CA2366263A1 - Squaraine dyes - Google Patents

Abstract

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 C1-C20 hydrocarbon or a group -L-G, or one of R1 and R2 is -OR5 or -NR6R7 or -COR7 or -NR6 COR7 or -N =R8 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 R5, R6, R7 and R8 is H or C1-C20 hydrocarbon or a group -L-G, R9 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
s 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 ~o 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 ~s 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 2o 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 2s 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 -NR'R2, where R' and R2 are the same or different and each is H or C1 - C2o hydrocarbon or a group -L -G, or one of R' and R2 is -OR5 or -NR6R' or -COR' or -NR6COR' or -N = R8 or -C(O)OR', or R1 and R2 together form a single ring or fused ring system, s saturated or unsaturated and unsubstituted or substituted, or an azacrown or a metal-binding group, Sq represents or ~ R9 m m O O
~o 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 Is 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 R5, R6, R' and R8 is H or C1 - C2o hydrocarbon or a group -L -G, 2o 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 2s 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 _3_ 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.
s 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.
R' and R2 may be C1 - C2o hydrocarbon, for example alkyl or aryl or aralkyl. If one or both of R' 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 ~s or unsaturated, for example pyrrolidine, piperidine, pyrrole, piperazine, or morpholine, or a fused ring system. Alternatively R' 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. R' or both of R1 and R2 zo may be a group -L-G which is discussed below. Examples of amine moieties A are:

H H alkyl / / /
- N - N - N
\ \ \
alkyl aryl alkyl /alkyl /aryl -N -N
~ aryl \
aryl O
- N~ aliphatic or aromatic ring -N
./
- N/ \X X = CH2, NH, O, CHCOOH
R = alkyl, aryl - N
i R
O - N/
\ \ O - ~\ _ N
R R R
R
-N
O-R

The wing moiety W is preferably .~ . . . \ \
R4n .' ' . R4n ' N+, ,sN .

R4n ;. ..~ R4n ,,. . ,, . . . N+. . N+.
Rs Rs where each of R3 and R4 is H or C1 - C2o or a group -L-G, s 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 C1 - C2o hydrocarbon as discussed above for R' and R2. Or R3 may be -L-G as discussed below.
to 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:
is X
R4n ~ ~ ~ or R4 \ N+ / X

N+

where X is O or S or -NR4 or -CR'°R" where each of R'°

and R" is C1 - C2° hydrocarbon or -L -G, or R'° and R1' together form a single ring of fused ring or heterocyclic or cycloaliphatic group.
Examples of dyes with different wing moieties W are:
R~ Ra O NwRz \ \s O_ \ \ O_ \ ~ ,.~ / X \
\ \ O O~N'R' ~. , , ' O~N~Rz Rz~ R'~
~ / N+J
I
Ra R' \ R' N - Rz \
O \ O N - R2 R+ / O_ /. ~ _ / CN / O
aX

O
N+ w , ~w~
O- ~ i ~NiR~
Rs Oy ~ ~ z z R
R
~N O
R' O O
R~ + ~R~ ~R~
s N- - / N\ N~ - / N
R ~_ Rz ~_ \Rz _7_ 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:
O
R' WING ~ N SOUARAINE
\R2 O
O O
R' WING ~ N / CROCAINE

RHODIZAINE
R' WI
Rj L is a linker chain of 0 to 30 or 60 moieties, preferably selected form alkylene, alkenylene and alkynylene or a branched or straight ~o 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 _g_ 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(O)CH21 and maleimide O
~N
O
s 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 ~o 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
Is 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.
2o 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 2s have the structure W'-Sq-A'-Sq-W2 or alternatively the structure A'-Sq-W'-Sq-A2, where A' and A2 each represent an amine moiety A, and W' and W2 each represent a wing moiety W. Where R' and R2 are part of a substituted fused ring system this ring system may contain _g_ 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 s were present energy transfer could take place.
Where A' and A2 (or W' 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:

N N
~~bs 528 nm 7~,e~" 542 nm cmaX 2~0 000 dm~mol-gem ~
Alternative scaffolds, to piperazine shown above are cyclam, dendrimers, adamantane (Tetrahedron Letters, 1999, 40, 223):
Linker Linker Dye Dye Dye Dye ~N ~ ~D a N N N Y
Linker Dye Dye/ N N ~D a N N ~ DYe Dye Y
Dye Where A1 and A2 (or W' 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 s 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 to invention and replaces fluorescein the lack of pH sensitivity, greater photostability and increased extinction coefficient could be advantageous.
Examples are _ O
N~'_-~~
O
~ dabs = 490 nm A 7~abs = 530 nm hem = 515 nm 7~em = 570 nm O
N
~~--~~~N H \
a \
/ \ ~ ~/ \
~ dabs = 490 nm A 7~abs = 550 nm hem = 515 nm ~,,em ~ 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 ~o 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, ~s 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.
2o 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 s nitrogen electron density.
Thus, when one of R' 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:
~o pH Sensors NHR2 NR'R2 Metal ion sensors N Chelate Redox Sensor (e.g. NADH) i i N~ _ N
O
s 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) alkyl =L
to ii) optionally subjecting the intermediate a) to hydrolysis to give an intermediate b) OH
= L' ~ O
' ~ m iii) reacting the intermediate a) or the intermediate b) with RiR2NH to give a final product c) ', ~-L'=Sq-NR1R2 s Reacting intermediate a) with R' R2NH gives a 1,2 squarate or analogue. Reacting intermediate b) with R' R2NH gives a 1,3 squarate or analogue.
Thus squaraine compounds according to the invention can be ~o, made by the following reaction sequence:-i) Squarate half-dye formation . - ~~ O O , . O
+ /~ NaOMe, MeOH ' Warm ~~ . N '\ O
R3 ~ ~ R3 O~
ii) Half-dye hydrolysis to acid form O
HCI (aq.), AcOH ~ O
. \ . .
R3 \ O Warm ~~ . N \ ~ O
O~ R3 OH
iii) Dye formation O - O
.. '\ R1 Solvent (eg. 1-butanol) . ' R1 N ~'~~O -H HN~R2 Heat ' t~ ~ N
R3 Rg / ~R2 OH O-The half dye methyl ester from i) also reacts with amines to give a 1,2-adduct:

iv) Dye formation O - O
\ R~ Solvent (eg. 1-butanol) ~ ~l ~ O -E HN v . '\
R3 ~Rp Heat ~ ~ \ O

R1 ~N~R2 Four more specific reaction schemes follow by way of example.
s a) / N+

_16_ b) O
--~ ~ ~ O ~ ~N\
N
O
O
OBu HO
O
N
O-c) CS
m N OBu O
OH
Cv, N+ ~ OH
O
N~
O
OH

d) a The invention also includes an assay or labelling method which comprises contacting a sample containing an amine with a s compound a) or a compound b) a) alkyl O
~ = L' ~ O
' ~ m O

_18_ b) OH
. - L, / O
' ~ m O
and observing dye formation by reaction of the amine with the s 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.
o The invention is further illustrated by the following examples.
Examples:
Amines:
U
~s Amino-acids:
~O
OH
OH

_19_ Water solubility:

,s o R

i N

\

The groups -NRiR2 may comprise an amino-sugar, or amine with additional quaternary ammonium groups.
Wavelength variations:

/ o os / o R1 ~ R1 \ + i \ ,~ _ i N ~ N N ~ N
R3 R2 R3 ~ R2 O O
/ S O / \ O
\ ~ +, R1 \ ~ ~ R1 i R3 / N R2 N ~ N

O_ O_ o / ~ /
\ +' N
N ~ N

O

Use of polyamines for multiple dye loading or energy-transfer cassettes:
/ o o \
/
\ N+ / N~ ~ ~N+
R3 _ R3 O O
O O / \
\ I N+~ ~N ~N+ I /
R3 / ~ \ 'R3 °_ °_ The amine function may be part of a metal chelating system e.g. azacrown:
+ ° / ° o I
R3 ~ \ R9 / N
O
O- O
o Chelation of an appropriate metal ion should alter fluorescence properties.
pH sensors:
/ O _H / O
\ I N+, NR1 ~ \ I N NR1 / ~ /
R2 + H ~ '\'-~ R2 O O
/ O _H / O
\ I N+, N R1 ~ \ I N-\ -N R1 /
R3 H + H ~ R3 15 ° °

Other amino compounds:
/ o N~~ N

O
/ O / O

N' ~ N R1 ~ N~ ~ N O

EXPERIMENTAL EXAMPLES

s 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 100m1 round-bottomed flask, with stirrer bar, was to added dimethyl squarate (3.55g, 25mmol) and dry methanol (l0ml). 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 ~s to crystallize out. Stirring was continued for l6hrs, then the mixture was cooled in the fridge for 2hrs. The title compound [1 a] was collected by filtration, washed with ice-cold methanol (3x5m1), then diethyl ether (4x20m1) and dried under vacuum. Yield = 6.34g (90%) as a yellow crystalline powder.
20 Amax (MeOH) = 422nm.
8H (300MHz, CDC13) 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).
2s 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 (20m1) and concentrated aqueous hydrochloric acid (5ml). The mixture was 3o 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. [1 a], Rf = 0.5, [1 b] Rf =
0.7). Once the reaction was complete, the solvent was evaporated under reduced pressure; the residue was co-evaporated with acetonitrile, twice s 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).
~0 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 (35m1) were mixed in a 100m1 flask, with stirrer. The mixture was heated at 90°C for l8hrs; a dark greenish solution containing a pale solid resulted. After cooling, the solid ~s was collected by vacuum filtration, washed with dichlorobenzene (2x20m1), then excess diethyl ether. It was dried under vacuum to give the title solid as a purple-tinged pale solid. Yield = 15.93g (87%).
8H (300MHz, CDC13) 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 [1d]
1-Ethyl-2,3,3-trimethylbenz(e)indolinium iodide [1 cJ (7.3g, 20mmol) was added to a solution of sodium hydroxide (4g, 100mmol) in water (150m1); this mixture was swirled briefly, then diethyl ether (100m1) 2s added and the mixture swirled again. The brown ether layer was collected;
the aqueous layer was extracted with fresh ether (50m1).
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 ~o dried under high vacuum to give the title compound as a brownish solid.
Yield = 4.77g (100%) 8H (300MHz, CDC13) 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).
s 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 100m1 flask, with stirrer. Dry methanol (35m1) was then added. This mixture was ~o heated at 50°C for l6hrs, 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 (2x15m1), then diethyl ether (4x30m1) and dried under high vacuum to give the title compound [1 a]. Yield = 5.448 (78%).
Amax (MeOH) = 440nm 8H (300MHz, CDC13) 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).
20 3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione [if]
3-Methoxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione [1 e] (3.47g, 1 Ommol) was mixed with acetic acid (50m1) in a 250m1 flask. To this was added a 2s solution of concentrated hydrochloric acid (2.5m1) in water (l0ml). The resulting mixture was incubated in a water bath at 60°C and the reaction monitored by t.l.c. (RPC18. Methanol, 90: water, 10. [1 e] Rf = 0.4, [1 f] Rf =
0.7). Once the reaction was complete, the solvent was evaporated under reduced pressure; the residue was co-evaporated twice with acetonitrile ~o and dried under high vacuum. The resulting foam was triturated with diethyl ether (25m1) to give a yellow-brown powder; petroleum ether 40-60°
(25m1) 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 [1 f]. Yield =
3.1 g (93%). This material was used directly to prepare dyes.
Amax (MeOH) = 442nm 8H (300MHz, CD30D) 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).
~0 1,1,2-Trimethylbenz(e)indole-5-sulphonate, potassium salt [ig]
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 ~s [1h]
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 25m1 flask, with stirrer. This mixture was heated to 120°C
20 (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 2s vacuum; this material was used directly without further purification.
Expected to contain 1 equivalent of KI.
8H (300MHz, CD30D) 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).
~o 3-Methoxy-4-(5-sul phonato-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 50m1 flask with stirrer, s followed by dry methanol (l5ml) and sodium methoxide (0.19g, 3.5mmol).
This mixture was stirred for 5mins, then dimethyl squarate (0.71 g, 5.Ommol) added. The resulting mixture was heated at around 60°C (sand bath, water condenser, silica gel guard tube) for l6hrs.
At the end of this time the solvent was evaporated under ~o 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 ~s dried under high vacuum to give the title compound as a yellow powder.
Yield = 640mg.
~maX (MeOH) = 442nm.
8H (300MHz, CD30D) 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 20 (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 [1 j]
3-Methoxy-4-(5-sulphonato-1-ethyl-3,3-dimethyl-2-2s benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione, sodium salt [1 i]
(640mg, 1.4mmol) was mixed with acetic acid (l0ml) 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. (RPC18.
o Methanol, 60: water, 40. Rf [1 i] = 0.6, Rf [1 j] = 0.8). Once the reaction appeared complete the solvent was evaporated under reduced pressure, _27_ the residue was co-evaporated twice with acetonitrile and finally dried under vacuum.
Purification by prep. HPLC (RPCiB. Water ~ methanol gradient). The appropriate fractions were combined and evaporated under s 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.
Amax (MeOH) = 442nm.
~0 1-(4-sulfonatobutyl)-2,3,3-trimethylbenz(e)indolinium hydroxide inner salt [1 k]
1,1,2-Trimethylbenz(e)indole (1.05g, 5mmol), 1,4-butanesultone (1.36g, 1 Ommol) and 1,2-dichlorobenzene (5ml) were mixed and heated at 120°C for 20hrs. The mixture was then allowed to cool ~s 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%).
8H (300MHz, CD30D) 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 20 (1 H, d), 8.22 (1 H, d) and 8.31 (1 H, d).
3-Methoxy-4-(1-(4-sulfonatobutyl)-3,3-dimethyl-2-benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione [11]
1-(4-Sulfonatobutyl)-2,3,3-trimethylbenz(e)indolinium 2s hydroxide inner salt [1 k] (1.38g, 4.Ommol), dimethyl squarate (0.57g, 4.Ommol) and methanol (l0ml) were mixed in a dry flask; to the resulting mixture was added a solution of sodium methoxide, 0.5M in methanol (B.OmI, 4.Ommol). A yellow colour soon formed. The mixture was left to stir overnight. The solvent was then evaporated under vacuum and the residue 3o purified by flash chromatography (silica. 15-25% methanol / chloroform).

Product fractions were combined and evaporated to give the title compound, 1.47g (77%) ~max (MeOH) = 440nm.
8H (300MHz, 10% CD30D in CD2C12) 1.80 (6H, s), 1.91 (4H, s 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]
~o This was prepared in the same manner as for [1 j], by reacting the methoxy half-dye [11] (1.45g, 3.Ommol) with 2M aqueous HCI (5ml) in acetic acid (l5ml) with warming. The reaction was followed by t.l.c. (RPCis.
Methanol, 60: water, 40. [1 I], Rf = 0.35; [1 m], Rf = 0.65). Once the reaction was complete, the solvent was evaporated under vacuum, the residue co-~s evaporated with acetonitrile three times and finally dried under high vacuum over phosphorus pentoxide. This material was used without further purification.

\ ~ o ~~R
[1 a] R = CH3 [ib]R=H
\
J
(1 c] [1 d]

_29_ ~R
[1 e] R = CH3 [1f] R=H
K+_OsS ~ ~ _OsS
/ N / N+
[1 g] [1 h]
Na~O~SO
ii O
_R
[1 i] R = CH3 [1j]R=H
N+
O=S=O
i_ O [1 k]

_30_ [1 I] R = CH3 [1m]R=H

s Synthesis of sguarate dues from indolenine-sguaric acid intermediates 2-(1-Piperidino)-4-(1,3,3-trimethyl-2-o indolinylidenemethyl)cyclobutenediylium-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 (40p1 = 34mg, 0.40mmol). This mixture was heated at around 100°C (sand bath, air ~s condenser). The colour darkened during this time (intense orange-yellow at the meniscus). The reaction was followed by t.l.c. (RPC18. Methanol, 90:
water, 10. [1 b] Rf = 0.7, [2a] Rf = 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 2o 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%) ~R
O=S=O
I
O~Na Amax (MeOH) = 470nm; ~max = 101,000 dm3 mol-1 cm-1.
Fluorescence (MeOH); ~,eX = 471 nm, ~.em = 495nm.
bH (300MHz, CDC13) 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 s (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]
to This was prepared in an analogous method to [2a], using morpholine (35.1 = 35mg, 0.40mmol) in place of piperidine. The title dye [2b] was isolated in the same manner. Yield = 91 mg (90%).
Amax (MeOH) = 476nm; Emax = 106,000 dm3 mol~' cm-' Fluorescence (MeOH); ~,eX = 474nm, hem = 498nm.
~s 8H (300MHz, CD30D) 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) 20 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%).
2s a,,nax (MeOH) = 506nm; Amax = 284,000 dm3 mol-' cm-' Fluorescence (MeOH); 7~eX = 506nm, ~,em = 520nm.
8H (300MHz, CDC13) 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), 611 (M+Na).
~o 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 (441 = 43mg, 0.40mmol) in place of piperidine. The title dye s [2d] was isolated in the same manner. Yield = 96mg (89%).
Amax (MeOH) = 496nm; Emax = 87,0OO dm3 mol-' cm-' Fluorescence (MeOH); ~,eX = 496nm, hem = 547nm.
8H (300MHz, CD30D) 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).
~o 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 ~s dipropylamine (55.1 = 40.5mg, 0.40mmol) in place of piperidine. The title dye [2e] was isolated in the same manner. Yield = 97mg (92%).
Amax (MeOH) = 474nm; ~max = 97,000 dm3 mol-' cm-'.
Fluorescence (MeOH); ~,ex = 474nm, ~,em = 494nm.
8H (300MHz, CD30D) 0.88 (6H, t), 1.56 (6H, s), 1.65 (4H, 2o 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) 2s 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 ~0 4hrs. The reaction produced two main products by t.l.c. (RPC18. Methanol, 95: water, 5. [1 b] Rf = 0.8, product 1 Rf = 0.65, product 2 Rf = 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]
(l0mg), presumably formed by the trapping of dimethylamine liberated from s the DMF. Product 1 eluted later and was the desired dye [2f], isolated yield = 22mg (19%).
[2f]
~max (MeOH) = 472nm Fluorescence (MeOH); ~,eX = 472nm, ~,e,~, = 498nm.
~0 8H (300MHz, CD30D) 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).
~ s [2g]
Amax (MeOH) = 468nm 8H (300MHz, CD30D) 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).
O

O
[2a] R1-R2 = -(CH2)s -[2b] R1-R2 = -(CH2)2-O-(CH2)2 -[2d] R1 = Ph, R2 = CH3 [2e] R1 = R2 = -CH2CH2CH3 [2f] R1-R2 = -(CH2)2-CH(C02H)-(CH2)2 -[2g] R1 = R2 = CH3 \ O O /
/ N+' N J ~N+ \
O O
[2c]
s EXAMPLE 3 Synthesis of sauarate dyes from benzindolenine-sauaric acid intermediates ~0 2-(1-Piperidino)-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [3a]
3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione [1 f] (1 OOmg, 0.30mmol) was dissolved in 1-butanol (3ml); to the resulting yellow solution was added ~s piperidine (40.1 = 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.
(RPCi8. Methanol, 90: water, 10. [1 f] Rf = 0.7, [3a] Rf = 0.25). After 1 hr the solvent was evaporated under reduced pressure; the residue was purified 2o 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 2s (91 %).
Amax (MeOH) = 490nm; ~max = 95,000 dm3 mol-' Cm-'.
Fluorescence (MeOH); 7~ex = 492nm, ~,em = 516nm.

8f., (300MHz, CD30D) 1.36 (3H, t), 1.78 (6H, broad s), 1.90 (6H, s), 4.02-4.11 (6.H, 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 (40p1 = 34mg, 0.40mmol) in hot ethanol (6ml) gave an alternative product. T.I.c. (RPC,8. Methanol, 90: water, 10. [1 e] Rf = 0.4, ~o product Rf = 0.4, cf [3a], Rf = 0.25) and (silica. Methanol, 2.5:
chloroform, 97.5. [1 a], Rf = 0.55, product Rf = 0.45, cf [3a], Rf = 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%).
Amax (MeOH) = 452nm, cf [3a], 490nm. Overall appearance is Is more like the half dye [1 e] than [3a].
8H (300MHz, CDC13) 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:
or The single-double bond conjugation is now more fixed so E- and Z-isomers are possible.
2s 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 (35.1 = 35mg, 0.40mmol) in place of piperidine. The title dye s [3b] was isolated in the same manner. Yield = 99mg (82%).
~max (MeOH) = 494nm; ~max = 93,000 dm3 mol-' cm-' Fluorescence (MeOH); ~,eX = 494nm, ~,em = 520nm.
8H (300MHz, CD30D) 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 ~o (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 ~ s [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 %).
~max (MeOH) = 528nm; ~max = 270,000 dm3 mol-' cm-1 2o Fluorescence (MeOH); 7~eX = 528nm, ~,em = 542nm.
8H (300MHz, CDC13) 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 (441 = 43mg, 0.40mmol) in place of piperidine. The title ~o dye [3d] was isolated in the same manner. Yield = 120mg (=100%).

~max (MeOH) = 516nm; ~max = 76,000 dm3 mol-' cm-'.
Fluorescence (MeOH); ~,ex = 522nm, ~,em = 563nm.
8H (300MHz, CD30D) 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 s (1 H, d).
Mass spectrum: (ES+) 423 (M+H), 445 (M+Na).
2-(N,N-Diphenylamino)-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [3e]
~o 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 %).
Amax (MeOH) = 540nm; ~max = 96,000 dm3 mol-' cm-'.
Fluorescence (MeOH); ~,ex = 542nm, 7~em = 588nm.
~s 8H (300MHz, CD30D) 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).
20 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, 0.40mmo1) in place of piperidine. The title dye [3f] was isolated in the same manner. Yield = 47mg (34%).
2s Amax (MeOH) = 530nm Fluorescence (MeOH); ~,eX = 533nm, ~,em = 566nm.
8H (300MHz, CD30D) 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).
~o Mass spectrum: (ES+) 589 (M+H), 611 (M+Na).

_38_ 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 [1 f] (1 OOmg, 0.30mmol) s 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. [1 f] Rf = 0.75, yellow spot. Product Rf =
0.5, orange spot). The solvent was then evaporated under vacuum; the residue was purified by flash chromatography (silica gel. 5-15% methanol in ~o 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%).
Amax (MeOH) = 492nm ~s Fluorescence (MeOH); ~,eX = 492nm, ~,em = 516nm.
8H (300MHz, CD30D) 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).
2o Mass spectrum: (ES+) 445 (M+H), 467 (M+Na).
22mg (50~mo1) of this carboxy dye was converted to the reactive N-hydroxysuccinimide ester [3h], by reaction with O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, l5mg, 2s 50~mo1), in DMF (0.5m1) containing N,N-diisopropylethylamine (9~.1). The activation was followed by t.l.c. (RPC18. Methanol, 95: water, 5. [3g] Rf =
0.5. Product Rf = 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 ~o a conjugate with structure [3i]. This product was isolated, purified by preparative t.l.c. (silica. Methanol, 25: chloroform, 75. Product isolated pure; Rf = 0.2 in this system) and characterized; it represents an example of an energy-transfer system.
Amax (MeOH) = 492, 552nm Mass spectrum: (ES+) 920 (M+), 921 (M+H), 943 s (M+Na).
Fluorescence (MeOH); ~,eX = 492, 551 nm, 7~em = 567nm. The Cy3 acceptor dye has ~,eX = 551 nm, ~,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 ~0 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 ~s 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]
2o This was prepared from the squaramide NHS ester ([3h], 0.1 mmol, prepared as above) and N-(2-aminoethyl)maleimide.HCl salt (2x20mg, 0.11 mmol), in anhydrous acetonitrile in the presence of N,N-diisopropylethylamine. The reaction was followed by t.l.c. (silica. Methanol, 5: dichloromethane, 95. Rf values: [3g] = 0.02, [3h] = 0.4, [3j] = 0.1.
2s 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 3o 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 s resulting labelled proteins were then analysed by a 2D-DICE method, as described in PCT patent application WO/96/33406.
Amax (MeOH) = 492nm 8H (300MHz, CD30D) 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), ~0 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) Is 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.
20 Amax (MeOH) = 494nm 8H (300MHz, CDC13) 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 CHC13), 7.332 (1 H, t), 7.493 (1 H, t), 7.82 (2H, m) and 8.10 (1 H, d).
2s 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]
3o 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%).
Amax (MeOH) = 526nm.
8f., (300MHz, CD30D) 1.48 (3H, t), 2.02 (6H, s), 4.05 (3H, s), s 4.25 (2H, q), 6.06 (1 H, s) and 7.47-8.27 (10H, m).
2-N-(4-carboxyphenyl)-N'-(tert-butoxycarbonyl)hydrazino-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)cyclobutenediylium-1,3-diolate [3m]
~o 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 %).
Amax (1"120) = 528nm ~s Fluorescence (H20); ~,eX = 532nm, ~,em = 566nm.
8H (300MHz, CD30D) 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), 611 (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 2s 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%).
Amax (MeOH) = 532nm 8H (300MHz, CDC13) 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 (1 H, d).

N

[3b] R1-R2 = -(CH2)2-O-(CH2)2 -[3d] R1 = Ph, R2 = CH3 [3e] R1 = R2 = Ph [3f] R1 = Ph, R2 = -NH-CO-CH3 O ~ ''O
N / N~
OH
O-[3g]
[3a] R1-R2 = -(CH2)s -I~
O
I O O
N+ N\-/ 'O-N
J o_ O
[3h]
N\-J 'O
NH
Nw w W N I / O
~~ S
[3i] o ~O
I~
O O O
N / N\-/ ' ~ N I
J o- ~ O
[3jJ
O
N O
~-O O-' ~J
[3k]

[3~l [3m]
O
N, O
_ N==~~OH
O
[3n]

Synthesis of sctuarate dyes from sulphonated benzindolenine-sctuaric acid intermediates s 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-~o benzindolinylidenemethyl) cyclobut-3-en-1,2-dione, sodium salt [1j]
(130mg, 0.30mmol), piperidine (401 = 34mg, 0.40mmol) and 1-butanol (5ml) were mixed and heated to 110°C; not all the solid dissolved, but an orange colour developed. The reaction was followed by t.l.c. (RPC,s.
Methanol, 60: water, 40. [1j] Rf = 0.8, [4a] Rf = 0.5). Once the reaction ~s appeared to have halted, another 20p1 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 (RPC18. Water -~ methanol gradient.); collected fractions that contained pure product were combined and evaporated, dried under high 2o vacuum and trituration with diethyl ether. The resulting orange powder was further dried under vacuum to give the title compound [4a], 99mg.
Amax (MeOH) = 490nm, (pH 7.0 phosphate buffer) = 488nm.
Fluorescence (pH 7.0 phosphate buffer) ;~,eX = 488nm, ~,em =
512nm.
2s 8H (300MHz, CD30D) 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.
3o Buffer solutions were bought pre-prepared from Radiometer Analytical S.A.;

pH 4.0 (potassium hydrogen phthalate, 50mmol / I) pH 7.0 (Na2HP04, 27.5mmol / I : KH2P04, 20.Ommol / I) pH 10.0 (NaHC03, 25mmol / I : Na2C03, 25mmol / I) s 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.
pH ~maX / Absorbanc ~,eX / ~,em / Rel. intensity nm a nm nm 4 488 0.202 488 512 75 7 488 0.186 488 512 68 488 0.186 488 512 57 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 ~s benzindolinylidenemethyl) 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 [1 j]
(130mg, 0.30mmol), isonipecotic acid (52mg, 0.40mmol) and acetic acid (2.5m1) were mixed and heated to 100°C; the reaction was followed by t.l.c.
(RPC18. Methanol, 40: water, 60. [1 j] Rf = 0.7, product Rf = 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.
~max (MeOH) = 492nm, (water) = 488nm.
Fluorescence (MeOH) ;~,eX = 492nm, ~,em = 517nm.

8H (300MHz, CD30D) 1.362 (3H, t), 1.90-1.95 (8H, m), 2.13-2.19 (2H, m), 2.73 (1 H, m), 3.55-3.65 (2H, m), 4.10 (2H, broad q), 4.65 (2H, m), 5.748 (1 H, s), 7.51 (1 H, d), 7.93 (1 H, dd), 7.99 (1 H, d), 8.21 (1 H, d) and 8.37 (1 H, d).
s Mass spectrum: (ES-) 523 (M-), 261 ((M-H) 2-/2).
NaO~S O
O
~J
[4a]
NaO~ i~
OS
/ O
O
N+ / N\-/
_ OH
O
[4b]

_48-Synthesis of squarate dyes using primary amines - pH sensitive dues If the amino compound used to form the final dye is a primary s 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 ~o 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) ~s cyclobutenediylium-1,3-diolate [5a]
This was prepared in an analogous method to [3a], using aniline (30p1 = 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 20 [5a] = 1 OOmg (81 %).
Amax (MeOH) = 532nm; Emax = 99,000 dm3 mol~' cm-'.
(MeOH + Bu4+ OH~) = 456nm, ~max = 53,000 dm3 mol-' cm-'.
Fluorescence (MeOH); 7~ex = 532nm, ~,em = 548nm.
(MeOH + Bu4+ OH~) = non-fluorescent.
2s 8H (300MHz, CDC13) 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), 8.11 (1 H, d) and 9.9 (1 H, broad).
Mass spectrum: (ES+) 409 (M+H), 431 (M+Na).
~o _49_ 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 (32g.1 = 24mg, 0.33mmol) in place of piperidine. The reaction s was followed by t.l.c. (RPC18. Methanol, 95: water, 5. [1 f], Rf = 0.8, [5b], Rf = 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 (MgS04), filtered and the solvent evaporated. Purified by flash chromatography (silica. 2-3%
~o methanol / chloroform), to give 84mg (72%) of the title compound.
Amax (MeOH) = 484nm; ~max = 85,000 dm3 mol'' cm''.
(MeOH+Bu4+ OH') = 434+388nm;
Amax ~ 33,000 dm3 mol-' cm'' (434nm) Fluorescence (MeOH); ~,eX = 483nm, ~,em = 511 nm.
~s (MeOH + Bu4+ OH') = non-fluorescent.
8H (300MHz, CDC13) 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) 2o Mass spectrum: (ES+) 389 (M+H), 411 (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 2s ammonium acetate (77mg, 1.Ommol) in place of piperidine. The reaction was followed by t.l.c. (RPC18. Methanol, 90: water, 10. [1f], Rf = 0.65, [5b], Rf = 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.
=~o ~max (MeOH) = 478nm (MeOH + Bu4+ OH') = 434nm.

_50_ Fluorescence (MeOH); ~,ex = 481 nm, ~,em = 506nm.
(MeOH + Bu4+ OH-) = non-fluorescent.
8H (300MHz, DMSO-d6) 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) s 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 ~o salt [5d]
3-Hydroxy-4-(1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl)-cyclobut-3-en-1,2-dione [1 f] (1 OOmg, 0.30mmol), metanilic acid (60mg, 0.35mmol), anhydrous sodium acetate (30mg, 0.37mmol) and acetic acid (2.5m1) were mixed and heated at around 100°C
~s 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 (RPC~B.
Water--methanol gradient). Final yield of the title compound was 92mg (60%).
20 ~max (MeOH) = 536nm For measurements in aqueous solution, a 1.OmMol solution in DMF was prepared; this was diluted 1:100 with 0.02Mo1 bis-tris propane/HCI aqueous buffer to give a 1 OpMol working solution.
(pH7 aq.) = 524nm, ~max = 75,000 dm3 mol-' cm-' 2s (pH10 aq.) = 460nm, ~max = 49,000 dm3 mol-' cm'1 Fluorescence (MeOH); ~,ex = 537nm, ~,em = 552nm 8H (300MHz, CD30D) 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 s [5d], using sulfanilic acid (60mg, 0.35mmol) in place of metanilic acid.
Yield =120mg (78%).
Amax (MeOH) = 540nm For measurements in aqueous solution, a 1.OmMol solution in DMF was prepared; this was diluted 1:100 with 0.02Mo1 bis-tris ~o propane/HCI aqueous buffer to give a 1 O~MoI working solution.
(pH7 aq.) = 530nm, ~max = 82,000 dm3 mol-' cm-' (pHlO aq.) = 468nm, Emax = 53,000 dm3 mol-' cm-' Fluorescence (MeOH); ~,ex = 540nm, ~,em = 555nm 8H (300MHz, CD30D) 1.450 (3H, t), 1.967 (6H, s), 4.31 (2H, ~s 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-2-2o 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 2s compound was isolated in 112mg yield (88%).
Amax (MeOH) = 540nm Fluorescence (MeOH); ~,ex = 537nm, ~,em = 553nm.
8H (300MHz, DMSO-ds) 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).
s 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 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], Rf = 0.65, yellow spot;
product, Rf =0.38, magenta spot). The mixture was cooled to 10°C, whereupon the product precipitated; the solid was collected, washed with cooled acetic Is acid, then diethyl ether and dried under vacuum. Yield of title compound =
67mg (46%).
Amax (MeOH) = 538nm, (pH7 aqueous buffer) = 524nm.
Fluorescence (MeOH); 7~ex = 541 nm, ~,em = 558nm.
8H (300MHz, DMSO-ds) 1.72-1.90 (4H, m), 1.916 (6H, s), 3.5 20 (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, 50p.mol) was reacted with TSTU (l5mg, 50p.mol), in DMF (200p1) in the presence of N,N-diisopropylethylamine (9pl). The reaction was followed by t.l.c. (RPC18. Methanol, 60: water, 40. [5g], Rf =
~0 0.4; NHS ester [5h], Rf = 0.32). The NHS ester was precipitated from solution by dilution with diethyl ether; the solid was isolated, washed with a methanol:ether 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 s primary amino groups.
2-Butylamino-4-[1-(4-sulfonatobutyl)-3,3-dimethyl-2-benzindolinylidenemethyl] cyclobutenediylium-1,3-diolate, sodium salt [5i]
~o This was prepared in an analogous method to [5g), using 1-butylamine (301, 0.3mmol) in place of 4-aminophenylacetic acid. The reaction was followed by t.l.c. (RPC18. Methanol, 60: water, 40. [1 m], Rf = 0.65; [5i], Rf = 0.24). After 5.5hrs the solvent was evaporated under vacuum; the residue was purified by preparative HPLC (RPC18.
~s Water~methanol gradient). Final yield of the title compound was 28mg (22%).
~max (MeOH) = 486nm, (pH7 aqueous buffer) = 480nm (MeOH + Bu4+ OH-) = 430nm, Fluorescence (MeOH); ~,eX = 487nm, ~,em = 512nm.
20 8H (300MHz, CD30D) 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, appal) 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%).

Amax (MeOH) = 552nm (pH7 aq.) = 542+510nm, (pHlO aq.) = 472nm Fluorescence (MeOH); ~,eX = 552nm, ~,em = 566nm.
~H (300MHz, CD30D) 1.445 (3H, t), 1.977 (6H, s), 4.29 (2H, s 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), 8.11 (1 H, dd), 8.23 (1 H, d) and 8.37 (1 H, d).
Mass spectrum: (ES-) 553 (M-) ~0 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, ~s 0.35mmol) in place of metanilic acid. Yield =113mg (67%).
Amax (MeOH) = 524nm (pH7 aq.) = 512nm, (pHlO aq.) = 458nm Fluorescence (MeOH); 7~ex = 530nm, ~,em = 566nm.
8H (300MHz, CD30D) 1.445 (3H, t), 1.973 (6H, s), 4.30 (2H, 2o 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-2-2s 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 ~o cold acetic acid, then with diethyl ether to furnish the product as a red solid.
Yield = 116mg (83%).

Amax (MeOH) = 536nm.
8H (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), 8Ø (2H, app d) and 8.20 (1 H, d).
s Mass spectrum: (ES+) 467 (M+H) R
N
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 H H
N N
O O O
O=S=O O=S=O
p, OH O, O N' l Na N ~a [59] [5h] O
O
N+"~~=_~ N H
O
O= =O
i O~Na [5i]

O', O-Na S' O
O
OH ~ ~ ~ S-O
N N~ p Na H H
[5J] [5k]
O
OH
N
H
[51]

_57_ Preparation of acylated sguarate dye examples Any of the dyes in example 5 can be acylated on the nitrogen s 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-~o benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [5b] (19.4mg, 50~.mol) was dissolved in dry pyridine (0.5m1) 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 ~s 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 2o the product dye, l6mg (74%).
Amax (CH2C12) = 536nm; (MeOH) = 506nm 8H (300MHz, CD2C12) 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 2s (1 H, d).
Mass spectrum: (ES+) 431 (M+H), 453 (M+Na), 469 (M+K).

_58_ 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-s benzindolinylidenemethyl)cyclobutene- diylium-1,3-diolate [5a] (20.4mg, 50~mo1) and acetyl chloride (1 drop). The title compound was isolated in 6mg yield (27%).
Amax (CH2C12) = 538nm; (MeOH) = 502nm 8H (300MHz, CD2C12) 1.53 (3H, t), 1.979 (6H, s), 2.617 (3H, ~o 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-('I-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate (6c]
~s 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] (l7mg, 50~mo1) 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:
2o dichloromethane, 95. [5c], Rf = 0.16 , yellow spot; product, Rf = 0.3, red spot). Isolated yield l2mg (55%).
~max (CH2C12) = 534nm; (MeOH) = 502nm 8H (300MHz, CD2C12) 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).
2s 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]
3o This was prepared and purified in an analogous method to [6c], using an excess of benzoyl chloride. (silica. Methanol, 5:

dichloromethane, 95. [5c], Rf = 0.16 , yellow spot ~ [6c], Rf = 0.3, red spot ~ [6d], Rf = 0.55, orange spot). Isolated yield 11 mg (41 %).
Amax (CH2C12) = 528nm; (MeOH) = 486nm bH (300MHz, CD2C12) 1.446 (3H, t), 1.848 (6H, s), 4.37 (2H, s 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) ~0 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 =
l3mg (70%).
~max (CH2C12) = 522+498nm; (MeOH) = 486nm 8H (300MHz, CD2C12) 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 20 (M+K) N

[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

Synthesis of sguarate dye examples with benzothiazole nuclei s 3-Methoxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut-3-en-1,2-dione [7a]
A 50m1 reaction flask was charged with dimethyl squarate (1.42g, l0mmol), dry methanol (l0ml) and N,N-diisopropylethylamine (1.29g, l0mmol). This mixture was heated to boiling until all the solid had ~o dissolved.
To the hot solution was added 3-ethyl-2-methylbenzothaizolium iodide (3.05g, 1 Ommol) - 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 ~s 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%).
Amax (MeOH) = 440nm 8H (300MHz, CDC13) 1.372 (3H, t), 4.04 (2H, q), 4.451 (3H, s), 20 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.I.c. (RPC18. Methanol, 95: water, 5. [7a], Rf = 0.5; minor pink impurity, Rf = 0.3).
Mass spectrum: (ES+) 288 (M+H), 310 (M+Na), 326 2s (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 ~o & 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, s 581-582).
Data for [7b]:
~max (MeOH) = 446nm 8H (300MHz, CD30D) 1.312 (3H, t), 4.05 (2H, q), 7.05 (1 H, t), ~0 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]:
Amax (MeOH) = 524nm 8H (300MHz, DMSO-ds) 1.276 (6H, t), 4.27 (4H, q), 5.865 (2H, s), 7.11-7.19 (2H, m), 7.32-7.39 (4H, m) and 7.74 (2H, d).
Mass spectrum: (ES+) 433 (M+H).
20 2-(1-Morpholino)-4-(3-ethyl-2-benzothiazolylidenemethyl)cyclobutenediylium-1,3-diolate [7d]
3-Hydroxy-4-(3-ethyl-2-benzothiazolylidenemethyl)-cyclobut-3-en-1,2-dione [7b] (82mg, 0.3mmol) and morpholine (35p1 = 35mg, 0.4mmol) were mixed in 1-butanol (3ml) and heated at 100°C for 2.5 hours.
2s The reaction was followed by t.l.c. (RPC18. Methanol, 95: water, 5. [7b], Rf = 0.8; [7d], Rf = 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.
Amax (MeOH) = 494nm; ~max = 114,000 dm3 mol~1 cm~'.
~o Fluorescence (MeOH); ~,eX = 492nm, ~,em = 518nm.

8H (300MHz, CDC13) 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).
s 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 (441 = 43mg, 0.40mmol) in place of piperidine. The title dye to [7e] was isolated in the same manner. Yield = 43mg (40%).
~max (MeOH) = 514nm; Emax = 105,000 dm3 mol'' cm''.
Fluorescence (MeOH); ~,ex = 518nm, ~,em = 565nm.
8H (300MHz, CDC13) 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).
~s 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-20 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 (1.5m1). 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 (3x2m1), then diethyl ether 2s and dried. Yield = 102mg (75%).
Amax (MeOH 50: H20 50) = 518nm; (+ Bu4+ OH') = 475nm.
Fluorescence (MeOH); 7~eX = 530nm, ~,em = 552nm.
8H (300MHz, DMSO-ds) 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).
~o Mass spectrum: (ES-) 427 (M-) O O O
\ ~ \ ~ S S
N \ ° \ ~ ~ ~ 1 /
N N
°~R J
[7a] R = CH3 [7c]
[7b] R = H
p , I S O
\ + - ~ \ + -/ N~% ~ / N
O O
[7d] [7e]
O'' O-Na S' O
S O
/ NH
O
[7f]

Synthesis of sguarate dye examples with 2-quinoline nuclei s 3-Methoxy-4-(1-ethyl-2-quinolylidenemethyl)-cyclobut-3-en-y,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 to 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%).
Amax (MeOH) = 462nm ~s bH (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).
20 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-quinolylidenemethyl)-cyclobut-3-en-1,2-dione [8a] (281 mg, 1 mmol) in a dilute aqueous HCI / acetic acid mix.
2s The reaction was followed by t.l.c. (RPCi8. Methanol, 95: water, 5. Samples dissolved in methanol spiked with triethylamine. [8a], Rf = 0.5, yellow spot;
[8b], Rf = 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 3o further purification.

Mass spectrum: (ES+) 268 (M+H), 290 (M+Na), 306 (M+K).
2-(1-Morpholino)-4-(1-ethyl-2-s 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 (35.1 = 35mg, 0.4mmol) in 1-butanol. T.I.c. (RPC18. Methanol, 95: water, 5. [8b], Rf = 0.7, orange spot;
~ o [8c], Rf = 0.4, magenta spot). Yield = 74mg (73%).
~max (MeOH) = 516nm; ~,nax = 65,000 dm3 mol-' cm-~.
8H (300MHz, DMSO-ds) 1.313 (3H, t), 3.74-3.80 (4H, m), 3.88-3.95 (4H, m), 4.22 (4H, broad q), 5.411 (1 H, s), 7.27 (1 H, t), 7.54-7.66 (4H, m) and 8.91 (1 H, d).
~s 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 20 [7d], using 3-hydroxy-4-(1-ethyl-2-quinolylidenemethyl)-cyclobut-3-en-1,2-dione [8b] (80mg, 0.30mmol) and N-methylaniline (44.1 = 43mg, 0.4mmol) in 1-butanol. T.I.c. (RPC18. Methanol, 95: water, 5. [8b], Rf = 0.7, orange spot; [8d], Rf = 0.4, deep pink spot). Yield = 85mg (79%).
Amax (MeOH) = 544nm; ~,nax = 67,000 dm3 mol-1 cm-' 2s 8H (300MHz, DMSO-ds) 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, appal) and 9.14 (1 H, d).
Mass spectrum: (ES+) 357 (M+H), 379 (M+Na).
~o 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 s anhydrous sodium acetate (30mg, 0.37mmol) were mixed with acetic acid (1.5m1). 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 (3x5m1), then diethyl ether and dried. Yield = 135mg 0100%).
Io Amax (MeOH 50: H20 50) = 540nm; (+ Bu4+ OH-) = 514nm.
8H (300MHz, DMSO-ds) 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-).
Is i I ~ o O.R
[8a] R = CH3 [8b] R = H
i I ~ O , ~ ~ p / \
N+ / ~ ~ N+ / \
J o_ J o_ [8~] [8d]

O', O-Na S' O
i I ~ p / \
W ~ +"~~=_:~~NH
O
[8e]

s 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 ~o [8a], using 1-ethyl-4-methylquinolinium iodide (900mg, 3mmol) in place of 1-ethyl-2-methylquinolinium iodide. Yield = 374mg (44%).
Amax (MeOH) = 528+498nm 8H (300MHz, DMSO-d6) 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).
~s 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]
2o 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~B. Methanol, 95: water, 5. Samples dissolved in methanol spiked with triethylamine. [9a], Rf = 0.5, pink spot;
2s [9b], Rf = 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.
s 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-~o dione [9b] (80mg, 0.3mmol). The reaction was followed by t.l.c. (RPC18.
Methanol, 95: water, 5. [9b], Rf = 0.65, red-pink spot; [9c], Rf = 0.4, deep purple spot).
Amax (MeOH) = 590+554nm 8H (300MHz, CD30D) 1.435 (3H, t), 1.76 (6H, broad s), 3.94 ~s (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)-2o 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 (44.1 = 43mg, 0.4mmol) in 1-butanol. T.Lc. (RPCiB. Methanol, 95: water, 5. [9b], Rf = 0.65, red-2s pinkspot; [9d], Rf = 0.4, deep blue spot). Yield = 73mg (68%).
Amax (MeOH) = 602nm 8H (300MHz, CDC13) 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).
~o Mass spectrum: (ES+) 357 (M+H), 379 (M+Na).

_69_ ~N \ O /~N \ O
I \ \ \ o I \ \ \ o ~ O ~ OH
[sa] [sb]
o ~ +. O
N ( N I
\ / N~ I \ /
o- ~ o-[sc] [sd]

_70_ Labelling of biological molecules s 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 25pg of protein in a final volume of 9~.1 lysate buffer (8M Urea, 4% (w/v) ~o CHAPS, 40mM Tris (pH8)). To this was added lOmM TCEP (lp.l 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 (2pl of a lOnmol/1~I DMF solution). Labelling was effected by incubation at 37°C for 30mins. The reaction was then quenched ~s by the addition of sample buffer (12.1 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 coniuqate 5-(3-(2-(4-amidopiperidino)-4-(1,3,3-trimethyl-2-benzindolinylidenemethyl) cVclobutenediylium-1,3-diolate)-allVl)-2'-deoxyuridine-5'-triphosphate.
5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate (1 mg) was 2s dissolved in 200 ~.I carbonate buffer pH 9.5. 2-(4-carboxypiperidino)-4-(1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate NHS ester [3h] in 100 ~.I 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 ~o reaction mixture was purified on a reverse phase prep. plate (1:1 acetonitrile : 0.1 M TEAB (triethylammonium bicarbonate buffer)). The _71 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 s (Rf = 0.22 ) was higher running than the starting material (0.05) and the carboxy derivative (0.14).
Mass spectrum: (ES+) 787 (M-P206), 865 (M-P03), 949 (M+H), 1046 (M+Et3NH).
~o Labelling of an oligonucleotide with 2-(4-O-N
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) cyclobutenediylium-1,3-diolate [4b] (15 mg, Is 0.027 mmol) was treated with O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU, 8.3mg, 30pmol), in DMF
(0.5m1) containing N,N-diisopropylethylamine (5pl). The activation was followed by t.l.c. (RPCis. Methanol, 40: water, 60. [4b] Rf = 0.48. Product Rf = 0.20); it was complete after 1 hour. Diethyl Ether was added to the 2o 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, l6mg (89%) (10a).
8H (300MHz, CD30D) 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 2s 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 ~o 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 1 Oa labelling with a cDNA reaction including reverse transcriptase (RT) (test).
s 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 ~o transcriptase (negative control) Method Labelling of cDNA with dye 10a was carried out as follows.
Eight cDNA reactions were set up each containing 2 ~.g of human skeletal ~s muscle mRNA, 2~1 of random primers (Nonamers, Amersham Pharmacia RPK0158), and 12 ~.I of water. For the no reverse transcriptase reactions 14 pl of water was added. The reactions were heated to 70 °C for 10 mins and then transferred to room temperature for 15 mins. 8~1 of 5x First strand buffer (Promega M531A) was added, 4~1 of 0.1 M DTT, 2~.1 of a nucleotide 2o mix (4mM dATP, dCTP, dGTP), 8~.1 of a 1 mM aminoallyl-dUTP (Sigma A0410) and 2~1 of reverse transcriptase (Promega M3682) to the relevant tubes. The reactions were incubated at 42 °C for 3 hours. To remove the mRNA, 2 ~I of 5M Sodium hydroxide was added and the tubes incubated at 42 °C for 10 min. The Sodium hydroxide was neutralised by adding 20 ~I
of 2s 2M HEPES. The free aminoallyl-dUTP was removed by adding 450 ~.I of water, placing in a Microcon 30 column (Millipore cat no 42410). The column was spun for 8 mins at l3krpm in a microfuge (MicroCentaur, MSE). 450 ~.I of water was added again to the column and spun through for 8 minutes as before. The 450 ~I water was added once again and the 3o column spun as previously. The purified cDNA was then eluted from the _ 73 -column by a 1 minute spin at l3krpm into a fresh tube.
The cDNA was dried down in a Speedvac. The cDNA was resuspended in 4.5.1 of water. 3.2x10'' moles of (~0.25mgs) of Squaramide NHS ester and Cy3-NHS ester (Amersham Pharmacia s PA23001 ) were each resuspended in 72 ~I 0.1 M Sodium bicarbonate pH9.
4.5 p.l 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. 4.5.1 of 4M hydroxylamine (Sigma H2391 ) was then added and incubation continued for a further l0minutes. The next step was to remove Io the free dye from the dye incorporated into the cDNA. 5008.1 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 l3krpm. 500p1 of buffer PE (Qiagen cat28106) was added to wash the column and the spin repeated. The wash ~s and spin step was repeated twice more. A further 1 minute spin was carried out before elution with 100 ~,I of elution buffer (Qiagen cat28106). The dye incorporation into the probes was then measured using a Cary UV/visible spectrophotometry.
2o Results Wavelength Absorbance Duplicate of measurement +RT Dye 10a 495nm 0.078 0.102 -RT Dye 10a 495nm 0.001 0.004 +RT Cy3 550nm 0.200 0.141 -RT Cy3 550nm 0.004 0.000 _74_ 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 s unincorporated dye is removed efficiently by the protocol used.

Claims (11)

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 C1-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(O)OR7, then the other of R1 or R2 is H or C1-C20 hydrocarbon or a group -L-G, 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' 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 R5, R6, R7 and R8 is H or C1 - C20 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 where each of R3 and R4 is H or C1 - C20 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 C1 - 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 CO2H, acid halide, anhydride, CO active ester, NCS, O
phosphoramidite, -NC(O)CH2I 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)

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).