Chiral indole intermediates and their fluorescent cyanine dyes containing functional groups

Description of equivalent WO 2004039894 A2

CHIRAL INDOLE INTERMEDIATES AND THEIR FLUORESCENT CYANINE DYES CONTAINING FUNCTIONAL GROUPS.

The present application claims benefit of U.S. provisional application Serial No. 60/379,107, filed May 10, 2002, the entire contents of which is incorporated herein by reference.

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of NIH Grant No. R01-NS-19353 and NSF Grant No. MCB-8920118.

This invention relates to the functionalized cyanine dyes and more particularly, to the synthesis of chiral 3-substituted 2,3'-dimethyl-3H-indole and its derivatives as intermediates for preparation of cyanine dyes, to methods of preparing these dyes and the dyes so prepared.

Highly fluorescent carboxy! containing indocyanines are useful as labeling reagents for biological investigations. Functional groups on the dyes permit covalent binding to biomaterials and/or other non-biological materials for purpose of fluorescence detection, while water soluble arylsulfonate groups reduce dye-dye aggregation and enhance fluorescence brightness (see US patents by Waggoner et. al U.S. Patent Nos. 5,268,486; 5,486,616; 5,569,587; 5,852,191 ; 5,981 ,747; 5,986,093).

The carboxylic and arylsulfonate groups occupy key positions in the heteroaromatic bases such as R-i and R₂ in (1), and thereby restrict the ability for dye modifications. <img file="WO2004039894A2_D0001.tif" />

wherein R-, is CH₂NH₂, CH₂COOH, SO₃H or H and R₂ is (CH₂)_mNH₂, (CH₂)_mCOOH, (CH₂)mSO₃H or (CH₂)mOH, and m is an integer ranging from 1 to 6.

Thus, it is of interest to synthesize compounds with functional groups at different sites in the indole bases. These new intermediates will enable chemists to synthesize a wide range of cyanines.

Chiral 2,3-dimethyl-3H indoles (2) are expected to possess exceptional advantages as a precursor of a family of cyanine dyes for use as covalently attached fluorescent labels for biological research. Disclosed herein is a convenient synthesis of various 3-substituted 2-butanones and their conversion to cyanine dyes (Figure 1).

These dyes are very similar to those described in the U.S. Patent No. 5,268,486 and other related publications of the inventor, such as, for example, listed herein which disclose luminescent 'mono- and polymethine- cyanine and related dyes such as merocyanine and styryl dyes which contain groups enabling these dyes to covalently attach to amine, hydroxyl, aldehyde and sulphydryl groups on a target material. The disclosed compounds fluoresce in the green, orange, red and near infrared regions of the spectrum.

<img file="WO2004039894A2_D0002.tif" />

R₁ = -CH₂NH_2ι , -CH₂COOH, -SO3H, H, -N0₂ or X (CI, Br, or I)

R₂, R_{3 =} -&lt;CH2)_mNH₂, -(CH₂)_mCOOH, -&lt;CH₂)_mS0₃H, -(CH₂)_mOH, -CH₂X (CI, Br, I), or H wherein m is an integer ranging from 1 to 6.

Rosenstock (Research Laboratories, The National Drug Company, NOTES (Dec. 1966 pp. 537-539) has described the synthesis of a plant growth hormone by means of Fischer indolization of levulinic acid phenylhydrazone. The indole is used for the development of various plant growth hormones.

WO 02/26891 discloses cyanine dyes incorporating similar structures.

A number of papers, such as Eggers et al (Angew. Chem. Int. Ed. Engl. (1997) 36, No. 8, 881-883), Eggers et al (Liebigs Ann. (1996), 979-983), Reichardt et al (Chem. Rev. 123, (1990), 565-581), Reichardt et al (Liebigs Ann. (1995) 329-340), and U.S. Patent Nos. 6,190,641 ; 6,183,726; 6,180,087; 6,180,086 and 6,180,085 have described the synthesis of chiral indoles such as (3). Cyanine dyes described in these papers are water insoluble and have no functional groups that react with biological specimens. The chiral indoles are obtained by direct alkylation of 2,3- dimethylindole as shown below. This method has very limited applications. Attempts to alkylate 2,3-dimethylindole with various alkylhalides (such as 6-iodo ethylhexanoate) have been unsuccessful.

<img file="WO2004039894A2_D0003.tif" />

Brief Description of the Drawings

FIGURE 1 : A General scheme for the synthesis of 3-substituted-2-butanone and its cyanine dyes.

FIGURE 2: Computer generated energy minimized structure of 6-(1 ,2-dimethyl-7- sulfohydrobenzo[e]indolyl)hexanoic acid. The hexanoic acid chain is almost perpendicular to the benzindole ring. Such a configuration of the chain prevents dye- dye aggregation.

FIGURE 3: ¹H NMR spectra in CDCI₃ of 6-(2,3-dimethyl-3H-indol-3-yl)hexanoic acid and 5-(2,3-dimethyl-3H-indol-3-yl)pentan-1-ol show identical aromatic signals. Some of the methylene protons of the hexanoic acid and pentanol alkyl chains show high field shift, suggesting shielding of those protons due to ring current effect of the indole.

FIGURE 4: ¹H NMR spectra of Cy3.18 and NS Cy3 in D₂O. Some of the methylene protons of the hexanoic acid chain in NSCy3 show high field shift, suggesting shielding of those protons due to ring current effect of the indole.

FIGURE 5: Absorption spectra of non-sulfonated Cy3 isomers and thejr antibody conjugates.

FIGURE 6: Relative fluorescence intensities of non-sulfonated Cy3 isomers NCy3,

Cy3.10 and Cy3.24. The absorbance for all dyes in methanol and PBS solution at 550 nm is 0.05 and the excitation wavelength is 514 nm

FIGURE 7: Absorption spectra of Cy7.18 free acid (dotted line) and its lgG conjugate (solid line) are shown in (A). Absorption spectra of NCy7 free acid (dotted line) and its lgG conjugates (solid line) are shown in (B). NSCy7 showed less dye-dye aggregation (indicated by low vibronic shoulder). FIGURE 8: Antibody brightness of NCy3. The brightness is a product of number of dyes per protein, extinction coefficient of the dye and quantum yield of the dye/antibody conjugates.

FIGURE 9: is a graph showing the emission spectra of Cy3.18.OH and the disulfonated dye (NSCy3) in methanol and phosphate buffer solution when solutions of equal concentrations were excited at 514 nm. NSCy3 is 20-25% brighter than Cy3.18 in both solutions.

The Figures 2-9 emphasize improved properties of the cyanine dyes of the present invention due to the modifications at the 3, 3' positions of the indolenine. Specifically, Figure 2 shows a computer generated, energy minimized structure of 6- (1 ,2-dimethyl-7-sulfo-3-hydrobenz[e]indolyl)hexanoic acid. The hexanoic acid chain is perpendicular to the plane of benzindole ring. Some of alkyl protons of the hexanoic acid chain are over the plane of the ring. This is also substantiated by the NMR spectrum of 5-(2,3-dimethyl-3/-/-indol-3-yl)pentan-1-ol (Example 9) and 6-(2,3- dimethyl-3 --indol-3-yl)hexanoic acid (Example 3) in Figure 3. The alkyl chain protons of hexanoic acid and pentanol are shielded due to indole ring and therefore appear at high field (0.5-0.7 ppm). In Figure 4, the NMR of NSCy3 shows similar up- field protons (shown by arrow). The hexanoic acid protons for Cy3.18 appear at 1.5 -2.0 ppm. Such a configuration prevents dye-dye aggregation thereby increasing the fluorescence brightness of the dye in aqueous solutions. This is a major advantage of these new intermediates of this invention. Figure 5 shows the absorbance spectra of three non-sulfonated Cy3 dyes and their lgG conjugates. Cy3.10- lgG conjugates (Figure 5C) show considerable deviation from the base line suggesting significant dye-dye aggregation. The spectrum Cy3.24 and its lgG conjugates (Figure 5B) and NCy3 and its lgG conjugates (Figure 5A) are almost identical. But NCy3 is 20-25% brighter than Cy3.24 in aqueous solution. This is shown in Figure 6. The equal concentration of the dyes in methanol and PBS solutions were excited at 514 nm; fluorescence intensities of the dyes in methanol are almost the equal. Because of high solubility in methanol, no dye-dye aggregation is observed. However, in a phosphate buffer solution, NSCy3 is 20-25% brighter than Cy3.24. The reduction of dye-dye aggregation in an antibody conjugated dyes is also evident from Figure 7. The absorption spectra of commercialized Cy7.18 and new NSCy7 are compared with their antibody conjugates. Cy7.18 aggregates more on antibody as seen from the increase in the vibronic shoulder.

The present invention provides molecules, such as compounds of formula (I)

<img file="WO2004039894A2_D0004.tif" />

0) which include a reactive or water solubilizing site at R₃ and/or R₄ in structure (I)

The present invention further provides a method of synthesizing, and synthesized ketones and indolenines wherein substituent R₃ may be -(CH₂)_tι A and substituent R₄ may be -(CH₂)t₂B, wherein ti and t₂ are an integer, preferably from 1- 22, and A and B are independently selected from -COOH, -NH₂, -SO₃, -OH, H and halogens as shown in (II).

<img file="WO2004039894A2_D0005.tif" />

The present invention also provides a method of synthesizing ketones and indolenines wherein R₃ and/or R , of formula (I) for example, is -(CH₂)_tCH₃, wherein t is an integer in the range of 0-22, preferably 16 to 22, to produce long alkyl chain dyes as membrane potential probes.

The present invention further relates to the synthesis of cyanine, merocyanine and styryl dyes that can be modified to create covalent labeling reagents dyes, such as shown herein, wherein functional or water solubilizing groups may be included.

The present invention therefore, provides intermediates for, and methods of synthesizing cyanine, merocyanine and styryl dyes, such as those shown below.

<img file="WO2004039894A2_D0006.tif" />

(III)

<img file="WO2004039894A2_D0007.tif" />

(IV)

<img file="WO2004039894A2_D0008.tif" />

STYRYL

(V) More specifically, the intermediates of the present invention provide methods of synthesizing intermediates and synthesed compounds from the same, such as the following polymethine cyanine type dyes:

<img file="WO2004039894A2_D0009.tif" />

CYANINE

(VI)

<img file="WO2004039894A2_D0010.tif" />

MEROCYANINE

(VII) <img file="WO2004039894A2_D0011.tif" />

STYRYL

(VIII)

<img file="WO2004039894A2_D0012.tif" />

CYANINE

<img file="WO2004039894A2_D0013.tif" />

MEROCYANINE

(X)

<img file="WO2004039894A2_D0014.tif" />

STYRYL

(XI) In these structures, Y is selected from the group consisting of O, S, -CH=CH-

&gt;C(CH₃)₂, and &gt;N-(CH₂)ι-ι₀Rι , wherein R is selected <img file="WO2004039894A2_D0015.tif" /> from -COOH, -NH₂, -S0₃^~, -OH and halogen; Z is selected from the group consisting of O and S; and n is an integer selected from the group consisting of 1 , 2, 3 and 4.

At least one, preferably only one, and possibly two or more of the Ri, R₂, R₃, R₄, R₅, Re , R₇, R-11, and Rι₂ groups in each molecule is a reactive group for attaching the dye to the labeled component. A reactive group of a compound according to formula (XIII) or (XIV) can react under suitable conditions with a complementary functional group of a component, such that the component becomes covalently labelled with the compound. For certain reagents, at least one of the R-t, R₂, R₃, R₄, R₅, R_δ, R₇, ₁1, and R12 groups on each molecule may also be a group that increases the solubility of the chromophore, or affects the selectivity of labeling of the labeled component or affects the position of labeling of the component by the dye.

In the above formulas, at least one of said Ra (if any), Rg (if any) and R₁₀ (if any) and R₁₃ (if any) groups comprises at least one sulfonate group. The term sulfonate is meant to include sulfonic acid because the sulfonate group is merely ionized sulfonic acid.

Reactive groups that may be attached directly or indirectly to the chromophore to form the Ri, R₂, R₃, R , R5, Re , R7, R11, and R12 groups may include reactive moieties such as groups containing isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal and aldehyde. Halogen and halo groups are selected from fluorine, chlorine, bromine and iodine.

Specific examples of the Rt, R₂, R₃, R4, R5, Re, R7, Rn, and R₁₂ groups that are especially useful for labeling components with available amino-, hydroxy-, and sulfhydryl groups include:

-NCS / V -NCS — (CH₂)_n— NCS

<img file="WO2004039894A2_D0016.tif" />

<img file="WO2004039894A2_D0017.tif" />

<img file="WO2004039894A2_D0018.tif" />

<img file="WO2004039894A2_D0019.tif" />

<img file="WO2004039894A2_D0020.tif" />

where n is 0 or an integer from 1-10, and at least one of Q or W is a leaving group such as I, Br, CI. Specific examples of R-i, R_2&gt; R3, R4, s, RΘ , R7 , R₁1, and Rι₂ groups that are especially useful for labeling components with available sulfhydryls which can be used for labeling antibodies in a two-step process:

-N <img file="WO2004039894A2_D0021.tif" />

where Q is a leaving group such as I, Br, CI.

<img file="WO2004039894A2_D0022.tif" />

<img file="WO2004039894A2_D0023.tif" />

<img file="WO2004039894A2_D0024.tif" />

where n is an integer from 1-10.

Specific examples of Ri, R₂, R₃, R₄, R5, RΘ , R₇ , R1₁ and Rι₂ groups that are especially useful for labeling components by light-activated cross linking include: <img file="WO2004039894A2_D0025.tif" />

For the purpose of increasing water solubility or reducing unwanted nonspecific binding of the labeled component to inappropriate components in a sample or to reduce the interactions between two or more reactive chromophores on the labeled component which might lead to quenching of fluorescence, any of the R-i, R₂, R₃, R₄, R5, e , R7 , R11, and R₁₂ groups can be selected from the well known polar and electrically charged chemical groups. Examples are -E-F where F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino and where E is a spacer group such as -(CH₂)_n- where n is 0, 1 , 2, 3 or 4. Useful examples include alkyl sulfonate; -(CH₂)₃-SO₃^"; and -(CH₂)₄-SO₃^".

The polymethine chain of the luminescent dyes of this invention may also contain one or more cyclic chemical groups that form bridges between two or more of the carbon atoms of the polymethine chain. These bridges might serve to increase the chemical or photo-stability of the dye and might be used to alter the absorption and emission wavelength of the dye or change its extinction coefficient or quantum yield. Improved solubility properties may be obtained by this modification.

The present invention provides compounds of the following general formula

(XII):

<img file="WO2004039894A2_D0026.tif" />

(XII) wherein: R_a and R_b are independently selected from V or L-V where L is a C₁-₂₂ , such as a C16-21, straight or branched alkyl chain, optionally containing 0, 1 or 2 unsaturations or unsaturated pendent or interchain groups selected from alkenyl, alkynyl and aryl groups; and

V is selected from hydrogen, halogen, -OH, -NH₂, -SH, -CN, trifluoromethyl, -SO₃-, phosphate, phosphonate, quaternary ammonium, -NO₂, mono- or di-nitro- substituted benzyl, -COOH, and -NHCOR_g , where R_g is Cι„₂₀ straight or branched alkyl; a target bonding group, reactive group, reactive moiety, or NHR_h where R_h is H, C₁-₂₀ straight or branched alkyl or COOH;

T is CH, L-V , wherein each L and V are independently as defined

X L-^"V " L-V above; and p is 0 or an integer from 1-4

The scope of reactive groups, reactive moieties and target bonding groups described and used herein will be appreciated from the present disclosure as well as, for example, U.S. Patent No. 6,133,445 and WO 02/26891 , the entire contents of each of which is incorporated herein by reference.

The present invention provides pH sensitive cyanines of the following formula

(XIII):

<img file="WO2004039894A2_D0027.tif" />

(XIII)

wherein R_c, Rd, R_e and Rf are any R_a as defined above; Y, T and R_b are as defined above; Z_a and Z_b are independently, fused <img file="WO2004039894A2_D0028.tif" />

and n is an integer from 1-4. Preferably, n is an integer from 1-3.

The present invention further provides compounds of the following formula

(XIV) wherein R_a, R_c, Rc Rf. T, Y, Z_a and Zb are as defined above.

<img file="WO2004039894A2_D0029.tif" />

More than one R_a may be contained on the noted ring and each such substituent may be the same or different. In one embodiment, R_a is a sulfonate group and p is 1 , where sulfonate includes sulfonic acid as the sulfonate group is merely an ionized sulfonic acid. Where Z_a and Z_b are fused rings, R_a R_c, R_d and R_f may be substituted around either ring and p, u, r and v are each independently an integer from 0-4.

In the above formulas, the number of methine groups determines, in part the excitation color. The cyclic azine structures can also determine in part the excitation color. Often, higher values of n contribute to increased luminescence and absorbance. At values of n above 4, the compound becomes unstable. Thereupon, further luminescence can be imparted by modifications at the ring structures. When n=2, the excitation wavelength is about 650 nm and the compound is very fluorescent. Maximum emission wavelengths are generally 15-100 nm greater than maximum excitation wavelengths.

The present invention relates to the other substituents, such as R₂ = H, and n is an integer from 0 to 4, in structure (I), which may be used to produce pH sensitive cyanine dyes, such as those described in copending U.S. Patent Application No. 09/589,502, filed June 8, 2000, which is incorporated herein by reference and discloses, for example, compounds similar but distinguishable from the following formula (XV).

<img file="WO2004039894A2_D0030.tif" />

(XV)

Specifically, for example, the corresponding compounds of the related application define the substituents at positions 1 and 2 of formula (XV) as &gt;C(C-ι - C₄alkyl)₂, sulfur or oxygen.

The present invention further provides dyes, such as the following rigid trimethine cyanine dyes. Similar but distinct compounds are described in the U.S. Patent No. 6,133,445. Compounds of the present invention include, for example, compounds of the following formula (XVI), intermediates and methods for synthesizing the same. <img file="WO2004039894A2_D0031.tif" />

(XVI)

The corresponding, yet distinct, compounds of U.S. Patent No. 6,133,445 define the substituents at positions 1 and 2 of formula (XVI) as the same or different and selected from bis-Ci -C alkyl and C -C₅ spiro alkyl substituted carbon, oxygen, sulphur, selenium, CH=CH, and N-W wherein N is nitrogen and W is selected from hydrogen, a group ~(CH2)_n R ² where n is an integer from 1 to 26 and R¹² is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary amino, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, phosphoryl, and sulphydryl groups. Compounds of the present invention are other than those disclosed in U.S. Patent No. 6,133,445.

Exemplary dyes according to the invention are as follows:

i) 2-{(1 E,3E,5E)-5-[3-(5-carboxypentyl)-3-methyl-5-sulfo-1 ,3-dihydro-2H-indol-2- ylidene]penta-1 ,3-dienyl}-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3/V-indolium (Example 13); ii) 3-(5-carboxypentyl)-2-[(1 E,3E,5£)-5-(5,7-dichloro-3,3-dimethyl-1 ,3-dihydro- 2/V-indol-2-ylidene)penta-1 ,3-dienyl]-3-methyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium (Example 14); iii) 2-{(1 E,3E,5E)-5-[3-(5-carboxypentyl)-3-methyl-5-sulfo-1 ,3-dihydro-2 -/-indol-2- ylidene]penta-1 ,3-dienyl}-5-chloro-3,3-dimethyl-1-(4-sulfobutyl)-3r -indolium

(Example 15); iv) 3-(5-carboxypentyl)-2-[(1 E,3E,5E)-5-(7-chloro-3,3-dimethyl-5-sulfo-1 ,3- dihydro-2H-indol-2-ylidene)penta-1 ,3-dienyl]-3-methyl-5-sulfo-1-(4-sulfobutyl)-3 - - indolium (Example 16); v) 3-(5-carboxypentyl)-3-methyi-2-{(1E,3E,5E)-5-[3-methyl-5-sulfo-3-(4- sulfobutyl)-1 ,3-dihydro-2H-indol-2-ylidene]penta-1 ,3-dienyl}-5-sulfo-1 -(4-sulfobutyl)-

3/-/-indoiium (Example 17); and vi) 6,7,9,10-tetrahydro-2,14-disulphonato-16,16,18-trimethyl-7aH,8aH- bisindolinium[3,2-a,3'2'-a]pyrano[3,2-c;5,6-c']dipyridin-5-ium-18-hexanoic acid

(Example 19).

The present invention relates to the energy transfer dyes wherein cyanine- cyanine dye conjugates are synthesized such as (XVll). Such cyanine-cyanine dye complexes have been described in the U.S. Patent Nos. 6,008,373 and 6,130,094.

<img file="WO2004039894A2_D0032.tif" />

Target bonding groups, reactive groups and reactive moieties include, amine, hydroxy, thiol, N-hydroxy-succinimidyl ester, N-hydroxy-sulfosuccinimidyl ester, isothiocyanate, anhydride, haloacetamide, isocyanate, monochlorotriazine, dichlorotriazine, optionally substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, imidoester, alkylimidate, hydrazide carbodiimide, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, aldehyde, maleimide, sulphonyl halide, phosphoramidite, acid halide, hydrazine and carbodimide groups, and groups reactive with amino, hydroxyl, aldehyde, phosphoryl, or sulphydryl groups which are, for example, covalently attached to proteins, nucleic acids, nucleosides, nucleotides, carbohydrates, sugars, cells, and combinations thereof, and other biological and non-biological materials, to make the same fluorescent and detectable, as described, for example in U.S. Patent No. 6,048,982.

V may also be a group which increases the solubility of the chromophone or affects the selectivity of labeling of the ultimately labeled component or affects the position of labeling of the labeled component by the dye.

The compounds or dyes of the present invention may be used to label, for example avidin, streptavidin, antibodies, DNA, RNA, nucleosides, nucleotides or lectins to detect, measure and/or quantify, for example, biotinylated materials, antigens, haptens, carbohydrate groups, DNA, RNA and complementary DNA or RNA, such as described therein.

In another embodiment, the present invention provides a component-labeled complex wherein the label is a compound of any one of formulas (lll)-(XVll) and the component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, oligosaccharide, polysaccharide, nucleic acid, derivatized deoxy nucleic acid, DNA fragment, RNA fragment, derivatized DNA fragment, derivatized RNA fragment, nucleoside, nucleotide, natural drug, synthetic drug, virus particle, bacterial particle, virus component, yeast component, blood cell, blood cell component, plasma component, serum component, biological cell, non-cellular blood component, bacteria, bacterial component, natural or synthetic lipid vesicle, poison, environmental pollutant, polymer, polymer particle, glass particle, glass surface, plastic particle, plastic surface, polymer membrane, conductor or semiconductor.

A cyanine dye or its derivative of the present invention preferably absorbs maximally in the range of 400-950 nm, preferably in the range of 420-470 nm, 520- 580 nm, 640-680 nm, 740-790 nm or 820-850 nm. In yet another embodiment, the present invention provides a method of fluorescent detection wherein the dye or derivative of the present invention is detected or when multiple fluorescent dyes are used, a dye or derivative of the present invention may be used in conjunction with dyes which do not either fluoresce at the same or similar pH conditions and/or at the wavelengths of the presently provided dyes or derivatives.

In a further embodiment, the compound of formula (Xlll) may be used in a fluorescence method for the qualitative and/or quantitative detection of pH. The method comprises contacting or mixing a dye of formula (Xlll) with a composition containing cells, tissues or biological fluid, and detecting the emitted fluorescence. The presence of a fluorescent signal is indicative of an acidic environment. In one embodiment, the method may be used for detecting intracellular environments, such as may be contained in subcellular compartments or structures. Compounds of formula (Xlll) or their membrane permeant derivatives may be actively or passively absorbed by cells where they may be detected by fluorescence detection. One of ordinary skill will appreciate the variability of cell permeability properties of compounds according to formula (Xlll) and will be able to routinely test for the same.

The methods according to the present invention may employ known devices for illumination and detection at separate defined wavelengths. Such devices include fluorescence spectrophotometers, fluorescence microscopes. Alternatively, changes in fluorescence intensity may be measured by means of a charge coupled device (CCD) imager (such as a scanning imager or an area imager) to image all of the wells of a microtitre plate. The present invention is further described by the following examples.

EXAMPLES

1. Synthesis of diethyl 2-acetyl-2-methyloctane-1 ,8-dioate

<img file="WO2004039894A2_D0033.tif" />

In a 1L three necked flask equipped with a mechanical stirrer and reflux condenser was placed dry toluene (300 ml). The system was flushed with argon and sodium hydride (Aldrich, 60% dispersion in mineral oil) (7.2 g, 0.18 mmol) was added. Ethyl-γ-methylacetoacetate (Aldrich, 21 g, 0.15 mole) was added with stirring over a 30 minutes. The resulting solution was heated under reflux for 2 hrs and cooled slightly. (Note: Reaction mixture becomes a thick paste and mechanical stirrer is essential). Ethyl 6-bromohexanoate (Aldrich, 33.5 g, 0.15 mol) was added (all at once) and the suspension was heated under reflux for 12 hrs, cooled, filtered and the solvent evaporated under pressure. The residue was distilled under vacuum to yield 20 g (46%) of colorless liquid, b.p. 110-115° C (0.1 mm). IR (neat): vcm^"1 = 1735 (s) and 1713 (s). H NMR in CDCI₃ δ, 4.1-4.2 (m, 4H, (O-CH₂)₂); 2.1 (t, J = 8.4Hz, 2H, CH2COO-); 2.15(s, 3H, CH₃CO); 1.5-2.0 (m, 4H, (CH₂)₂); 1.2-1.4 (m, 13H, 2 CH₂ and three -CH₃). Synthesis of 7-acetyloctanoic acid (or 7-methyl-8-oxononanoic acid)

<img file="WO2004039894A2_D0034.tif" />

A mixture of diester from Example 1 (15 g, 0.052 mol) in dilute hydrochloric acid (40-50%) (100 ml) was heated to reflux for 12 hrs. The reaction mixture was cooled and extracted with ethyl acetate (3 x 100 ml). The organic phase was dried over sodium sulfate, filtered, and ethyl acetate was removed. The residue was distilled under reduced pressure, to yield 7.7 g (80%), colorless liquid, b.p. 120-125 °C, IR (neat): vcm^'1 = 1736 (s) and 1710 (s). ¹H NMR, CDCI₃, δ, 2.45-2.55 (q, 1H, J =6.6 Hz,7-H); 2.35 (t, J = 7.3 Hz, 2H, -CH₂-COOH); 2.125 (s, 3H, COCH₃); 1.65- 1.1.75 (m, 2H, -CH₂); 1.23-1.4 (m, 6H, (-CH₂)₃); 1 -1 (d, J = 5.9Hz, 7-CH₃).

Synthesis of 6-,2,3-dimethyl-3H-indol-3-yl)hexanoic acid

<img file="WO2004039894A2_D0035.tif" />

To a stirred solution of phenylhydrazine hydrochloride (Aldrich, 7.2 g, 0.05 mol) in acetic acid (50 ml) was added 7-acetyloctanoic acid (11 g, 0.06 mol). The reaction mixture was heated under reflux for 4 hrs. The solution was cooled. (No clear precipitate was observed). Acetic acid was removed under reduced pressure. The resulting yellow liquid was chromatographed on a silica gel column using chloroform/methanol mixture as solvent. Pure 6-(2,3-dimethyl-3H-indol-3-yl)hexanoic acid (8.0 g, 61 %) was obtained as pale yellow oil which crystallized on standing, m.p. 115-118 °C; IR v cm ^"1 = 2927, 2860, 2526, and 1709. ¹H NMR, CDCI₃, δ, 7.6 (d, 1 H, J = 7 Hz, 4-H); 7.2-7.4 (m, 3H, aromatic protons); 2.25 (s, 3H, - CH₃); 2.1-2.2( t, - CH₂COOH); 1.72-1.94 (m 2H, -CH₂); 1.4-1.45 (m, 2H, -CH₂); 1.23-1.05 (m, singlet merged in m, 5H, -CH₂, -CH ); 0.55-0.85 (m, 2H, -CH₂). TLC, Rf = 0.22 (silica gel, methanol-chloroform, 5:95).

4. Synthesis of 3-(5-carboxypentyl -2-{(1 E,3E -3-r3-(5-carboxypentvn-1-ethyl-3- methyl-1.3-dihvdro-2/- -indol-2-ylidenelprop-1 -enyl)-1 -ethyl-3-methyl-3/-/-indolium iodide ,NCy3)

<img file="WO2004039894A2_D0036.tif" />

4.1 3-(5-CarboxypentvP-1 -ethyl-2,3-dimethyl-3H-indolium iodide

6-(2,3-Dimethyl-3-hydroindole-3-yl)hexanoic acid (518 mg, 2 mmol) was suspended in acetonitrile (5 ml) and ethyl iodide (1.2g, 10 mmol) and the mixture was heated to reflux with stirring for 1hr. More ethyl iodide (2 g) was added and heating continued for an additional 12 hrs. The mixture was then cooled, and acetonitrile and excess ethyl iodide were removed on a rotary evaporator. The sticky mass was washed several times with ice-cooled diethyl ether and dried to yield pink amorphous powder (0.45 g, 78%). The product was used for the next reaction without further purification.

4.2 To a stirred solution of 6-(1 ethyl-2,3-dimethyl-3-hydroindol-3-yl)hexanoic acid (288 mg, 0.95 mmol) in pyridine (10 ml) at 120 °C was added dropwise, triethyl orthoformate (100 mg, 67 mmol) over 30 minutes. After 2 hrs the reaction mixture was cooled and triturated with diethyl ether. The product was purified on a silica gel column using chloroform/methanol mixture as solvent. The major compound (Rf = 0.45, tic, silica gel, methanol in chloroform 5%) was obtained as a pink solid (251 mg, 35%); 1 H NMR, CDCI₃, d, 8.2 (t, H, J = 7Hz, β-H); 7.1-7.6 (m, 8H, aromatic); 6.4 (d, 2H, J = 7Hz, α-H, '-H); 4.2 (m, 4H, 2-NCH₂); 2.2-2.3 (m, 4H, 2-CH₂COOH, 2-CH₂); 1.8 (s, 6H, (-CH₃)₂); 1 -43 (t, 6H, J = 6.5Hz, 2-CH₃); 1.1-1.2 (m, 4H, 2-CH₂); 0.5-0.7 (m, 4H, 2-CH₂); λmax: 550 nm, methanol.

5. Preparation of 6-(2,3-dimethyl-5-sulfo-3H-indol-3-yl)hexanoic acid

CH3COOH, reflux <img file="WO2004039894A2_D0037.tif" /><img file="WO2004039894A2_D0038.tif" />

To a stirred solution of 4-hydrazinobenzenesulfonic acid (Aldrich, 11.25 g, 0.06 mol) in acetic acid (50 ml) was added 7-acetyioctanoic acid (16.7 g, 0.09 mol). The reaction mixture was heated under reflux for 12 hrs. Acetic acid was removed under reduced pressure. The resulting solid was dissolved in methanol and reprecipitated with a saturated solution of potassium hydroxide in isopropanol. The solid was filtered, washed with isopropanol and dried, (8 g, 40%). The analytical sample was obtained by C18 reversed phase column chromatography using water/methanol mixture as solvent., m.p. 250 °C dec; IR v cm ^~1 = 2930, 2597, and 1719. ¹H NMR, D₂O, δ, 7.8-7.9 (m, 2H, 4-H and 6-H of aromatic protons); 7.6 (d, J = 7Hz, 1 H, 7-H of aromatic); 2.2 (t, J =7Hz, 2H, -CH₂-COOH); 1.9-2.1 (m, 2H, alkyl); 1.2-1.6 (a singlet merged in a multiplet, 7H, -CH₃ &amp; (-CH₂)₂) ; 0.6-0.9 (m, 2H, alkyl.). 6. Synthesis of 3-(5-carboxypentvn-2-((1 E,3E)-3-.3-(5-carboxypentvn-1-ethyl-3- methyl-5-sulfo-1 ,3-dihvdro-2H-indol-2-ylidene1prop-1 -envP-1 -ethyl-3-methyl-3H- indolium-5-sulfonate (NSCy3)

<img file="WO2004039894A2_D0039.tif" /> 3-(5-Carboxypentyl)1 -ethyl-2,3-dimethyl-3/τ'-indolium-5-sulfonate was synthesized following the procedure described in Example 4.1. 6-(2,3-Dimethyl-5- sulfo-3-hydroindole-3-yl)hexanoic acid (680 mg, 2 mmol) was suspended in acetonitrile (10 ml) and ethyl iodide (1.2g, 10 mmol) and the mixture was heated to reflux with stirring for 1 r. More ethyl iodide (2 g) was added and heating continued for additional 12 hrs. The mixture was then cooled and solvent removed on a rotary evaporator. The sticky mass was dissolved in methanol (5ml). A solution of potassium acetate in isopropanol was added until the reaction product was alkaline to pH. The solid obtained was filtered and washed several times with isopropanol to yield a gray amorphous powder (500 mg, 61 %). The product was used for the next reaction without further purification.

To a stirred solution of 3-(5-carboxypentyl)1-ethyl-2,3-dimethyl-3H-indolium-5- sulfonate (potassium salt) (400 mg, 0.98 mmol) in pyridine (10 ml) at 100 °C was added triethyl orthoformate (100 mg, 66 mmol) over 30 minutes. After 2 hours the reaction mixture was cooled and diluted with several volumes diethyl ether. A product separated as red powder from which supernatant fluid was removed by decantation. It was dissolved in methanol and reprecipitated with isopropanol containing potassium acetate. The crude dye was collected on a filter paper and dried (380 mg, 90%). It was purified by C18, column chromatography using water- methanol mixture for elution. A pure dye was obtained as pink solid (251 mg), λmax 555 nm; ¹H NMR (D₂O) δ, 8.42 (t, 1 H, J=13 Hz, β-H of the bridge); 7.6-7.8 (m, 4 H, 4-H, 4'-H, 6-H, 6'-4); 7.2 (d, 2H, J=7.7 Hz, 7-H, 7'-H); 6.5 (d, 2H, J=13Hz, α, α'-H);4.1 (broad signal, 4H, (N-CH₂)₂); 1.8-2.2 (m, 8H, alkyl and CHgCOOH); 1.2-1.7 (m, 24H, 6-CH₂, 4-CH₃); 0.45-0.8 (m, 4H, alkyl).

7. Synthesis of ethyl 2-acetyl-7-acetyloxy-2-methylheptanoate

NaH, Toluene Br-(CH₂)₅-OCOCH₃

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In a 1 L three necked flask equipped with a mechanical stirrer and reflux condenser was placed dry toluene (300 ml). The system was flushed with argon and sodium hydride (Aldrich, 60% dispersion in mineral oil) (7.2 g, 1.2 eq.) was added. Ethyl- -methylacetoacetate (Aldrich, 21 g, 0.15 mole) was added with stirring over a 30 minutes. The resulting solution was heated under reflux for 2 hours and cooled slightly. (Note: Reaction mixture becomes a thick paste and a mechanical stirrer is essential). 5-Bromopenty!acetate (Aldrich, 31.5 g, 15 mol) was added (all at once) and the suspension was heated under reflux for 12 hr, cooled, filtered and the solvent evaporated under reduced pressure. The residue was distilled under vacuum to yield 20 g (45%) of colorless liquid, b.p 90-92 (0.5 mm). ¹H NMR in CDCI3 δ, 4.1-4.2 (m, 2H, O-CH₂CH₃): 3.5 (t, J = 7Hz, 2H, CH₂OCO-); 2.2 (s, 3H, CH₃CO); 2.1 (s, 3H, - COCH₃); 1.5-2.0 (m, 10H, 4-(CH₂)₂, C-CJHg, OCH?CH₃).. 8. Synthesis of 8-hydroxy-3-methyloctan-2-one or 6-acetyl-octanol

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A mixture of diester (15 g, 0.06 mol) in a dilute hydrochloric acid (40-50%) (100 ml) was heated to reflux for 12 hrs. The reaction mixture was cooled and extracted with ethyl acetate (3 x 100 ml). The organic phase was dried over sodium sulfate, filtered, and ethylacetate was removed. The residue was distilled in vacuum, to yield 7.19 g (80%) colorless liquid, b.p. 90-97 °C at 0.7 mm , IR (neat): v cm^"1 = 1736; ¹H NMR, CDCI₃, δ, 3.55 (t, 2H, J = 6.6 Hz, -CH₂OH); 2.45 (m, 1 H, 7-H); 2.15 (s, 3H, COCH₃); 1.65-1.82 (m, 4H, (-CH₂)₂); 1.2-1.55 (m, 4H, (- CH₂)₂); 1.1 (d, J = 7.3 Hz, -CH-CH₃).

9. Synthesis of 5-.2.3-dimethyl-3/-.-indol-3-yl)pentan-1-ol

<img file="WO2004039894A2_D0043.tif" /> To a stirred solution of phenylhydrazine hydrochloride (Aldrich, 7.2 g, 0.05mol) in acetic acid (50 ml) was added 8-hydroxy-3-methyloctan-2-one (12 g, 0.07mol). The reaction mixture was heated under reflux for 4 hrs. The solution was cooled. (No clear precipitate was observed). Acetic acid was removed under reduced pressure. The resulting yellow liquid was chromatographed on a silica gel column using chlorofornr.methanol mixture as solvent. Pure 6-(2,3-dimethyl-3-hydroindol-3- yl)pentanol (8.0 g, 60%) was obtained as pale yellow oil; ¹H NMR, CDCI₃, δ, 7.8 (d, J = 7 Hz, 4-H aromatic); 7.2-7.6 (m, 3H, aromatic protons); 3.5 (t, J = 6.5 Hz, -CH₂OH; 2.6 (s,3H, 2-CH₃ of indole); 1.8-2.12 (m, 2h, alkyl), 1.3-1.7 (singlet merged in multiplets, 7H, 2 alkyl and -CH₃ of indole); 0.6-0.9 (m, 2H, alkyl).

10. Synthesis of 6-(1 ,2-dimethyl-6.8-disulfo-1rY-benzo.e1indol-1-v0hexanoic acid

<img file="WO2004039894A2_D0044.tif" />

(5,7-Disulfo-2-naphthyl)hydrazinium chloride (2.0g), 7-methyl-8-oxononanoic acid (1.5g) and acetic acid (15ml) were heated from 80-140°C for a total of 24hrs. After evaporation of the solvent under vacuum, the residue was triturated with 2- propanol to give a pink solid. This solid was collected by filtration, washed with 2- propanol, then excess diethyl ether and dried under vacuum over phosphorus pentoxide. Yield of crude product = 1.96g. This was purified as required by preparative HPLC (RPC18. Water/MeCN/TFA) to give pure product. H-nmr (D₂O) δ 0.25-0.4 (1H, broad m), 0.5-0.65 (1 H, broad m), 1.00 (2H, m), 1.25 (2H, m), 1.78 (3H, s), 2.06 (2H, t), 2.35-2.5 (1 H, broad m), 2.65-2.8 (1 H, broad m), 2.90 (3H, s), 8.05 (1 H, d), 8.58 (1 H, d), 8.70 (1 H, d) and (8.97 (1 H, d). MS (LCMS) MH+ 470. Ace. Mass : MH+ 470.0931 (-2.6ppm for C₂₀H₂₄NO₈S₂).

11. Synthesis of 3-(5-carboχypentyl)-2-f(1E,3E. 5E. 7E)-7-.3-(5-carboxypentyl)-1 ■ ethyl-3-methyl-5sulfo-1.3-dihvdro-2 -/-indol-2-ylidene1hepta-1 ,3,5-trienvD-l -ethyl-3- methyl-3 -/-indolium-5-sulfonate (NSCv7)

<img file="WO2004039894A2_D0045.tif" />

Glutaconaldehyde dianil hydrochloride (143 mg, 0.5 mmol) was dissolved in a heated mixture of acetic anhydride (4 ml) and pyridine (1 ml). The intermediate 6-(1- ethyl-2,3-dimethyl-5-sulfo-3-hydroindol-3-yl)hexanoic acid (368 mg, 1 mmol) was added and mixture was heated to reflux for an additional 10 min. and then cooled. The dye was precipitated with diethyl ether. The supernatant liquid was separated. Solid was redissolved in a minimum volume of methanol and reprecipitated with isopropanol. The purple solid was collected on a filter paper and dried (160 mg, yield 18%). The pyridinium salt was converted into its potassium salt and purified by C18 column chromatography using water-methanol mixture as eluent. λ max = 750 nm.

1 H NMR (D₂O) δ, 7.7 (m, 6H, 4-H, 4'-H, 6-H, 6'-H and γ γ^* protons of the bridge); 7.4

(t, 1 H, J = 13 Hz, δ-proton of the bridge): 7.2 (d, 2H, J = 7.7 Hz, 7-H, 7'-H); 6.35 (t. 2H, J = 13 Hz, β, and β' protons of the bridge, 6.15 (d, 2H, J = 7 Hz, α '-H ); 4.1 (broad signal, 4H, (N-CH₂) ); 1 -8-2.2 (m, 8H, alkyl and CH₂COOH); 1.2-1.7 (m, 24H, 6 -CH2, 4 -CH₃); 0.45-0.8 (m, 4H, alkyl). Rf = 0.26 (RP-C18, water/methanol- 7.5:2.5). 12. Succinimidyl Esters of Carboxyalkyl Cyanine Dyes

The following general procedure was used to prepare succinimidyl esters of Cy3.24.OH, Cy3.10.OH, NCy3, NSCy3, Cy7.18.OH, and NSCy7. A procedure for making succinimidyl active ester using disuccinimidyl carbonate (DSC) has been described by Ogura, H., Kobayashi, T., Keiko, S., Kawabe. K. and Takeda R. (1979) A novel active ester synthesis reagent (N,N'- Disuccinimidyl carbonate). Tetrahedron Lett. 4745-4746.

In a typical experiment carboxyalkyl indocyanine was dissolved in mixture of dry DMF (2 mL/100 mg dye) and dry pyridine (0.1 mL/100 mg dye). Disuccinimidyl carbonate (DSC) (1.5 eq/carboxyl group) was added and the mixture was stirred at

55-60° C for 90 min. under nitrogen. After diluting the mixture with dry ethyl ether, the supernatant was decanted. The product was either washed repeatedly with solvent or dissolved in DMF and reprecipitated. Nearly quantitative yields of cyanine active esters were obtained. The formation of the active succinimidyl ester was confirmed by its reaction with benzylamine in DMF or its reaction with taurine in a pH 9.4 bicarbonate buffer. Reversed phase C18 TLC spotted with the conjugate, the succinimidyl ester and hydrolyzed carboxylate product for comparison and developed with water-methanol mixture. Since activation was sometimes incomplete, reverse- phase HPLC was also used to determine the percentage of fluorochrome in the active ester form. The sample was eluted through an Alltech Econosphere 250 mm x 4.6 mm C18 RP column using a mixture of 25% acetonitrile and 75% water containing 0.1% trifluoroacetic acid. The percentages of activated and unactivated cyanine fluorohore were determined by integration of the absorbance signals from a Varian UV/VIS detector.

Alternative procedures for preparing N-hydroxysuccinimidyl esters of carboxyalkyl cyanine dyes are described hereinafter, for example, see Examples 13 - 16. 13. Synthesis and activation of 2-{(1 E,3E.5E)-5-r3-(5-carboxypentvπ-3-methyl-5- sulfo-1.3-dihvdro-2 -/-indol-2-ylidene1penta-1.3-dienyll-3.3-dimethyl-5-sulfo-1-(4- sulfobutvπ-3/-/-indolium

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13.1 6-(2,3-Dimethyl-5-sulfo-3/-7-indol-3-yl)hexanoic acid

4-Hydrazinobenzenesulfonic acid (1.88g, 10mmol), 7-methyl-8-oxononanoic acid (2.8g, 15mmol) and glacial acetic acid (10ml) were mixed and heated under reflux for 6hrs. The solvent was then evaporated under vacuum and the residue triturated with diethyl ether until a solid was obtained. This was dried under vacuum to give crude product, 3.4g (100%). This was purified by preparative HPLC as required (RPC18. Water+0.1 %TFA→MeCN+0.1 %TFA gradient). UV/Vis

(Water+0.1 %TFA): 274, 229, 204nm. ¹H-nmr (D₂O) δ 0.6-0.9 (2H, broad m), 1.10- 1.25 (2H, broad m), 1.35-1.50 (2H, m), 1.60 (3H, s), 2.10-2.40 (2H, broad m + 2H, t), 7.77 (1 H, d), 7.97 (1 H, dd) and 8.06 (1 H, d) MS (MALDI-TOF) MH+ 340.

13.2 4-(2.3.3-Trimethyl-5-sulfo-3H-indolium-1-yl)butane-1-sulfonate A mixture of potassium 2,3,3-trimethyl-3 -/-indole-5-sulfonate (5.50g, 20mmol), 1 ,4-butanesultone (4.0ml, 40mmol) and 1 ,2-dichlorobenzene (100ml) was mixed and heated at 140°C for 24 hrs and then 175°C for 6hrs. After cooling, the magenta solid was collected by filtration, washed with ethyl acetate and dried under vacuum to give 7.97g (97%) of product. Used without further purification. ¹H-nmr (D₂O) δ 1.62 (6H, s), 1.9-2.0 (2H, m), 2.1-2.2 (2H, m), 2.89 (2H,t), 4.55 (2H, t), 7.98 ( H, d), 8.08 (1 H, d) and 8.12 (1 H, d).

13.3 4-f2-r(1 E,3EV4-Anilinobuta-1 ,3-dienyl1-3,3-dimethyl-5-sulfo-3r7-indolium-1 - yl)butane-1 -sulfonate

A mixture of 4-(2,3,3-trimethyl-5-sulfo-3H-indolium-1 -yl)butane-1 -sulfonate (6.20g), malonaldehyde bis(phenylimine) monohydrochloride (7.8g), triethylamine (5ml) and acetic acid (50ml) was heated at 120°C for 18hrs to give a dark brown-red solution. The solvent was evaporated under vacuum and the residue semi-purified by flash chromatography (silica. Acetic acid/ethanol/water mixtures). Final purification was by HPLC (RPC18. Water+0.1 %TFA-&gt;MeCN+0.1 %TFA gradient). UV/Vis (Water, 50: MeCN, 50: TFA, 0.1) 521 nm. ¹H-nmr (DMSO) δ 1.68 (6H, s), 1.7- 1.9 (4H, broad m), 2.60 (2H, t), 4.14 (2H, t), 6.36 (1 H, t), 6.56 (1 H, d), 7.21 (1H, m), 7.3-7.5 (5H, m), 7.65 (1 H, m), 7.83 (1 H, s), 8.39 (1 H, t), 8.83 (1 H, t) and 11.7 (1 H, d). MS (MALDI-TOF) M+ 504.

13.4 2-((1E.3E.5E)-5-r3-(5-Carboxypentyl)-3-methyl-5-sulfo-1.3-dihvdro-2/-/-indol-2- ylidene1penta-1.3-dienyl)-3.3-dimethyl-5-sulfo-1-(4-sulfobutylV3H-indolium

4-{2-[(1 E,3E)-4-anilinobuta-1 ,3-dienyl]-3,3-dimethyl-5-sulfo-3H-indolium-1 - yl}butane-1 -sulfonate (250mg) and 6-(2,3-dimethyl-5-sulfo-3H-indol-3-yl)hexanoic acid (312mg) were mixed in pyridine (9ml): acetic acid (9ml) : acetic anhydride (2ml) and stirred at ambient temperature overnight. After evaporation of solvent under vacuum, the residue was then purified twice by preparative HPLC (RPC18. Water+0.1 %TFA-»MeCN+0.1 %TFA gradient). The appropriate fractions were pooled and evaporated under vacuum. The residue was redissolved in water and freeze-dried to give the purified dye (135mg). Observed pKa in aqueous phosphate buffers (via fluorescence): 7.0. UV/Vis (Water+0.1%TFA): 645nm (ε = 2.0x10⁵ l mol^"¹). MS (MALDI-TOF) MH+ 751. ¹H-nmr (D₂O) δ 0.5-0.65 (1 H, broad), 0.75-0.9 (1 H, broad), 1.1 92H, m), 1.35 (2H, m), 1.5 (3H, s), 1.65 (6H, 2xs), 1.75-2.05 (6H, m), 2.1 (2H, t), 2.95 (2H, t), 4.05 (2H, broad t), 6.0 (1H, d), 6.2 (1H, d), 6.5 (1 H, t), 7.2 (1 H, d), 7.3 (1 H, d) and 7.1-8.1 (6H, m).

13.5 2-{(1E,3E.5E)-5-r3-(5-Carboxypentyl)-3-methyl-5-sulfo-1.3-dihvdro-2 -/-indol-2- ylidene1penta-1.3-dienyl)-3.3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3rV-indolium, N- hydroxysuccinimidyl ester

<img file="WO2004039894A2_D0047.tif" />

Carboxy dye (30mg) was dissolved in anhydrous DMF (300μl); to this were added O-(N-succinimidyl)-N,N,N\N'-tetramethyleneuronium hexafluorophosphate (HSPyU, 60mg) and diisopropylethylamine (42μl). The resulting solution was mixed for 2hrs, whereupon TLC (RPC18. Water/MeCN/AcOH) revealed complete reaction. The reaction mix was evaporated under vacuum and the residue was purified by preparative HPLC (Water+0.1 % AcOH -&gt; MeCN+0.1 % AcOH gradient). Fractions containing the principal dye peak were pooled and evaporated under vacuum; the residue was redissolved in water and freeze-dried. UV/Vis (Water, 50: MeCN, 50: TFA, 0.1 ) 646nm. MS (MALDI-TOF) MH+ -850. 14. Synthesis and activation of 3-,5-carboxypentyl)-2-..1 ,3E,5E)-5-(5,7-dichloro- 3.3-dimethyl-1 ,3-dihvdro-2r7-indol-2-ylidene)penta-1.3-dienvn-3-methyl-5-sulfo-1 -(4- sulfobutyl)-3r.'-indoliuιτι

<img file="WO2004039894A2_D0048.tif" />

14.1 4-f3-(5-Carboxypentyl)-2.3-dimethyl-5-sulfo-3H-indolium-1-yl1butane-1- sulfonate

6-(2,3-Dimethyl-5-sulfo-3H-indol-3-yl)hexanoic acid (340mg) and sodium acetate (100mg) were dissolved in methanol (5ml), then the solvent was evaporated under vacuum. The residue was treated with 1 ,4-butanesultone (680mg) and 1 ,2- dichlorobenzene (5ml); this mixture was heated at 150°C for 18hrs under nitrogen. A further aliquot of 1 ,4-butanesultone (500mg) was added and heating continued for 4hrs. After cooling, the solid product was collected by vacuum filtration, washed with dichlorobenzene and diethyl ether and dried. Crude yield 557mg. After purification by preparative HPLC (RPC18. Water+0.1 %TFA -&gt; MeCN+0.1 %TFA gradient), the desired product was isolated. ¹H-nmr (DMSO) δ 0.6-0.8 (2H, m), 1.05-1.12 (2H, m), 1.43 (2H, app. quin), 1.64 (3H, s), 1.95 (2H, aqq. quin), 2.05-2.42 (4H, m + 2H, t), 3.01 (2H, t), 4.60 (2H, t), 7.97 (1 H, d), 8.09 (1 H, dd) and 8.13 (1 H, d). LCMS : MH+ 476 14.2 4-(2-r(1 E.3E -4-Anilinobuta-1 ,3-dienvn-3-(5-carboxypentyl)-3-methyl-5-sulfo- 3/-/-indolium-1 -yl)butane-1 -sulfonate

4-[3-(5-Carboxypentyl)-2,3-dimethyl-5-sulfo-3H-indolium-1-yl]butane-1- sulfonate (410mg), malonaldehyde bis(phenylimine) monohydrochloride (500mg), triethylamine (0.3ml) and acetic acid (3ml) were heated at 120°C for 18hrs to give a dark brown-red solution. The solvent was evaporated under vacuum and the residue semi-purified by HPLC (RPC18. Water+0.1%TFA→MeCN+0.1 %TFA gradient). The product was then used directly. UV/Vis (Water, 50: MeCN, 50: TFA, 0.1) 523nm. MS (MALDI-TOF) MH+ 605.

14.3 5,7-Dichloro-2,3.3-trimethyl-3H-indole

2,4-Dichlorophenylhydrazine.HCI (3.0g), 3-methyl-2-butanone (3.0ml) and acetic acid (25ml) were mixed and heated at 120°C for 3hrs. A dark orange solution resulted. The solvent was evaporated under vacuum; the residue was partitioned between ethyl acetate and aqueous sodium hydrogen carbonate solution. The organic phase was collected, dried (MgSO ), filtered and the solvent evaporated to give a dark oil. Purification by flash chromatography (silica: ethyl acetate/hexane) gave the product as a light orange oil, which solidified on standing. ¹H-nmr (CDCI₃) δ 1.31 (6H, s), 2.32 (3H, s), 7.15 (1H, d), 7.26 (1H, s) and 7.32 (1H, d).

14.4 3-(5-carboxypentyl)-2-r(1E.3E.5E)-5-(5J-dichloro-3₁3-dimethyl-1.3-dihydro- 2/-/-indol-2-ylidene)penta-1.3-dienvn-3-methyl-5-sulfo-1-(4-sulfobutyl)-3/-/-indolium

4-{2-[(1 E,3E)-4-Anilinobuta-1 ,3-dienyl]-3-(5-carboxypentyl)-3-methyl-5-sulfo- 3H-indolium-1-yl}butane-1 -sulfonate (250mg) and 5,7-dichloro-2,3,3-trimethyl-3/-/- indole (230mg) were mixed with acetic anhydride (1.0ml) and DMF (10.0ml). The mixture was stirred under nitrogen for 18hrs, then at 50°C for 24hrs. The solvent was then evaporated under vacuum; the residue triturated with ether, dried and purified by preparative HPLC (RPC18. Water+0.1%TFA-»MeCN+0.1%TFA gradient). The appropriate fractions were pooled and evaporated under vacuum to give the product dye, 84mg. Observed pKa in aqueous phosphate buffers (via fluorescence): 6.05 UV/Vis (Water, 50: MeCN, 50: TFA, 0.1) 646nm. MS (MALDI-TOF) MH+740.

14.5 3-(5-carboxypentvn-2-r(1E.3E.5a-5-(5.7-dichloro-3.3-dimethyl-1 ,3-dihvdro- 2/-/-indol-2-ylidene)penta-1.3-dienvn-3-methyl-5-sulfo-1 -(4-sulfobutvO-3H-indolium, N- hydroxysuccinimidyl ester

Carboxy dye (24mg) was dissolved in anhydrous DMF (2ml) and evaporated under vacuum to ensure dryness. O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, 30mg) was added, along with diisopropylethylamine (25μl) and DMF (2ml). The red solution was allowed to stand for 1 hr, whereupon TLC (RPC18. Water/MeCN/AcOH) revealed complete reaction. The reaction was quenched by addition of acetic acid (50μl) and the solvent evaporated under vacuum. The residue was purified by preparative HPLC (Water+0.1 % AcOH - MeCN+0.1 % AcOH gradient). Fractions containing the principal dye peak were pooled and evaporated under vacuum; the residue was redissolved in water and freeze-dried. UV/Vis (Water, 50: MeCN, 50: TFA, 0.1 ) 646nm. MS (MALDI-TOF) MH+ 837.

15. Synthesis and activation of 2-f(1 E.3E.5E)-5-r3-(5-carboxypentvn-3-methyl-5- sulfo-1.3-dihvdro-2/-/-indol-2-ylidenelpenta-1.3-dienylV5-chloro-3.3-dimethyl-1-(4- sulfobutv0-3/-/-indolium

<img file="WO2004039894A2_D0049.tif" /> 15.1 5-Chloro-2.3.3-trimethyl-3H-indole

4-Chlorophenylhydrazine.HCI (5.4g), 3-methyl-2-butanone (6.4ml) and acetic acid (70ml) were mixed and heated to 80°C initially, to give a solution. The temperature was then raised to 120°C over 2hrs, during which time a solid separated. TLC analysis (RPC18. Water/MeCN/TFA) indicated consumption of the hydrazine starting material and generation of a single main product. After evaporation of the solvent under vacuum, the residue was partitioned between ethyl acetate and aqueous sodium hydrogen carbonate solution. The organic phase was collected, dried (MgSO₄), filtered and the solvent evaporated to give the crude indolenine. Purification by flash chromatography (silica. Ethyl acetate/hexane) gave 4.66g (80%) of pure product. MS (MALDI-TOF) M+ 193, 195. ¹H-nmr (CDCI₃) δ 1.30 (6H, s), 2.27 (3H, s), 7.23-7.29 (2H, m) and 7.44 (1 H, d).

15.2 4-(5-Chloro-2.3,3-trimethyl-5-sulfo-3/-/-indolium-1-vnbutane-1-sulfonate

5-Chloro-2,3,3-trimethyl-3H-indole (1.94g) and 1 ,4-butanesultone (5.0g) were mixed in 1 ,2-dichlorobenzene (15ml). The resulting solution was heated under nitrogen to 140°C for 18hrs, during which time a solid separated. After cooling to ambient temperature, this solid was collected by filtration, washed with dichlorobenzene followed by excess diethyl ether and then dried under vacuum to give the desired product. Yield 2.85g (86%). MS (MALDI-TOF) MH+ 330, 332. ¹H-nmr (CD₃OD) δ 1.61 (6H, s), 1.95-1.98 (2H, m), 2.08-2.18 (2H, m), 2.88.(2H, t), 4.53 (2H, t), 7.65 (1 H, dd), 7.86 (1 H, d) and 7.93 (1 H, d)

15.3 4-I2-IY1 E.3E)-4-Anilinobuta-1 ,3-dienyll-5-chloro-3.3-dimethyl-3/-/-indolium-1 - yl) butane-1 -sulfonate

4-(5-Chloro-2,3,3-trimethyl-5-sulfo-3H-indolium-1-yl)butane-1 -sulfonate (0.99g), malonaldehyde bis(phenylimine).HCI (1.55g), triethylamine (1.0ml) and acetic acid (10.0ml) were mixed and heated at 120°C for 18hrs to give a dark purple solution. After evaporation of solvent, the crude reaction product was purified by flash chromatography (silica: MeOH/DCM) to give 1.12g of pure product. UV/Vis (EtOH): 525nm. MS (MALDI-TOF) M+ 458, 460.

15.4 2-((1E.3E.5E -5-r3-(5-Carboxypentyl)-3-methyl-5-sulfo-1.3-dihvdro-2H-indol-2- ylidenelpenta-1.3-dienyl&gt;-5-chloro-3.3-dimethyl-1-(4-sulfobutvπ-3/-/-indolium

4-{2-[(1 E,3E)-4-Anilinobuta-1 ,3-dienyl]-5-chloro-3,3-dimethyl-3H-indolium-1 - yl}butane-1 -sulfonate (50mg), 6-(2,3-dimethyl-5-sulfo-3fV-indol-3-yl)hexanoic acid (50mg), pyridine (2.25ml), acetic acid (2.25ml) and acetic anhydride (0.50ml) were mixed and incubated at 90°C for 1 hr, giving a deep blue solution. The solvent was then evaporated under vacuum and the residue triturated with diethyl ether. Purification by preparative HPLC (RPC18. Water/MeCN/TFA) yielded two blue components, the first-eluting component having the desired molecular mass and pH- sensitivity. Fractions containing this component were pooled and evaporated under vacuum to give the product dye, 16mg. Observed pKa in aqueous phosphate buffers (via fluorescence): 7.44. UV/Vis (Water, 50: MeCN, 50: TFA, 0.1) 645nm. MS (MALDI-TOF) M+ 704, 706.

15.5 2-.(1E.3E,5E)-5-r3-(5-carboχypentvn-3-methyl-5-sulfo-1.3-dihvdro-2H-indol-2- ylidenelpenta-1 ,3-dienyl)-5-chloro-3,3-dimethyl-1 -(4-sulfobutvπ-3/- -indolium. N- hydroxysuccinimidyl ester

<img file="WO2004039894A2_D0050.tif" />

The carboxy dye (13.5mg) was dissolved in anhydrous DMF (2ml) and evaporated under vacuum to ensure dryness. O-(N-Succinimidyl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TSTU, 30mg) was added, together with diisopropylethylamine (25μl) and DMF (2ml). The orange solution was allowed to stand for 1hr, whereupon TLC (RPC18. Water/MeCN/AcOH) revealed complete reaction. The reaction was quenched by addition of acetic acid (5Oμl) and the solvent evaporated under vacuum. The residue was purified by preparative HPLC (Water+0.1 % AcOH → MeCN+0.1 % AcOH gradient). Fractions containing the principal dye peak were pooled and evaporated under vacuum; the residue was redissolved in water and freeze-dried. UV/Vis (Water, 50: MeCN, 50: TFA, 0.1) 645nm. MS (MALDI-TOF) M+ 801 , 803.

16. Synthesis and activation of 3-(5-carboχypentyl)-2-r(1E3E,5E)-5-(7-chloro-3.3- dimethyl-5-sulfo-1.3-dihvdro-2H-indol-2-ylidene enta-1 ,3-dienvn-3-methyl-5-sulfo-1- (4-sulfobutvπ-3H-indolium

<img file="WO2004039894A2_D0051.tif" />

16.1 7-Chloro-2,3,3-trimethyl-3H-indole-5-sulfonic acid

2-Chioroaniline-4-sulfonic acid (2.5 g, 9.2 mmol) was dissolved in HCI (cone;

40 ml) and cooled to 0 °C. A solution of sodium nitrite (1.26 g, 18.3 mmol) in H₂O (10 ml) was transferred to a dropping funnel and added dropwise to the reaction vessel over 2 h. After stirring for 2 h at 0 °C, stannous chloride (8.3 g, 36.9 mmol; dissolved in 10 ml cone. HCI) was added dropwise over 2 h. The reaction mixture was then allowed to warm to room temperature and stirred overnight. The reaction mixture was then filtered and the filtrate discarded. The isolated solid was then transferred to a round-bottomed flask and treated with acetic acid (15 ml), potassium acetate (3 g) and 3-methyl-2-butanone (3 ml). After heating the vessel at 140 °C for 4 h, the reaction mixture was concentrated in vacuo and the resultant gum purified by RP- HPLC (Phenomenex Synergi; 10u hydro-RP 80, 250 x 21.20 mm; MeCN:H₂O; 0- 100% MeCN 30 min; 10 ml/min, RT = 14 min) to isolate the desired product.(550 mg; 2 mmol, 22%). MS (MALDI-TOF) MH⁺ 273. 16.2 3-(5-Carboxypentyl)-2-r(1E.3E.5E)-5-(7-chloro-3.3-dimethyl-5-sulfo-1.3- dihvdro-2H-indol-2-ylidene)penta-1 ,3-dienvn-3-methyl-5-sulfo-1-(4-sulfobutvO-3H- indolium

To a solution of 7-chloro-2,3,3-trimethyl-3H-indole-5-sulfonic acid (50 mg; 0.18 mmol) in acetic acid/pyridine/acetic anhydride (4.5:4.5:1 ; 10 ml) was added (2E)-3-(5- carboxypentyl)-7-chloro-3-methyl-1-pentyl-2-[(2E,4E)-4-(phenylimino)but-2- enylidene]indoline-5-sulfonate (110 mg; 0.18 mmol). The reaction mixture was heated at 60 °C for 4 h then concentrated in vacuo to yield the crude dye. The resultant gum was purified by prep RP-HPLC (Phenomenex Synergi; 10u hydro-RP 80, 250 x 21.20 mm; MeCN:H₂O; 0-100% MeCN 30 min; 10 ml/min, RT = 16 min) to yield the desired product (10 mg; 0.01 mmol; 7%). MS (MALDI-TOF) MH⁺ 783

16.3 3-(5-Carboxypentvn-2-r(1E.3E.5EV5-(7-chloro-3.3-dimethyl-5-sulfo-1.3- dihvdro-2/-.-indol-2-ylidene)penta-1 ,3-dienvn-3-methyl-5-sulfo-1-(4-sulfobutvπ-3/-/- indolium, N-hydroxysuccinimidyl ester

<img file="WO2004039894A2_D0052.tif" />

To a stirred solution of 6-[(2E)-2-[(2E,4E)-5-(7-chloro-3,3-dimethyl-5-sulfo-3H- indol-2-yl)penta-2,4-dienylidene]-3-methyl-5-sulfo-1-(4-sulfobutyl)-2,3-dihydro-1/-.- indol-3-yrjhexanoic acid (9 mg; 0.01 mmol) in DMF (3 ml) was added DIPEA (2 ml) followed by O-(N-succinimidyl-N,N,N',N'-bis-tetramethylene)uronium hexafluorophosphate (14 mg; 0.05 mmol). After 4 h, the reaction mixture was concentrated in vacuo and the resultant gum purified by RP-HPLC (Phenomenex Synergi; 10u hydro-RP 80, 250 x 21.20 mm; MeCN:H₂O; 0-100% MeCN 30 min; 10 ml/min, RT = 17 min) to isolate the product (6 mg; 0.007 mmol; 53%). Observed pKa in aqueous phosphate buffers (via fluorescence): 6.2. MS (MALDI-TOF) M+Na⁺ 902

17. Synthesis of 3-(5-carboxypentvn-3-methyl-2~f(1 E.3E.5EV5-[3-methyl-5-sulfo- 3-(4-sulfobutyl)-1 ,3-dihvdro-2/-/-indol-2-ylidene1penta-1 ,3-dienyl)-5-sulfo-1 -(4- sulfobutvD-3H-indolium

<img file="WO2004039894A2_D0053.tif" />

17.1 5-Methyl-6-oxoheptane-1 -sulfonic acid

Ethyl 2-methylacetoacetate (43.2g) in N,N-dimethylformamide (25ml) was added to a suspension of sodium hydride (12.0g of 60% NaH in mineral oil) in N,N- dimethylformamide (100ml), dropwise with ice-bath cooling. This mixture was allowed to warm to ambient temperature for 30mins before re-cooling. 1 ,4- butanesultone (40.8g) in N,N-dimethylformamide (25ml) was then added dropwise. The final mixture was heated at 50°C for 18hrs, then quenched with 50ml of water. The solvent was evaporated under vacuum; the residue was partitioned between water and diethyl ether. The aqueous layer was collected, washed with fresh diethyl ether and evaporated under vacuum; final drying was under high vacuum over phosphorus pentoxide. A yield of 103g of 5-(ethoxycarbonyl)-5-methyl-6- oxoheptane-1 -sulfonic acid was obtained.

This intermediate was dissolved in concentrated hydrochloric acid (200ml) and reacted at 90°C for 3hrs, then the solvent was evaporated under vacuum. The residue was purified by flash chromatography (silica. Dichloromethane - ethanol). Fractions containing the product were pooled and evaporated under vacuum to give the title compound, 49.6g. ¹H-nmr (D₂O) 61.05 (3H, d), 1.3-1.8 (6H, m), 2.20 (3H, s), 2.65 (1 H, m), and 2.90 (2H, m)

17.2 2.3-Dimethyl-3-(4-sulfobutyl)-3 -/-indole-5-sulfonic acid

4-Hydrazinobenzenesulfonic acid (1.88g), 5-methyl-6-oxoheptanesulfonic acid (2.5g) and acetic acid (50ml) were mixed and heated under reflux for 6hrs. The solvent was evaporated under vacuum, then the residue was triturated with 2- propanol to yield the crude product as a yellow solid. This was purified by HPLC as required (RPC₁₈. Water + 0.1 % TFA). ¹ H-nmr (D₂O) δ0.8-1.0 (2H, m), 1.55-1.65 (5H, =3H, s + 2H, m), 2.16 (1 H, ddd), 2.29 (1 H, ddd), 2.75 (2H, m), 2.81 (partially d- exchanged methyl singlet), 7.71 (1 H, d), 7.94 (1 H, d) and 8.01 (1 H, d). UV/Vis (Water+0.1 %TFA): 269, 229nm. MS (LCMS) : MH⁺ 362. Ace. Mass: Found, 362.0729. MH⁺ = C₁₄H₂oNO₆S₂ requires 362.0732 (-O.δppm).

17.3 3-(5-Carboxypentyl)-3-methyl-2-{(1 E,3E,5E)-5-r3-methyl-5-sulfo-3-(4- sulfobutyl)-1 ,3-dihvdro-2/-/-indol-2-ylidenelpenta-1 ,3-dienyl)-5-sulfo-1 -(4-sulfobutvO- 3/-/-indolium

4-{2-[(1E,3E)-4-Anilinobuta-1 ,3-dienyl]-3-(5-carboxypentyl)-3-methyl-5-sulfo- 3H-indolium-1-yl}butane-1 -sulfonate (made as in Example 14.2, 50mg) and 2,3- dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid (50mg) were mixed in pyridine (2.25ml), acetic acid (2.25ml) and acetic anhydride (0.50ml) and stirred at ambient temperature under nitrogen for 18hrs, then for 2hrs at 60°C. The resulting green- blue solution was evaporated under vacuum. The residue was purified by HPLC (RPC-_I8. Water / MeCN / TFA gradient). Fractions containing the principal dye peak were collected, pooled and evaporated under vacuum to give the title dye, 22mg. UV/Vis (Water+0.1%TFA): 651 nm. MS (MALDI-TOF): M+ 872.

18. Synthesis of 6-,9-(5-carboxypentylV5,7-dimethyl-5,11-disulfo- 1.2.9.14.15.16.15a.16a.2a-nonahvdroindolo-f2".1"-1',2'lisoquinolinor7',6'- 4,3]pyridinoπ .2-a1indolin-7-yl/hexanoic acid (rigid Cv3)

<img file="WO2004039894A2_D0054.tif" />

To a stirred solution of 6-(2,3-dimethyl-5-sulfo-3-hydroindol-3-yl)hexanoic acid (150 mg, 0.45 mmol) in ethanol (20 ml) at ambient temperature was added hydrobromic acid (3ml, 40% aqueous solution). After 1 hr the reaction solvent was removed in vacuo. The hydrobromide salt was redissolved in acetonitrile (20 ml) and acetic acid (200 ml) and acrolein diethyl acetal (10 g, 75 mmol) was added. The reaction mixture was heated to 70 °C. for 20 minutes. The solution was cooled and the solvent removed in vacuo. The product was rapidly purified on reverse phase C18 column using 1 :1 water/acetonitrile (containing 0.1 % TFA) as solvent. All fractions were monitored by RP C18 TLC. Fractions containing product were pooled. Evaporation of the solvent gave a sticky mass (120 mg, 36%). It was immediately used for the next reaction. To a stirred solution of 6-[1-(3,3-diethoxypropyl)-2,3-dimethyl-5-sulfo-3- hydroindole-3 yljhexanoic acid (100 mg, 0.21 mmol) in pyridine (5 ml) at 120 °C was added dropwise, triethyl orthoformate (100 mg, 67 mmol) over 30 minutes. After 2 hrs the reaction mixture was cooled. The product was purified by reversed phase C18 column using water-acetonitrile mixture (containing 0.1 % TFA) as solvent. The product was obtained as a pink solid (45 mg, 40%); λ max: 560 nm water, λem 570 nm, φ0.09

To a stirred solution of 1 ,1-di-(3,3-diethoxypropyl)-3-methyl-3'(6- carboxypentenyl)-indocarbocyanine (40 mg, 0.04 mmol) in ice water was added 50% aqueous sulphuric acid (2 mi). The reaction mixture was heated at 40 °C for 30 minutes. The mixture was cooled and neutralized with triethylamine. Solvent was removed under vacuum on a flash evaporator was obtained as red solid, λmax 565 nm, λem 584nm, φ 0.8.

19. Synthesis of 6,7.9, 10-tetrahvdro-2. 14-disulphonato-16,16,18-trimethyl-

7aH.8aH-bisindQliniumr3,2-a.3'2'-alPyranof3.2-c:5.6-c'1dipyridin-5-ium-18-hexanoic acid

<img file="WO2004039894A2_D0055.tif" /> 19.1 1-(3.3-Diethoxypropyl)-3-(6-ethoxy-6-oxohexyl)-2.3-dimethyl-3H-indolium-5- sulfonate

<img file="WO2004039894A2_D0056.tif" />

6-(2,3-Dimethyl-5-sulfo-3/V-indol-3-yl)hexanoic acid was synthesised as described in Example 5. To a stirred solution of 6-(1-ethyl-2,3-dimethyl-5-sulfσ-3/-/- indol-3-yl)hexanoic acid (1.8g, 5.3 mmol) in ethanol (200ml) at ambient temperature was added hydrobromic acid (10ml 48% aqueous solution). After 1 Hr the reaction solvent was removed in vacuo. The hydrobromide salt was re-dissolved in acetonitrile (150ml) and acetic acid (1.54ml) and acrolein diethyl acetal (6.6g, 51 mmol). The reaction mixture was heated to 70°C for 30 minutes. The solution was cooled and the solvent removed in vacuum. The product was rapidly purified by preparative HPLC on reverse phase C18 column using gradient elution of water containing 10% acetonitrile to acetonitrile (containing 0.1% TFA) as solvent over 30 minutes. All fractions were monitored by MALDI -TOF mass spectrometry (M/Z = 498). Fractions containing product mass were pooled. Evaporation of the solvent gave a sticky mass (162mg 11 %). It was immediately used for the next reaction. 19.2 1-(3.3-Diethoxypropyl)-2.3.3-trimethyl-3H-indolium-5-sulphonate

<img file="WO2004039894A2_D0057.tif" />

Potassium 2,3,3-trimethyl-3/-/-indole-5-sulfonate was synthesised following the procedure described in Mujumdar, R, B Bioconjugate Chem., (1993), 4(2), 105-111. To a stirred solution of potassium 2,3,3-trimethyl-3/-/-indole-5-sulfonate (5g, 18mmol) in ethanol (250ml) at ambient temperature was added hydrobromic acid (50ml 48% aqueous solution). After 1 Hr the reaction solvent was removed in vacuo. The hydrobromide salt was re-dissolved in acetonitrile (200ml) and acetic acid (5ml) and acrolein diethyl acetal (42.25g, 325mmol). The reaction mixture was heated to 70 °C for 60 minutes. The solution was cooled and the solvent removed in vacuum. The product was rapidly purified by preparative HPLC on reverse phase C18 column using gradient elution of water containing 10% acetonitrile to acetonitrile over 60 minutes (containing 0.1% TFA) as solvent. All fractions were monitored by MALDI - ToF mass spectrometry (M/Z 369). Fractions containing product mass were pooled. Evaporation of the solvent gave a sticky mass (1.8g 36%). It was immediately used for the next reaction.

19.3 1 -(3.3-Diethoxypropyl)-2-{(1 E,3E,5Z)-5-.1 -(3,3-diethoxypropylV3.3-dimethyl-5- sulfonato-1 ,3-dihvdro-2/-/-indol-2-ylidene1penta-1.3 dienyl)-3-(6-ethoxy-6-oxohexy0-3- methyl-3fV-indolium-5-sulfonate

<img file="WO2004039894A2_D0058.tif" />

To 120mg (0.325mmol) of 1 -(3,3-diethoxypropyl)-3-(6-ethoxy-6-oxohexyl)-2,3- dimethyl-3H-indolium-5-sulponate was added pyridine (5ml) to dissolve. To 162mg (0.325mmol) 1 -(3,3-diethoxypropyl)-2,3,3-trimethyl-3H-indolium-5-sulphonate was added pyridine (5ml) to dissolve. The contents of the above flasks were combined, stirred and triethylorthoformate (385mg, 2.6mmol) added. The reaction mixture was heated to 120 °C for 5 Hr. The solution was cooled and the solvent removed in vacuum. The product was purified by preparative HPLC on reverse phase C18 column using gradient elution of water to acetonitrile over 90 minutes (containing 0.1% TFA) as solvent. All fractions were monitored by MALDI -TOF mass spectrometry (M/Z=1006). Fractions containing product mass were pooled. Evaporation of the solvent gave a pink solid (78 mg 13%). It was immediately used for the next reaction. 19.4 6,7,9, 10-tetrahvdro-2, 14-disulphonato-16.16.18-trimethyl-7aH.8aH- bisindoliniumr3.2-a.3'2'-a1pyranor3.2-c;5.6-c'1dipyridin-5-ium-18-hexanoic acid

78mg (8.88 x10^"5 mol) of 1-(3,3-diethoxypropyl)-2-{(1 ,3E,5Z)-5-[1-(3,3- diethoxypropyl)-3,3-dimethyl-5-sulfonato-1 ,3-dihydro-2rV-indol-2-ylidene]penta-1 ,3 dienyl}-3-(6-ethoxy-6-oxohexyl)-3-methyl-3/-/-indolium-5-sulfonate was dissolved in sulphuric acid (2ml 48% aqueous solution) and stirred at ambient temperature for 4 hours. The solvent was removed under vacuum. The product was purified by preparative HPLC on reverse phase C18 column using gradient elution over 90 minutes of water to acetonitrile (containing 0.1 % TFA) as solvent. All fractions were monitored by MALDI -TOF mass spectrometry. Fractions containing the product mass were pooled. Evaporation of the solvent gave a pink solid (27mg 35%).

20. General protein labeling procedure

The following protein labeling procedure was followed to label sheep lgG with NHS ester of Cy3.10, Cy3.24, NSCy3, Cy7.18 and NSCy7. Stock solutions of the succinimidyl active esters were made in dry DMF (0.3-1.0 mg active ester/100 μL) and are stable for days when stored at 4 °C. The active esters are also stable in distilled water for several hours provided the pH of the solution is not basic. Aqueous solutions of the dyes can be used for labeling antibodies if the use of DMF is not suitable for certain antibodies. The concentration of cyanine fluorophore in the stock solution was determined by measuring the absorbance of an aliquot of the appropriately diluted stock solution in phosphate-buffered saline (PBS) and using the extinction coefficient of the dye. The stock solution concentration of cyanine fluorophore in the active ester form was then determined by reverse-phase HPLC (typically 50-95% but sometimes as low as 35%). The antibody labeling was carried out in 0.1 M carbonate-bicarbonate buffer (pH 9.4) for 15 minutes at room temperature. The sheep lgG (1 mg, 6.45 mM) was dissolved in 0.25 -1 mL buffer solution and the desired amount of dye (e.g., 20 μL of stock containing 0.35mg active ester/100 μL DMF) was added during vigorous vortex mixing. Unconjugated dye was separated from the labeled protein by gel permeation chromatography (0.7 X 20 cm column of Sephadex G-50) using pH 7 buffer solution as eluent.

21. Determination of dye-to-antibody ratio

A simple method for estimating dye/protein (d/p) ratios involves direct measurement of the protein absorbance at 280 nm and dye absorbance at the absorption maximum. Specifically, the dye/protein ratio is calculated using the equation below with measured values of the absorbance of the labeled dye (Cy3 at 550 nm or Cy7 at 750 nm) and the absorbance of protein at 280 nm. The extinction coefficients of Cy3 and Cy7 are approximately 150,000 and 250,000 respectively. The extinction coefficient of the lgG antibody at 280 nm was taken to be 170,000 L/mol-cm. The factor "X" in the denominator accounts for dye absorption at 280 nm which is 0.05 (for both Cy3) and 0.08 (for Cy7) of the absorption of the dye at its maximum absorption (A&lt;j_ve).

D Λiye Eprot

P ^~ (A₂₈₀ - 0.05 A_dye) * E_dye

A more accurate method is needed when the labeling reagent shows significant spectral changes when bound to the antibody molecule. For example, this approach is needed when both dimers and monomers of dye, which have different absorption peaks and different extinction coefficients, are present on the protein at higher d/p ratios. In this case, the labeled protein is dissolved in formamide for absorption spectroscopy and the extinction coefficients of the dye determined independently for the calculation 22. Measurement of pKa values of pH-sensitive cvanine dves

The general method of pKa determination was performed as follows. Purified dye was dissolved in water to give a bulk stock solution (if necessary, a limited amount of acetonitrile was added to ensure solubility). By experiment, the volume of this stock solution that was required to be added to 10.0ml of water + 0.1%v/v trifluoroacetic acid, in order to give an absorbance of 0.5±0.02 at the dye peak wavelength, was determined. This volume of bulk stock solution was then added to 10.0ml of water, to be used as the working stock solution.

Aqueous phosphate buffers were prepared covering the region of pH 4-9 {see "Data for Biochemical Research", p.432, 3^rd edition, R.M.C Dawson, D.C.Elliott, W.H.Elliott &amp; K.M.Jones, 1987 Oxford University Press. Buffers outside the listed range were made by adding in orthophosphoric acid or sodium hydroxide}. Plastic fluorescence cuvettes were then charged with 3.5ml of each buffer. A fixed volume of dye working stock solution (in the range 50-250μl) was added to a cuvette of buffer, the solution mixed and the fluorescence signal measured immediately {Perkin Elmer LS55 fluorimeter, excitation 640nm. Emission spectrum was scanned and fluorescence intensity value at 680nm recorded}. This was repeated for all cuvettes. The graph of fluorescence intensity versus pH was plotted using PRISM, the data fitted to a sigmoidal dose-response curve and the pKa value extracted as the calculated IC50 value from the fitting.

Table 1 lists the pKa values of the carboxy dyes prepared in the above four examples, along with a selection of non-functional dyes (dyes 5-9) that were prepared using the same methodology. These extra examples help to explain the effect of substitution on the observed pKa value of the dye chromophore, and show that this figure can be tuned to any value across the physiological pH range. Table 1

<img file="WO2004039894A2_D0059.tif" /> Table 1 (continued)

<img file="WO2004039894A2_D0060.tif" />

Comparison of dyes 5 and 6 show that substitution of H for sulfonate, in the position para- to the unquaternized nitrogen, has no measurable effect on dye pKa. Furthermore, dye 1 shows that addition of the functional arm onto dye 6 has no measurable effect on dye pKa.

Comparison of the above dye set with dyes 7,4 and 2 shows the effect of incorporating an electron-withdrawing group, such as CI, onto the unquaternized indolenine unit. Substitution of CI para- to the unquaternized nitrogen lowers the dye pKa by 0.3-0.4 units (dye 1-»dye 7). Substitution of CI ortho- to the unquaternized nitrogen lowers the dye pKa by -0.8 units (dye 1-→-dye 4). Incorporating both substitutions gives an additive effect, lowering the pKa by -1.0 unit (dye 1-κJye 2). Dye 8 shows that another electron-withdrawing group, cyano, has a similar effect to chloro. Fluoro and trifluoromethyl act similarly.

Comparison of dyes 9 and 3 with dyes 6, 7 and 1 shows the effect of incorporating an electron-withdrawing group, such as CI, onto the quaternized indolenine unit. Substitution of CI para- to the quaternized nitrogen raises the dye pKa by 0.3-0.4 units. Hence the pKa of these pH-sensitive dyes can be adjusted from that of the base structures, at pKa =7.0, in either direction by the considered placement of electron-withdrawing groups such as chloro (fluoro, cyano, trifluoromethyl).

The contents of references cited herein are incorporated herein by reference in their entirety.