FLUORESCENT DYES AND COMPLEXES

Field of the Invention This invention relates to fluorescent dyes comprising a xanthene- derived fluorophore, complexes comprising these dyes, and use of such dyes and complexes in cell and other analysis procedures.

Background of the Invention

Fluorescent dyes, when bonded to target molecules such as proteins, are commonly used to probe the properties of living cells. In particular, such dyes are often used to probe the pH of individual cell compartments. This is commonly achieved by comparing the intensity of fluorescence of a particular dye when it is within a particular cell compartment with a calibration curve of intensity versus pH for that dye. In this way, information may be obtained about the migration of molecules throughout a cell and/or kinetics of reactions taking place in different compartments of the cell.

One example of a fluorescent dye used for this purpose is fluorescein which has the formula

The fluorescence response of fluorescein decreases as pH decreases, and while it is useful in providing information on kinetics of reactions taking place within cell compartments, the pH of the cell compartments can not be accurately determined through its use. As a result, it is desirable to provide novel fluorescent dyes with different pH responses, in

order to provide a more accurate determination of the internal pH of cell compartments, and for other analysis procedures. Summary of the Invention

Surprisingly, it has now been found that, contrary to what is suggested in the literature, the pH response of fluorescein and its derivatives may be altered by providing an additional substituent on the benzoic acid moiety of the molecule, despite the belief that this moiety takes no part in the conjugation from which fluorescence is derived. I refer in this regard to Weidgans et al, The Analayst (2004)129:645-650, which describes the spectral properties of fluorescein as governed by the substitution pattern of the xanthene moiety, and to The Handbook of Fluorescent Probes and Reasearch Products (9 ^th Edition), by Richard Haugland, which shows that inclusion of amino and isothiocyanate groups in either the five- or six- position on the benzoic acid moiety of fluorescein make very little difference to its pKi.

According to a first aspect of the present invention, therefore, a novel fluorescent dye has the formula (I)

wherein the group X is selected from aliphatic, alicyclic and aromatic amine groups.

The novel fluorescent dyes have a fluorescence response which increases with pH up to a maximum fluorescence and then decreases as pH continues to increase.

The present invention also extends to complexes of the above- described fluorescent dyes with target molecules, and the use of the dyes and

complexes to determine and/or monitor the pH of living cells or cell compartments, and in other analysis procedures. Detailed Description of the Invention

It is envisaged that the group X may be selected from a wide variety of amine groups, which may be aliphatic, alicyclic or aromatic in nature, or a combination of any of these. Typically, the group X is represented by -NR2, in which the R groups are independently selected from hydrogen and alkyl, aryl, aryalkyl and alkryl groups, provided that both R groups are not hydrogen, or where both R groups together form an imine linkage. Preferably, neither R group is hydrogen. Included in the above definition for the group X are groups -NR ₂ which form a ring structure by bonding onto the aryl group of the benzoic acid moiety of the molecule, wherein the ring may be aromatic or non- aromatic in nature. Preferred groups -NR ₂ are the dialkylamino groups, in which the alkyl groups may be the same or different, and typically contain up to 10 carbon atoms. A particularly preferred -NR ₂ group is a dimethylamino group.

One or more of the aryl rings of the fluorescent dyes of the present invention may be optionally substituted, to vary the pH response and/or to aid in bonding, or coupling, to a target molecule or cell analysis procedures. For instance, substituents may be included on the aryl rings of the xanthene- derived moiety, as is the case in known fluorescent dye dichlorofluorescein. For instance, the fluorescent dye may have the formula (II).

wherein X is as defined above and each Y is a halogen atom which may be the same as or different to one another, and is typically selected from

chlorine and fluorine. One example of such a dye is 2',7'-dichloro-4- (dimethylamino) fluorescein.

The fluorescent dyes of the present invention can be prepared by any combination of standard chemical synthetic steps, and in particular those conventionally used in the manufacture of fluorescein and its derivatives.

The dyes may be functionalised so that they may be bonded to a range of target molecules, for introduction into a living cell or cell compartment. If desired, a plurality of different dyes, having fluorescence responses at different pH's, may be bonded to a single target molecule. This may be particularly useful for analysing the kinetics of migration of a particular target molecule through different regions of a cell, or through different cell compartments.

In the context of the present Application, a living cell includes eukaryotic cells, prokaryotic cells and plant cells. Further, while the present invention is described in the context of use of the novel fluorescent dyes with living cells, the present invention may find use in other applications.

There is a wide variety of functionalities that may be included in the dyes of the present invention to achieve bonding to target molecules, typically on one or more of the aryl rings, and these are referred to in what follows as "bonding groups". For clarity, bonding may be achieved by direct reaction between a dye and its target molecule, herein referred to as "direct bonding". Alternatively, "indirect bonding" may be achieved with the aid of a coupling agent, or "activator", which activates the dye by forming a complex therewith, and which is typically displaced after reaction with the target molecule. Depending on the nature of the coupling agent, however, a moiety derived therefrom may become incorporated in the final bonded complex. A wide variety of coupling reactions is known in the prior art, and may be applicable to the present invention.

The nature of the bonding group, or groups, will depend upon the nature of the functional group(s) on the target molecule available for reaction with the dye. For instance, where the target molecule contains pendant amino or amine groups, the bonding group may be selected from isocyanates;

isothiocyanates; and carbonyl groups containing a leaving group, for instance acid chloride, sulphonate, carboxylate, and so-called "active" esters, i.e. containing very good leaving groups including, for instance, nitrophenyl and N-hydroxysuccinimide, all of which are capable of direct bonding to said amino or amine groups on the target molecule. Alternatively, the bonding group may comprise a carboxylic acid group, an aldehyde or a ketone which, on activation with a coupling agent, are capable of reaction with said amino or amine groups. When the bonding group is an aldehyde or ketone, the reaction is typically conducted under reducing conditions to give rise to an amino-alkyl chain linkage in the final dye/target molecule complex.

Where the target molecule comprises a thiol group available for reaction with the dye, the bonding group may be selected from many of the groups mentioned above, disulphide and thiols.

For clarity, many of the above bonding groups will react with and bond to a wide variety of target functional groups on target molecules, i.e. other than amino, amine and thiol groups, optionally through the use of a coupling agent.

The dyes and complexes of the present invention may be used, either alone or in combination with other dyes or complexes, to establish the pH of a living cell or cell compartment, or to analyse the kinetics of migration of the dyes or complexes into a living cell or cell compartment, or from location to location within a cell.

By "a cell compartment" typically we mean one of the many organelles suspended in the cell cytoplasm. The pH of a cell or cell compartment can be measured by introducing a dye or complex into a cell or cell compartment, irradiating the dye or complex with a suitable light source, and observing the intensity of fluorescein of the dye or complex. The observed fluorescence intensity can then be used to determine pH by a variety of methods known in the field, selected according to the method of accumulation of the dye or complex. For instance, the observed fluorescence may be compared to a known standard, for example a calibration curve of fluorescence intensity versus pH, or to fluorescence intensity measurements indicative of the total

dye or complex present. Any conventional fluorometric equipment can be used to irradiate the sample, and to measure the resulting fluorescent response.

Typically, the dyes and complexes are introduced into a living cell or cell compartment by mixing with a sample comprising a cell or cell compartment, and then leaving the mixture to stand for a time interval adequate to allow entry of the dye or complex into the cell or cell compartment. During this time interval, the dye or complex diffuses towards a cell or cell compartment within the sample. The dye or complex then attaches itself to the membrane of a cell or cell compartment. In the case of complexes, target molecules are generally cell or cell compartment specific, hence a specific complex generally attaches to only one kind of cell or cell compartment. Once attached to a cell or cell compartment, the dye or complex may diffuse through a membrane of that cell or cell compartment or be trafficked to a specific cell compartment by receptor-mediated endocytosis, hence exposing itself to the internal pH of the cell or cell compartment.

Alternatively, the dyes may be incorporated into other reporting molecules, and the observed fluorescence used as a measure of a secondary response. As mentioned above, use of the dyes of the present invention is not limited to cell analysis procedures. Instead, the dyes find use in a wide variety of direct analysis procedures, for instance where it may be desirable to determine pH or whether the pH of a substance falls within an acceptable range. Examples include analysis of soil or urine. The present invention is now further described with reference to the accompanying drawings.

Figure 1 shows the pH titration curve for dichlorofluorescein, and the structure of this molecule is shown to the right of the curve.

Figure 2 shows the pH titration curve for 2',7'-dichloro-(4- dimethylamino)fluorescein, which includes a dimethylamino group ortho to the carboxyl group of the benzoic acid moiety, as shown in the formula to the right of the curve.

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The effect on the pH response by adding the dimethylamino group ortho to the carboxyl group of the benzoic acid moiety is clearly seen, converting a simple "positive" pH response (ie. where fluorescence increases with pH) to a bell-shaped response. Figure 3 shows pH titration curves of fluorescein and three derivatives thereof. Curve A represents dichlorofluorescein, curve B fluorescein, curve C 2',7'-dichloro-4-(dimethylamino)fluorescein and curve D 4-dimethylamino- fluorescein. The pK's of these compounds were obtained by iterative curve fitting of non-cooperative protonation with floating maximum signal, and are given in Table 1 , below, where PK ₁ is for the "positive" response to pH and PK ₂ is for the negative response to pH (ie. where fluorescence decreases with increasing pH). Table 1

The titrations were performed at the same molarity and sensitivity settings for each compound. The data show that the pKi value is essentially unaffected by the addition of the dimethylamino substituent, and appears to be dependent solely on the substituents on the xanthene moiety. In contrast, the value of pK ₂ is essentially independent of the substituents on the xanthene moiety.

The invention is now further illustrated by reference to the following Example. Example 3-(dimethylamino)phthalic acid 3-nitrophthalic acid (10 g) was hydrogenated in ethanol containing 1% acetic acid (200 ml) and 10% Pt on charcoal (500 mg) until uptake of hydrogen was nearly complete (6I). Formaldehyde solution (35%, 20 ml) was added and the hydrogenation continued until hydrogen uptake ceased (ca. 3

I). The catalyst was removed by filtration and washed with aqueous ethanol. Evaporation and filtration gave the required product (4.1 g, 41%).

2' ₁ 7'-dichloro-4-(dimethylamino)fluorescein The 3-dimethylaminophthalic acid (2.09g 1OmMoI) was dissolved in trifluoroacetic anhydride (10 ml) overnight, evaporated to dryness from toluene. 4-chlororesorcinol (3.6 g, 25 mMol) was added and heated with stirring in methanesulphonic acid (20 ml) at 80 ⁰ C overnight. The dark solution was cooled and poured into water (100 ml) and the solid product collected by filtration. (1.4 g, 32%). The product was virtually single spot tic, and was dissolved in water with the minimum of sodium hydroxide and precipitated at pH 3 with hydrochloric acid, recovered by filtration and dried to give a bright orange solid.

An alternative to the use of methansulphonic acid is fusion with zinc chloride at approximately 180 ⁰ C for approximately 30 minutes.

2',7'-dichloro-4-(dimethylamino)fluorescein diacetate

The diacetate was prepared by dissolution of the free 2',7'-dichloro-4- (dimethylamino)fluorescein (200 mg) in pyridine (2 ml) with excess acetic anhydride (0.5 ml) overnight. The excess anhydride was destroyed by slow addition of water with cooling and the product precipitated with water to 5 ml and collected by filtration, washed with water and dried to give the product (230 mg) as an off-white solid.

There were two possible products from the synthesis, the 4- and the 7- dimethylamino compounds shown as acetates below. The NMR and NOESY spectra given in Figure 4 confirm the product as solely the 4 -isomer as described above.

-dimethylamino 7-dimethylamino