GB2373508A - Long wavelength fluorescent dyes with 2-(2-{2-chloro-3-[2-(2,3-dihydro-1H-indol-2-yliden)ethylidene]cyclohex-1-enyl}eth-1-enyl)-1H-benzo[e]indolium skeleton - Google Patents

Abstract

The present invention relates to compounds comprising:<BR> ```Such compounds may include hydroxyl, carboxyl, isothiocyanate, amine, alcohol, thiol or acid groups. The worked example discloses a compound of formula:<BR> ```These compounds are useful as long wavelength dyes suitable as fluorescent labels for biological/non-biological assays. A composition comprising the compounds above and either a biological molecule or a non-biological molecule are also claimed. The biological molecule may be selected from the group consisting of: proteins; peptides; polypeptides; enzyme; enzyme substrates; hormones; antibodies; antigens; haptens; avidin; streptavidin; carbohydrates; lipids; oligosaccharides; polysaccharides; microbial materials; toxins; virus, bacteria; drugs; nucleic acids; deoxy nucleic acids; fragments of DNA or RNA; cells; synthetic combinations of biological fragments such as peptide nucleic acids (PNAs) and certain other biological molecules; and biological cells and fragments thereof. Such labelled conjugates make possible a variety of assays for biomolecules and biological components, including fluorescent immunoassays, receptor labelling and fluorescence hybridisation assays.

Description

Long wavelength fluorescent dyes

The present invention relates to a new chemical used primarily as a long wavelength fluorescent dye and methods of using the chemical in assays.

Long wavelength dyes can be arbitrarily defined as all fluorophores which emit at wavelengths above 600nm. The principal advantage of working in the long wavelength region is the absence or significant reduction of background signals (e. g. Rayleigh and Raman scattering) that are prevalent in the Ultraviolet/Visible (UV/VIS) region of the spectrum. In addition to the absence or reduction of background signals, working in this region also avoids the use of conventional xenon light sources requiring special power light sources and photomultiplier detectors, which are large, delicate and also require high tension supplies.

In recent years with the advance in optical and electronic instrumentation (LED, laser diodes, CCD, photodiodes and photodiode arrays), instruments have been developed that can excite fluorescence molecules in the 600-900nm range. These instrument are considerably cheaper than the fluorescence instruments used to excite fluorescent materials in the UV/VIS regions of the spectrum i. e.

300-580nm. Although there are a large number of fluorescent materials that can be excited in the UV and VIS region of the spectrum there is a large spectral window between 580nm and 900nm where new labelling reagents need to become available for labelling biological and non-biological materials for analysis with the newly available instrumentation.

Fluorescent dyes with longer wavelength absorbance and emission are particularly useful in conjunction with materials of biological origin such as blood, urine, cells and tissues, where background or inherent fluorescence or absorbance often interferes with detection of a shorter wavelength fluorescent

dye. Furthermore, biological specimens often have decreasing levels of both absorbance and fluorescence emission as the illumination energy approaches the infrared. In addition, numerous biological and non-biological applications of long wavelength dyes exist, including use as laser dyes, using relatively low cost illumination sources such as laser diodes. Consequently, dyes that possess these spectral properties have potential utility in biological and non-biological applications.

It is known to provide water soluble long wavelength dyes with activated functionalities e. g. aryl sulfonated cyanine dyes that are covalently attached to proteins and other biological and non-biological materials to make these materials fluorescent so that they can be detected. The labelled materials have been used in assays employing excitation sources and luminescence detectors.

US-A-5268486, US-A-5627027, US-A-5486616, US-A-5569587, US-A5569766, US-A-6048982 have described the use of aryl sulfonate cyanine dyes as labels for biomolecules. For example, US-A-5268486 discloses and claims fluorescent arylsulfonated cyanine dyes having large extinction coefficients and quantum yields for the purpose of detection and quantification of labelled components. Further dyes are disclosed in US-A-6086737, "Comparison of covalent and non covalent labelling with near-infrared dyes for the highperformance liquid chromatographic determination of human serum albumin" Williams et al, Anal. Chem. (1993), 65,601-605 and Lakowicz et al"The synthesis, spectral properties and photostabilities of symmetrical and unsymmetrical squarines: a new class of fluorophores with long-wavelength excitation and emission"Analytical Chimica Acta, (1993), 282,633-641.

"Synthesis of squarine-N-Hydroxysuccinimide Esters and their biological application as long wavelength fluorescent labels"Terpetsching et al, Analytical Biochemistry (1994), 217,194-204 have also described the use heptamethine cyanines dyes and squarine dyes as labels for the qualitative and quantitative detection of biomolecules. Another group of long wavelength fluorophores dibenzopyrrometheneboron difluorid compounds with emissions ranging from

645nm to 730nm as biolabels have been US-A-5433896. Although the dyes described above i. e. cyanines, squarines and dibenzopyrrometheneboron difluorid compounds are useful, additional long wavelength dyes are sought.

The present invention seeks to provide further long wavelength dyes.

According to the present invention there is provided a new chlorocyclohexene dye useful in labelling biological and non-biological molecules. The dye of the present invention is preferably highly coloured, and can also be detected visually or colorimetrically. Alternatively or additionally the dye can be detected using its fluorescence properties. To observe fluorescence, the sample is first excited by a light source such as a visible lamp, a laser, or LEDs capable of producing light at or near the wavelength of maximum absorption of the dye. Preferably the dye is excited at a wavelengths greater than about 750nm. The fluorescence of the dye is detected qualitatively or quantitatively by detection of the resultant light emission at a wavelength of greater than about 800 nm.

The emission can be detected by methods that include visible inspection, photographic film, or the use of current instrumentation such as fluorimeters, quantum counters, plate readers, fluorescence microscopes and flow cytometers, or by any means for amplifying the signal, such as a photomultiplier or charge-coupled device (CCD).

The preferred compounds and compositions of this invention have sufficient solubility in aqueous solutions that once attached to a soluble biomolecule, the biomolecule retains its solubility. The preferred compounds also have good solubility in organic media, which provides considerable versatility in synthetic approaches to the labelling of desired materials.

A preferred embodiment of the present invention relates to a dye which is highly light absorbing with a molar extinction coefficient greater than 100, 000 litres per mole centimetre and very luminescent with quantum yields of at least 0.2.

As luminescence involves highly sensitive optical techniques, the presence of these dyes as labels can be detected and quantified even when the label is present in very low amounts. Thus the dye labelling reagents can be used to measure and quantify the material that has been labelled.

The present invention describes an alternative chlorocyclohexene dye that can be used as fluorescent labels for biological and non-biological materials including proteins, cells, nucleic acid or DNA, drugs, toxins, microbial materials, particles.

The dyes of the present invention can be employed to determine or quantify the concentration of a variety of proteins or other materials in a system by labelling all of a mixture of the proteins in the system and then separating the labelled proteins by any means, such as chromatography or by heterogeneous means in an immunoassay. The amount of separated proteins that are luminescent can then be determined in a detection system.

A preferred dye compound of the present invention has a maximum light absorbance which occurs near 790nm a. Readily available solid state excitation sources e. g. laser diodes (780nm, 790nm), super bright LED that are compact, reliable and inexpensive and emit light at this wavelength can be used to excite the dyes of the present invention. Suitable commercially available lasers can be obtained, for example, from Photonics Products and Roithnen Lasertechnik.

According to a second aspect of the present invention there is provided a composition comprising the compound of the first aspect of the invention linkable to a (non) biological molecule. This is normally found by the covalent reaction of a highly luminescent and highly tight absorbing chlorocyctohexene dye molecules with the hydroxyl, carboxyl and isothiocyanate groups (i. e. functional groups) to provide linking groups for attachment to a wide variety of (non) biologically important molecules. Examples of biological molecules include proteins, peptides, enzyme substrates, hormones, antibodies, antigens,

haptens, avidin, streptavidin, carbohydrates, lipids, oligosaccharides, polysaccharides, microbial materials, toxins, drugs, nucleic acids, deoxynucleic acids, fragments of DNA or RNA, cells and synthetic combinations of biological fragments such as peptide nucleic acids (PNAs) and certain other biological molecules, biological cells. The dye may also be linked in a similar fashion to non-biological materials such as soluble polymers, polymeric particles and other particles and surfaces.

The dyes of the present invention can be covalently attached to a desired biological or non-biological material to form a conjugate. As used herein, a conjugate is any molecule, substance or material that can be attached to the dye to yield a dye-conjugate. Preferably, because of the relative ease of synthesis, dyes that are substituted by amines, alcohols, thiols or acids are generally utilised to prepare the labelled conjugates. Selected conjugates include, but are not limited to, antibodies, amino acids, proteins, peptides, polypeptides, enzymes, enzyme substrates, hormones, Iymphokines, metabolites, antigens, haptens, lectins, avidin, streptavidin, toxins, poisons, environmental polluants, carbohydrates, oligosaccharides, polysaccharides, glycoproteins, glycolipids, nucleotides, oligonucleotides, nucleic acids and derivatized nucleic acids (including deoxyribo-and ribonucleic acids), DNA, and RNA fragments and derivatized fragments (including single and multi-stranded fragments), natural and synthetic drugs, receptors, virus particles, bacterial particles, virus components, biological cells, cellular components (including cellular membranes and organelles), natural and synthetic lipid vesicles, polymers, polymer particles, polymer membranes, and glass and plastic surfaces and particles.

Such labelled conjugates make possible a variety of assays for biomolecules and biological components, including fluorescent immunoassays, receptor labelling and fluorescence hybridisation assays.

In an additional embodiment of the invention, the dye of the present invention is used to label polymer microparticles. The polymer microparticle can be prepared from a variety of polymers including, but not limited to nitrocellulose, polystyrene (including high density polystyrene latexes such as brominated polystyrene) etc.

A preferred embodiment of the invention will now be described with reference to the accompanying drawings, in which: Fig. 1 shows the formula of a preferred dye species.

The compound of this invention has the general formula shown in Fig. 1. A synthesis and analysis of a preferred compound of the invention is shown below.

Synthesis of 2- ( (E)-2-2-chloro-3Y)-2-r1, 3, 3-trimethyl-2, 3-dihydro- H-indol2-yliden) ethylidenel-cyclohex- I-enyijeth-I-enyl)-3-ethyl-1, 1-dimethyl-IH benzolelindolium Synthesis N-Ethyl-2, 3, 3-trimethyl-4, 5-benzo-indolium iodide (0.52g, 1. 44mmol) was added to a solution of 2-Chloro-1-formyl-3-hydroxymethylene cyclohexene (0. 25g, 1. 44mmol) in a 60: 40 mixture of butanol : toluene (40ml). The reaction solution was purged with nitrogen and then refluxed at 140oC with a Dean-Stark trap for 2 hours. After this time the reaction was allowed to cool to room temperature and a slurry of 1, 2, 3, 3 tetramethyl-3H-indolium iodide (0. 45g, 1. 50mmol) in the reaction solvent mixture was added. The reaction was returned to the heat and refluxed for a further 12 hours. The reaction was allowed to cool and the solvents were removed under reduced pressure to give a dark oil. Purification over silica gel (DCM: methanol 95: 5)

afforded the title compound as a purple/green metallic solid (0. 239g, 24%) AH (CDCI3) was complex with two isomers of the compound present 1.39 (3H, dt, 3 and 7 CH3), 1.73 (6H, s, CH3), 1. 93 (2H, quintet, CH2), 2.01 (6H, d, J 2 CH3), 2. 77 (4H, m, CH2), 3. 70 (3H, s, CH3), 3. 76 (3H, s, CH3), 4.34-4. 45 (4H, 2 x q, J 7, CH2), 6.11-6. 34 (2H, m, CH), 7.16 - 7. 67 (6H, m, Ph), 7.94-8. 02 (2H, m, Ph), 8.14 (2H, d, J 8, Ph), 8.278.56 (2H, m, CH) ; duc (CDCI3) uninterpretable.

Table 1 below shows the spectroscopic properties of the exemplified dye of the present invention.

Dye Spectrophotometry Fluorescence Amax (nm) s (M 11 Excitation (nm) Emission (nm) Compound IV 793 152, 753 793 814 Measurements carried out in methanol

------ The above synthesis can be simply adapted to yield the other compounds of the invention as will be readily apparent to the skilled person. Other known synthetic methods can also be used easily modified by selection of appropriate starting materials and conditions in order to prepare the compounds of the invention.

Claims (15)

1. CLAIMS : 1. A compound comprising :
2. 2. The compound according to any one of the preceding claims, wherein the compound is substantially water soluble.
3. 3. The compound according to any one of the preceding claims, wherein the compound is highly light absorbing and/or with a molar extinction coefficient greater than 100, 000 litres per mole centimetre
4. 4. The compound according to any one of the preceding claims, wherein the compound has is luminescent with quantum yields of at least 0.2.
5. 5. The compound according to any one of the preceding claims, wherein the compound is fluorescent.
6. 6. The compound according to claim 5, wherein the compound is fluorescently excitable at wavelengths greater than about 750nm.
7. 7. The compound according to claim 5 or claim 6, wherein the compound is detectable by resultant light emissions at a wavelength greater than about 800nm in methanol.
8. 8. The compound according to any one of the preceding claims, wherein the compound comprises a fluorescent dye.
9. 9. A composition comprising the compound according to any one of the preceding claims and either a biological molecule or a non-biological molecule.
10. 10. The composition according to claim 9, wherein the biological molecule is selected from the group consisting of: proteins; peptides; polypeptides ; enzyme; enzyme substrates; hormones; antibodies; antigens; haptens; avidin; streptavidin; carbohydrates; lipids ; oligosaccharides ; polysaccharides ; microbial materials ; toxins; virus, bacteria; drugs; nucleic acids; deoxy nucleic acids; fragments of DNA or RNA; cells ; synthetic combinations of biological fragments such as peptide nucteic ads (PNAs) and certain othc. biolocical molecules ; and biological cells and fragments thereof.
11. 11. The composition according to claim 9, wherein the non-biological molecule is selected from the group consisting of: soluble polymers ; polymeric particles ; and other particles and surfaces.
12. 12. The composition according to any one of claims 9 to 11, wherein the molecule is covalently attached to the compound to form a conjugate.
13. 13. The composition according to claim 12, wherein compound comprises a substituted amine, alcohol, thiol, or acid.
14. 14. A compound as hereinbefore described with reference to and/or as illustrated by the accompanying drawing.
15. 15. A composition as hereinbefore described with reference to and/or as illustrated by the accompanying drawing.