WO2000058405A2 - Squaraine dyes - Google Patents

Abstract

Compounds having the structure: W = Sq - A where A has the formula: -NR1R2, where R?1 and R2¿ are the same or different and each is H or C¿1?-C20 hydrocarbon or a group -L-G, or one of R?1 and R2 is -OR5 or -NR6R7¿ or -COR?7 or -NR6 COR7¿ or -N =R?8 or R1 and R2¿ together form a single ring or fused ring system, saturated or unsaturated and unsubstituted or substituted, or an azacrown or a metal-binding group, Sq represents formula (A) or formula (B) where m is 1, 2 or 3, W represents a wing moiety having structure (C), L' is a linker of 0-3 moieties selected from carbon atoms and arylene groups, the dotted line represents a single ring or fused ring system, aromatic or heterocyclic, unsubstituted or substituted, containing or joined to a tertiary or quaternary N atom, and having unsaturation co-ordinated with that of L' = Sq, each of R?5, R6, R7 and R8¿ is H or C¿1?-C20 hydrocarbon or a group -L-G, R?9¿ is a negative charge or a group -L-G, L is a linker chain of 0 to 30 moieties, branched or unbranched which optionally contains one or more arylene groups, or O or N or N+ or S or S+ or P or P+ or Se atoms, and G is a functional or a reactive group by means of which the compound may be covalently linked to a biomolecule, or a group which enhances or reduces water solubility or provides electron donating or withdrawing properties to modify the spectral characteristics of the compound, and homo-dimers and -oligomers and hetero-dimers and -oligomers of the compounds.

Description

SQUARAINE DYES

The development of automated fluorescent methods has now become routine for detection in a range of biological applications e.g. flow cytometry, sequencing and arrays. The following are illustrative of the range of fluorescent dyes that can be used in such applications. Benzophenoxazine dyes have been described in Amersham International's WO 97/29154. Waggoner et al US 5,268,486 have described the properties of some conjugates of cyanine dyes, and Middendorf US 5,230,781 and Patonay EP 0 670 374 have described the use of various cyanine dyes in DNA sequencing. Berger et al EP 0 214 847 has described the use of other cyanine dyes some of which contain squarate groups in assays which involve a specific binding partner. Other squarate dyes are described by Pease et al USP 4,830,786, by A J G Mank et al in Anal. Chem. 1995, 67, 1742-8, and by Amersham International in WO 97/40104. Cushman et al WO 93/09172 and Krutak et al WO 94/19387 have described cyanine dyes containing squarate groups for use in thermoplastics and inks. There is still a need for fluorescent dyes with improved physical characteristics such as photostability and families of dyes with good spectral resolution for multiplexing biological samples. For example, fluorescein (as its reactive derivatives) is one of the original fluorescent labels for biomolecules and continues to be widely used with a 488nm laser as an excitation light source. It is far from ideal, however, for it has a low extinction coefficient, it is fluorescent only in a certain pH range, and it has poor chemical stability and photostability and solubility characteristics.

The present invention provides squaraine compounds having the structure

W = Sq - A where A has the formula -NR¹R², where R¹ and R² are the same or different and each is H or Ci - C₂o hydrocarbon or a group -L -G, or one of R¹ and R² is -OR⁵ or -NR⁶R⁷ or -COR⁷ or -NR⁶COR⁷ or -N = R⁸ or -C(O)OR⁷, or R¹ and R² together form a single ring or fused ring system, saturated or unsaturated and unsubstituted or substituted, or an azacrown or a metal-binding group,

Sq represents

where m is 1 , 2 or 3,

W represents a wing moiety having the structure

-L'=

L' is a linker of 0 - 3 moieties selected from carbon atoms and arylene groups, the dotted line represents a single ring or fused ring system, aromatic or heterocyclic, unsubstituted or substituted, containing or joined to a tertiary or quaternary N atom, and having unsaturation coordinated with that of L' = Sq, each of R⁵, R⁶, R⁷ and R⁸ is H or Ci - C₂o hydrocarbon or a group -L -G, R⁹ is a negative charge or a group -L -G,

L is a linker chain of 0 to 60 moieties, branched or unbranched which optionally contains one or more arylene groups, or O or N or N⁺ or S or S⁺ or P or P⁺ or Se atoms, and G is a functional or a reactive group by means of which the compound may be covalently linked to a biomolecule, other small molecule e.g. a dye or a group which enhances or reduces water solubility or provides electron donating or withdrawing properties to modify the spectral characteristics of the compound, and homo-dimers and -oligomers and hetero-dimers and -oligomers of the compounds. These compounds are characterised by having Sq joined, by

1 ,2-bonds or more preferably 1 ,3-bonds, directly to a carbon atom of W and to a nitrogen atom of A. The substituent groups including the ring structure (represented by a dotted line) and the various R groups are generally those described in prior publications, including those listed in the introduction, in connection with related dyes.

R¹ and R² may be Ci - C₂o hydrocarbon, for example alkyl or aryl or aralkyl. If one or both of R¹ and R² is hydrogen, then the compound will be pH-sensitive e.g. having fluorescent properties at one pH and not at another. Or R¹ and R² may together form a ring system, which is saturated or unsaturated, for example pyrrolidine, piperidine, pyrrole, piperazine, or morpholine, or a fused ring system. Alternatively R¹ and R² may together with the N atom to which they are joined form an aza crown or other metal- binding group such as EDTA or other metal chelating moiety containing various combinations of N, O and S ligand atoms. R¹ or both of R¹ and R² may be a group -L-G which is discussed below. Examples of amine moieties A are:

H H alkyl

^■N ^•N alkyl aryl alkyl

alkyl aryl

^■N ^■N

\ aryl aryl

aliphatic or aromatic ring

/ \

N X X = CH₂, NH, O, CHCOOH R = alkyl, aryl

\ /

^■N

\ O — R The wing moiety W is preferably

1ST

X R^{^}-

where each of R³ and R⁴ is H or Ci - C₂o or a group -L-G, and n is 0 - 3.

R³ may be H, in which case the compound may be pH sensitive, for example exhibiting fluorescent properties at one pH and not at another. Or R³ may be Ci - C₂o hydrocarbon as discussed above for R¹ and R². Or R³ may be -L-G as discussed below.

The dotted line preferably represents a heterocyclic ring system, either single ring or fused ring and generally unsaturated, for example pyrrole or pyridine or indole or benzindole or benzoxazole or benzothiazole or quinolyl. Preferably the ring system represented by the dotted line has the structure:

where X is O or S or -NR⁴ or -CR¹⁰R¹¹ where each of R¹⁰ and R¹¹ is Ci - C₂o hydrocarbon or -L -G, or R¹⁰ and R¹¹ together form a single ring of fused ring or heterocyclic or cycloaliphatic group.

Examples of dyes with different wing moieties W are:

In the Sq moiety, the integer m may be 1 or 2 or 3. When m is 1 , these are squaric compounds; when n is 2 they are croconic compounds; when m is 3 they are rhodizonic compounds. Thus for example:

SQUARAINE

CROCAINE

RHODIZAINE

L is a linker chain of 0 to 30 or 60 moieties, preferably selected form alkylene, alkenylene and alkynylene or a branched or straight chain of up to 30 carbon atoms optionally incorporating one to six O or N or N⁺ or S or S⁺ or P or P⁺ or Se atoms or arylene groups.

G may be a functional or a reactive group by means of which the squaraine compound may be covalently linked to a biomolecule or other molecule. Examples of G as a functional group are such nucleophiles as NH2, OH, SH. Examples of G as a reactive group are -COOH, activated carboxyl such as acid halide or anhydride, CO active ester, -NCS, O phosphoramidite, -NC(0)CH₂l and maleimide

Preferred biomolecules are nucleosides, nucleotides and analogues thereof, oligonucleotides and nucleic acids, and also amino acids, peptides, proteins, antibodies, polysaccharides, lipids, sugars and other small molecules. Another example of a biomolecule in this context is a cyclodextrin. Cyclodextrin - fluorophore conjugates have been shown to possess greatly enhanced photostability in aqueous solution (Tetrahedron Letters, Volume 38, No 35, pages 6167-6170, 1997).

Alternatively G may be a group which enhances water solubility such as sulphonate, phosphate, quaternary ammonium, sugar and polyether; or that reduces water solubility e.g. alkyl. Alternatively G may be a group which provides electron donating or withdrawal properties to modify the spectral characteristics of the squaraine compound; for example halogen, alkoxy, nitro or cyano. See the Chemistry of Synthetic Dyes, Venkataraman, Academic Press, New York, 1971 , 4, Chapter 5 Part iiic, pages 228-240 particularly Table 1 on page 230. Although the compounds of this invention have interesting fluorescent properties, they are also in general coloured and can be used as conventional dyes e.g. in colorimetric assays.

Also envisaged according to the invention are dimers and oligomers of the compounds defined above. For example a dimer may have the structure W¹-Sq-A¹-Sq-W² or alternatively the structure

A¹-Sq-W¹-Sq-A², where A¹ and A² each represent an amine moiety A, and W¹ and W² each represent a wing moiety W. Where R¹ and R² are part of a substituted fused ring system this ring system may contain one or more amino groups capable of forming a dye species. In such cases the substituted ring system is acting as a scaffold upon which dye molecules can be formed. If all the dye species are the same then an enhancement of the fluorescent signal would result. If different dye species were present energy transfer could take place.

Where A¹ and A² (or W¹ and W²) are the same, the compounds are expected to have more intense dye properties than the monomers. An example of such a dimer is:

λem 542 nm ε_maχ 270 000 dm mol^"1cm^"1

Alternative scaffolds, to piperazine shown above are cyclam, dendrimers, adamantane (Tetrahedron Letters, 1999, 40, 223):

Where A¹ and A² (or W¹ and W²) are different, then an energy-transfer cassette is possible, comprising a donor dye and an acceptor dye, to provide increased separation between the absorption wavelength and the emission wavelength of the compound. Trimers and higher oligomers may be made and used in similar fashion.

As well as the above the dyes of the invention can be used to form energy transfer cassettes with known dyes such as cyanine, rhodamine, etc. In such cases where the donor dye is a dye of the invention and replaces fluorescein the lack of pH sensitivity, greater photostability and increased extinction coefficient could be advantageous. Examples are

D ^λabs = 490 nm A λ_abs = 530 nm λ_om = 515 nm λ_pm = 570 nm

D λ_abs = 490 nm A λ 'a. bs = 550 nm λ„_m = 515 nm = 570 nm

The energy transfer cassettes are but one means of providing an energy transfer system. The dyes of the invention can also be used in simple FRET assays where energy transfer is facilitated by bringing the donor and acceptor to a distance where energy transfer becomes viable. Such systems can be based solely on the dyes described above or use known dyes as one of the donor acceptor pair.

Another aspect of a FRET assay is the use of quencher dyes which initially result in no signal when the donor is in close proximity to the quencher. When an event occurs that results in a greater physical separation between the two dyes the quencher dye is no longer able to quench the fluorescence of the second dye and thus a signal is produced. It is reported that N0₂ groups attached to squarate dyes (Dye and Pigment, Vol 35, 331 , 1997) greatly reduce the fluorescence of squarate based dyes. By such means quencher dyes from the invention dyes can be produced to enable dyes to be produced for such FRET assays. Mixing and matching with other known dyes as well as invention dyes can provide a range of pairs for multiplexing in such arrays. With a nitrogen atom of A being directly covalently bound to the Sq moiety which forms an integral part of the chromophore, perturbation of its electronic density via protonation or metal chelation or as part of a redox system will affect the properties of the dye. These changes can be used to give indication of the events that caused the change in the nitrogen electron density.

Thus, when one of R¹ and R³ is H, the compounds may be pH sensors. When A comprises a metal chelating moiety, the compounds may act as metal ion sensors. When A comprises a NADH system, the compounds may act as redox sensors. Examples are:

pH Sensors

Metal ion sensors

Redox Sensor (e.g. NADH)

The invention also includes a method of making the compounds as defined, which method comprises the steps of i) reacting the wing moiety W with a dialkyl squarate or analogue to give an intermediate a)

ii) optionally subjecting the intermediate a) to hydrolysis to give an intermediate b)

iii) reacting the intermediate a) or the intermediate b) with

R¹R²NH to give a final product c)

-L'=Sq-NR¹R²

Reacting intermediate a) with R¹R²NH gives a 1 ,2 squarate or analogue. Reacting intermediate b) with R¹R²NH gives a 1 ,3 squarate or analogue.

Thus squaraine compounds according to the invention can be made by the following reaction sequence:-

i) Squarate half-dye formation

ii) Half-dye hydrolysis to acid form

iii) Dye formation

The half dye methyl ester from i) also reacts with amines to give a 1 ,2-adduct: iv) Dye formation

Four more specific reaction schemes follow by way of example.

b)

The invention also includes an assay or labelling method which comprises contacting a sample containing an amine with a compound a) or a compound b)

a)

b)

and observing dye formation by reaction of the amine with the compound a) or b). The amine may be in solution or on a solid phase, and may be for example a peptide or a 5'-amino derivatised oligonucleotide. The assay may be qualitative or quantitative and may be designed to identify a particular amine by reference to the spectral characteristics of the resulting dye.

The invention is further illustrated by the following examples.

Examples:

Amines:

Amino-acids:

Water solubility:

The groups -NR¹R² may comprise an amino-sugar, or amine with additional quaternary ammonium groups.

Wavelength variations:

Use of polyamines for multiple dye loading or energy-transfer cassettes:

The amine function may be part of a metal chelating system e.g. azacrown:

Chelation of an appropriate metal ion should alter fluorescence properties.

pH sensors:

Other amino compounds:

EXPERIMENTAL EXAMPLES

EXAMPLE 1

Preparation of intermediates for dye synthesis

3-Methoxy-4-(1 ,3,3-trimethyl-2-indolinylidenemethyl)-cyclobut-3-en- 1 ,2-dione [1a]

To a dried 100ml round-bottomed flask, with stirrer bar, was added dimethyl squarate (3.55g, 25mmol) and dry methanol (10ml). This was stirred until all the solid had dissolved.

To the resulting solution was then added 2-methylene-1 ,3,3- trimethylindoline (vacuum distilled before use, 4.4g, 25.4mmol); a strong yellow colour formed immediately. After about 20mins a yellow solid started to crystallize out. Stirring was continued for 16hrs, then the mixture was cooled in the fridge for 2hrs. The title compound [1 a] was collected by filtration, washed with ice-cold methanol (3x5ml), then diethyl ether (4x20ml) and dried under vacuum. Yield = 6.34g (90%) as a yellow crystalline powder. λ_max (MeOH) = 422nm. δ_H (300MHz, CDCI₃) 1 .60 (6H, s), 3.34 (3H, s), 4.49 (3H, s), 5.32 (1 H, s), 6.87 (1 H, d), 7.04 (1 H, d) and 7.22-7.28 (2H, m).

Mass spectrum: (ES+) 284 (M+H), 306 (M+Na), 322 (M+K).

3-Hydroxy-4-(1 ,3,3-trimethyl-2-indolinylidenemethyl)-cyclobut-3-en- 1 ,2-dione [1 b]

3-Methoxy-4-(1 ,3,3-trimethyl-2-indolinylidenemethyl)- cyclobut-3-en-1 ,2-dione [1 a] (630mg, 2.2mmol) was mixed with acetic acid (20ml) and concentrated aqueous hydrochloric acid (5ml). The mixture was warmed gently with a hot air gun to around 40-50°C; after a few minutes at this temperature the solid all dissolved to give a deep yellow solution. The mixture was held at this temperature while the reaction progressed; it was monitored by t.l.c. (RPC₁₈. Methanol, 90: water, 10. [1a], R_f = 0.5, [1 b] R_f = 0.7). Once the reaction was complete, the solvent was evaporated under reduced pressure; the residue was co-evaporated with acetonitrile, twice and then dried under high vacuum. Trituration with diethyl ether gave the title compound as a yellow powder. Yield = 590mg (98%). This material was used directly to prepare dyes.

Mass spectrum: (ES+) 270 (M+H).

1-Ethyl-2,3,3-trimethylbenz(e)indolinium iodide [1c]

1 ,1 ,2-Trimethylbenz(e)indole (10.47g, 50mmol), iodoethane (11.7g, 75mmol) and 1 ,2-dichlorobenzene (35ml) were mixed in a 100ml flask, with stirrer. The mixture was heated at 90°C for 18hrs; a dark greenish solution containing a pale solid resulted. After cooling, the solid was collected by vacuum filtration, washed with dichlorobenzene (2x20ml), then excess diethyl ether. It was dried under vacuum to give the title solid as a purple-tinged pale solid. Yield = 15.93g (87%). δ_H (300MHz, CDCI₃) 1.63 (3H, t), 1.85 (6H, s), 3.21 (3H, s), 4.86 (2H, q), 7.62-7.74 (2H, m), 7.81 (1 H, app. d) and 8.02-8.11 (3H, m).

1 -Ethyl-2-methylene-3,3-dimethylbenz(e)indoline [1 d]

1 -Ethyl-2,3,3-trimethylbenz(e)indolinium iodide [1c] (7.3g, 20mmol) was added to a solution of sodium hydroxide (4g, lOOmmol) in water (150ml); this mixture was swirled briefly, then diethyl ether (100ml) added and the mixture swirled again. The brown ether layer was collected; the aqueous layer was extracted with fresh ether (50ml).

The ether extracts were combined, washed twice with water, then dried (MgS0₄), filtered and the ether evaporated under reduced pressure. The resulting brown oil soon crystallized on standing; this was dried under high vacuum to give the title compound as a brownish solid. Yield = 4.77g (100%) δ_H (300MHz, CDCI3) 1.28 (3H, t), 1.72 (6H, s), 3.73 (2H, q), 4.01 (2H, approx. s), 7.04 (1 H, s), 7.24 (1 H, t), 7.44 (1 H, m), 7.74-7.83 (2H, m) and 8.02 (1 H, d).

3-Methoxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)- cyclobut-3-en-1,2-dione [1e]

1 -Ethyl-2-methylene-3,3-dimethylbenz(e)indoline [1 d] (4.75g, 20mmol) and dimethyl squarate (2.84g, 20mmol) were added to a 100ml flask, with stirrer. Dry methanol (35ml) was then added. This mixture was heated at 50°C for 16hrs, then it was allowed to cool to ambient temperature. After further cooling in the fridge, the precipitated yellow solid was collected by vacuum filtration, washed with ice-cold methanol (2x15ml), then diethyl ether (4x30ml) and dried under high vacuum to give the title compound [1e]. Yield = 5.44g (78%). λmax (MeOH) = 440nm δ_H (300MHz, CDCI₃) 1.37 (3H, t), 1.88 (6H, s), 3.98 (2H, q), 4.52 (3H, s), 5.41 (1 H, s), 7.20 (1 H, d), 7.36 (1 H, m), 7.52 (1 H, m), 7.84 (2H, m) and 8.09 (1 H, m).

3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)- cyclobut-3-en-1 ,2-dione [1f]

3-Methoxy-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1 e] (3.47g, 10mmol) was mixed with acetic acid (50ml) in a 250ml flask. To this was added a solution of concentrated hydrochloric acid (2.5ml) in water (10ml). The resulting mixture was incubated in a water bath at 60°C and the reaction monitored by t.l.c. (RPCι₈. Methanol, 90: water, 10. [1 e] R_f = 0.4, [1f] R_f = 0.7). Once the reaction was complete, the solvent was evaporated under reduced pressure; the residue was co-evaporated twice with acetonitrile and dried under high vacuum. The resulting foam was triturated with diethyl ether (25ml) to give a yellow-brown powder; petroleum ether 40-60° (25ml) was added at this point. After cooling in the fridge the solid was collected by vacuum filtration, washed with more diethyl ether : petroleum ether 1 :1 mixture, and dried under vacuum to give the title compound [1f]. Yield = 3.1 g (93%). This material was used directly to prepare dyes. λ_max (MeOH) = 442nm δ_H (300MHz, CD₃OD) 1.37 (3H, t), 1.89 (6H, s), 4.10 (2H, q), 5.48 (1 H, s), 7.33-7.44 (2H, m), 7.50-7.56 (1 H, m), 7.90 (2H, app. d) and 8.14 (1 H, app. d).

1,1 ,2-Trimethylbenz(e)indole-5-sulphonate, potassium salt [1g]

This was prepared as described in EP 0288076 B1 (Richard L. Parton, Eastman Kodak Co.)

1 -Ethyl-2,3,3-trimethylbenz(e)indolinium-5-sulphonate internal salt [1 h]

1 ,1 ,2-Trimethylbenz(e)indole-5-sulphonate, potassium salt [1 g] (ground to a fine powder using a pestle and mortar before use; 1.64g, 50mmol), iodoethane (2g, 12.5mmol) and 1 ,2-dichlorobenzene (8ml) were mixed in a 25ml flask, with stirrer. This mixture was heated to 120°C (Wood's metal bath, water condenser, silica gel guard tube); it was allowed to react over 3 days, with periodic addition of extra 1 ml aliquots of iodoethane every few hours. At the end of this period, the mixture was cooled to ambient temperature and the suspended solid collected by vacuum filtration. This was washed with diethyl ether and dried under high vacuum; this material was used directly without further purification. Expected to contain 1 equivalent of Kl. δ_H (300MHz, CD₃OD) 1.32 (3H, t), 1.78 (6H, s), 4.67 (2H, q), 8.00-8.08 (2H, m), 8.17 (1 H, d), 8.29 (1 H, d) and 8.38 (1 H, d). 3-Methoxy-4-(5-sulphonato-1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione, sodium salt [1 i]

1 -Ethyl-2,3,3-trimethylbenz(e)indolinium-5-sulphonate internal salt [1 h] (1.56g, 3.2mmol) was added to a dry 50ml flask with stirrer, followed by dry methanol (15ml) and sodium methoxide (0.19g, 3.5mmol). This mixture was stirred for 5mins, then dimethyl squarate (0.71 g, δ.Ommol) added. The resulting mixture was heated at around 60°C (sand bath, water condenser, silica gel guard tube) for 16hrs.

At the end of this time the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica gel. 20-25% methanol in chloroform). Fractions containing pure product were combined and evaporated under reduced pressure; the residue was redissolved in 10% methanol in chloroform, filtered and re-evaporated. The resulting orange oil was triturated with diethyl ether to give a solid. This was dried under high vacuum to give the title compound as a yellow powder. Yield = 640mg. λ_max (MeOH) = 442nm. δ_H (300MHz, CDgOD) 1.36 (3H, t), 1.88 (6H, s), 4.11 (2H, q), 4.54 (3H, s), 5.55 (1 H, s), 7.51 (1 H, d), 7.94 (1 H, dd), 8.01 (1 H, d), 8.21 (1 H, d) and 8.37 (1 H, d).

3-Hydroxy-4-(5-sulphonato-1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione, sodium salt [1j]

3-Methoxy-4-(5-sulphonato-1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione, sodium salt [1 i]

(640mg, 1.4mmol) was mixed with acetic acid (10ml) and 2M aqueous HCI (5ml). This mixture was heated with a hot air gun until all the solid had dissolved; the resulting intensely yellow solution was allowed to stir with further periodic warming. The reaction was followed by t.l.c. (RPCiβ. Methanol, 60: water, 40. R_f [1 i] = 0.6, R_{ [1 j] = 0.8). Once the reaction appeared complete the solvent was evaporated under reduced pressure, the residue was co-evaporated twice with acetonitrile and finally dried under vacuum.

Purification by prep. HPLC (RPCι₈. Water → methanol gradient). The appropriate fractions were combined and evaporated under reduced pressure; the residue was again co-evaporated twice with acetonitrile before drying under high vacuum to give the title compound. Yield = 376mg (61%). Used directly. λ_max (MeOH) = 442nm.

1-(4-sulfonatobutyl)-2,3,3-trimethylbenz(e)indolinium hydroxide inner salt [1k]

1 ,1 ,2-Trimethylbenz(e)indole (1.05g, 5mmol), 1 ,4-butanesultone (1.36g, 10mmol) and 1 ,2-dichlorobenzene (5ml) were mixed and heated at 120°C for 20hrs. The mixture was then allowed to cool to ambient temperature and the precipitated solid collected by filtration, washed with dichlorobenzene followed by diethyl ether, and dried under vacuum at 50°C to give the title compound, 1.70g (99%). δ_H (300MHz, CD₃OD) 1.827 (6H, s), 1.97 (2H, m), 2.21 (2H, m), 2.92 (2H, t), 4.658 (2H, t), 7.69 (1 H, m), 7.79 (1 H, m), 8.06 (1 H, d), 8.14 (1 H, d), 8.22 (1 H, d) and 8.31 (1 H, d).

3-Methoxy-4-(1-(4-sulfonatobutyl)-3,3-dimethyl-2- benzindoiinylidenemethyl)-cyclobut-3-en-1 ,2-dione [11]

1-(4-Sulfonatobutyl)-2,3,3-trimethylbenz(e)indolinium hydroxide inner salt [1 k] (1.38g, 4.0mmol), dimethyl squarate (0.57g, 4.0mmol) and methanol (10ml) were mixed in a dry flask; to the resulting mixture was added a solution of sodium methoxide, 0.5M in methanol (8.0ml, 4.0mmol). A yellow colour soon formed. The mixture was left to stir overnight. The solvent was then evaporated under vacuum and the residue purified by flash chromatography (silica. 15-25% methanol / chloroform). Product fractions were combined and evaporated to give the title compound, 1.47g (77%) λ_max (MeOH) = 440nm. δ_H (300MHz, 10% CD₃OD in CD₂CI₂) 1.80 (6H, s), 1.91 (4H, m), 2.88 (2H, t), 3.97 (2H, broad), 4.49 (3H, s), 5.44 (1 H, s), 7.27-7.37 (2H, m), 7.50 (1 H, m), 7.83 (2H, m) and 8.05 (1 H, d).

3-Hydroxy-4-(1-(4-sulfonatobutyl)-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1 m]

This was prepared in the same manner as for [1j], by reacting the methoxy half-dye [11] (1.45g, 3.0mmol) with 2M aqueous HCI (5ml) in acetic acid (15ml) with warming. The reaction was followed by t. I.e. (RPC-ι₈. Methanol, 60: water, 40. [11], R_f = 0.35; [1 m], R_f = 0.65). Once the reaction was complete, the solvent was evaporated under vacuum, the residue co- evaporated with acetonitrile three times and finally dried under high vacuum over phosphorus pentoxide. This material was used without further purification.

[1a] R = CH₃ [1 b] R = H

[1 c] [1d]

[1 e] R = CH₃ [1f] R = H

[ig] [1h]

[1i]R = CH₃ [1j] R = H

[1 I] R = CH₃ [1 m] R = H

EXAMPLE 2

Synthesis of squarate dyes from indolenine-squaric acid intermediates

2-(1 -Piperidino)-4-(1 ,3,3-trimethyl-2- indolinyiidenemethyl)cyciobutenediylium-1 ,3-diolate [2a]

3-Hydroxy-4-(1 ,3,3-trimethyl-2-indolinylidenemethyl)-cyclobut- 3-en-1 ,2-dione [1 b] (81 mg, 0.30mmol) was dissolved in 1 -butanol (3ml); to the resulting yellow solution was added piperidine (40μl = 34mg, 0.40mmol). This mixture was heated at around 100°C (sand bath, air condenser). The colour darkened during this time (intense orange-yellow at the meniscus). The reaction was followed by t.l.c. (RPC-ι₈. Methanol, 90: water, 10. [1 b] R_f = 0.7, [2a] R_f = 0.4). After 4hrs the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica gel. 2-5% methanol in chloroform). Fractions containing the major product were combined and evaporated; the residue was redissolved in chloroform, filtered and re-evaporated. Trituration with diethyl ether : petroleum ether 40-60° mixtures gave the title dye [2a] as a powder, after drying under high vacuum. Yield = 93mg (92%) λma (MeOH) = 470nm; ε_maχ = 101 ,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 471 nm, λ_em = 495nm. δ_H (300MHz, CDCI₃) 1 .67 (6H, s), 1 .68-1 .78 (6H, m), 3.35 (3H, s), 4.05 (4H, m), 5.56 (1 H, s), 6.85 (1 H, d), 7.01 (1 H, t) and 7.20-7.27 (2H, m).

Mass spectrum: (ES+) 337 (M+H), 359 (M+Na)

2-(1 -Morpholino)-4-(1 ,3,3-trimethyl-2- indolinylidenemethyl)cyclobutenediylium-1 ,3-diolate [2b] This was prepared in an analogous method to [2a], using morpholine (35μl = 35mg, 0.40mmol) in place of piperidine. The title dye [2b] was isolated in the same manner. Yield = 91 mg (90%). λmax (MeOH) = 476nm; ε_maχ = 106,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 474nm, λ_em = 498nm. δ_H (300MHz, CD₃OD) 1 .64 (6H, s), 3.45 (3H, s), 3.86

(4H, app. t), 4.06 (4H, app. t), 5.63 (1 H, s), 7.06-7.12 (2H, m) and 7.27-7.37 (2H, m).

Mass spectrum: (ES+) 339 (M+H)

1 ,4-Bis-[2-(1 -3,3-trimethyl-2-indolinylidenemethyl)cyclobutenediylium- 1 ,3-diolate]piperazine [2c]

This was prepared in an analogous method to [2a], using piperazine (12.9mg, 0.15mmol) in place of piperidine. The title dye [2c] was isolated in the same manner. Yield = 79mg (89%). λmax (MeOH) = 506nm; ε_maχ = 284,000 dm³ mol^'1 cm^"1.

Fluorescence (MeOH); λ_ex = 506nm, λ_em = 520nm. δ_H (300MHz, CDCI₃) 1 .68 (12H, s), 3.44 (6H, s), 4.29 (8H, s), 5.68 (2H, s), 6.93 (2H, d), 7.08 (2H, t) and 7.22-7.31 (4H, m).

Mass spectrum: (ES+) 589 (M+H), 61 1 (M+Na). 2-(N-Methylanilino)-4-(1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [2d]

This was prepared in an analogous method to [2a], using N- methylaniline (44μl = 43mg, 0.40mmol) in place of piperidine. The title dye [2d] was isolated in the same manner. Yield = 96mg (89%). λmax (MeOH) = 496nm; ε_maχ = 87,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 496nm, λ_em = 547nm. δ_H (300MHz, CD₃OD) 1.66 (6H, s), 3.53 (3H, s), 3.96 (3H, s), 5.77 (1 H, s), 7.12-7.19 (2H, m) and 7.30-7.47 (7H, m). Mass spectrum: (ES+) 359 (M+H), 381 (M+Na).

2-(N,N-Dipropylamino)-4-(1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [2e]

This was prepared in an analogous method to [2a], using dipropylamine (55μl = 40.5mg, 0.40mmol) in place of piperidine. The title dye [2e] was isolated in the same manner. Yield = 97mg (92%). λmax (MeOH) = 474nm; ε_max = 97,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 474nm, λ_em = 494nm. δ_H (300MHz, CD₃OD) 0.88 (6H, t), 1.56 (6H, s), 1.65 (4H, hex), 3.33 (3H, s), 3.74 (4H, t), 5.52 (1 H, s), 6.96-7.01 (2H, m) and 7.16- 7.26 (2H, m).

Mass spectrum: (ES+) 353 (M+H), 375 (M+Na).

2-(4-Carboxypiperidino)-4-(1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [2f]

3-Hydroxy-4-(1 ,3,3-trimethyl-2-indolinylidenemethyl)-cyclobut- 3-en-1 ,2-dione [1 b] (81 mg, 0.30mmol) was dissolved in N,N-dimethylformamide (3ml); to the resulting yellow solution was added isonipecotic acid (52mg, 0.40mmol). This mixture was heated at 100°C for 4hrs. The reaction produced two main products by t.l.c. (RPCι₈. Methanol, 95: water, 5. [1 b] R_f = 0.8, product 1 R_f = 0.65, product 2 R_f = 0.5). After cooling and evaporation of solvent under reduced pressure, the two products were isolated by flash chromatography (silica gel. 2.5-20% methanol in chloroform. Product 2 eluted first; this proved to be dye [2g] (10mg), presumably formed by the trapping of dimethylamine liberated from the DMF. Product 1 eluted later and was the desired dye [2f], isolated yield = 22mg (19%). [2f] λmax (MeOH) = 472nm

Fluorescence (MeOH); λ_ex = 472nm, λ_em = 498nm. δ_H (300MHz, CD₃OD) 1.64 (6H, s), 1.82-1.94 (2H, m), 2.11-

2.17 (2H, m), 2.65-2.74 (1 H, m), 3.43 (3H, s), 3.46-3.61 (2H, m), 4.60-4.64 (2H, m), 5.61 (1 H, s), 7.05-7.10 (2H, m) and 7.26-7.36 (2H, m).

Mass spectrum: (ES+) 381 (M+H).

[2g] λmax (MeOH) = 468nm δ_H (300MHz, CD₃OD) 1.64 (6H, s), 3.41 (3H, s), 3.49 (6H, s), 5.59 (1 H, s), 7.04-7.09 (2H, m) and 7.25-7.35 (2H, m).

Mass spectrum: (ES+) 297 (M+H).

[2a] R1-R2 = -(CH₂)₅ -

[2b] R1-R2 = -(CH₂)₂-0-(CH₂)₂ -

[2d] R1 = Ph, R2 = CH₃

[2e] R1 = R2 = -CH₂CH₂CH₃

[2f] R1 -R2 = -(CH₂)₂-CH(C0₂H)-(CH₂)₂

[2g] R1 = R2 = CH₃

[2c]

EXAMPLE 3

Synthesis of squarate dyes from benzindolenine-squaric acid intermediates

2-(1-Piperidino)-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3a]

3-Hydroxy-4-(1-ethy!-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1f] (100mg, 0.30mmol) was dissolved in 1 -butanol (3ml); to the resulting yellow solution was added piperidine (40μl = 34mg, 0.40mmol). This mixture was heated at around 100°C (sand bath, air condenser). The colour darkened during this time (intense orange-yellow at the meniscus). The reaction was followed by t.l.c. (RPCι₈. Methanol, 90: water, 10. [1f] R_f = 0.7, [3a] R_f = 0.25). After I hr the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica gel. 0-4% methanol in chloroform).

Fractions containing the major product were combined and evaporated; the residue was redissolved in chloroform, filtered and re-evaporated. Trituration with diethyl ether : petroleum ether 40-60° mixtures gave the title dye [3a] as a powder, after drying under high vacuum. Yield = 110mg (91%). λmax (MeOH) = 490nm; ε_maχ = 95,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 492nm, λ_e = 516nm. δ_H (300MHz, CD₃OD) 1.36 (3H, t), 1.78 (6H, broad s), 1.90 (6H, s), 4.02-4.11 (6H, m), 5.70 (1 H, s), 7.33-7.44 (2H, m), 7.52 (1 H, app. t), 7.89 (2H, app. d) and 8.14 (1 H, d).

Mass spectrum: (ES+) 401 (M+H), 423 (M+Na).

Reaction of 3-methoxy-4-(1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1 e] (105mg, 0.30mmol) with piperidine (40μl = 34mg, 0.40mmol) in hot ethanol (6ml) gave an alternative product. T.l.c. (RPCι₈. Methanol, 90: water, 10. [1 e] R_f = 0.4, product R_f = 0.4, cf [3a], R_f = 0.25) and (silica. Methanol, 2.5: chloroform, 97.5. [1e], R_f = 0.55, product R_f = 0.45, cf [3a], R_f = 0.4). This new product was isolated by evaporation of the ethanol under reduced pressure and trituration of the residue with diethyl ether. Yield = 120mg (100%). λ _ax (MeOH) = 452nm, cf [3a], 490nm. Overall appearance is more like the half dye [1 e] than [3a]. δπ (300MHz, CDCI₃) looks like a mixture of two isomers, neither of which matched exactly the spectrum of [3a]. This new product was expected to be the 1 ,2-adduct, 3-(1-piperidino)-4-(1 -ethyl-3,3-dimethyl- 2-benzinolinylidenemethyl)-cyclobut-3-en-1 ,2-dione:

The single-double bond conjugation is now more fixed so E- and Z- isomers are possible.

Mass spectrum: (ES+) 401 (M+H), 423 (M+Na). 2-(1-Morpholino)-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3b]

This was prepared in an analogous method to [3a], using morpholine (35μl = 35mg, 0.40mmol) in place of piperidine. The title dye [3b] was isolated in the same manner. Yield = 99mg (82%). λ_max (MeOH) = 494nm; ε_maχ = 93,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 494nm, λ_em = 520nm. δ_H (300MHz, CD₃OD) 1 .17 (3H, t), 1 .91 (6H, s), 3.87 (4H, app. t), 4.06-4.16 (6H, m), 5.75 (1 H, s), 7.38 (1 H, app. t), 7.46 (1 H, d), 7.55 (1 H, m), 7.90 (2H, app. d) and 8.15 (1 H, d).

Mass spectrum: (ES+) 403 (M+H).

1 ,4-Bis-[2-(1 ,3,3-trimethyl-2- benzindolinylidenemethyl)cyclobutenediylium-1 ,3-diolate]piperazine [3c]

This was prepared in an analogous method to [3a], using piperazine (12.9mg, 0.15mmol) in place of piperidine. The title dye [3c] was isolated in the same manner. Yield = 98mg (91 %). λmax (MeOH) = 528nm; ε_maχ = 270,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 528nm, λ_em = 542nm. δ_H (300MHz, CDCI₃) 1 .39 (6H, t), 1 .57 (12H, s), 4.08 (4H, q), 4.31 (8H, s), 5.76 (2H, s), 7.27 (2H, m), 7.41 (2H, app. t), 7.55 (2H, app. t), 7.86 (4H, app. t) and 8.12 (2H, d).

Mass spectrum: (ES+) 717 (M+H), 739 (M+Na).

2-(N-Methylanilino)-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3d]

This was prepared in an analogous method to [3a], using N-methylaniline (44μl = 43mg, 0.40mmol) in place of piperidine. The title dye [3d] was isolated in the same manner. Yield = 120mg (=100%). λmax (MeOH) = 516nm; ε_maχ = 76,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 522nm, λ_em = 563nm. δ_H (300MHz, CD₃OD) 1.40 (3H, t), 1.93 (6H, s), 3.96 (3H, s), 4.22 (2H, q), 5.90 (1 H, s), 7.34-7.60 (8H, m), 7.92-7.96 (2H, m) and 8.18 (1 H, d).

Mass spectrum: (ES+) 423 (M+H), 445 (M+Na).

2-(N,N-Diphenylamino)-4-(1-ethyl-3-3-dimethyl-2- benzindolinyiidenemethyl) cyclobutenediylium-1 ,3-diolate [3e] This was prepared in an analogous method to [3a], using

N,N-diphenylamine (68mg, 0.40mmol) in place of piperidine. The title dye [3e] was isolated in the same manner. Yield = 60mg (41%). λma (MeOH) = 540nm; ε_max = 96,000 dm³ mol^"1 cm^"1. Fluorescence (MeOH); λ_ex = 542nm, λ_e = 588nm. δ_H (300MHz, CD3OD) 1.44 (3H, t), 1.94 (6H, s), 4.30 (2H, q),

6.07 (1 H, s), 7.21-7.26 (4H, m), 7.34-7.49 (7H, m), 7.56-7.62 (2H, m), 7.98 (2H, app. t) and 8.20 (1 H, d).

Mass spectrum: (ES+) 485 (M+H), 507 (M+Na).

2-(N-phenyl-N'-(acetyl)hydrazino)-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3f]

This was prepared in an analogous method to [3a], using 1- acetyl-2-phenylhydrazine (60mg, 0.40mmol) in place of piperidine. The title dye [3f] was isolated in the same manner. Yield = 47mg (34%). λmax (MeOH) = 530nm

Fluorescence (MeOH); λ_ex = 533nm, λ_e = 566nm. δ_H (300MHz, CD3OD) 1 .41 (3H, t), 1 .93 (6H, s), 2.16 (3H, s), 4.29 (2H, q), 6.04 (1 H, s), 7.24 (1 H, m), 7.37-7.49 (5H, m), 7.56-7.61 (2H, m), 7.96 (2H, app. t) and 8.19 (1 H, d). Mass spectrum: (ES+) 589 (M+H), 61 1 (M+Na). 2-(4-Carboxypiperidino)-4-(1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3g]

3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1f] (100mg, 0.30mmol) and isonipecotic acid (52mg, 0.40mmol) were mixed with acetic acid (3ml); the resulting mixture was heated to 100°C for 5hrs, with monitoring by t.l.c. (RPC18. Methanol, 95: water, 5. [1f] R_f = 0.75, yellow spot. Product R_f = 0.5, orange spot). The solvent was then evaporated under vacuum; the residue was purified by flash chromatography (silica gel. 5-15% methanol in chloroform). Fractions containing the major product were combined and evaporated; the residue was redissolved in chloroform, filtered and re- evaporated. Trituration with diethyl ether gave the title dye as an orange solid, 105mg (79%). λmax (MeOH) = 492nm Fluorescence (MeOH); λ_ex = 492nm, λ_em = 516nm. δ_H (300MHz, CD₃OD) 1 .662 (3H, t), 2.13-2.21 (6H, s + 2H, m), 2.42-2.48 (2H, m), 3.02 (1 H, m), 3.82-3.92 (2H, m), 4.43 (2H, broad m), 4.91 -4.96 (2H, m), 6.024 (1 H, s), 7.663 (1 H, t), 7.70 (1 H, d), 7.83 (1 H, t), 8.20 (2H, app. d) and 8.45 (1 H, d). Mass spectrum: (ES+) 445 (M+H), 467 (M+Na).

22mg (50μmol) of this carboxy dye was converted to the reactive N-hydroxysuccinimide ester [3h], by reaction with 0-(N- succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, 15mg, 50μmol), in DMF (0.5ml) containing N,N-diisopropylethylamine (9μl). The activation was followed by t.l.c. (RPCι₈. Methanol, 95: water, 5. [3g] R_f = 0.5. Product R_f = 0.45); it was complete after 20minutes.

The NHS ester [3h] was then reacted directly with an indotrimethinecyanine dye bearing a pendant primary amino group, to give a conjugate with structure [3i]. This product was isolated, purified by preparative t.l.c. (silica. Methanol, 25: chloroform, 75. Product isolated pure; R_f = 0.2 in this system) and characterized; it represents an example of an energy-transfer system.

Mass spectrum: (ES+) 920 (M+), 921 (M+H), 943 (M+Na).

Fluorescence (MeOH); λ_ex = 492, 551 nm, λ_em = 567nm. The Cy3 acceptor dye has λ_ex = 551 nm, λ_em = 567nm; excitation of the Cy3 amino dye at 492nm results in an emission intensity at 567nm of only 20% of that induced by excitation at 551 nm. For the conjugate [3i], excitation at 492nm gives an emission intensity which is 65% of that induced by excitation at 551 nm. The squaramide donor emission peak at 517nm is almost completely suppressed in the conjugate [3i], upon excitation at its peak of 492nm, so its contribution to 567nm emission is negligible. Thus conjugation of the squaramide donor to the Cy3 dye causes an increase of over three-fold in emission, when excitation is at 492nm, over the Cy3 dye alone.

2-(4-((2-Maleimidoethyl)aminocarbonyl)piperidino)-4-(1 ,3,3-trimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3j] This was prepared from the squaramide NHS ester ([3h],

O.l mmol, prepared as above) and N-(2-aminoethyl)maleimide.HCI salt (2x20mg, 0.1 1 mmol), in anhydrous acetonitrile in the presence of N,N- diisopropylethylamine. The reaction was followed by t.l.c. (silica. Methanol, 5: dichloromethane, 95. R_f values: [3g] = 0.02, [3h] = 0.4, [3j] = 0.1 . Once the reaction was deemed to be complete, the mixture was partitioned between dichloromethane and 0.1 M aqueous HCI. The organic phase was collected, dried (MgS0₄), filtered and evaporated under vacuum. The residue was purified by flash chromatography (silica. 2.5-4% methanol/dichloromethane). The product fractions were combined and evaporated. The sticky residue was triturated with acetonitrile, which afforded a fine solid. The mixture was carefully diluted with ether, then the solid was collected by filtration, washed with ether and dried to give the title dye, 40mg (71 %).

This is a maleimide-functionalized dye, which is reactive towards thiol groups. It has been used to label proteins in E. Coli lysate; the resulting labelled proteins were then analysed by a 2D-DIGE method, as described in PCT patent application WO/96/33406. λmax (MeOH) = 492nm δ_H (300MHz, CD₃OD) 1 .373 (3H, t), 1 .80-2.03 (10H, m), 2.491 (1 H, m), 3.36-3.47 (4H, m), 3.63 (2H, m), 4.13 (2H, broad q), 4.76 (2H, m), 5.734 (1 H, s), 6.822 (2H, s), 7.38 (1 H, t), 7.45 (1 H, d), 7.54 (1 H, m), 7.91 (2H, app. d) and 8.16 (1 H, d).

Mass spectrum: (ES+) 567 (M+H), 589 (M+Na),

(ES-) 565 (M-H)

2-(1 -aza-18-crown-6)-4-(1 ,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [3k]

This was prepared in an analogous method to [3a], using 1 -aza-18-crown-6 (87mg, 0.33mmol) in place of piperidine. The title dye [3k] was isolated in the same manner. λma (MeOH) = 494nm δ_H (300MHz, CDCI₃) 1 .346 (3H, t), 1 .942 (6H, s), 3.64 (16H, app. s), 3.854 (4H, t), 4.01 (2H, broad q), 4.240 (4H, t), 5.695 (1 H, s), 7.18- 7.3 (m, partially obscured by CHCI3), 7.332 (1 H, t), 7.493 (1 H, t), 7.82 (2H, m) and 8.10 (1 H, d). Mass spectrum: (ES+) 579 (M+H), 601 (M+Na), 617

(M+K).

2-N-(4-carboxyphenyl)-N-methylamino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)cyclobutenediylium-1 ,3-diolate [31] This was prepared in an analogous method to [3a], using 4- methylamino benzoic acid (50mg, 0.33mmol) in place of piperidine. Acetic acid was used as alternative solvent. The title dye [31] was isolated in the same manner. Yield = 54mg (39%). λma (MeOH) = 526nm. δ_H (300MHz, CD₃OD) 1 .48 (3H, t), 2.02 (6H, s), 4.05 (3H, s), 4.25 (2H, q), 6.06 (1 H, s) and 7.47-8.27 (1 OH, m).

2-N-(4-carboxyphenyl)-N'-(tert-butoxycarbonyl)hydrazino-4-(1-ethyl- 3,3-dimethyl-2-benzindolinylidenemethyl)cyclobutenediylium-1 ,3- diolate [3m] This was prepared in an analogous method to [3a], using 1 - tert-butyl-2-phenylhydrazine (80mg, 0.32mmol) in place of piperidine. Acetic acid was used as alternative solvent. The title dye [3m] was isolated in the same manner. Yield = 29mg (21 %). λmax (H₂O) = 528nm Fluorescence (H₂0); λ_ex = 532nm, λ_em = 566nm. δ_H (300MHz, CD3OD) 1 .28 (3H, t), 1 .48 (9H, s), 1.99 (6H, s), 4.29 (2H, q), 6.04 (1 H, s), 6.73 (2H, d) 7.22 (1 H, app. t), 7.47-7.76 (2H, m), 7.82 (2H, d) and 7.98-8.27 (3H, m).

Mass spectrum: (ES+) 589 (M+H), 61 1 (M+Na).

2-(N-(phenyl)-N'-(levulinic acid) hydrazone)-4-(1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)cyclobutenediylium-1 ,3-diolate [3n]

This was prepared in an analogous method to [3a], using levulinic acid phenylhydrazone lactam (37mg, 0.20mmol) in place of piperidine. Acetic acid was used as alternative solvent. The title dye [3n] which was the minor product was isolated in the same manner. Further purification on a preparative plate was carried out (Silica; 10% MeOH/ 90% DCM). Yield = 2mg (3%). λ_max (MeOH) = 532nm δ_H (300MHz, CDCI3) 1 .25 (3H, s), 1 .43 (3H, t), 1 .57 (4H, broad s), 2.01 (6H, s), 4.19 (2H, q), 5.92 (1 H, s), 7.17-7.85 (8H, m), 7.89- 7.94 (2H, m) and 8.19 (1H,d).

[3a] R1-R2 = -(CH₂)₅-

[3b] R1-R2 = -(CH₂)₂-0-(CH₂)₂-

[3d] R1 = Ph, R2 = CH₃

[3e] R1 = R2 = Ph

[3f] R1 = Ph, R2 = -NH-CO-CH₃

[3c]

[3j]

[3k]

10

EXAMPLE 4

Synthesis of squarate dyes from sulphonated benzindolenine-squaric acid intermediates

2-(1 -Piperidino)-4-(5-sulphonato-1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, sodium salt [3a]

3-Hydroxy-4-(5-sulphonato-1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobut-3-en-1 ,2-dione, sodium salt [1 j]

(130mg, 0.30mmol), piperidine (40μl = 34mg, 0.40mmol) and 1 -butanol (5ml) were mixed and heated to 1 10°C; not all the solid dissolved, but an orange colour developed. The reaction was followed by t.l.c. (RPCι₈- Methanol, 60: water, 40. [1 j] R_f = 0.8, [4a] R_f = 0.5). Once the reaction appeared to have halted, another 20μl of piperidine was added along with 2ml ethanol, and heating resumed. After another 4hrs, the solvent was evaporated under reduced pressure. The residue was purified by prep. HPLC (RPC-18. Water → methanol gradient.); collected fractions that contained pure product were combined and evaporated, dried under high vacuum and trituration with diethyl ether. The resulting orange powder was further dried under vacuum to give the title compound [4a], 99mg. λmax (MeOH) = 490nm, (pH 7.0 phosphate buffer) = 488nm. Fluorescence (pH 7.0 phosphate buffer) ;λ_ex = 488nm, λ_em = 512nm. δ_H (300MHz, CD₃OD) 1 .36 (3H, t), 1 .80 (6H, broad s), 1 .91

(6H, s), 4.06 (6H, m), 5.73 (1 H, s), 7.50 (1 H, d), 7.93 (1 H, d), 7.99 (1 H, d), 8.21 (1 H, d) and 8.37 (1 H, s). Evidence of some piperidinium cations. Mass spectrum: (ES-) 479 (M^~).

A study on the effect of pH on fluorescence was conducted. Buffer solutions were bought pre-prepared from Radiometer Analytical S.A.; pH 4.0 (potassium hydrogen phthalate, 50mmol / 1) pH 7.0 (Na₂HPO₄, 27.5mmol / I : KH₂P0₄, 20.0mmol / I) pH 10.0 (NaHCO₃, 25mmol / 1 : Na₂C0₃, 25mmol / 1)

A stock solution of dye [4a] in methanol was diluted with these buffers, to give solutions of dye of equal concentration (approximately 2x10^"6 M). The absorbance and fluorescence spectra of these solutions were then recorded.

It can be seen from this that the fluorescence is not quenched in mildly acidic or basic solution.

2-(4-Carboxypiperidino)-4-(5-sulfonato-1 ,3,3-trimethyl-2- benzindoiinylidenemethyl) cyclobutenediylium-1 ,3-diolate [4b]

3-Hydroxy-4-(5-sulphonato-1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobut-3-en-1 ,2-dione, sodium salt [1j] (130mg, 0.30mmol), isonipecotic acid (52mg, 0.40mmol) and acetic acid (2.5ml) were mixed and heated to 100°C; the reaction was followed by t.l.c. (RPC18. Methanol, 40: water, 60. [1j] R_f = 0.7, product R_f = 0.5). After 5.5hrs the solvent was evaporated under vacuum and co-evaporated with acetonitrile, twice. The residue was triturated with diethyl ether. Purification was by dilution of the methanolic solution of this solid with water; 53mg of the title compound were isolated as the sodium salt. λ_max (MeOH) = 492nm, (water) = 488nm.

Fluorescence (MeOH) ;λ_ex = 492nm, λ_e = 517nm. δ_H (300MHz, CD₃OD) 1.362 (3H, t), 1.90-1.95 (8H, m), 2.13- 2.19 (2H, m), 2.73 (1H, m), 3.55-3.65 (2H, m), 4.10 (2H, broad q), 4.65 (2H, m), 5.748 (1H, s), 7.51 (1H, d), 7.93 (1H, dd), 7.99 (1H, d), 8.21 (1H, d) and 8.37 (1H,d).

Mass spectrum: (ES-) 523 (M^~), 261 ((M-H) ²72).

[4a]

EXAMPLE 5

Synthesis of squarate dyes using primary amines - pH sensitive dyes

If the amino compound used to form the final dye is a primary amine (i.e. has an -NH₂ group), then the product squarate dye has a nitrogen which is incompletely alkylated, having a hydrogen atom attached instead of a third carbon atom. This hydrogen can be removed under basic conditions, causing a marked change in visible absorption and fluorescence properties; the dye becomes pH-sensitive. The nature of the original amine provides a means of controlling the pKa value of this change, i.e. the dye can be "tuned" to change from one form to the other over a controlled pH range. The following examples illustrate this effect.

2-Phenylamino-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [5a]

This was prepared in an analogous method to [3a], using aniline (30μl = 31 mg, 0.33mmol) in place of piperidine. The title dye [5a] crystallized from the reaction mixture on cooling; the solid was collected, washed with 1 -butanol and diethyl ether, and dried under vacuum. Yield of [5a] = 100mg (81 %). λmax (MeOH) = 532nm; ε_max = 99,000 dm³ mol^"1 cm^"1.

(MeOH + Bu₄ ⁺ OH^") = 456nm, ε_max = 53,000 dm³ mol^"1 cm^"1.

Fluorescence (MeOH); λ_ex = 532nm, λ_em = 548nm.

(MeOH + Bu ⁺ OH^") = non-fluorescent. δ_H (300MHz, CDCI₃) 1 .391 (3H, t), 1 .978 (6H, s), 4.12 (2H, broad q), 5.880 (1 H, s), 7.130 (1 H, t), 7.25 (1 H, d), 7.34-7.40 (3H, m), 7.49- 7.55 (1 H, m), 7.81 -7.93 (4H, m), 8.1 1 (1 H, d) and 9.9 (1 H, broad).

Mass spectrum: (ES+) 409 (M+H), 431 (M+Na). 2-Butylamino-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [5b]

This was prepared in an analogous method to [3a], using 1 -butylamine (32μl = 24mg, 0.33mmol) in place of piperidine. The reaction was followed by t.l.c. (RPC_l8. Methanol, 95: water, 5. [1 f], R_f = 0.8, [5b], R_f = 0.4). After 7hrs the solvent was evaporated under vacuum; the residue was partitioned between 0.1 M aqueous HCI and chloroform. The organic phase was collected, washed with water, then dried (MgSO₄), filtered and the solvent evaporated. Purified by flash chromatography (silica. 2-3% methanol / chloroform), to give 84mg (72%) of the title compound. λma (MeOH) = 484nm; ε_maχ = 85,000 dm³ mol^"1 cm^"1. (MeOH+Bu₄ ⁺ OH^") = 434+388nm; ε_maχ « 33,000 dm³ mol^"1 cm^"1 (434nm) Fluorescence (MeOH); λ_ex = 483nm, λ_em = 51 1 nm. (MeOH + Bu ⁺ OH^") = non-fluorescent. δ_H (300MHz, CDCI₃) 1 .003 (3H, t), 1 .373 (3H, t), 1.50 (2H, app. hex) 1 .8 (2H, m), 1 .971 (6H, s), 3.90 (2H, q), 4.03 (2H, q), 5.691 (1 H, s), 7.21 (1 H, d), 7.35 (1 H, m), 7.51 (1 H, m), 7.84 (2H, app t), 8.10 (1 H, d) and 9.20 (1 H, broad) Mass spectrum: (ES+) 389 (M+H), 41 1 (M+Na).

2-Amino-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [5c]

This was prepared in an analogous method to [3a], using ammonium acetate (77mg, 1 .Ommol) in place of piperidine. The reaction was followed by t.l.c. (RPC₁₈. Methanol, 90: water, 10. [1f], R_f = 0.65, [5b], R_f = 0.35). Once the reaction was complete the solvent was evaporated under vacuum; the residue was purified by flash chromatography (silica. 2- 5% methanol / chloroform), to give the title compound. λ_max (MeOH) = 478nm

(MeOH + Bu₄ ⁺ OH^") = 434nm. Fluorescence (MeOH); λ_ex = 481 nm, λ_em = 506nm. (MeOH + Bu ⁺ OH^") = non-fluorescent. δ_H (300MHz, DMSO-de) 1.255 (3H, t), 1.870 (6H, s), 4.05 (2H, q), 5.555 (1 H, s), 7.37 (1 H, m), 7.55 (2H, m), 7.95 (2H, app d), 8.16 (1 H, d) and 9.47 (1 -2H, broad).

Mass spectrum: (ES+) 333 (M+H), 355 (M+Na).

2-(3-Sulfonatophenyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, sodium salt [5d]

3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)-cyclobut-3-en-1 ,2-dione [1f] (100mg, 0.30mmol), metanilic acid (60mg, 0.35mmol), anhydrous sodium acetate (30mg, 0.37mmol) and acetic acid (2.5ml) were mixed and heated at around 100°C for 3hrs; during this time the colour of the mixture turned from yellow to deep magenta. The solvent was then evaporated under vacuum.

The crude product was purified by preparative HPLC (RPCie- Water→methanol gradient). Final yield of the title compound was 92mg (60%). λmax (MeOH) = 536nm

For measurements in aqueous solution, a LOmMol solution in DMF was prepared; this was diluted 1 :100 with 0.02Mol bis-tris propane/HCI aqueous buffer to give a 10μMol working solution.

(pH7 aq.) = 524nm, ε_maχ « 75,000 dm³ mol^"1 cm^"1 (pH10 aq.) = 460nm, ε_maχ = 49,000 dm³ mol^"1 cm^"1

Fluorescence (MeOH); λ_ex = 537nm, λ_em = 552nm δ_H (300MHz, CD₃OD) 1.438 (3H, t), 1.959 (6H, s), 4.29 (2H, q), 6.017 (1 H, s), 7.44-7.65 (5H, m), 7.95-8.07 (4H, m) and 8.23 (1 H, d)

Mass spectrum: (ES-) 487 (M^~). 2-(4-Sulfonatophenyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, sodium salt [5e]

This was prepared and purified in an analogous method to [5d], using sulfanilic acid (60mg, 0.35mmol) in place of metanilic acid. Yield =120mg (78%). λmax (MeOH) = 540nm

For measurements in aqueous solution, a I .OmMol solution in DMF was prepared; this was diluted 1 :100 with 0.02Mol bis-tris propane/HCI aqueous buffer to give a 10μMol working solution. (pH7 aq.) = 530nm, ε_max - 82,000 dm³ mol^"1 cm^"1 (pH10 aq.) = 468nm, ε_maχ « 53,000 dm³ mol^"1 cm^"1 Fluorescence (MeOH); λ_ex = 540nm, λ_em = 555nm δ_H (300MHz, CD₃OD) 1.450 (3H, t), 1.967 (6H, s), 4.31 (2H, q), 6.046 (1 H, s), 7.48 (1 H, t), 7.60-7.66 (2H, m), 7.78-7.86 (4H, m), 7.97 (2H, app.t) and 8.24 (1 H, d).

Mass spectrum: (ES-) 487 (M^~).

2-(3-Hydroxyphenyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, [5f]

This was prepared in an analogous method to [3a], using 3-aminophenol (39mg, 0.35mmol) in place of piperidine. The product precipitated from the reaction mix on cooling; this was collected, washed with 1-butanol and diethyl ether, then dried under vacuum. The title compound was isolated in 112mg yield (88%). λmax (MeOH) = 540nm

Fluorescence (MeOH); λ_ex = 537nm, λ_em = 553nm. δ_H (300MHz, DMSO-d₆) 1.307 (3H, t), 1.916 (6H, s), 4.210 (2H, broad q), 5.755 (1 H, s), 6.55 (1 H, d), 7.154 (1 H, t), 7.37-7. 67 (3H, m), 7.589 (1 H, t), 7.67 (1 H, d), 7.99-8.02 (2H, m), 8.20 (1 H, d), 9.626 (s, partial exch.) and 11.368 (s, partial exch.).

Mass spectrum: (ES+) 425 (M+H), 447 (M+Na).

2-((4-Carboxymethyl)phenyl)amino-4-[1-(4-sulfonatobutyl)-3,3- dimethyl-2-benzindolinylidenemethyl] cyclobutenediylium-1 ,3-diolate, sodium salt [5g]

3-Hydroxy-4-(1 -(4-sulfonatobutyl)-3,3-dimethyl-2- benzindolinylidenemethyl) -cyclobut-3-en-1 ,2-dione [1 m] (116mg, 0.25mmol) and 4-aminophenylacetic acid (40mg, 0.26mmol) were mixed in acetic acid (3ml) and heated to 100°C for 1 hr. The reaction was followed by t.l.c. (RPC1₈. Methanol, 60: water, 40. [1 m], R_f = 0.65, yellow spot; product, R_f =0.38, magenta spot). The mixture was cooled to 10°C, whereupon the product precipitated; the solid was collected, washed with cooled acetic acid, then diethyl ether and dried under vacuum. Yield of title compound = 67mg (46%). λm_ax (MeOH) = 538nm, (pH7 aqueous buffer) = 524nm.

Fluorescence (MeOH); λ_ex = 541 nm, λ_em = 558nm. δ_H (300MHz, DMSO-de) 1.72-1.90 (4H, m), 1.916 (6H, s), 3.5 (m, partially obscured by HOD), 3.553 (2H, s), 4.15 (2H, broad s), 5.756 (1 H, s), 7.26 (2H, d), 7.42 (1 H, t), 7.59 (1 H, t), 7.71 (1 H, d), 7.83 (2H, d), 8.00 (2H, d), 8.21 (1 H, d), 1 1.49 (s, partially exch.) and 12.3 (broad s, partially exch.)

Mass spectrum: (ES-) 573 (M^~).

A sample of this material was converted to the reactive NHS ester. [5g] (30mg, 50μmol) was reacted with TSTU (15mg, 50μmol), in DMF (200μl) in the presence of N,N-diisopropylethylamine (9μl). The reaction was followed by t.l.c. (RPCι₈. Methanol, 60: water, 40. [5g], R_f = 0.4; NHS ester [5h], R_f = 0.32). The NHS ester was precipitated from solution by dilution with diethyl ether; the solid was isolated, washed with a methanokether mix, then ether, and dried under vacuum.

This material was coupled successfully with 1 -butylamine, demonstrating that this material could be used to label molecules bearing primary amino groups.

2-Butylamino-4-[1-(4-sulfonatobutyl)-3,3-dimethyl-2- benzindolinylidenemethyl] cyclobutenediylium-1 ,3-diolate, sodium salt [5i] This was prepared in an analogous method to [5g], using

1 -butylamine (30μl, 0.3mmol) in place of 4-aminophenylacetic acid. The reaction was followed by t.l.c. (RPCι₈. Methanol, 60: water, 40. [1 m], R_f = 0.65; [5i], R_f = 0.24). After 5.5hrs the solvent was evaporated under vacuum; the residue was purified by preparative HPLC (RPCι₈. Water→methanol gradient). Final yield of the title compound was 28mg (22%). λmax (MeOH) = 486nm, (pH7 aqueous buffer) = 480nm.

(MeOH + Bu₄ ⁺ OH^") = 430nm,

Fluorescence (MeOH); λ_ex = 487nm, λ_e = 512nm. δ_H (300MHz, CD₃OD) 0.992 (3H, t), 1 .45 (2H, hex), 1 .69 (2H, quin), 1.88-1 .97 (10H, m), 2.89 (2H, broad t), 3.774 (2H, t), 4.13 (2H, broad q), 5.757 (1 H, s), 7.368 (1 H, t), 7.55 (2H, m), 7.91 (2H, app.d) and 8.16 (1 H, d).

Mass spectrum: (ES-) 495 (M^~).

2-(8-Hydroxy-6-sulfonato-2-naphthyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, sodium salt [5j]

This was prepared and purified in an analogous method to [5d], using 6-amino-4-hydroxy-2-naphthalenesulfonic acid monohydrate (90mg, 0.35mmol) in place of metanilic acid. Yield =61 mg (35%). λmax (MeOH) = 552nm

(pH7 aq.) = 542+51 Onm, (pH10 aq.) = 472nm

Fluorescence (MeOH); λ_ex = 552nm, λ_em = 566nm. δ_H (300MHz, CD₃OD) 1.445 (3H, t), 1.977 (6H, s), 4.29 (2H, q), 6.024 (1 H, s), 7.26 (1 H, d), 7.46 (1 H, m), 7.55 (1 H, d), 7.62 (1 H, m), 7.82 (1 H, s), 7.91 -8.00 (3H, m), 8.1 1 (1 H, dd), 8.23 (1 H, d) and 8.37 (1 H, d).

Mass spectrum: (ES-) 553 (M ).

2-(6-Sulfonato-1-naphthyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate, sodium salt [5k]

This was prepared and purified in an analogous method to [5d], using 5-amino-2-naphthalenesulfonic acid monohydrate (78mg, 0.35mmol) in place of metanilic acid. Yield =113mg (67%). λmax (MeOH) = 524nm

(pH7 aq.) = 512nm, (pH10 aq.) = 458nm

Fluorescence (MeOH); λ_e = 530nm, λ_em = 566nm. δ_H (300MHz, CD₃OD) 1.445 (3H, t), 1.973 (6H, s), 4.30 (2H, q), 6.044 (1 H, s), 7.47 (1 H, t), 7.56-7.68 (3H, m), 7.90 (2H, app. t), 7.97- 8.02 (3H, m), 8.22-8.29 (2H, m) and 8.40 (1 H, d).

Mass spectrum: (ES-) 537 (M^~).

2-N-((4-carboxymethyl)phenyl)amino-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [51]

This was prepared in an analogous method to [3a], using 4-aminophenylacetic acid (50mg, 0.33mmol) in place of piperidine. Acetic acid was used as alternative solvent. After heating, the mixture was cooled down and the title dye [31] was isolated by filtration, washing with a little cold acetic acid, then with diethyl ether to furnish the product as a red solid. Yield = 116mg (83%). λmax (MeOH) = 536nm. δ_H (300MHz, DMSO) 1.31 (3H, t), 1.90 (6H, s), 3.55 (2H, s), 4.18 (2H, q), 5.76 (1 H, s), 7.25 (2H, d), 7.40 (1 H, t), 7.56 (1 H, t), 7.65 (1 H, d), 7.81 (2H, d), 8.0. (2H, app d) and 8.20 (1 H, d).

Mass spectrum: (ES+) 467 (M+H)

[5a] R = Ph

[5b] R = CH₂CH₂CH₂CH₃

[5c] R = H

[5d] R = (3-S03Na)Ph

[5e] R = (4-S03Na)Ph

[5f] R = (3-OH)Ph

EXAMPLE 6

Preparation of acylated squarate dye examples

Any of the dyes in example 5 can be acylated on the nitrogen with acyl halide reagents, as demonstrated by the following examples:

2-(N-Butylacetamido)-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [6a]

2-Butylamino-4-(1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [5b] (19.4mg, 50μmol) was dissolved in dry pyridine (0.5ml) to give a yellow solution. To this was added acetyl chloride (1 drop); a red colour formed immediately. After 30 minutes stirring at ambient temperature the solvent was evaporated under vacuum; the residue was co-evaporated twice with acetonitrile, then purified by preparative t.l.c. (silica. Methanol,

5: dichloromethane, 95). The orange-red product band was collected, the dye extracted with the eluant mix and the resulting filtered solution evaporated under vacuum. The residue was triturated with a mix of 4:1 light petroleum spirit : ether to give a solid; this was dried under vacuum to give the product dye, 16mg (74%). λm_ax (CH₂CI₂) = 536nm; (MeOH) = 506nm δ_H (300MHz, CD₂CI₂) 0.93 (3H, t), 1.38 (2H, hex), 1.518 (3H, t), 1.60 (2H, m), 2.034 (6H, s), 2.624 (3H, s), 4.134 (2H, t), 4.37 (2H, q), 6.228 (1 H, s), 7.48-7.57 (2H, m), 7.66 (1 H, m), 7.98-8.03 (2H, m) and 8.24 (1 H, d).

Mass spectrum: (ES+) 431 (M+H), 453 (M+Na), 469 (M+K). 2-(N-Phenylacetamido)-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [6b]

This was prepared and purified in an analogous method to [6a], using 2-phenylamino-4-(1 -ethyl-3,3-dimethyl-2- benzindolinylidenemethyl)cyclobutene- diylium-1 ,3-diolate [5a] (20.4mg, 50μmol) and acetyl chloride (1 drop). The title compound was isolated in 6mg yield (27%). λmax (CH₂CI₂) = 538nm; (MeOH) = 502nm δ_H (300MHz, CD₂CI₂) 1.53 (3H, t), 1.979 (6H, s), 2.617 (3H, s), 4.41 (2H, q), 6.333 (1 H, s), 7.28 (2H, m), 7.36-7.58 (5H, m), 7.66 (1 H, t), 8.01 (2H, m) and 8.19 (1 H, d).

2-Benzoylamido-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [6c] This was prepared and purified in a slightly modified method to [6a], using 2-amino-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutene- diylium-1 ,3-diolate [5c] (17mg, 50μmol) and benzoyl chloride (20% v/v solution in pyridine). The acid halide solution was added in 1 drop aliquots, monitoring the reaction by t.l.c. (silica. Methanol, 5: dichloromethane, 95. [5c], R_f = 0.16 , yellow spot; product, R_f = 0.3, red spot). Isolated yield 12mg (55%). λmax (CH₂CI₂) = 534nm; (MeOH) = 502nm δ_H (300MHz, CD₂CI₂) 1.45 (3H, t), 1.941 (6H, s), 4.32 (2H, q), 6.204 (1 H, s), 7.40-7.62 (7H, m), 7.90-7.98 (4H, m) and 8.14 (1 H, d). Mass spectrum: (ES+) 437 (M+H), 459 (M+Na),

475 (M+K)

2-(Dibenzoylamido)-4-(1-ethyl-3,3-dimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [6d] This was prepared and purified in an analogous method to

[6c], using an excess of benzoyl chloride, (silica. Methanol, 5: dichloromethane, 95. [5c], R_f = 0.16 , yellow spot → [6c], R_f = 0.3, red spot

→ [6d], R_f = 0.55, orange spot). Isolated yield 1 1 mg (41%). λmax (CH₂CI₂) = 528nm; (MeOH) = 486nm δ_H (300MHz, CD2CI2) 1.446 (3H, t), 1.848 (6H, s), 4.37 (2H, q), 6.273 (1 H, s), 7.33-7.62 (1 OH, m), 7.79-7.82 (3H, m), 7.95 (2H, m) and

8.12 (1 H, d).

Mass spectrum: (ES+) 541 (M+H), 563 (M+Na), 579

(M+K)

2-Acetamido-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate [6e]

This was prepared and purified in an analogous method to [6c], using acetyl chloride in place of benzoyl chloride. Isolated yield = 13mg (70%). λmax (CH₂CI₂) = 522+498nm; (MeOH) = 486nm δ_H (300MHz, CD2CI₂) 1.408 (3H, t), 2.026 (6H, s), 2.543 (3H, s), 4.37 (2H, q), 6.227 (1 H, s), 7.47-7.57 (2H, m), 7.63-7.69 (1 H, m), 7.98- 8.03 (2H, m), 8.24 (1 H, d) and 8.8 (1 H, broad s).

Mass spectrum: (ES+) 375 (M+H), 397 (M+Na), 413 (M+K)

[6a] R1 = CH₂CH₂CH₂CH₃; R2 = CO.CH₃ [6b] R1 = Ph; R2 = CO.CH₃ [6c] R1 = CO.Ph; R2 = H [6d] R1 = R2 = CO.Ph [6e] R1 = CO.CH₃; R2 = H EXAMPLE 7

Synthesis of squarate dye examples with benzothiazole nuclei

3-Methoxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut-3-en-1 ,2- dione [7a]

A 50ml reaction flask was charged with dimethyl squarate (1.42g, 10mmol), dry methanol (10ml) and N,N-diisopropylethylamine (1.29g, 10mmol). This mixture was heated to boiling until all the solid had dissolved.

To the hot solution was added 3-ethyl-2- methylbenzothaizolium iodide (3.05g, 10mmol) - an immediate orange-red colour formed as the solid dissolved, then an orange solid began to crystallize out. Heating was continued for another 10 minutes, then the mixture was allowed to cool to ambient temperature before final cooling in the fridge. The precipitated solid was collected by filtration, washed with ice-cold methanol, then diethyl ether and dried under vacuum. 2.35g (82%). λmax (MeOH) = 440nm δ_H (300MHz, CDCI₃) 1.372 (3H, t), 4.04 (2H, q), 4.451 (3H, s), 5.445 (1 H, s), 7.06 (1 H, d), 7.15 (1 H, app. t), 7.33 (app. t) and 7.48 (1 H, dd).

T.l.c. (RPC₁₈. Methanol, 95: water, 5. [7a], R_f = 0.5; minor pink impurity, R_f = 0.3).

Mass spectrum: (ES+) 288 (M+H), 310 (M+Na), 326 (M+K)

3-Hydroxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut-3-en-1,2- dione [7b]

This was prepared using a literature method (E.Terpetschnig & J.R. Lacowicz; Dyes and Pigments (1993), 21 , 227-234). 1.44g of [7a] gave a crude yield = 1.24g. 500mg of this material was purified further by flash chromatography (silica. Dichloromethane → methanol gradient). 258mg of pure [7b] was isolated, along with 30mg of the pink impurity described above - this proved to be the 1 ,2-bis-adduct [7c], a known compound (V.H.E. Sprenger & W.Ziegenbein; Angew. Chem., (1967), 79, 581 -582).

Data for [7b]: λ_max (MeOH) = 446nm δ_H (300MHz, CD₃OD) 1 .312 (3H, t), 4.05 (2H, q), 7.05 (1 H, t), 7.15 (1 H, d), 2.27 (1 H, ~t) and 7.46 (1 H, d). Squaric methine proton not evident, but this can exchange under acidic conditions.

Mass spectrum: (ES+) 274 (M+H), 296 (M+Na).

Data for [7c]: λmax (MeOH) = 524nm δ_H (300MHz, DMSO-d₆) 1 .276 (6H, t), 4.27 (4H, q), 5.865 (2H, s), 7.1 1 -7.19 (2H, m), 7.32-7.39 (4H, m) and 7.74 (2H, d).

Mass spectrum: (ES+) 433 (M+H).

2-(1 -Morpholino)-4-(3-ethyl-2- benzothiazo!ylidenemethyl)cyclobutenediylium-1 ,3-diolate [7d]

3-Hydroxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut- 3-en-1 ,2-dione [7b] (82mg, 0.3mmol) and morpholine (35μl = 35mg, 0.4mmol) were mixed in 1-butanol (3ml) and heated at 100°C for 2.5 hours. The reaction was followed by t.l.c. (RPC-ie- Methanol, 95: water, 5. [7b], R_f = 0.8; [7d], R_f = 0.4). The solvent was then evaporated under vacuum and the residue purified by flash chromatography (silica. 2-4% methanol / dichloromethane) to give 94mg (92%) of the title compound. λmax (MeOH) = 494nm; ε_maχ = 114,000 dm³ mol^"1 cm^"\ Fluorescence (MeOH); λ_ex = 492nm, λ_em = 518nm. δ_H (300MHz, CDCI₃) 1.368 (3H, t), 3.81 -3.85 (4H, m), 4.00- 4.11 (6H, m), 5.72 (1 H, s), 7.07 (1 H, d), 7.13 (1 H, t), 7.32 (1 H, t) and 7.47 (1 H, d).

Mass spectrum: (ES+) 343 (M+H).

2-(N-Methylanilino)-4-(3-ethyl-2-benzothiazolylidenemethyl) cyclobutenediylium -1 ,3-diolate [7e]

This was prepared in an analogous method to [7d], using N- methylaniline (44μl = 43mg, 0.40mmol) in place of piperidine. The title dye [7e] was isolated in the same manner. Yield = 43mg (40%). λmax (MeOH) = 514nm; ε_maχ = 105,000 dm³ mol^"1 cm^'1.

Fluorescence (MeOH); λ_ex = 518nm, λ_em = 565nm. δ_H (300MHz, CDCI₃) 1 .405 (3H, t), 3.915 (3H, s), 4.15 (2H, q), 5.900 (1 H, s), 7.12-7.23 (4H, m), 7.33-7.43 (4H, m) and 7.50 (1 H, d). Mass spectrum: (ES+) 363 (M+H), 385 (M+Na).

2-(4-Sulfonatophenyl)amino-4-(3-ethyl-2-benzothiazolylidenemethyl)- cyclobutenediylium-1 ,3-diolate, sodium salt [7f]

3-Hydroxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut- 3-en-1 ,2-dione [7b] (82mg, 0.3mmol), sulfanilic acid (60mg, 0.35mmol) and anhydrous sodium acetate (30mg, 0.37mmol) were mixed with acetic acid (1 .5ml). This mixture was heated at 100°C for 2hours, whereupon the colour reddened and a solid precipitated. After cooling, this solid was collected by filtration, washed with acetic acid (3x2ml), then diethyl ether and dried. Yield = 102mg (75%). λmax (MeOH 50: H₂0 50) = 518nm; (+ Bu₄ ⁺ OH^") = 475nm. Fluorescence (MeOH); λ_ex = 530nm, λ_em = 552nm. δ_H (300MHz, DMSO-d₆) 1 .388 (3H, t), 4.45 (2H, q), 5.998 (1 H, s), 7.21 (1 H, d), 7.40 (1 H, t), 7.55-7.83 (5H, m) and 8.04 (1 H, d). Mass spectrum: (ES-) 427 (M^~)

R = CH₃ [7c] [7b] R = H

[7d] [7^β]

EXAMPLE 8

Synthesis of squarate dye examples with 2-quinoline nuclei

3-Methoxy-4-(1 -ethyl-2-quinolylidenemethyl)-cyclobut-3-en-1 ,2-dione [8a]

This was prepared in an analogous method to [7a], using 1 -ethyl-2-methylquinilinium iodide (900mg, 3mmol) in place of 3-ethyl-2- methylbenzothiazolium iodide. Work-up involved evaporation of the solvent and partitioning between chloroform and dilute aqueous HCI. The organic phase was collected, washed with water and dried (MgS0₄); after filtration and concentration it was purified by flash chromatography (silica. 2.5-3% methanol / chloroform). Yield of the title compound was 570mg (68%). λ_max (MeOH) = 462nm δ_H (300MHz, DMSO-d₆) 1.309 (3H, t), 4.26 (2H, broad q),

4.383 (3H, s), 5.245 (1 H, s), 7.295 (1 H, t), 7.57-7.70 (4H, m) and 8.37 (1 H, d).

Mass spectrum: (ES+) 282 (M+H), 304 (M+Na).

3-Hydroxy-4-(1 -ethyl-2-quinolylidenemethyl)-cyclobut-3-en-1 ,2-dione [8b]

This was prepared in an analogous method to [1 b], involving hydrolysis of 3-hydroxy-4-(1 -ethyl-2-quinoIylidenemethyl)-cyclobut-3-en- 1 ,2-dione [8a] (281 mg, 1 mmol) in a dilute aqueous HCI / acetic acid mix. The reaction was followed by t.l.c. (RPdβ- Methanol, 95: water, 5. Samples dissolved in methanol spiked with triethylamine. [8a], R_f = 0.5, yellow spot; [8b], R_f = 0.7, orange spot). The colour of the product is reversibly lost in acidic solution, possibly due to protonation on the heteroaromatic ring.

The dried reaction mixture was used to prepare dyes without further purification. Mass spectrum: (ES+) 268 (M+H), 290 (M+Na), 306 (M+K).

2-(1 -Morpholino)-4-(1 -ethyl-2- quinolylidenemethyl)cyclobutenediylium-1 ,3-diolate [8c]

This was prepared and purified in an analogous method to [7d], using 3-hydroxy-4-(1 -ethyl-2-quinolylidenemethyl)-cyclobut-3-en-1 ,2- dione [8b] (80mg, 0.30mmol) and morpholine (35μl = 35mg, 0.4mmol) in 1 -butanol. T.l.c. (RPCι₈. Methanol, 95: water, 5. [8b], R_f = 0.7, orange spot; [8c], R_f = 0.4, magenta spot). Yield = 74mg (73%). λmax (MeOH) = 516nm; ε_maχ = 65,000 dm³ mol^"1 cm^"1. δ_H (300MHz, DMSO-de) 1 .313 (3H, t), 3.74-3.80 (4H, m), 3.88-3.95 (4H, m), 4.22 (4H, broad q), 5.41 1 (1 H, s), 7.27 (1 H, t), 7.54-7.66 (4H, m) and 8.91 (1 H, d). Mass spectrum: (ES+) 337 (M+H), 359 (M+Na).

2-(N-Methylanilino)-4-(1-ethyl-2-quinolylidenemethyl)- cyclobutenediylium-1 ,3-diolate [8d]

This was prepared and purified in an analogous method to [7d], using 3-hydroxy-4-(1 -ethyl-2-quinolylidenemethyl)-cyclobut-3-en-1 ,2- dione [8b] (80mg, 0.30mmol) and N-methylaniline (44μl = 43mg, 0.4mmol) in 1 -butanol. T.l.c. (RPC₁₈. Methanol, 95: water, 5. [8b], R_f = 0.7, orange spot; [8d], R_f = 0.4, deep pink spot). Yield = 85mg (79%). λmax (MeOH) = 544nm; ε_maχ = 67,000 dm³ mol^"1 cm^"1. δ_H (300MHz, DMSO-de) 1 .376 (3H, t), 3.836 (3H, s), 4.39 (2H, broad q), 5.669 (1 H, s), 7.21 (1 H, m), 7.36-7.44 (5H, m), 7.66-7.76 (2H, m), 7.81 (2H, app.d) and 9.14 (1 H, d).

Mass spectrum: (ES+) 357 (M+H), 379 (M+Na). 2-(4-Sulfonatophenyl)amino-4-(1-ethyl-2-quinolylidenemethyl)- cyclobutenediylium-1 ,3-diolate, sodium salt [8e]

3-Hydroxy-4-(1 -ethyl-2-quinolylidenemethyl)-cyclobut-3-en- 1 ,2-dione [8b] (81 mg, 0.3mmol), sulfanilic acid (60mg, 0.35mmol) and anhydrous sodium acetate (30mg, 0.37mmol) were mixed with acetic acid (1.5ml). This mixture was heated at 100°C for 2hours, whereupon the colour reddened and a solid precipitated. After cooling, this solid was collected by filtration, washed with acetic acid (3x5ml), then diethyl ether and dried. Yield = 135mg (-100%). λmax (MeOH 50: H₂0 50) = 540nm; (+ Bu₄ ⁺ OH^") = 514nm. δ_H (300MHz, DMSO-d₆) 1.403 (3H, t), 4.45 (2H, broad q), 5.754 (1 H, s), 7.40-7.52 (3H, m), 7.65-7.79 (4H, m), 7.85-7.95 (2H, m) and 9.22 (1 H, d).

Mass spectrum: (ES-) 421 (M^~).

[8a] R = CH₃ [8b] R = H

[8c] [8d]

EXAMPLE 9

Synthesis of squarate dye examples with 4-quinoline nuclei

3-Methoxy-4-(1-ethyl-4-quinolylidenemethyl)-cyclobut-3-en-1 ,2-dione [9a]

This was prepared and purified in an analogous method to [8a], using 1 -ethyl-4-methylquinolinium iodide (900mg, 3mmol) in place of 1 -ethyl-2-methylquinolinium iodide. Yield = 374mg (44%). λma (MeOH) = 528+498nm δ_H (300MHz, DMSO-de) 1 .320 (3H, t), 4.24 (2H, q), 4.381 (3H, s), 5.938 (1 H, s), 7.38 (1 H, m), 7.66-7.80 (4H, m) and 8.18 (1 H, d). Mass spectrum: (ES+) 282 (M+H), 304 (M+Na),

320 (M+K)

3-Hydroxy-4-(1-ethyl-4-quinolylidenemethyl)-cyclobut-3-en-1 ,2-dione [9b] This was prepared in an analogous method to [1 b], involving hydrolysis of 3-methoxy-4-(1 -ethyl-4-quinolylidenemethyl)-cyclobut-3-en- 1 ,2-dione [9a] (281 mg, 1 mmol) in a dilute aqueous HCI / acetic acid mix. The reaction was followed by t.l.c. (RPC-iβ- Methanol, 95: water, 5. Samples dissolved in methanol spiked with triethylamine. [9a], R_f = 0.5, pink spot; [9b], R_f = 0.65, red-pink spot). The colour of both the starting material and the product is reversibly lost in acidic solution, possibly due to protonation on the heteroaromatic ring.

The dried reaction mixture was used to prepare dyes without further purification.

2-(1-Piperidino)-4-(1-ethyl-4-quinolylidenemethyl)-cyclobutenediylium- 1 ,3-diolate [9c]

This was prepared and purified in an analogous method to [3a] using 3-hydroxy-4-(1-ethyl-4-quinolylidenemethyl)-cyclobut-3-en-1 ,2- dione [9b] (80mg, 0.3mmol). The reaction was followed by t.l.c. (RPCι₈. Methanol, 95: water, 5. [9b], R_f = 0.65, red-pink spot; [9c], R_f = 0.4, deep purple spot). λmax (MeOH) = 590+554nm δ_H (300MHz, CD₃OD) 1.435 (3H, t), 1.76 (6H, broad s), 3.94 (4H, broad s), 4.27 (2H, q), 6.217 (1 H, s), 7.40 (1 H, m), 7.53 (1 H, d), 7.63- 7.68 (2H, m), 8.17 (1 H, d) and 8.26 (1 H, d).

Mass spectrum: (ES+) 335 (M+H).

2-(N-Methylanilino)-4-(1-ethyl-4-quinolylidenemethyl)- cyclobutenediylium-1 ,3-diolate [9d]

This was prepared and purified in an analogous method to [7d], using 3-hydroxy-4-(1-ethyl-4-quinolylidenemethyl)-cyclobut-3-en-1 ,2- dione [9b] (80mg, 0.30mmol) and N-methylaniline (44μl = 43mg, 0.4mmol) in 1-butanol. T.l.c. (RPCι₈. Methanol, 95: water, 5. [9b], R_f = 0.65, red- pinkspot; [9d], R_f = 0.4, deep blue spot). Yield = 73mg (68%). λmax (MeOH) = 602nm δ_H (300MHz, CDCI₃) 1.468 (3H, t), 3.923 (3H, s), 4.16 (2H, q), 6.521 (1 H, s), 7.17-7.43 (8H, m), 7.60 (1 H, m), 8.27 (1 H, d) and 8.67 (1 H, d). Mass spectrum: (ES+) 357 (M+H), 379 (M+Na).

[9a] [9b]

EXAMPLE 10

Labelling of biological molecules

Labelling of a Protein Lysate with Dye 2-(4-((2-

Maleimidoethyl)aminocarbonyl)piperidino)-4-(1,33-trimethyl-2- benzindolinylidenemethyl) cycobutenediylium 1 ,3-diolate [3j]

An aliquot of E. coli lysate was prepared such that there was 25μg of protein in a final volume of 9μl lysate buffer (8M Urea, 4% (w/v) CHAPS, 40mM Tris (pH8)). To this was added 10mM TCEP (1μl of an aqueous solution) and the mixture incubated at 37°C for one hour. The resulting reduced protein was then labelled by the addition of a DMF solution of dye 3j (2μl of a 10nmol/1μl DMF solution). Labelling was effected by incubation at 37°C for 30mins. The reaction was then quenched by the addition of sample buffer (12μl of a buffer comprising of 8M Urea, 4% (w/v) CHAPS, 20mg/ml DTT, 4% IPG buffer) and the protein subjected to a 2D gel analysis i.e. IEF page followed by SDS page. The resultant gel was the visualised by a fluorescent scanner for protein positions on the gel as indicated by the dye labelled proteins.

Synthesis of the nucleotide dye conjugate 5-(3-(2-(4-amidopiperidino)- 4-(1 ,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium- 1 ,3-diolate)-allyl)-2'-deoxyuridine-5'-triphosphate.

5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (1 mg) was dissolved in 200 μl carbonate buffer pH 9.5. 2-(4-carboxypiperidino)-4-

(1 ,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate NHS ester [3h] in 100 μl acetonitrile was added to the mixture. The solution was stirred at room temperature in which the reaction mixture initially went turbid, then clear on further stirring. After 5 hours stirring the reaction mixture was purified on a reverse phase prep, plate (1 :1 acetonitrile : 0.1 M TEAB (triethylammonium bicarbonate buffer)). The product band was scraped off and extracted with the same eluent system, filtered, evaporated and redissolved in 50 acetonitrile:water and filtered through a cotton wool plug. The solution was evaporated and reverse phase TLC (1 :1 CH₃CN:0.1 M TEAB) after purification indicated the product (Rf = 0.22 ) was higher running than the starting material (0.05) and the carboxy derivative (0.14).

Mass spectrum: (ES+) 787 (M-P₂O₆), 865 (M-PO₃), 949 (M+H), 1046 (M+Et₃NH).

Labelling of an oligonucleotide with 2-(4-0-Λ/- succinimidylcarbonylpiperidino)-4-(5-sulfonato-1 ,3,3-trimethyl-2- benzindolinylidenemethyl) cyclobutenediylium-1 ,3-diolate (10a)

2-(4-Carboxypiperidino)-4-(5-sulfonato-1 ,3,3-trimethyl-2- benzindolinylidenemethyl) cyclobutenediyiium-1 ,3-diolate [4b] (15 mg, 0.027 mmol) was treated with 0-(/V-succinimidyl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TSTU, 8.3mg, 30μmol), in DMF (0.5ml) containing N,N-diisopropylethylamine (5μl). The activation was followed by t.l.c. (RPCι₈. Methanol, 40: water, 60. [4b] R_f = 0.48. Product R_f = 0.20); it was complete after 1 hour. Diethyl Ether was added to the mixture and the product precipitated out. The solvent was decanted and the product was triturated twice more with ether to yield the NHS derivative as an orange solid, 16mg (89%) (10a). δ_H (300MHz, CD₃OD) 1.362 (3H, t), 1.90-1.95 (8H, m), 2.13- 2.19 (2H, m), 2.73 (1 H, m), 2.82 (4H, s), 3.55-3.65 (2H, m), 4.20 (2H, broad q), 4.68 (2H, m), 5.85 (1 H, s), 7.60 (1 H, d), 7.93 (1 H, dd), 8.01 (1 H, d), 8.30 (1 H, d) and 8.46 (1 H, d).

Mass spectrum: (ES+) 644 (M+H), 666 (M+Na).

The above NHS ester derivative was then used to label a piece of cDNA that had had a modified nucleotide with an aminoallyl group in the 5 position of uridine incorporated into it. Eight labelling reactions were set up, two each of the following consisting of duplicates and alternative Cy 3 dye as a control: 1 ) A Squaraine dye 10a labelling with a cDNA reaction including reverse transcriptase (RT) (test). 2) A Squaraine dye 10a labelling with cDNA reaction with no reverse transcriptase (negative control).

3) A Cy3 labelling with cDNA reaction including reverse transcriptase (positive control).

4) A Cy3 labelling with cDNA reaction with no reverse transcriptase (negative control)

Method

Labelling of cDNA with dye 10a was carried out as follows. Eight cDNA reactions were set up each containing 2 μg of human skeletal muscle mRNA, 2μl of random primers (Nonamers, Amersham Pharmacia RPK0158), and 12 μl of water. For the no reverse transcriptase reactions 14 μl of water was added. The reactions were heated to 70 °C for 10 mins and then transferred to room temperature for 15 mins. 8μl of 5x First strand buffer (Promega M531 A) was added, 4μl of 0.1 M DTT, 2μl of a nucleotide mix (4mM dATP, dCTP, dGTP), 8μl of a 1 mM aminoallyl-dUTP (Sigma A0410) and 2μl of reverse transcriptase (Promega M3682) to the relevant tubes. The reactions were incubated at 42 °C for 3 hours. To remove the mRNA, 2 μl of 5M Sodium hydroxide was added and the tubes incubated at 42 °C for 10 min. The Sodium hydroxide was neutralised by adding 20 μl of 2M HEPES. The free aminoallyl-dUTP was removed by adding 450 μl of water, placing in a Microcon 30 column (Millipore cat no 42410). The column was spun for 8 mins at 13krpm in a microfuge (MicroCentaur, MSE). 450 μl of water was added again to the column and spun through for 8 minutes as before. The 450 μl water was added once again and the column spun as previously. The purified cDNA was then eluted from the column by a 1 minute spin at 13krpm into a fresh tube.

The cDNA was dried down in a Speedvac. The cDNA was resuspended in 4.5μl of water. 3.2x10^"7 moles of (~0.25mgs) of Squaramide NHS ester and Cy3-NHS ester (Amersham Pharmacia PA23001) were each resuspended in 72 μl 0.1 M Sodium bicarbonate pH9. 4.5 μl of the appropriate dye solutions were then added to the cDNA solution. The reactions were incubated at room temperature in the dark for 1.5 hours. 4.5μl of 4M hydroxylamine (Sigma H2391 ) was then added and incubation continued for a further l Ominutes. The next step was to remove the free dye from the dye incorporated into the cDNA. 500μl of buffer PB (Qiagen, Qiaquick kit cat28106) was then added and the probes added to a Qiaquick column (Qiagen cat 28106). The columns were spun in a MicroCentaur microfuge for 1 minute at 13krpm. 500μl of buffer PE (Qiagen cat28106) was added to wash the column and the spin repeated. The wash and spin step was repeated twice more. A further 1 minute spin was carried out before elution with 100 μl of elution buffer (Qiagen cat28106). The dye incorporation into the probes was then measured using a Cary UV/visible spectrophotometry.

Results

The absorbance reading for the dye 10a and Cy3 plus reverse transcriptase labelling reactions compared to those obtained for the no reverse transcriptase reactions confirms that dye 10a has labelled the cDNA molecules. The no reverse transcriptase results confirm that the unincorporated dye is removed efficiently by the protocol used.

Claims

C LAI MS

1 . Compounds having the structure

W = Sq - A

where A has the formula -NR¹R², where R¹ and R² are the same or different and each is H or d - C₂₀ hydrocarbon or a group -L-G, or one of R¹ and R² is -OR⁵ or -NR⁶R⁷ or -COR⁷ or -NR⁶COR⁷ or -N = R⁸ or -C(0)OR⁷, or R¹ and R² together form a single ring or fused ring system, saturated or unsaturated and unsubstituted or substituted, or an azacrown or a metal-binding group, Sq represents

where m is 1 , 2 or 3, W represents a wing moiety having the structure

-L':

L' is a linker of 0 - 3 moieties selected from carbon atoms and arylene groups, the dotted line represents a single ring or fused ring system, aromatic or heterocyclic, unsubstituted or substituted, containing or joined to a tertiary or quaternary N atom, and having unsaturation coordinated with that of L' = Sq, each of R⁵, R⁶, R⁷ and R⁸ is H or Ci - C₂o hydrocarbon or a group -L-G, R⁹ is a negative charge or a group -L-G,

L is a linker chain of 0 to 60 moieties, branched or unbranched which optionally contains one or more arylene groups, or O or N or N⁺ or S or S⁺ or P or P⁺ or Se atoms, and G is a functional or a reactive group by means of which the compound may be covalently linked to a biomolecule, other small molecule e.g. a dye or a group which enhances or reduces water solubility or provides electron donating or withdrawing properties to modify the spectral characteristics of the compound, and homo-dimers and -oligomers and hetero-dimers and - oligomers of the compounds.

2. The compounds of claim 1 , wherein W is

R⁴ R⁴ n

N N I 3

FT R

where each of R and R⁴ is H or Ci - C₂o or a group -L-G, and n is 0 - 3.

3. The compounds of claim 1 or claim 2, wherein the dotted line represents

where X is O or S or NR⁴ or CR¹⁰R¹¹ where each of R¹⁰ and

R¹¹ is Ci - C₂₀ hydrocarbon or -L-G, or R¹⁰ and R¹¹ together form a single ring or fused ring aromatic or heterocyclic or cycloaliphatic group.

4. The compounds of any one of claims 1 to 3, wherein there is present at least one group G which is a functional or reactive group by means of which the compound may be covalently linked to a biomolecule.

5. The compounds of any one of claims 1 to 4, wherein G is a functional group selected from NH₂, OH, SH, or a reactive group selected from C0₂H, acid halide, anhydride, CO active ester, NCS, O phosphoramidite, -NC(0)CH l and

6. Dimers and oligomers of the compounds of any one of claims 1 to 4, having the structure A- Sq = W )_a where A is an aromatic or alicyclic group having at least a nitrogen atoms and a is 2 to 6.

7. The compounds of any one of claims 1 to 6, wherein Sq is

8. A complex of a biomolecule and the compound according to any one of claims 1 to 7, wherein the biomolecule is covalently linked to a functional or reactive group G of the compound.

9. The complex of claim 8, wherein the biomolecule is an oligo- or poly-peptide or a nucleotide or an oligo- or poly-nucleotide.

10. A method of making the compounds of any one of claims 1 to

7, which method comprises the steps of i) reacting the wing moiety W with a dialkyl squarate or analogue to give an intermediate a)

ii) optionally subjecting the intermediate a) to hydrolysis to give an intermediate b)

iii) reacting intermediate a) or the intermediate b) with R¹R²NH to give a final product c)

^■ — L'=Sq-NR¹R²

11. An assay or labelling method which comprises contacting a sample containing an amine with a compound a) or a compound b)

and observing dye formation by reaction of the amine with the compound a) or b).