent No. 5,321,130 to Yue, et al. which is incorporated herein by reference. In addition to the positively charged substituents described in U.S. Patent No. 5,321,130, R ₁₂ is also intended to include aminoalkyi chains including a positively charged cyclic aminoalkyi group having 1-5 positively charged nitrogen atoms.

In a preferred embodiment, R^ has the general formula -R ₂₈ N(R ₂₉ R ₃₀ R ₃₁ ) where R ₂₈ is a C,^ alkyl and R , R _∞ , and R ₃₁ are each independently a C _1-10 alkyl.

Table 2 provides examples of some of the preferred cyclized cyanine dyes. It should be understood, however, that the dyes listed in Table 2 are intended only to exemplify the cyclized cyanine dyes of the present invention and are not intended to be limiting.

TABLE 2

TABLE 2 (cont.)

12/1 -

TABLE 2 (cont.)

- 12/2 -

TABLE 2 (cont.)

-13

TABLE 2 (cont.)

13/1 -

TABLE 2 (cont.)

- 13/2

The present invention also relates to fluorescent cyanine dyes having a positively charged substituent attached to the positively charged nitrogen on the benzothiazole portion of the cyanine dye. These fluorescent cyanine dyes are represented by General Formula VI

where n is 0, 1 or 2; Y may be either S or O;

R ₁₂ is a positively charged alkyl substituent, more preferably a positively charged aminoalkyi substituent;

R ₁₃ , R ₁₄ and R ₁₅ may each independently be either hydrogen, C, - C, ₀ alkyl, C, - C ₁₀ alkoxy, or C, - C ₁₀ alkylthio; R ₁₂ and R ₁₃ may optionally be taken together to form a 5,

6, 7 or 8 membered ring;

R _1β may be a C ₁ - C _∞ alkyl, preferably substituted with one or more polar substituents which preferably includes one or more positively charged atoms, or a cyclized fluorescent cyanine dye of the present invention, i.e., where R _1β is a linker between two cyclized fluorescent cyanine dyes;

R ₁₇ and R _1β may each independently be either H or C,. ₁₀ alkyl, or may be taken together to form a 5 or 6 membered ring, most

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preferably a 5 or 6 membered aromatic ring, optionally substituted with C^ alkyl or C, - C ₁₀ alkoxy groups;

R ₁₉ and R _w may each independently be either H or C,.,,, alkyl, or may be taken together to form a 5 or 6 membered ring, most preferably a 5 or 6 membered aromatic ring, optionally substituted with C ₁₄ alkyl or C, - C ₁₀ alkoxy groups; and

R ₂₁ may be either H, C _M alkyl, C, - C ₁₀ alkoxy or a fused benzene. As used above, alkyl and alkoxy refer to any substituent having a carbon backbone having the specified range of carbon atoms. The carbon backbone may form a straight chain, may be branched or may be cyclic. The alkyl and alkoxy groups may be optionally substituted by a wide variety of substituents including, for example, alcohols, amines, thiols, phosphates, halides, ethers, esters, ketones, aldehydes, carboxylic acids, amides, cydoalkyls, and aromatic rings.

With regard to n, it is noted that n may equal 1. Accordingly, an embodiment of the present invention includes cyanine dyes having the General Formula VII (i.e. where n = 1)

where Y, R ₁₂ , R, ₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R _M and R ₂₁ are as specified above.

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With regard to dyes having General Formula VI or VII, Y may be either S or O and is most preferably S.

R ₁₂ can be an aminoalkyi chain containing a backbone of 3-42 carbons and 1-5 positively charged nitrogen atoms as described in U.S. Patent No. 5,321 ,130 to Yue, et al. which is incorporated herein by reference. In addition to the positively charged substituents described in U.S. Patent No. 5,321,130, R ₁₂ is also intended to include aminoalkyi chains including a positively charged cyclic aminoalkyi group having 1-5 positively charged nitrogen atoms.

In a preferred embodiment, R ₁₂ has the general formula ^N^ _M R- _JO R^) where R _2β is a C^ alkyl and R _w , R _x , and R ₃₁ are each independently a C,. ₁₀ alkyl.

In an alternate preferred embodiment, R ₁₂ and R ₁₃ are taken together to form a 5, 6, 7 or 8 membered ring where the ring includes a positively charged alkyl substituent, more preferably an aminoalkyi chain containing a backbone of 3-42 carbons and 1-5 positively charged nitrogen atoms as described in

U.S. Patent No. 5,321 ,130 to Yue, et al. Dyes of this embodiment may be generally represented by General Formula VIM

where R ₁₂ and R ₁₃ represents the atoms necessary to form a 5, 6, 7 or 8 membered ring and R ₂₇ is a positively charged substituent, as specified above with regard to R ₁₂ , which may be attached to any atom used to form the 5, 6, 7 or 8 membered ring as represented by R ₁₂ and R ₁₃ . In this regard, these dyes are equivalent to the dyes described above having the General Formula V.

16

R, ₄ and R ₁₅ may each independently be either hydrogen, C,-C ₁₀ alkyl, 0,-0, ₀ alkylthio, and are preferably H.

R, ₆ may be a C,-C _3o alkyl. Since DNA and RNA to which the cyclized cyanine dyes bind contain negatively charged phosphate backbones, it is preferred that R ₁₆ be substituted with one or more polar substituents. It is most preferred that R, ₆ include one or more positively charged atoms in the polar substituent, such as is specified with regard to R, ₂ above. The cyanine dyes according to General Formula

VI, i.e., dyes where a positively charged substituent is positioned off the nitrogen of the benzothiazole portion of the dye, provide the advantage over previous cyanine dyes of exhibiting a significantly larger net fluorescence enhancement with DNA than cyanine dyes where a positively charged substituent is positioned at R, ₆ alone.

The use of intercalating dyes for staining cell nuclei requires that the dye itself be membrane-permeable or that a membrane permeabilizing step be incorporated into the sample preparation. Methods for enabling charged molecules and very large molecules into cells include the use of chemicals, such as digitonin, freeze-thaw cell lysis steps, or the use of non-ionic detergents such as TRITON X-100. For speed and simplicity, it is preferred to add approximately 9mM TRITON X-100.

The presence of a detergent solution (TRITON X- 100) causes significant fluorescence enhancement of the dyes as compared to in PBS buffer. An increase in detergent-enhanced fluorescence CFJHΠ _O H /? _}>& ) has the effect of decreasing the net DNA enhanced fluorescence over detergent-enhanced background fluorescence (F _DNA /F _TRrroN ) . The detergent-enhanced fluorescence is believed to increase with increasing hydrophobicity.

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N

* Compounds 5 and 7 are taught in U.S. Patent No. 5,321,130 to Yue, et al.

^# Compound 6 is taught in U.S. Patent No.4,957,870 to Lee, et al.

Fluorescence enhancement of the dyes upon binding to an excess of DNA was found to be fairly constant regardless of how the quinolinium ring side chain was modified (R _1β ). Advantageously, however, it was found that indusion of a positively charged substituent off the positively charged nitrogen of the benzothiazole portion of the dye (General Formula VI) causes the dye to exhibit a significantly larger net DNA-enhancement than the positioning of a positively charged substituent at R _1β alone. As a result, smaller concentrations of nudeic adds can be detected using cyanine dyes having General Formula VI.

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For example, Table 3 compares the fluorescence ratios of dyes in a saline buffer, a detergent (TRITON X-100) and in a DNA solution. Dye solutions (1.0 μM) were prepared in phosphate buffered saline (PBS), in PBS with TRITON X-100 (9mM), and in PBS with double-stranded DNA (100μM). Table 3 shows the effect of various side chains on the fluorescence background in TRITON X-100 (9mM). As illustrated in Table 3, the net DNA enhanced fluorescence over detergent-enhanced background fluorescence (F _ONA /F _TTOTON ) was found to be a factor of 5 greater In dye 8 than in dye 7. This result is unexpected since the net charge of 3+ is the same for both dyes 7 and 8. It appears that both the location and quantity of charges affect the fluorescence enhancement of the dyes.

The cyanine dyes according to General Formula VI preferably absorb light at a wavelength of at least about 640 nm, more preferably at least about 649 nm and emit fluorescence at a wavelength of at least about 650 nm, more preferably at least about 663 nm. The cyanine dyes also preferably have a positive Stoke ' s shift (λEmJssJon-λAbs.) of at least 11 nm. Table 4 provides examples of some of the preferred cyanine dyes having General Formula VI. It should be understood, however, that the dyes listed in Table 4 are intended only to exemplify the cyanine dyes of the present invention and are not intended to be limiting.

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TABLE 4

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TABLE 4 (cont.)

- 20/1 -

TABLE 4 (cont.)

20/2 -

TABLE 4 (cont.)

-21

TABLE 4 (cont.)

21/1-

TABLE 4 (cont.)

- 21/2 -

The present invention also relates to the use of the cyanine dyes having General Formulas I, II, IV, V, VI, VII or VIII to form compositions for detecting the presence of nucleic acids in a sample. In general, the compositions include a cyanine dye according to the present invention non-covalently bound to a nucleic acid, i.e., DNA or RNA.

The fluorescence of the cyanine dyes of the present invention significantly increase when bound to a nucleic acid. As a result, it is possible to qualitatively or quantitatively determine the presence of nucleic acids in a sample by monitoring the change in the fluorescence intensity of the dye at a wavelength corresponding to the composition of the dye bound to the nucleic acids. Use of cyanine dyes in general for detecting the presence of nucleic acids in a sample is known in the art. A discussion regarding the use of cyanine dyes to detect the presence of nucleic acids in a sample is provided in U.S. Patent No. 5,321,130 to Yue, et al. which is incorporated herein by reference.

The present invention also relates to a method for detecting nucleic acids by contacting the nucleic acids with a cyanine dye of the present invention. According to the method, a sample of nucleic acids are contacted with a cyanine dye of the present invention in order to form the composition of a cyanine dye non-covalently bound to a nucleic acid sequence. After the dye- nucleic acid sequence composition is formed, the bound dye is exposed to light having a wavelength near an absorbance maximum of the dye when bound to a nucleic acid sequence. The resulting fluorescence emission of the dye is then detected in order to qualitatively or quantitatively determine the presence of nucleic acids in the sample.

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Example 1 : Preparation of Compound 4

1a. Preparation of 2,3-Tetramethylenenaphth[2,1-d]thiazolium Bromide

2-Aminonaphthalene-1-thiol was prepared by the method of Ambrogi, et al. (Ambrogi, V.; Grandolini, G.; Perioli, L.; Rossi, C. Synthesis, 1992, 7, 656- 8.) 2-Aminonaphthalene-1-thiol (0.14 g, 0.8 mmol) and bromovaleryl chloride (0.48 g, 2.4 mmol) were combined and heated to 100° for 1 h, then to 50°C overnight. The resulting solid was washed with acetone and air-dried to provide a white solid (0.16 g, 0.5 mmol, 60% yield).

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1b. Preparation of lodoNAPδ

4-(2"-Acetanilidovinyl)-1'-(3 ^, -iodopropyl)-quinolinium iodide (prepared by the general method of Brooker, et al. J. Am. Chem. Soc. 1941, 63, 3192-3203; 32 mg, 63 μmol), 2,3-tetramethylenenaphth[2,1-d]thiazolium bromide (20 mg, 63 μmol), triethylamine (40 uL) and ethanol (1 mL) were combined and refluxed for 2.Q-min. The dark blue solid was recryεtallized sequentially from fεopropanol and ethanol to provide a purple solid (12 mg, 30% yield). HPLC analysis on a C8 reverse-phase column using gradient elution of 40% to 80% acetonitriie vs. 0.1 M triethylammonium acetate buffer showed one major peak at 16 min.

1 c. Preparation of Compound 4

lodoNAP6 (2 mg, 3 μmol) was dissolved in dimethytformamide. Trimethylamine was bubbled through the solution. The reaction was monitored by thin layer chromatography on silica gel with methanol as the eluant The Rf values of IodoNAP6 and compound 4 were 0.5 and zero, respectively. After 30 min, reaction was complete. The solvent was evaporated and the residue partitioned between methylene chloride (CHjClj) and water. The aqueous layer was washed with 2 x 1 mL CH ₂ CI ₂ and concentrated to dryness. HPLC

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analysis with the same gradient that was used with iodoNAP6 showed one broad peak at 7.2 min with no apparent starting material. The absorbance maximum of compound 4 in methanol was at 667 nm.

Example 2- Preparation of Compound 8

2a. Preparation of T,1 "-(3',3"Bisiodopropyl)-thia-4-carbocyanine Iodide

1'3(e'-Iodopropyl)-2-(2"-acentanilidovinyl)- benzothiazium iodide (15 mg, 26μmol) , l ^« -3(3'-iodopropyl)- quinolinium iodide (15 mg, 34 μmol) , triethylamine (50 μL) and methanol (1 mL) were combined at room temperature. A blue precipitate formed immediately. The reaction mixture was centrifuged and the residue washed with methanol and isopropanol and air-dried to provide a dark solid (15 mg, 20μmol, 77% yield) .

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b. Preparation of Compound 8

1', l"-(3', 3'-Bisiodopropyl)-thia-4- carbocyanine iodide (15 mg, 20 μmol) was dissolved in DMF and trimethylamine bubbled through the solution. The reaction progress was monitored by TLC on reverse-phase plates with 1:1 dimethylformamide:4 M NaCl as eluant. The Rf'ε of the bisiodo starting material and the bisammonium salt were 0 and 0.8, respectively. The intermediate monoammonium salts could also be resolved, at Rf's of 0.7 and 0.6. After 30 min the reaction was complete. The solvent was evaporated. The absorbance maximum of compound 8 in DMSO was at 639 nm.

While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, which modifications will be within the spirit of the invention and the scope of the appended claims.

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