JP2002540279A - Square rain dye - Google Patents

Abstract

(57) Abstract: A compound having the structure: W = Sq-A, where A has the formula -NR ¹ R ² , wherein R ¹ and R ² are the same or different and each is H or C ₁ -C _20. a hydrocarbon or a group -L-G, or one of ^{R 1} and ^{R 2} are -OR ⁵ or ^-NR 6 ^{R 7} or -COR ⁷ or ^-NR 6 COR ⁷ or -N = ^{R 8,} or R ¹ and R ² together form a saturated or unsaturated, unsubstituted or substituted monocyclic or condensed ring, or an azacrown or metal binding group, and Sq is represented by formula (A) or formula (B ) Embedded image Wherein m is 1, 2 or 3; and W is a structure (C). L ′ is a linker of a moiety of 0-3 selected from a carbon atom or an arylene group, and a dotted line represents a monocyclic or fused ring, an aromatic or heterocyclic ring, an unsubstituted or substituted, Or an unsaturation coordinated to the unsaturation of L ′ = Sq, wherein each of R ⁵ , R ⁶ , R ⁷ and R ⁸ is H or C ₁ -C ₂₀ carbon atom or group -LG, R ⁹ is a negative charge or group -LG, L is a connecting chain of 0 to 30 moieties, and one or more arylene groups; Or branched or unbranched, optionally containing O or N or N ⁺ or S or S ⁺ or P or P ⁺ or Se atoms, and G is a functional or reactive group, whereby the compound Is water soluble with biomolecules Additive or decreasing, also it can be covalently linked to the groups that provide electron donating or withdrawing alter the spectral characteristics of the compound, and homodimers and oligomers and hetero-dimers and oligomers of the compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

Today, the development of autofluorescence has become routine for detection in a range of biological applications such as, for example, flow cytometry, sequencing and arrays. The following are illustrative of the range of fluorescent dyes that can be used in such applications. Benzophenoxazine dyes are available from Amersham International's WO 97 /
29154. Waggoner et al., US 5,268,486, describe the properties of some conjugated cyanine dyes, see Middendorf, US 5,230,781 and Patonay, EP.
0 670 374 describes the use of various cyanine dyes in DNA sequencing. EP 0 214 847 of Berger et al. Describes other cyanine dyes, some of which contain a squarate group in assays involving specific binding partners. Other squarate dyes are USP 4,830,786 from Pease et al.
Anal. Chem. 1995, 67, 1742-8 by Mank et al., And Amersham Internatio
nal in WO 94/40104. WO 93/09172 to Cushman et al. And WO 94/19387 to Krutak et al. Describe cyanine dyes having squarate groups for use in thermoplastics and inks. Even now, there is a need for fluorescent dyes with improved physical properties, such as a family of dyes with photostability and excellent spectral resolution for a variety of biological samples. For example, fluorescein (as well as its reactive derivatives) is one of the first fluorescent labels on biomolecules and has been widely used with 488 nm lasers as an excitation light source. However, it is far from ideal because it has a low extinction coefficient, is a fluorescent material only within a certain pH range, and has poor chemical and photostability and solubility properties .

The present invention relates to a compound of the formula: W = Sq-A} wherein A has the formula —NR ¹ R ² , wherein R ¹ and R ² are the same or different, each being H or C ₁ -C ₂₀ is a hydrocarbon or a group -L-G, or ^{R 1} and one of ^{R 2} is -OR ⁵ or ^-NR 6 ^{R 7} or -COR ⁷ or -
NR ⁶ COR ⁷ or —N ＝ R ⁸ or —C (O) OR ⁷ , or R ¹ and R ² together are saturated or unsaturated, unsubstituted or have a substituent, or azacrown or metal Form a single or fused ring with a linking group. A squaraine compound having the formula:

Embedded image (Where m is 1, 2 or 3), and W is a structural formula

Embedded image L ′ is a linker consisting of 0-3 moieties selected from a carbon atom or an arylene group, and the dotted line is an aromatic or heterocyclic, unsubstituted or substituted, A monocyclic ring or a condensed ring containing or bonding a quaternary or quaternary nitrogen atom and having an unsaturated bond coordinated to an unsaturated moiety of L ′ = Sq, R ⁵ , R ⁶ , R ⁷ and each ^{R 8} is H or _C 1 _{-C 20} carbon atoms or a group -L-G, ^{R 9} is a negative charge or group -L-G, L is a linker of 0-60, one Or more than one arylene group, or branched or unbranched optionally containing O or N or N ⁺ or S or S ⁺ or P or P ⁺ or Se atoms; and G is a functional or reactive group. so Ri, whereby the compound biomolecules,
Can be covalently linked to other small molecules, such as dyes, or groups that increase or decrease water solubility or provide electron donating or withdrawing properties that alter the spectral properties of the compound, and homodimers and oligomers of the compound And a squaraine compound having a heterodimer and an oligomer｝.

[0003] These compounds are characterized by having Sq directly attached to the carbon atom of W and to the nitrogen atom of A by a 1,2-bond or, more preferably, a 1,3-bond. Substituents and various R groups included in the ring structure (indicated by dotted lines)
Generally, those described in previous publications, including those listed in the introduction, in relation to the like dyes.

[0004] R ¹ and R ² may be C ₁ -C ₂₀ hydrocarbons, such as alkyl, aryl or aralkyl. If one or both of R ¹ and R ² are hydrogen,
The compound will be pH sensitive, for example, having fluorescent properties at one pH and not at others. Alternatively, R ¹ and R ² may together form a saturated or unsaturated monocyclic or fused ring system, for example, pyrrolidine, piperidine, pyrrole, piperazine or morpholine. Alternatively, R ¹ and R ² may comprise other metal bonding groups such as azacrown or EDTA, or various combinations of N, O, S coordination atoms, together with the nitrogen atom to which they are attached. A metal chelate portion may be formed. R ¹ or both R ¹ and R ² may be a group —LG, discussed below. Examples of amine moieties A are:

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[0005] The wing portion W is preferably

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Here, each of R ³ and R ⁴ is H or C ₁ -C ₂₀ or a group —LG, and n is 0-3.

[0007] R ³ can be H, in which case the compound can be pH sensitive, eg, exhibiting fluorescent properties at one pH and not at others. R ³ can be a _C 1 _{-C 20} hydrocarbon as was Ronze ^{R 1} and ^{R 2} above.

The dotted line preferably indicates a heterocycle, which is either a single ring or a fused ring, and is generally unsaturated, for example, pyrrole or pyridine or indole or benzoindole or benzoxazole or benzothiazole or Quinolyl. The ring indicated by the dotted line preferably has the structure:

Embedded image Having.

Wherein X is O or S or —NR ⁴ or —CR ¹⁰ R ¹¹ , wherein each of R ¹⁰ and R ¹¹ is a C ₁ -C ₂₀ hydrocarbon or —LG, or R ¹⁰ and R ¹¹ together form a condensed ring, a heterocyclic ring or a cycloaliphatic monocyclic ring.

Examples of dyes having different wing portions W are:

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In the Sq part, the integer m can be 1 or 2 or 3. When m is 1,
These are squaric acid compounds; when n is 2, they are croconic acid compounds; when m is 3, they are rhodizonic acid compounds. So, for example:

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L is a linking chain of 0 to 30 or 60 moieties, preferably alkylene, alkenylene and alkynylene of the chosen form, or 1 to 6 O or N or N ⁺ or S or S ⁺ or P Or P ⁺ or Se
It is a branched or straight chain having up to 30 carbon atoms, optionally incorporating atoms or arylene groups.

G can be a functional or reactive group, which allows the squaraine compound to be covalently linked to biological or other molecules. Examples of G as a functional group include NH ₂ , OH
, SH and nucleophiles. Examples of G as a reactive group are -COOH, activated carbonyls such as acid halides or anhydrides, CO-activated esters, -NC
S, O phosphoramidite, -NC (O) CH ₂ I and maleimide

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[0014] Preferred biomolecules are nucleosides, nucleotides and their analogs, 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 cyclodextrin. Cyclodextrin-fluorophore conjugates have been shown to have greatly increased photostability in aqueous solutions (Tetrah
edron Letters, Volume 38, No 35, pages 6167-6170, 1997).

Alternatively, G may be a group that increases water solubility, such as sulfonates, phosphates, quaternary ammoniums, sugars and polyethers; or a group that decreases water solubility, such as, for example, alkyl. Alternatively, G may be a group that provides an electron donating or attracting property that alters the spectral properties of the squaraine compound; for example, a halogen, alkoxy, nitro or cyano group. the Chemis
try of Synthetic Dyes, Venkataraman, Academic Press, New York, 1971, 4,
Chapter 5 Part iiic, 228-240 See especially Table 1 on page 230.

Although the compounds of the invention have interesting fluorescent properties, they are also generally colored and can be used, for example, as traditional dyes in colorimetric assays.

Dimers and oligomers of the compounds defined above are also contemplated by the present invention.
Have been. For example, a dimer has the structure W¹-Sq-A¹-Sq-W²Or teens
Structure A¹-Sq-W¹-Sq-A²May be provided. Where A¹And A² Each represents an amine moiety A;¹And W²Indicates a wing portion W, respectively. R ¹ And R²Is part of a substituted fused ring system that forms a dye species
It can contain one or more amino groups that can be In such a case,
The substituted ring system serves as a scaffold from which the dye molecule can be formed. Also
However, if all dye species are the same, this will result in increased fluorescent signal.
Would. If different pigment species are present, energy transfer will occur
.

Here, if A ¹ and A ² (or W ¹ and W ² ) are the same, the compound is expected to have even stronger dye properties than the monomer. Examples of such dimers are:

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Alternative scaffolds for piperazine shown above are cyclam, dendrimer and adamantane (Tetrahedron Letters, 1999, 40, 223):

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Where A ¹ and A ² (or W ¹ and W ² ) are different, there may be an energy-transfer cassette consisting of a donor dye and an acceptor dye, the increase between the absorption and emission wavelengths of the compound. To provide separation. Trimers and higher oligomers can be made and used in a similar manner.

As described above, the dyes of the present invention can be used to form energy transfer cassettes with known dyes such as cyanine, rhodamine, and the like. If the donor dye is the dye of the present invention and replaces fluorescein, which lacks pH sensitivity, it will benefit from greater photostability and increased extinction coefficient. Examples are

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An energy transfer cassette is just one means of providing an energy transfer system. The dyes of the invention can also be used in simple FRET analysis,
It is facilitated by bringing the donor and acceptor into a distance where energy transfer is possible. Such systems can be based solely on the dyes described above, or they can use dyes known as one of the donor-acceptor pairs.

Another aspect of FRET analysis is the use of a quencher, which initially results in no signal when the donor is very close to the quencher. When a greater physical separation occurs between the two dyes, the quenching dye can no longer quench the fluorescence of the second dye, resulting in a signal. It has been reported that NO ₂ groups attached to squarate dyes (Dye and Pigment, Vol 35, 331, 1997) significantly reduce the fluorescence of squarate-based dyes. By such methods, quenching dyes from the present invention can be manufactured to yield dyes for such FRET analysis. The formulation and harmonization of other known dyes, similar to the dyes of the present invention, can provide a series of diverse sets in such analysis.

Perturbation of the electron density by protonation or metal chelation or as part of a redox system to the nitrogen atom of A, which is covalently bonded directly to the Sq moiety forming an integral part of the chromophore ) Affects the properties of the dye.
These changes can be used to provide an indication of the event that caused the change in electron density of the nitrogen atom.

Thus, when ^one of R ¹ and R ³ is H, the compound can be a pH sensor. When A contains a metal chelating moiety, it can act as a metal ion sensor. If A contains the NADH system, the compound can act as a redox sensor. Examples are: pH sensor

Embedded image Metal ion sensor

Embedded image Redox sensor (eg NADH)

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The invention also includes a method of making a compound as disclosed, wherein the method comprises:

I) reacting the wing portion W with a dialkyl squarate or the like to obtain an intermediate a
)give

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Ii) optionally subjecting intermediate a) to hydrolysis to give intermediate b)

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Iii) Reaction of intermediate a) or intermediate b) with R ¹ R ² NH gives the final product c
)give

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Reaction of intermediate a) with R ¹ R ² NH gives 1,2 squarate or the like. Reaction of intermediate b) with R ¹ R ² NH gives 1,3 squarate or the like.

Thus, squaraine compounds according to the present invention can be made by the following reaction procedure:

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The half-dye methyl ester from i) also reacts with the amine to give the 1,2-adduct:

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Four further specific reaction schemes follow the example method

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The present invention also includes an analytical or labeling method comprising contacting a sample containing an amine with compound a) or compound b).

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And observing dye formation due to the reaction of the amine with compound a) or b). The amine may be in solution or on a solid phase, and may be, for example, a peptide or 5'-amino oligonucleotide derivative. The analysis 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 present invention is further illustrated by the following examples. Example amine:

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[0037] amino acids

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Water solubility:

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The group —NR ¹ R ² may include an amino sugar or an amine with an additional quaternary ammonium group.

Variation of wavelength

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Use of polyamines for complex dye loading or energy transfer cassettes:

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The amino group may be part of a metal chelate system, for example, an azacrown:

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Naturally, the chelation of the appropriate metal ion changes the fluorescent properties.

[0044] pH sensor

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Other amino compounds

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EXAMPLES Example 1 Synthesis of Intermediate for Dye Synthesis 3-Methoxy-4- (1,3,3-trimethyl-2-indolinylidenemethyl)
-Cyclobut-3-ene-1,2-dione [1a] Dimethylsquarate (3.55 g, 25 mmol) and dried methanol (10 ml) were placed in a dry 100 ml round bottom flask equipped with a stir bar. This was stirred until all solids were dissolved.

Next, 2-methylene-1,3,3-trimethylindoline (distilled under reduced pressure before use, 4.4 g, 25.4 mmol) was added to the solution; it immediately turned deep yellow. . After about 20 minutes, a yellow solid began to precipitate. Stirring was continued for 16 hours, after which the mixture was cooled in the refrigerator for 2 hours. The title compound [1a] was collected by filtration, ice-cooled methanol (3 × 5 ml), then diethyl ether (4 × 20 ml).
After washing in l), it was dried under reduced pressure. Yield = 6.34 as a clear yellow powder.
g (90%).

Λ _max (MeOH) = 422 nm.δ _H (300 MHz, CDCl ₃ ) 1.60 (6H, s), 3.34 (3H, s), 4.49 (3H, s), 5.32 (1H
, s), 6.87 (1H, d), 7.04 (1H, d) and 7.22-7.28 (2H, m). Mass spec: (ES +) 284 (M + H), 306 (M + Na), 322 (M + K).

3-hydroxy-4- (1,3,3-trimethyl-2-indolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1b] 3-methoxy-4- (1,3 , 3-Trimethyl-2-indolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1a] (630 mg, 2.2 mmol
l) was mixed with acetic acid (20 ml) and concentrated aqueous hydrochloric acid (5 ml). The mixture was slowly warmed to about 40-50 ° C with a hot air gun. After several minutes at this temperature, all the solids had dissolved, forming a deep yellow solution. The mixture was kept at this temperature as the reaction proceeded; the reaction was followed by tlc (RPC _18, methanol,
90: water, 10. [1a], Rf = 0.5, [1b] Rf = 0.7). After the reaction was completed, the solvent was evaporated under reduced pressure. The residue is azeotroped twice with acetonitrile,
Thereafter, it was dried under a high vacuum. Trituration with diethyl ether gave the title compound as a yellow powder. Yield = 590 mg. This material was used directly for the synthesis of the dye. Mass spec: (ES +) 270 (M + H).

1-ethyl-2,3,3-trimethylbenz (e) indolinium iodide [1
c] 1,1,2-trimethylbenz (e) indole (10.47 g, 50 mmol
l), ethane iodide (11.7 g, 75 mmol) and 1,2-dichlorobenzene (35 ml) were mixed with a stirrer in a 100 ml flask. The mixture was heated to 90 ° C. for 18 hours; it resulted in a dark greenish solution containing a pale solid. After cooling, the solid was collected by suction filtration and dichlorobenzene (2
x20 ml) and then washed with excess diethyl ether. This was dried under reduced pressure to give the title solid as a pale purple solid. Yield = 15.93 g (87
%). δ _H (300MHz, CDCl ₃ ) 1.63 (3H, t), 1.85 (6H, s), 3.21 (3H, s), 4.86 (2H,
q), 7.62-7.74 (2H, m), 7.81 (1H, clear 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) indolium [1c]
] (7.3 g, 20 mmol) in sodium hydroxide (4 g,
(100 mmol) in water; the mixture was stirred briefly, then diethyl ether (100 ml) was added and the mixture was stirred again. The brown ether layer was collected; the aqueous layer was extracted with fresh ether (50 ml).

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

3-methoxy-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1e] 1-ethyl-2-methylene -3,3-dimethylbenz (e) indoline [1d
(4.75 g, 20 mmol) and dimethyl squarate (2.84 g, 20 m)
mol) was added to a 100 ml flask with a stirrer. Thereafter, dried methanol was added. The mixture was heated at 50 ° C. for 16 hours and then allowed to cool to room temperature. After further cooling in a refrigerator, the precipitated yellow solid was collected by suction filtration, ice-cooled methanol (2 × 15 ml) and then diethyl ether (4 × 30 ml).
) And dried under high vacuum to give the title compound [1e]. Yield =
5.44 g (78%) λ _max (MeOH) = 440 nm δ _H (300 MHz, CDCl ₃ ) 1.37 (3H, t), 1.88 (6H, s), 3.98 (2H, q), 4.52 (3H,
s), 5.41 (1H, s), 7.20 (1H, d), 7.36 (1H, m), 7.52 (1H, m), 7.84 (2H, m)
and 8.09 (1H, m).

3-hydroxy-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1f] 3-methoxy-4- (1 -Ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1e] (3.47 g)
, 10 mmol) were mixed with acetic acid (50 ml) in a 250 ml flask. To this was added a solution of concentrated hydrochloric acid (2.5 ml) in water (10 ml). The resulting mixture was kept warm in a water bath at 60 ° C. and the reaction was monitored by tlc (RPC ₁₈ .Methanol, 90: water, 10. [1e] Rf = 0.4, [1f] Rf = 0.7. ). When the reaction was completed, the solvent was evaporated under reduced pressure; the residue was azeotroped twice with acetonitrile and dried under high vacuum. The resulting foam was triturated with diethyl ether (25 ml) to give a yellow-brown powder; at this point 40-60 ° of petroleum ether was added. After cooling in a refrigerator, the solid was collected by suction filtration, further washed with a mixture of diethyl ether: petroleum ether 1: 1 and dried under reduced pressure to obtain the title compound [1f]. Yield = 3.1 g (93%). This material was used directly for the synthesis of the dye. λ _max (MeOH) = 442 nm δ _H (300 MHz, CD ₃ OD) 1.37 (3H, t), 1.89 (6H, s), 4.10 (2H, q), 5.48 (1H
, s), 7.33-7.44 (2H, m), 7.50-7.56 (1H, m), 7.90 (2H, clear d) and 8.14
(1H, clear d).

1,1,2-Trimethylbenz (e) indole-5-sulfonate, potassium salt [1 g] This was synthesized as described in EP 0288076 B1 (Richard L. Parton, Eastman Kodak Co.). .

1-ethyl-2,3,3-trimethylbenz (e) indolinium-5-sulfonate inner salt [1h] 1,1,2-trimethylbenz (e) indole-5-sulfonate, potassium salt [ 1 g] (granulate to a fine powder with a pestle and mortar before use; 1.64 g, 5
0 mmol), ethane iodide (2 g, 12.5 mmol) and 1,2-dichlorobenzene (8 ml) were mixed with a stirrer in a 25 ml flask. Heat the mixture to 120 ° C. (Wood metal bath, water cooler, silica gel protective tube);
Ethane iodide was further divided into 1 ml portions every few hours, and periodically added.
Allowed to react over days. At the end of this period, the mixture was cooled to room temperature and the suspended solid was collected by suction filtration. This was washed with diethyl ether and dried under high vacuum. This material was used directly without further purification. It is believed to include one equivalent of potassium iodide. δ _H (300MHz, CD ₃ OD) 1.32 (3H, t), 1.78 (6H, s), 4.67 (2H, q), 8.00-8.08
(2H, m), 8.17 (1H, d), 8.29 (1H, d) and 8.38 (1H, d).

3-methoxy-4- (5-sulfonate-1-ethyl-3,3-dimethyl-2-
Benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione, sodium salt [1i] 1-ethyl-2,3,3-trimethylbenz (e) indolinium-5-sulfonate inner salt [1h ] (1.56 g, 3.2 mmol) was placed in a dried 50 ml flask with a stirrer and then dried methanol (15 m
l) and sodium methoxide (0.19 g, 3.5 mmol) were added. The mixture was stirred for 5 minutes before dimethyl squarate (0.71 g, 5.0 mm
ol) was added. This mixture was heated at about 60 ° C. (sand bath, water cooling device, silica gel protective tube) for 16 hours.

At the end of this time, the solvent was evaporated under reduced pressure; the residue was subjected to flash chromatography (silica gel, 20-25% methanol in chloroform). The clean product fractions were combined and evaporated under reduced pressure; the residue was redissolved in 10% methanol in chloroform, filtered and evaporated again. 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 = 640 mg λ _max (MeOH) = 442 nm.δ _H (300 MHz, CD ₃ OD) 1.36 (3H, t), 1.88 (6H, s), 4.11 (2H, q), 4.54 (3H
, s), 5.55 (1H, s), 7.51 (1H, d), 7.94 (1H, dd), 8.01 (1H, d), 8.21 (1H,
d) and 8.37 (1H, d).

3-hydroxy-4- (5-sulfonate-1-ethyl-3,3-dimethyl-2
-Benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione;
Sodium salt [1j] 3-methoxy-4- (5-sulfonate-1-ethyl-3,3-dimethyl-2
-Benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione;
The sodium salt [1i] (640 mg, 1.4 mmol) was mixed with acetic acid (10 ml) and 2M aqueous hydrochloric acid (5 ml). The mixture was heated with a hot air gun until the solids dissolved; the resulting dark yellow solution was stirred with periodic heating. The reaction was followed by tlc. . _{(RPC 18} methanol, 60: water, 40.R
f [1i] = 0.6, Rf [1j] = 0.8). At the end of the reaction, the solvent was evaporated under reduced pressure, the residue was azeotroped twice with acetonitrile and finally dried under reduced pressure.

The purification was performed according to prep. Performed by HPLC (RPC _18, water → methanol gradient). The appropriate fractions were combined and evaporated under reduced pressure; the residue was again azeotroped twice with acetonitrile before drying under high vacuum to give the title compound. Yield = 376 mg (61%). Used directly. λ _max (MeOH) = 442 nm.

1- (4-sulfonatobutyl) -2,3,3-trimethylbenz (e) indolinium hydroxide inner salt [1k] 1,2,2-trimethylbenz (e) indole (1.05 g) , 5 mmol)
1,4-butanesultone (1.36 g, 10 mmol)
And 1,2-dichlorobenzene (5 ml) were mixed and heated to 120 ° C. for 20 hours. The mixture was then allowed to cool to room temperature and the precipitated solid was collected by filtration,
The extract was washed with dichlorobenzene and then with diethyl ether, and dried under reduced pressure at 50 ° C. to obtain 1.70 g (99%) of the title compound. δ _H (300MHz, CD ₃ OD) 1.827 (6H, s), 1.97 (2H, s), 2.21 (2H, m), 2.92 (2H,
t), 4.658 (2H, t), 7.69 (1H, m), 7.79 (1H, m), 8.06 (1H, d), 8.14 (1H,
d), 8.22 (1H, d) and 8.31 (1H, d).

3-methoxy-4- (1- (4-sulfonatobutyl) -3,3-dimethyl-2
-Benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [
1l] 1- (4-sulfonatobutyl) -2,3,3-trimethylbenz (e) indolinium hydroxide inner salt [1k] (1.38 g, 4.0 mmol), dimethyl squarate (0.57 g) , 4.0 mmol) and methanol (10 m
l) was mixed in a dried flask;
M sodium methoxide solution (8.0 ml, 4.0 mmol) was added. It quickly turned yellow. This was allowed to stir overnight. Thereafter, the solvent was removed under reduced pressure, and the residue was purified by flash chromatography (silica; 15-25% methanol / chloroform). The product fractions were collected and distilled under reduced pressure to give the title compound 1.4.
7 g (77%) were obtained. λ _max (MeOH) = 440 nm.δ _H (300 MHz, CD ₃ OD in CD ₂ Cl ₂ ) 1.80 (6H, s), 1.91 (4H, m), 2.88 (2H, t),
3.97 (2H, broad), 4.49 (3H, s), 5.44 (1H, s), 7.27-7.37 (2H, m), 7.50
(1H, m), 7.83 (2H, m) and 8.05 (1H, d).

3-hydroxy-4- (1- (4-sulfonatobutyl) -3,3-dimethyl-
2-Benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1m] This was synthesized in the same way as [1j], and in acetic acid (15 ml), the methoxy half-dye [11] ( (1.45 g, 3.0 mmol) with a 2 M aqueous hydrochloric acid solution (5 ml) by heating. The reaction was followed by tlc (RPC ₁₈ .
Methanol, 60: water, 40. [1l], Rf = 0.35, [1m], Rf = 0.
65). When the reaction has ended, the solvent is evaporated under reduced pressure and the residue is treated with acetonitrile for 3 hours.
It was azeotropically dried and finally dried under high vacuum in the presence of phosphorus pentoxide. This material was used without further purification.

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Example 2 Synthesis of Squarate Dye from Indolenine-Squaric Acid Intermediate 2- (1-Piperidino) -4- (1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium -1,3-diolate [2a] 3-hydroxy-4- (1,3,3-trimethyl-2-indolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1b] (81 mg, 0 .30mm
ol) was dissolved in 1-butanol (3 ml); to the resulting yellow solution was added piperidine (40 μl = 34 mg, 0.40 mmol). About 100 ° C (
(Sand bath, air cooling device). During this time the color darkened (meniscus deep orange yellow). The reaction was monitored by tlc (RPC _18, methanol, 90: water, 10. [1
b], Rf = 0.7, [2a], Rf = 0.4). After 4 hours, the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica gel; 2-5% methanol / chloroform). Fractions containing the main product were collected and evaporated; the residue was dissolved in chloroform, filtered and evaporated again. Trituration with a mixture of diethyl ether: petroleum ether 40-60 ° gave the title dye [2a] as a powder after drying under high vacuum. Yield = 93 mg (92%) λ _max (MeOH) = 470 nm; ε _max = 101,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 471 nm, λ _em = 495 nm. Δ _H (300 MHz, CDCl ₃ ) 1.67 (6H, s), 1.68-1.78 (6H, m), 3.35 (3H, s), 4.0
5 (4H, m), 5.56 (1H, s), 6.85 (1H, d), 7.01 (1H, t) and 7.20-7.27 (2H, m
Mass spectrometry: (ES +) 337 (M + H), 359 (M + Na).

2- (1-morpholino) -4- (1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium-1,3-diolate [2b] This is similar to [2a] And morpholine (35 μl = 35 mg, 0.40 mmol) was used instead of piperidine. The title dye [2b] was isolated in the same way. Yield = 91 mg (90%) λ _max (MeOH) = 476 nm; ε _max = 106,000 dm ³ mol ⁻¹ cm ⁻¹ . Fluorescence (MeOH); λ _ex = 474 nm, λ _em = 498 nm. Δ _H (300 MHz, CD ₃ OD) 1.64 (6H, s), 3.45 (3H, s), 3.86 (4H, clear t), 4
.06 (4H, clear t), 5.63 (1H, s), 7.06-7.12 (2H, m) and 7.27-7.37 (2H, m
). Mass spectrometry: (ES +) 339 (M + H).

1,4-bis- [2- (1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediyl-1,3-diolate] piperazine [2c] This comprises [2a] It was synthesized in a similar manner, using piperazine (12.9 mg, 0.15 mmol) instead of piperidine. The title dye [2c] was isolated in the same way. Yield = 79 mg (89%) λ _max (MeOH) = 506 nm; ε _max = 284,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 506 nm, λ _em = 520 nm. Δ _H (300 MHz, CDCl ₃ ) 1.68 (12H, s), 3.44 (6H, s), 4.29 (8H, s), 5.68 (2
H, s), 6.93 (2H, d), 7.08 (2H, t) and 7.22-7.31 (4H, m). Mass spec: (ES +) 589 (M + H), 611 (M + Na).

2- (N-methylanilino) -4- (1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediyl-1,3-diolate [2d] which is similar to [2a] And N-methylaniline (44 μl = 43 mg, 0.40 mmol) was used instead of piperidine. Title dye [2
d] was isolated in the same manner. Yield = 96 mg (89%) λ _max (MeOH) = 496 nm; ε _max = 87,000 dm ³ mol ⁻¹ cm ⁻¹ . Fluorescence (MeOH); λ _ex = 496 nm, λ _em = 547 nm. Δ _H (300 MHz, CD ₃ OD) 1.66 (6H, s), 3.53 (3H, s), 3.96 (3H, s), 5.77 (1H
, s), 7.12-7.19 (2H, m) and 7.30-7.47 (7H, m). Mass spectrometry: (ES +) 359 (M + H), 381 (M + Na).

2- (N, N-dipropylanilino) -4- (1,3,3-trimethyl-2-indolinylidenemethyl) cyclobutenediylium-1,3-diolate [2e] It was synthesized in a similar manner to [2a], using diisopropylamine (55 μl = 40.5 mg, 0.40 mmol) instead of piperidine. The title dye [2e] was isolated in the same way. Yield = 97 mg (92%). λ _max (MeOH) = 474 nm; ε _max = 97,000 dm ³ mol ⁻¹ cm ⁻¹ . Fluorescence (MeOH); λ _ex = 474 nm, λ _em = 494 nm. δ _H (300 MHz, CD ₃ OD) 0.88 (6H, t ), 1.56 (6H, s), 1.65 (4H, hex), 3.33 (
3H, s), 3.74 (4H, t), 5.52 (1H, s), 6.96-7.01 (2H, m) and 7.16-7.26 (2H,
m). Mass spec: (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-ene-1,2-dione [1b] (81 mg, 0.30 mm
ol) was dissolved in N, N-dimethylformamide (3 ml); to the resulting yellow solution was added isonipecotic acid (52 mg, 0.40 mmol). The mixture was heated at 100 ° C. for 4 hours. The reaction gave two major products at tlc. . _{(RPC 18} methanol, 95:. Water, 5 [1b], Rf = 0.8, product 1
Rf = 0.65, product 2 Rf = 0.5). After cooling and evaporation of the solvent under reduced pressure, the two compounds were flash chromatographed (silica gel; 2.5-20% methanol in chloroform; product 2 eluted first; this was the dye [2 g] (
10 mg), probably formed by supplementation of dimethylamine released from DMF. ). Product 1 eluted later and was the desired dye [2f], isolated yield = 22 mg (19%). [2f] λ _max (MeOH) = 472 nm Fluorescence (MeOH); λ _ex = 472 nm, λ _em = 498 nm. Δ _H (300 MHz, CD ₃ OD) 1.64 (6H, s), 1.82-1.94 (2H, m), 2.11-2.17 (2H, m)
, 2.65-2.74 (1H, m), 3.43 (3H, s), 3.46-3.61 (2H, m), 4.60-4.64 (2H, m),
5.61 (1H, s), 7.05-7.10 (2H, m) and 7.26-7.36 (2H, m). [2 g] λ _max (MeOH) = 468 nm δ _H (300 MHz, CD ₃ OD) 1.64 (6H, s) , 3.41 (3H, s), 3.49 (6H, s), 5.59 (1H
, s), 7.04-7.09 (2H, m) and 7.25-7.35 (2H, m).

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Example 3 Synthesis of Squarate Dye from Benzindolenin-Squaric Acid Intermediate 2- (1-Piperidino) -4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) Cyclobutenediyl-1,3-diolate [3a] 3-hydroxy-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-en-1,2-dione [ 1f] (100m
g, 0.30 mmol) was dissolved in 1-butanol (3 ml); to the resulting yellow solution was added piperidine (40 μl = 34 mg, 0.40 mmol). This was heated to about 100 ° C. (sand bath, air cooling device). During this time the color darkened (dark orange with meniscus). The reaction was monitored by tlc (RPC _18, methanol, 90:
Water, 10. [1f], Rf = 0.7, [3a] Rf = 0.25). After 1 hour, the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica gel; 0-4% methanol in chloroform). Fractions containing the main product were collected and evaporated. The residue was dissolved in chloroform, filtered and evaporated again. Trituration with a mixture of diethyl ether: petroleum ether 40-60 ° gave the title dye [3a] as a powder after drying under high vacuum. Yield = 110 mg (91
%) Λ _max (MeOH) = 490 nm; ε _max = 95,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 492 nm, λ _em = 516 nm. Δ _H (300 MHz, CD ₃ OD) 1.36 (3H , t), 1.78 (6H, broad s), 1.90 (6H, s),
4.02-4.11 (6H, m), 5.70 (1H, s), 7.33-7.44 (2H, m), 7.52 (1H, clear t)
, 7.89 (2H, clear d) and 8.14 (1H, d). Mass spectrometry: (ES +) 401 (M + H), 423 (M + Na).

3-Methoxy-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1e] (105 mg) in heated ethanol , 0.30 mmol) and piperidine (40 μl = 34 m
g, 0.40 mmol) to give an alternative product. tlc (RP
_C18 . Methanol, 90: water, 10. [1e], Rf = 0.4, Rf of product =
0.4, reference [3a] Rf = 0.25) and (silica. Methanol, 2.5: chloroform, 97.5. [1e], Rf = 0.55, product Rf = 0.45, Rf of reference [3a] = 0.4). The new product was isolated by evaporation of the ethanol under reduced pressure and trituration of the residue with diethyl ether. Yield = 120 mg
(100%).

Λ _max (MeOH) = 452 nm. Reference [3a], 490 nm. Overall, it looks more like a semi-dye [1e] than [3a].

At δ _H (300 MHz, CDCl ₃ ), it appears to be a mixture of the two isomers, but neither exactly matches the spectrum of [3a]. The new product is the 1,2-adduct, 3- (1-piperidino) -4- (1-ethyl-3,3-dimethyl-2-benzynolinylidenemethyl) cyclobut-3-ene-1,2. -Zeon:

Embedded image Here, the conjugation of one double bond is further fixed so that E- and Z-isomers are possible. Mass spec: (ES +) 401 (M + H), 423 (M + Na).

2- (1-morpholino) -4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [3b] And morpholine (35 μl = 35 mg, 0.40 mmol) was used in place of piperidine. The title dye [3b] was isolated in the same way. Yield = 99 mg (82%) λ _max (MeOH) = 494 nm; ε _max = 93,000 dm ³ mol ⁻¹ cm ⁻¹ . Fluorescence (MeOH); λ _ex = 494 nm, λ _em = 520 nm. Δ _H (300 MHz, CD ₃ OD) 1.17 (3H, t), 1.91 (6H, s), 3.87 (4H, clear t), 4
.06-4.16 (6H, m), 5.75 (1H, s), 7.38 (1H, clear t), 7.46 (1H, d), 7.55
(1H, m), 7.90 (2H, clear d) and 8.15 (1H, d). Mass spectrometry: (ES +) 401 (M + H).

1,4-bis- [2- (1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate] piperazine [3c] This is [3a] And piperazine (12.9 mg, 0.15 mmol) was used instead of piperidine. The title dye [3c] was isolated in the same way. Yield = 98 mg (91%) λ _max (MeOH) = 528 nm; ε _max = 270,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 528 nm, λ _em = 542 nm. Δ _H (300 MHz, CDCl ₃ ) 1.39 (6H, t), 1.57 (12H, s), 4.08 (4H, q), 4.31 (8
H, s), 5.76 (2H, s), 7.27 (2H, m), 7.41 (2H, clear t), 7.55 (2H, clear
t), 7.86 (4H, clear t) and 8.12 (2H, d). Mass spectrometry: (ES +) 717 (M + H), 739 (M + Na).

2- (N-methylanilino) -4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3
d] This was synthesized in a similar manner to [3a], using N-methylaniline (44 μl = 43 mg, 0.40 mmol) instead of piperidine. Title dye [3
d] was isolated in the same manner. Yield = 120 mg (≒ 100%) λ _max (MeOH) = 516 nm; ε _max = 76,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 522 nm, λ _em = 563 nm. Δ _H (300 MHz, CD ₃ OD) 1.40 (3H, t), 1.93 (6H, s), 3.96 (3H, s), 4.22 (2H
, q), 5.90 (1H, s), 7.34-7.60 (8H, m), 7.92-7.96 (2H, m) and 8.18 (1H, d
). Mass spectrometry: (ES +) 423 (M + H), 445 (M + Na).

2- (N, N-diphenylamino) -4- (1-ethyl-3,3-dimethyl-2
-Benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3e] This is synthesized in an analogous manner to [3a], instead of piperidine, N, N-diphenylamine (68 mg, 0.40 mmol) Was used. Title dye [3e]
Was isolated in the same manner. Yield = 60 mg (41%) λ _max (MeOH) = 540 nm; ε _max = 96,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 542 nm, λ _em = 588 nm. Δ _H (300 MHz, CD ₃ OD) 1.44 (3H, t), 1.94 (6H, s), 4.30 (2H, q), 6.07 (1H
, s), 7.21-7.26 (4H, m), 7.34-7.49 (7H, m), 7.56-7.62 (2H, m), 7.98 (2H,
Clear t) and 8.20 (1H, d). Mass spec: (ES +) 485 (M + H), 507 (M + Na).

2- (N-phenyl-N ′-(acetyl) hydrazino) -4- (1-ethyl-3
, 3-Dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3f] This was synthesized in an analogous manner to [3a], and 1-acetyl-2- instead of piperidine. Phenylhydrazine (60 mg, 0.40 mmol) was used. The title dye [3f] was isolated in the same way. Yield = 47 mg (34%) λ _max (MeOH) = 530 nm Fluorescence (MeOH); λ _ex = 533 nm, λ _em = 566 nm. Δ _H (300 MHz, CD ₃ OD) 1.41 (3H, t), 1.93 (6H, s) ), 2.16 (3H, s), 4.29 (2H
, q), 6.04 (1H, s), 7.24 (1H, m), 7.37-7.49 (5H, m), 7.56-7.61 (2H, m),
7.96 (2H, clear t) and 8.19 (1H, d). MS: (ES +) 589 (M + H), 611 (M + Na).

2- (4-carboxypiperidino) -4- (1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [3
g] 3-hydroxy-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) -cyclobut-3-ene-1,2-dione [1f] (100 m
g, 0.30 mmol) and isonipecotic acid (52 mg, 0.40 mmol)
) Was mixed with acetic acid (3 ml); the resulting mixture was heated to 100 ° C. for 5 hours and tlc (RPC ₁₈ .methanol, 95: water, 5. [1f], Rf = 0.75.
, Yellow spot. Product Rf = 0.5, orange spot). then,
The solvent was evaporated under reduced pressure; the residue was flash chromatographed (silica gel.
(5-15% methanol in chloroform). Fractions containing the main product were collected and evaporated. The residue was dissolved in chloroform, filtered and evaporated again. Trituration with diethyl ether gave the title dye 105 as an orange solid.
mg (79%) were obtained. λ _max (MeOH) = 492 nm Fluorescence (MeOH); λ _ex = 492 nm, λ _em = 516 nm.δ _H (300 MHz, CD ₃ OD) 1.662 (3H, t), 2.13-2.21 (6H, s + 2H, m) , 2.42-2
.48 (2H, m), 3.02 (1H, m), 3.82-3.92 (2H, m), 4.43 (2H, broad m), 4
.91‐4.96 (2H, m), 6.024 (1H, s), 7.663 (1H, t), 7.70 (1H, d), 7.83 (1H,
t), 8.20 (2H, clear t) and 8.45 (1H, d). Mass spec: (ES +) 445 (M + H), 467 (M + Na).

22 mg (50 μmol) of this carboxy dye was converted to highly reactive N-hydroxysuccinimide ester [3h]. The reaction is O- (N-succinimidyl)
-N, N, N ', N'-tetramethyluronium tetrafluoroborate (TSTU
, 15 mg, 50 μmol) and N, N-diisopropylethylamine (9 μl)
In DMF (0.5 ml) containing Activation was monitored by tlc _{(RPC 1 8} methanol, 95:.. Water, 5 [3g], Rf = 0.5, product Rf = 0.45
); Completed after 20 minutes.

Then, the NHS ester [3h] immediately reacted with the indotrimethine cyanine dye having a pendant primary amino group to obtain a conjugated compound having the structural formula [3i]. The product was isolated and prepared tlc (silica. Methanol, 25: chloroform, 75. The product was isolated as a single entity. Rf =
0.2) and had the following characteristics; this represents an example of an energy-transfer system: λ _max (MeOH) = 492, 552 nm Mass spectrometry: (ES +) 920 (M +), 921 (M + H) ), 943 (M + Na).

The Cy3 accepting dye has λ _ex = 551 nm, λ _em = 567 nm; excitation of the Cy3 amino dye at 492 nm results in an emission intensity at 567 nm of only 20% of that caused by excitation at 551 nm. Become. For the conjugate [3i], the excitation at 492 nm is 65% of the emission intensity caused by the excitation at 551 nm.
Release intensity. The emission peak of the squalamide donor at 517 nm is 49
Excitation at the 2 nm peak is almost completely suppressed at the conjugate [3i] and its contribution to 567 nm emission is negligible. Thus, when a squalamide donor is conjugated to a Cy3 dye, emission is increased by a factor of 3 or more when the excitation is at 492 nm compared to when the Cy3 dye is used alone.

2- (4-((2-maleimidoethyl) aminocarbonyl) piperidino) -4-
(1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3j] This is prepared by reacting squalamide NHS ester in anhydrous acetonitrile in the presence of N, N-diisopropylethylamine. [3h], 0.1 mmol, synthesized above)
And N- (2-aminoethyl) maleimide hydrochloride (2 × 20 mg, 0.11 m
mol). The reaction was monitored by tlc (silica, methanol, 5:
Dichloromethane, 95. Rf value: [3g] = 0.02, [3h] = 0.4, [3j]
] = 0.1).

When the reaction was deemed complete, the mixture was partitioned between dichloromethane and 0.1 M aqueous hydrochloric acid. The organic layer was collected, dried (MgSO ₄ ), filtered and evaporated under reduced pressure. The residue is purified by flash chromatography (silica, 2 in dichloromethane).
. (5-4% methanol). Product fractions were collected and evaporated. The sticky residue was triturated with acetonitrile to give a fine solid. The mixture was carefully diluted with ether, then the solid was collected by filtration, washed with ether and dried to give 40 mg (71%) of the title dye.

This is a dye having a maleimide group and has high reactivity with a thiol group. It is E. has been used as a labeled protein in E. coli lysates; the resulting labeled protein was subsequently analyzed by the 2D-DIGE method as described in PCT patent application WO / 96/33406. λ _max (MeOH) = 492 nm δ _H (300 MHz, CD ₃ OD) 1.373 (3H, t), 1.80-2.03 (10H, m), 2.491 (1H, m)
, 3.36-3.47 (4H, m), 3.63 (2H, m), 4.13 (2H, broad q), 4.76 (2H, m),
5.734 (1H, s), 6.822 (2H, s), 7.38 (1H, t), 7.45 (1H, d), 7.54 (1H, m),
7.91 (2H, clear d) and 8.16 (1H, d). Mass spec: (ES +) 567 (M + H), 589 (M + Na), (ES-) 565 (MH).

2- (1-aza-18-crown-6) -4- (1,3,3-trimethyl-2-
Benzindolidenemethyl) cyclobutenediyl-1,3-diolate [3
k] This is synthesized in an analogous manner to [3a], where 1-aza-
18-Crown-6 (87 mg, 0.33 mmol) was used. Title dye [3
k] was isolated in the same manner. λ _max (MeOH) = 494 nm δ _H (300 MHz, CDCl ₃ ) 1.346 (3H, t), 1.942 (6H, s), 3.64 (16H, clear s)
, 3.854 (4H, t), 4.01 (2H, broad q), 4.240 (4H, t), 5.695 (1H, s), 7.18
-7.3 (m, partially observed by chloroform), 7.332 (1H, t), 7.493 (
1H, t), 7.82 (2H, m), and 8.10 (1H, d). Mass spec: (ES +) 579 (M + H), 601 (M + Na), 617 (M + K).

2-N- (4-carboxyphenyl) -N-methylamino-4- (1-ethyl-
3,3-Dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [31] This is synthesized in a similar manner to [3a], and instead of piperidine 4-methylaminobenzoate The acid (50 mg, 0.33 mmol) was used. Acetic acid was used as an alternative solvent. The title dye [31] was isolated in the same way. Yield = 54 mg
(39%) λ _max (MeOH) = 526 nm δ _H (300 MHz, CD ₃ OD) 1.48 (3H, t), 2.02 (6H, s), 4.05 (3H, s), 4.25 (2H
, q), 6.06 (1H, 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] This is synthesized in an analogous manner to [3a], with 1-ter instead of piperidine.
t-Butyl-2-phenylhydrazine (80 mg, 0.32 mmol) was used. Acetic acid was used as an alternative solvent. The title dye [3m] was isolated in the same way. Yield = 29 mg (21%) λ _max (H ₂ O) = 528 nm Fluorescence (H ₂ O); λ _ex = 532 nm, λ _em = 566 nm. Δ _H (300 MHz, CD ₃ OD) 1.28 (3H, t), 1.48 (9H, s), 1.99 (6H, s), 4.29 (2H
, q), 6.04 (1H, s), 6.73 (2H, d), 7.22 (1H, clear t), 7.47-7.76 (2H, m)
, 7.82 (2H, d), and 7.98-8.27 (3H, m). Mass spec: (ES +) 589 (M + H), 611 (M + Na).

2-N- (phenyl) -N ′-(levulinic acid) hydrazone-4- (1-ethyl-
3,3-Dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3n] This is synthesized in a similar manner to [3a], and phenylhydrazolate levulinate is used instead of piperidine. Lactam (37 mg, 0.20 mmol) was used. Acetic acid was used as an alternative solvent. The by-product, the title dye [3n], was isolated in the same manner. Furthermore, purification was performed on a preparative plate. Yield = 2 mg (
Λ _max (MeOH) = 532 nm δ _H (300 MHz, CDCl ₃ ) 1.25 (3H, s), 1.43 (3H, t), 1.57 (4H, broad s),
2.01 (6H, s), 4.19 (2H, q), 5.92 (1H, s), 7.17-7.85 (8H, m), 7.89-7.94
(2H, m), and 8.19 (1H, d).

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Example 4 Synthesis of Squarate Dye from Sulfonated Benzindolenine-Squaric Acid Intermediate 2- (1-Piperidino) -4- (5-sulfonato-1-ethyl-3,3-dimethyl- 2-benzindolinylidenemethyl) cyclobutenediyl-1,3-
Diolate [3a] 3-hydroxy-4- (5-sulfonate-1-ethyl-3,3-dimethyl-
2-benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione, sodium salt [1j] (130 mg, 0.30 mmol), piperidine (40
μl = 34 mg, 0.40 mmol) and 1-butanol (5 ml) were mixed and heated to 110 ° C .; all solids did not dissolve, but turned orange. The reaction was monitored by tlc (RPC _18, methanol, 60: water, 40. [1]
j], Rf = 0.8, [4a] Rf = 0.5). When the reaction appeared to have stopped, another 20 μl of piperidine was added with 2 ml of ethanol and heating was resumed. After a further 4 hours, the solvent was evaporated under reduced pressure. prep. HPLC (R
PC ₁₈ . Water-methanol gradient); the fractions containing the pure product were collected, evaporated, dried under high vacuum and triturated with diethyl ether; the orange powder obtained was further purified under high vacuum. After drying, 99 mg of the title dye [4a] was obtained as a powder. λ _max (MeOH) = 490 nm, (pH 7.0 phosphate buffer) = 488 nm. Fluorescence (pH phosphate buffer); λ _ex = 488 nm, λ _em = 512 nm. δ _H (300 MHz, CD ₃ OD) 1.36 (3H, t), 1.80 (6H, broad s), 1.91 (6H, s),
4.06 (6H, m), 5.73 (1H, s), 7.50 (1H, d), 7.93 (1H, d), 7.99 (1H, d), 8
.21 (1H, d) and 8.37 (1H, s). Evidence for some piperidinium cations. Mass ^{spectrometry: (ES-) 479 (M -} ).

A study was conducted on the effect of pH on fluorescence. Buffer is Radiometer Ana
pH4.0 (potassium hydrogen phthalate, 50 mmol / l) pH 7.0 (Na ₂ HPO ₄ , 27.5 mmol / l: KH ₂ PO ₄ , 20.0 mm)
ol / l) pH 10.0 (NaHCO ₃ , 25 mmol / l: Na ₂ CO ₃ , 25 mmol)

A standard solution of Dye [4a] in methanol was diluted with these buffers to give a solution of the dye at equal concentration (about 2 × 10 ⁻⁶ M). Next, the absorption and fluorescence spectra of these solutions were recorded.

[Table 1]

It can be seen from this that the fluorescence is not lost in a weakly acidic or alkaline solution.

2- (4-carboxypiperidino) -4- (5-sulfonate-1,3,3-
Trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1
, 3-diolate [4b] 3-hydroxy-4- (5-sulfonate-1-ethyl-3,3-dimethyl-
2-benzoindolinylidenemethyl) -cyclobut-3-ene-1,2-dione, sodium salt [1j] (130 mg, 0.30 mmol), isonipecotic acid (52 mg, 0.40 mmol) and acetic acid (2.5 ml) At 100 ° C
The reaction was monitored by tlc (RPC _18, methanol, 40: water,
60. [1j], Rf = 0.7, product Rf = 0.5). After 5.5 hours, the solvent was evaporated under reduced pressure and azeotroped twice with acetonitrile. The residue was triturated with diethyl ether. Purified by diluting this solid methanol solution with water; 53 m
g of the title compound was isolated as the sodium salt. λ _max (MeOH) = 492 nm, (water) = 488 nm. fluorescence (MeOH); λ _ex = 492 nm, λ _em = 517 nm. δH (300MHz, CD ₃ OD) 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) a
nd 8.37 (1H, d) Mass spectrometry:. (ES-) 523 (M -), 261 ((M-H) 2- / 2)

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Example 5 Synthesis of a Squarate Dye Using a Primary Amine-pH Sensitive Dye If the amine compound used to form the final dye is a primary amine (ie, has a —NH ₂ group) For example, the resulting squarate dye will have a partially alkylated nitrogen atom with the hydrogen atom attached in place of the third carbon atom. This hydrogen atom can be removed under basic conditions, causing significant changes in visible absorption and fluorescence properties; the dye becomes pH sensitive. The intrinsic nature of the amine is to provide a means of controlling this change in pKa value. That is, the dye can be "tuned" to change from one form to another within a controlled pH range.

2-phenylamino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [5a] which is similar to [3a] Aniline (instead of piperidine)
30 μl = 31 mg, 0.33 mmol). The title dye [5a] crystallized on cooling the reaction mixture; the solid was collected, washed with 1-butanol and diethyl ether and dried under reduced pressure. Yield of [5a] = 100 mg (81
%) Λ _max (MeOH) = 532 nm; ε _max = 99,000 dm ³ mol ⁻¹ cm ⁻¹ . (MeOH + Bu ₄ ⁺ OH ⁻ ) = 456 nm, ε _max = 53,000 dm ³ mol-'cm- '. Fluorescence (MeOH) λ _ex = 532 nm, λ _em = 548 nm. (MeOH + Bu ₄ ⁺ OH ⁻ ) = non-fluorescent. δ _H (300 MHz, CDCl ₃ ) 1.391 (3H, t), 1.978 (6H, s), 4.12 (2H, broad q),
5.880 (1H, s), 7.130 (1H, t), 7.25 (1H, d), 7.34-7.40 (3H, m), 7.49-7.5
5 (1H, m), 7.81-7.93 (4H, m), 8.11 (1H, d) and 9.9 (1H, broad). Mass spectrometry: (ES +) 409 (M + H), 431 (M + Na).

2-butylamino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [5b] which is similar to [3a] 1-butylamine (32 μl = 24 mg, 0.33 mmol) was used instead of piperidine. The reaction was monitored by tlc (RPC _18, methanol, 95: water, 5. [1f], Rf = 0.8, [5
b] Rf = 0.4). After 7 hours, the solvent was evaporated under reduced pressure; the residue was partitioned between 0.1M aqueous hydrochloric acid and chloroform. Collect the organic layer, wash with water, dry (Mg
SO ₄ ), filtered and the solvent was evaporated. Purification by flash chromatography (silica, 2-3% methanol / chloroform) yielded 84 mg of the title compound (
72%). λ _max (MeOH) = 484 nm; ε _max = 85,000 dm ³ mol-'cm ”. (MeOH + Bu ₄ ⁺ OH ⁻ ) = 434 + 388 nm; ε _max ≒ 33,000 dm ³ mol ⁻¹ cm ⁻¹ (434 nm) Fluorescence _{. (MeOH);. λ ex} = 483nm, λ em = 511nm (MeOH + Bu 4 + OH -) = non-fluorescent _{δ H (300MHz, CDCl 3)} 1.003 (3H, t), 1.373 (3H, t), 1.50 (2H, clear hex)
1.8 (2H, m), 1.971 (6H, s), 3.90 (2H, q), 4.03 (2H, q), 5.691 (1H, s), 7
.21 (1H, d), 7.35 (1H, m), 7.51 (1H, m), 7.84 (2H, app t), 8.10 (1H, d)
and 9.20 (1H, broad) mass spec: (ES +) 389 (M + H), 411 (M + Na).

2-amino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [5c] This is similar to [3a]. Synthesized by the method, using ammonium acetate (77 mg, 1.0 mmol) instead of piperidine. The reaction was followed by tlc (RP
_C18 . Methanol, 90: water, 10. [1f], Rf = 0.65, [5b] Rf
= 0.35). After the reaction was completed, the solvent was evaporated under reduced pressure; the residue was purified by flash chromatography (silica; 2-5% methanol / chloroform) to give the title compound. _{.. λ max (MeOH) =} 478nm (MeOH + Bu 4 + OH -) = 434nm fluorescence _{(MeOH);. λ ex =} 481 nm, λ em = 506nm (MeOH + Bu 4 + OH -) = non-fluorescent [delta] _H (300MHz, DMSO-d6) 1.255 (3H, t), 1.870 (6H, s), 4.05 (2H, q), 5.555
(1H, s), 7.37 (1H, m), 7.55 (2H, m), 7.95 (2H, app d), 8.16 (1H, d) and
9.47 (1-2H, broad). Mass spec: (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-ene-1,2-dione [1f] (100 m
g, 0.30 mmol), metanilic acid (60 mg, 0.35 mmol), sodium acetate anhydride (30 mg, 0.37 mmol) and acetic acid (2.5 ml) were mixed and heated at about 100 ° C. for 3 hours; Changed from yellow to dark purple-red. Thereafter, the solvent was evaporated under reduced pressure.

The crude product was purified by preparative HPLC (RPC _18, water → methanol gradient). Final yield of the title compound was 92 mg (60%).

Λ _max (MeOH) = 536 nm A 1.0 mMol solution in DMF was prepared for measurement in aqueous solution; this was diluted 1: 100 with 0.02 Mol bis-trispropane / HCl aqueous buffer. Thus, a working solution of 10 μMol was obtained. (pH 7 aqueous solution) = 524 nm, ε _max ≒ 75,000 dm ³ mol ^-1 cm ^-1 (pH 10 aqueous solution) = 460 nm, ε _max ≒ 49,000 dm ³ mol ^-1 cm ^-1 Fluorescence (MeOH); λ _ex = 537 nm, λ _em = 552nm δ _H (300MHz, CD ₃ OD) 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 (1H, d) Mass ^{spectrometry: (ES-) 487 (M -} ).

2- (4-sulfonatophenyl) amino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate, sodium salt [5e This was synthesized and purified in a similar manner to [5d], using sulfanilic acid (60 mg, 0.35 mmol) instead of metanilic acid. Yield = 120 mg (
78%) λ _max (MeOH) = 540 nm A 1.0 mM solution in DMF was prepared for measurement in aqueous solution; this was diluted 1: 100 with 0.02 Mol bis-trispropane / HCl aqueous buffer. Thus, a working solution of 10 μMol was obtained. λ _max (MeOH) = 540 nm

2- (3-hydroxyphenyl) amino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate, [5f] , [3a], using 3-aminophenol (39 mg, 0.35 mmol) instead of piperidine. Upon cooling, the product precipitated from the reaction; it was collected, washed with 1-butanol and diethyl ether, and then dried under reduced pressure. The title compound was isolated in a yield of 112 mg (88%). (pH 7 aqueous solution) = 530 nm, ε _max ≒ 82,000 dm ³ mol ^-1 cm ^-1 (pH 10 aqueous solution) = 468 nm, ε _max ≒ 53,000 dm ³ mol ^-1 cm ^-1 fluorescence (MeOH); λ _ex = 540 nm, λ _em = 555nm δ _H (300MHz, CD ₃ OD) 1.450 (3H, t), 1.967 (6H, s), 4.31 (2H, q), 6.046 (1
H, s), 7.48 (1H, t), 7.60-7.66 (2H, m), 7.78-7.86 (4H, m), 7.97 (2H, distinct t) and 8.24 (1H, d). ES-) 487 (M ^-).

2-((4-carboxymethyl) phenyl) amino-4- [1- (4-sulfonatobutyl) -3,3-dimethyl-2-benzindolinylidenemethyl] cyclobutenediylium-1, 3-diolate, sodium salt [5 g] 3-hydroxy-4- (1- (4-sulfonatebutyl) -3,3-dimethyl-2-benzoindolinylidenemethyl) -cyclobut-3-ene-1,2- Dione [1m] (116 mg, 0.25 mmol) and 4-aminophenylacetic acid (40
mg, 0.26 mmol) in acetic acid (3 ml) and heated at 100 ° C. for 1 hour. The reaction was monitored by tlc (RPC _18, methanol, 60: water, 40.
1 m], Rf = 0.65, yellow spot; product Rf = 0.38, purple-red spot). The mixture was cooled to 10 ° C. where the product precipitated; the solid was collected, washed with cold acetic acid, then with diethyl ether and dried in vacuo. Yield of the title compound = 67 mg (46%). λ _max (MeOH) = 538 nm, (pH 7 aqueous buffer) = 524 nm. Fluorescence (MeOH); λ _ex = 541 nm, λ _em = 558 nm. δ _H (300 MHz, DMSO-d6) 1.72-1.90 (4H, m) , 1.916 (6H, s), 3.5 (m, partially hidden by HOD), 3.553 (2H, s), 4.15 (2H, broad s), 5.756 (1H,
s), 7.26 (2H, d), 7.42 (1H, t), 7.59 (1H, t), 7.71 (1H, d), 7.83 (2H, d)
, 8.00 (2H, d), 8.21 (1H, d), 11.49 (s, partial replacement) and 12.3 (broad
s, partial exchange) Mass ^{spectrometry: (ES-) 573 (M -} ).

A sample of this material was converted to a highly reactive NHS ester. [5g] (30m
g, 50 μmol) in the presence of N, N-diisopropylethylamine (9 μl)
Reaction with TSTU (15 mg, 50 μmol) in DMF (200 μl).
The reaction was monitored by tlc (RPC _18, methanol, 60: water, 40. [5 g],
Rf = 0.4; NHS ester [5h] Rf = 0.32). The NHS ester precipitated from solution upon dilution with diethyl ether; the solid was isolated, washed with a methanol: ether mixture, then with ether and dried under reduced pressure. This material binds successfully to 1-butylamine, proving that this material can be used as a labeling molecule with a primary amino group.

2-butylamino-4- [1- (4-sulfonatobutyl) -3,3-dimethyl-2-benzindolinylidenemethyl] cyclobutenediylium-1,3-diolate, sodium salt [5i This was synthesized in a similar manner to [5g], using 1-butylamine (30 μl, 0.3 mmol) instead of 4-aminophenylacetic acid. The reaction was followed by tlc (RPC ₁₈ ; methanol, 60: water, 40. [1 m], Rf = 0.65;
[5i] Rf = 0.24,). After 5.5 hours, the solvent was evaporated under reduced pressure;
Purified by preparative HPLC (RPC _18, water → methanol gradient). Final yield of the title compound was 28 mg (22%). λ _max (MeOH) = 486 nm, (pH 7 aqueous buffer) = 480 nm. (MeOH + Bu ₄ ⁺ OH ⁻ ) = 430 nm, fluorescence (MeOH); λ _ex = 487 nm, λ _em = 512 nm. δ _H (300 MHz, CD ₃ OD) 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 (1H, s), 7.368 (1H, t), 7.55 (2H, m), 7.91 (2H, clear d) and
. 8.16 (1H, d) Mass ^{spectrometry: (ES-) 495 (M -} ).

2- (8-hydroxy-6-sulfonato-2-naphthyl) amino-4- (1-
Ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate, sodium salt [5j] This is synthesized in an analogous manner to [5d] and replaces metanilic acid. 6-amino-4-hydroxy-2-naphthalenesulfonic acid monohydrate (90 mg, 0.35 m
mol). Yield = 61 mg (35%) λ _max (MeOH) = 552 nm (pH 7 aqueous solution) = 542 + 510 nm, (pH 10 aqueous solution) = 472 nm Fluorescence (MeOH); λ _ex = 552 nm, λ _em = 566 nm. Δ _H (300 MHz, CD ₃ OD) 1.445 (3H, t), 1.977 (6H, s), 4.29 (2H, q), 6.024 (1
H, s), 7.26 (1H, d), 7.46 (1H, m), 7.55 (1H, d), 7.62 (1H, m), 7.82 (1H,
. s), 7.91-8.00 (3H, m), 8.11 (1H, dd), 8.23 (1H, d) and 8.37 (1H, d) Mass ^{spectrometry: (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 synthesized and purified in an analogous manner to [5d], replacing 5-methy-2-naphthalenesulfonic acid monohydrate (78 mg, 0.35 mmol) in place of metanilic acid.
) Was used. Yield = 113 mg (67%) λ _max (MeOH) = 524 nm (pH 7 aqueous solution) = 512 nm, (pH 10 aqueous solution) = 458 nm fluorescence (MeOH); λ _ex = 530 nm, λ _em = 566 nm. Δ _H (300 MHz, CD ₃ OD) ) 1.445 (3H, t), 1.973 (6H, s), 4.30 (2H, q), 6.044 (1
H, s), 7.47 (1H, t), 7.56-7.68 (3H, m), 7.90 (2H, clear t), 7.97-8.02 (
. 3H, m), 8.22-8.29 ( 2H, m) and 8.40 (1H, d) Mass ^{spectrometry: (ES-) 537 (M -} ).

2-N-((4-carboxymethyl) phenyl) amino-4- (1-ethyl-3
, 3-Dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [51] This was synthesized in a similar manner to [3a], and instead of piperidine, 4-aminophenylacetic acid ( (50 mg, 0.33 mmol). Acetic acid was used as an alternative solvent. After heating, the mixture was cooled and the title dye [3l] was isolated by filtration and washed with a small amount of cold acetic acid followed by diethyl ether to give the product as a red solid. Yield _{= 116mg (83%) λ max} (MeOH) = 536nm. Δ H (300MHz, DMSO) 1.31 (3H, t), 1.90 (6H, s), 3.55 (2H, s), 4.18 (2H, q
), 5.76 (1H, s), 7.25 (2H, d), 7.40 (1H, t), 7.56 (1H, t), 7.65 (1H, d),
7.81 (2H, d), 8.0. (2H, app d) and 8.20 (1H, d). Mass spectrometry: (ES +) 467 (M + H).

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Example 6 Synthesis of Acylated Squarate Dye Samples Any of the dyes in Example 5 can undergo acylation at a nitrogen atom by an acyl halide reagent, as shown in the following example:

2- (N-butylacetamide) -4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [
6a] 2-butylamino-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [5b] (1
9.4 mg, 50 μmol) was dissolved in dry pyridine (0.5 ml) to give a yellow solution. To this was added acetyl chloride (1 drop); it immediately turned red. After stirring at room temperature for 30 minutes, the solvent was evaporated under reduced pressure; the residue was azeotroped twice with acetonitrile and then purified by preparative tlc (silica; methanol, 5: dichloromethane, 95). The orange-red product band was collected, the dye was extracted with an eluent, and the resulting filtrate was evaporated under reduced pressure. The residue was triturated with a gas oil spirit: ether 4: 1 mixture to give a solid; it was dried under reduced pressure to give 16 mg (74%) of the product dye. λ _max (CH ₂ Cl ₂ ) = 536 nm; (MeOH) = 506 nm δ _H (300 MHz, CD ₂ Cl ₂ ) 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
(1H, s), 7.48-7.57 (2H, m), 7.66 (1H, m), 7.98-8.03 (2H, m) and 8.24 (1
H, d). Mass spec: (ES +) 431 (M + H), 453 (M + Na), 469 (M + K).

2- (N-phenylacetamide) -4- (1-ethyl-3,3-dimethyl-2-
[Benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [6b] This was synthesized and purified in an analogous manner to [6a] to give 2-phenylamino-
4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [5a] (20.4 mg, 50 μm
ol) and acetyl chloride (1 drop). The title compound was obtained in a yield of 6 mg (
27%). λ _max (CH ₂ Cl ₂ ) = 538 nm; (MeOH) = 502 nm δ _H (300 MHz, CD ₂ Cl ₂ ) 1.53 (3H, t), 1.979 (6H, s), 2.617 (3H, s), 4.41 (2
H, q), 6.333 (1H, s), 7.28 (2H, m), 7.36-7.58 (5H, m), 7.66 (1H, t), 8.0
1 (2H, m) and 8.19 (1H, d).

2-Benzoylamido-4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [6c] This reduces [6a] slightly. Synthesized and purified in an altered manner to give 2-amino-4-
(1-Ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutene-diylium-1,3-diolate [5c] (17 mg, 50 μmol)
And benzoyl chloride (20% v / v solution in pyridine). The acid halide solution was added dropwise and the reaction was monitored by tlc (silica, methanol, 5
: Dichloromethane, 95. [5c], Rf = 0.16, yellow spot; product R
f = 0.3, red spot). Isolated yield 12 mg (55%). λ _max (CH ₂ Cl ₂ ) = 534 nm; (MeOH) = 502 nm δ _H (300 MHz, CD ₂ Cl ₂ ) 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 (1H, d). Mass spectrometry: (ES +) 437 (M + H), 459 (M + Na), 475 (M + K).

2- (dibenzoylamide) -4- (1-ethyl-3,3-dimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,3-diolate [6
d] It was synthesized and purified in a manner analogous 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%). λ _max (CH ₂ Cl ₂ ) = 528 nm; (MeOH) = 486 nm δ _H (300 MHz, CD ₂ Cl ₂ ) 1.446 (3H, t), 1.848 (6H, s), 4.37 (2H, q), 6.273 (
1H, s), 7.33-7.62 (10H, m), 7.79-7.82 (3H, m), 7.95 (2H, m) and 8.12 (1H
, d). Mass spectrometry: (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] which is similar to [6c] Synthesized and purified by the method, using acetyl chloride instead of benzoyl chloride. Isolated yield = 13 mg (70%). λ _max (CH ₂ Cl ₂ ) = 522 + 498 nm; (MeOH) = 486 nm δ _H (300 MHz, CD ₂ Cl ₂ ) 1.408 (3H, t), 2.026 (6H, s), 2.543 (3H, s), 4.37 (
2H, q), 6.227 (1H, s), 7.47-7.57 (2H, m), 7.63-7.69 (1H, m), 7.98-8.03 (
2H, m), 8.24 (1H, d) and 8.8 (1H, broad s). Mass spec: (ES +) 375 (M + H), 397 (M + Na), 413 (M + K).

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Example 7 Synthesis of a squarate dye sample having a benzothiazole nucleus

3-Methoxy-4- (3-ethyl-2-benzothiazolylidenemethyl) -cyclobut-3-ene-1,2-dione [7a] In a 50 ml reaction flask, dimethyl squarate (1.42 g) was added. , 10mmo
l), dried methanol (10 ml) and N, N-diisopropylethylamine (1.29 g, 10 mmol) were added. The mixture was heated and boiled until all solids dissolved.

The heated solution was added to 3-ethyl-2-methylbenzothiazolium iodide (3.0
(5 g, 10 mmol) was added and the color immediately turned orange-red as the solid dissolved, after which an orange solid began to precipitate. Heating was continued for a further 10 minutes, then the mixture was allowed to cool to room temperature before final cooling in the refrigerator. The precipitated solid was collected by filtration, washed with ice-cooled methanol, then with diethyl ether, and dried under reduced pressure to give 2.35 g (82%). λ _max (MeOH) = 440 nm δ _H (300 MHz, CDCl ₃ ) 1.372 (3H, t), 4.04 (2H, q), 4.451 (3H, s), 5.445 (1
H, s), 7.06 (1H, d), 7.15 (1H, clear t), 7.33 (clear t) and 7.48 (1H,
dd).

3-Hydroxy-4- (3-ethyl-2-benzothiazolylidenemethyl) -cyclobut-3-ene-1,2-dione [7b] This is described in the literature (E. Terpetschnig & JR Lacowicz; Dyes). and Pigments (1
993), 21, 227-234). 1.44 g of [7
In a), the yield of the crude product = 1.24 g was obtained. 500 mg of this material was further purified by flash chromatography (silica; dichloromethane → methanol gradient). 258 mg of pure [7b] were isolated, and 30 mg of the pink impurity described above, which was identified as a known compound, the 1,2-bis-adduct [7c]. (VHE Sprenger & W. Ziegenbein; Angew. Chem.,
(1967), 79, 581-582) [7b] data λ _max (MeOH) = 446 nm δ _H (300 MHz, CD ₃ OD) 1.312 (3H, t), 4.05 (2H, q), 7.05 (1H, t) ), 7.15 (1H,
d), 2.27 (1H, ~ t) and 7.46 (1H, d). Although the methine proton of squaric acid is not clear, it can be exchanged under acidic conditions. Mass spectrometry: (ES +) 274 (M + H), 296 (M + Na). Data of [7c] λ _max (MeOH) = 524 nm δ _H (300 MHz, DMSO-d6) 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 spec: (ES +) 433 (M + H).

2- (1-morpholino) -4- (3-ethyl-2-benzothiazolylidenemethyl) cyclobutenediyl-1,3-diolate [7d] 3-hydroxy-4- (3-ethyl- 2-benzothiazolylidenemethyl) -cyclobut-3-ene-1,2-dione [7b] (82 mg, 0.3 mmol) and morpholine (35 μl = 35 mg, 0.4 mmol) were added to 1-butanol (3 m
1) and heated at 100 ° C. for 2.5 hours. The reaction was followed by tlc (R
PC ₁₈ . Methanol, 95: water, 5. [7b], Rf = 0.8; [7d], Rf
= 0.4). Then the solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (silica; 2-4% methanol / dichloromethane) to give 94 mg (92%) of the title compound. λ _max (MeOH) = 494 nm; ε _max = 114,000 dm ³ mol ^-1 cm ^-1 . Fluorescence (MeOH); λ _ex = 492 nm, λ _em = 518 nm. δ _H (300 MHz, CDCl ₃ ) 1.368 (3H, t) , 3.81-3.85 (4H, m), 4.00- 4.11 (6H, m)
, 5.72 (1H, s), 7.07 (1H, d), 7.13 (1H, t), 7.32 (1H, t) and 7.47 (1H, d
). Mass spectrometry: (ES +) 343 (M + H).

2- (N-methylanilino) -4- (3-ethyl-2-benzothiazolylidenemethyl) cyclobutenediylium-1,3-diolate [7e] This is synthesized in a similar manner to [7d]. N-methylaniline (44 μl = 43 mg, 0.40 mmol) was used instead of piperidine. Title dye [7e
] Were isolated in the same manner. Yield = 43 mg (40%). λ _max (MeOH) = 514 nm; ε _max = 105,000 dm ³ mol ^-1 cm ^-1. Fluorescence (MeOH); λ _ex = 518 nm, λ _em = 565 nm. δ _H (300 MHz, CDCl ₃ ) 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 (1H, d). Mass spectrometry: (ES +) 363 (M + H), 385 (M + Na).

2- (4-sulfonaphthophenyl) amino-4- (3-ethyl-2-benzothiazolylidenemethyl) -cyclobutenediylium-1,3-diolate, sodium salt [7f] 3-hydroxy- 4- (3-ethyl-2-benzothiazolylidenemethyl) -cyclobut-3-ene-1,2-dione [7b] (82 mg, 0.30 mmol),
Sulfaninic acid (60 mg, 0.35 mmol) and anhydrous sodium acetate (3
0 mg, 0.37 mmol) were mixed in acetic acid (1.5 ml). The mixture was heated at 100 ° C. for 2 hours, whereupon the color became red and a solid precipitated. After cooling, the solid was collected by filtration, washed with acetic acid (3 × 2 ml) and then with diethyl ether,
Dried. Yield = 102 mg (75%). λ _max (MeOH 50: H ₂ O 50) = 518 nm; (+ Bu ₄ ⁺ OH ⁻ ) = 475 nm. Fluorescence (MeOH); λ _ex = 530 nm, λ _em = 552 nm. δ _H (300 MHz, DMSO-d6) 1.388 (3H, t), 4.45 (2H, q), 5.998 (1H, s), 7.21 (1
. H, d), 7.40 ( 1H, t), 7.55-7.83 (5H, m) and 8.04 (1H, d) Mass ^{spectrometry: (ES-) 427 (M -} )

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Example 8 Synthesis of squarate dye sample having 2-quinoline nucleus 3-methoxy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobute-3
-Ene-l, 2-dione [8a] This is synthesized in a similar manner to [7a], except that 3-ethyl-2-methylbenzothiazolium iodide is replaced by 1-ethyl-2-methylquini iodide. Linium (900
mg, 3 mmol). Workup required evaporation of the solvent and separation with chloroform and dilute hydrochloric acid. The organic layer was collected, washed with water, dried (MgSO _4). After filtration and concentration, flash chromatography (silica. 2.5-3%
(Methanol / chloroform). The yield of the title compound was 570 mg (6
8%). λ _max (MeOH) = 462 nm δ _H (300 MHz, DMSO-d6) 1.309 (3H, t), 4.26 (2H, broad q), 4.383 (3H, s
), 5.245 (1H, s), 7.295 (1H, t), 7.57-7.70 (4H, m) and 8.37 (1H, d). Mass spectrometry: (ES +) 282 (M + H), 304 (M + Na).

3-Hydroxy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobute-
3-ene-1,2-dione [8b] This was synthesized in an analogous manner to [1b], and was prepared in a dilute hydrochloric acid / acetic acid mixture.
Xy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobut-3-ene-
Requires hydrolysis of 1,2-dione [8a]. The reaction was followed by t.l.c. (RPC ₁₈ . Methanol, 95: water, 5. Triethyl on sample dissolved in methanol
Luamine was added. [8a], Rf = 0.5, yellow spot; [8b] Rf = 0
. 7, orange spot). In acidic solutions, the product color is lost reversibly
, Probably due to the protonation of the heteroaromatic ring.

The dried reaction mixture was used for the synthesis of the dye without further purification.
Mass spec: (ES +) 268 (M + H), 290 (M + Na), 306 (M + K).

2- (1-morpholino) -4- (1-ethyl-2-quinolylidenemethyl) -cyclobutenediyl-1,3-diolate [8c] This is synthesized in an analogous manner to [7d]. And purified in 3-butanol
Hydroxy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobut-3-
Ene-1,2-dione [8b] (80 mg, 0.30 mmol) and morpholine (35 μl = 35 mg, 0.4 mmol) were used. .. tlc _{(RPC 18} methanol, 95: water, 5 [8b], Rf = 0.7, orange spot; [8d
Rf = 0.4, purple-red spot). Yield = 74 mg (73%) λ _max (MeOH) = 516 nm; ε _max = 65,000 dm ³ mol ^-1 cm ^-1 . Δ _H (300 MHz, DMSO-d6) 1.313 (3H, t), 3.74-3.80 (4H, m), 3.88-3.95 (4H, m
), 4.22 (4H, broad q), 5.411 (1H, s), 7.27 (1H, t), 7.54-7.66 (4H, m)
and 8.91 (1H, d). Mass spec: (ES +) 377 (M + H), 359 (M + Na).

2- (N-methylanilino) -4- (1-ethyl-2-quinolylidenemethyl) cyclobutenediyl-1,3-diolate [8d] This was synthesized and synthesized in an analogous manner to [7d]. Purified, 3-butanol in 1-butanol
Hydroxy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobut-3-
Ene-1,2-dione [8b] (80 mg, 0.30 mmol) and N-methylaniline (44 μl = 43 mg, 0.4 mmol) were used. tlc (.. _{RPC 1 8} methanol, 95: water, 5 [8b], Rf = 0.7, orange spot; [8d] Rf = 0.4, dark pink spot). Yield = 85 mg (79%
) Λ _max (MeOH) = 544 nm; ε _max = 67,000 dm ³ mol ^-1 cm ^-1 . Δ _H (300 MHz, DMSO-d6) 1.376 (3H, t), 3.836 (3H, s), 4.39 (2H , Broad
q), 5.669 (1H, s), 7.21 (1H, m), 7.36-7.44 (5H, m), 7.66-7.76 (2H, m), 7
.81 (2H, clear d) and 9.14 (1H, d). MS: (ES +) 357 (M + H), 379 (M + Na).

2- (4-sulfonaphthophenyl) amino-4- (1-ethyl-2-quinolylidenemethyl) -cyclobutenediyl-1,3-diolate, sodium salt [8
e] 3-Hydroxy-4- (1-ethyl-2-quinolylidenemethyl) -cyclobut-3-ene-1,2-dione [8b] (81 mg, 0.3 mmol), sulfanic acid (60 mg, 0.1 mg). 35 mmol) and anhydrous sodium acetate (30 mg, 0
. 37 mmol) were mixed in acetic acid (1.5 ml). The mixture was heated at 100 ° C. for 2 hours, whereupon the color became red and a solid precipitated. After cooling, the solid was collected by filtration, washed with acetic acid (3 × 5 ml), then with diethyl ether and dried.
Yield = 135 mg (~ 100%). λ _max (MeOH 50: H ₂ O 50) = 540 nm; (+ Bu ₄ ⁺ OH ⁻ ) = 514 nm. δ _H (300 MHz, DMSO-d6) 1.403 (3H, t), 4.45 (2H, broad q), 5.754 (1H, s
), 7.40-7.52 (3H, m), 7.65-7.79 (4H, m), 7.85-7.95 (2H, m) and 9.22 (1H, m
. d) mass ^{spectrometry: (ES-) 421 (M -} ).

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Example 9 Synthesis of squarate dye having 4-quinoline nucleus 3-methoxy-4- (1-ethyl-4-quinolylidenemethyl) -cyclobute-3
-Ene-1,2-dione [9a] This was synthesized and purified in an analogous manner to [8a] to give 1-ethyl-2-iodide
Instead of methylquinolinium, 1-ethyl-4-methylquinolinium iodide (9
00 mg, 3 mmol). Yield = 374 mg (44%). λ _max (MeOH) = 528 + 498 nm δ _H (300 MHz, DMSO-d6) 1.320 (3H, t), 4.24 (2H, q), 4.381 (3H, s), 5.938
(1H, s), 7.38 (1H, m), 7.66-7.80 (4H, m) and 8.18 (1H, d). Mass spectrometry: (ES +) 282 (M + H), 304 (M + Na), 320 (M + K).

3-Hydroxy-4- (1-ethyl-4-quinolylidenemethyl) -cyclobute-
3-ene-1,2-dione [9b] This was synthesized in an analogous manner to [1b] and was prepared in a dilute hydrochloric acid / acetic acid mixture using 3-methoxy-4- (1-ethyl-4-quinolylidenemethyl). ) -Cyclobut-3-ene-
Requires hydrolysis of 1,2-dione [9a] (281 mg, 1 mmol). The reaction was monitored by tlc (RPC _18, methanol, 95: water, 5. Triethylamine was added to the sample dissolved in methanol. [9a], Rf = 0.5, pink spot; [9b] Rf = 0.65, red-pink spot). In acidic solutions, both starting and product colors are reversibly lost, probably due to protonation of the heteroaromatic ring. The dried reaction mixture was used for the synthesis of the dye without further purification.

2- (1-Piperidino) -4- (1-ethyl-4-quinolylidenemethyl) -cyclobutenediyl-1,3-diolate [9c] This is synthesized in an analogous manner to [3a]. And purified, 3-hydroxy-4- (
1-Ethyl-4-quinolilidenemethyl) -cyclobut-3-ene-1,2-dione [9b] (80 mg, 0.3 mmol) was used. The reaction was followed by tlc (R
PC ₁₈ . Methanol, 95: water, 5. [9b], Rf = 0.65, red-pink spot; [9c] Rf = 0.4, dark purple spot). λ _max (MeOH) = 590 + 554 nm δ _H (300 MHz, CD ₃ OD) 1.435 (3H, t), 1.76 (6H, broad s), 3.94 (4H, broad s), 4.27 (2H, q), 6.217 ( 1H, s), 7.40 (1H, m), 7.53 (1H, d), 7.63-7
.68 (2H, m), 8.17 (1H, d) and 8.26 (1H, d). Mass spectrometry: (ES +) 335 (M + H).

2- (N-methylanilino) -4- (1-ethyl-2-quinolilidenemethyl) cyclobutenediylium-1,3-diolate [9d] This is synthesized in a similar manner as [7d]. 3-hydroxy-4- (1-ethyl-4-quinolylidenemethyl) -cyclobut-3-ene-1, in 1-butanol;
2-dione [9b] (80 mg, 0.30 mmol) and N-methylaniline (
44 μl = 43 mg, 0.4 mmol) was used. tlc _{(RPC 18} methanol, 95:.. water, 5 [9b], Rf = 0.65, red - pink spot; [9
d] Rf = 0.4, dark blue spot). Yield = 73 mg (68%). λ _max (MeOH) = 602 nm δ _H (300 MHz, CDCl ₃ ) 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 (1H, m), 8.27 (1H, d) and 8.67 (1H, d). Mass spectrometry: (ES +) 357 (M + H), 379 (M + Na). .

Embedded image 0

Example 10 Labeling of Biomolecules Dye 2- (4-((2-maleimidoethyl) aminocarbonyl) piperidino)-
E. Labeling of protein lysates with 4- (1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate [3j] E. coli lysate was split and 9 μl lysate buffer (8 M urea, 4% (w
/ V) adjusted to have 25 μg of protein in the final volume of CHAPS, 40 mM Tris (pH 8). To this was added 10 mM TCEP (1 μl aqueous solution) and the mixture was kept at 37 ° C. for 1 hour. Next, the obtained reduced protein was mixed with the dye 3j (2 μl of a 10 nmol / 1 μl DMF solution) in DM
Labeled by adding F solution. Labeling is achieved by incubation at 37 ° C. for 30 minutes. Then, sample buffer (8 M urea, 4% (w / v) CHAPS, 20
The reaction was stopped by the addition of 12 μl of a buffer consisting of mg / ml DTT, 4% IPG buffer) and the protein was subjected to 2D gel analysis, ie, IEF page followed by SDS page. The resulting gel was indicated by the dye-labeled protein, so that the location of the protein on the gel could be seen with a fluorescence scanner.

The nucleotide dye conjugate 5- (3- (2- (4-amidopiperidino) -4- (1
Synthesis of 3,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolato) -allyl) -2'-deoxyuridine-5'-triphosphate 5- (3-aminoallyl) -2'-Deoxyuridine-5'-triphosphate (1 mg) was dissolved in 200 µl of a pH 9.5 carbonate buffer. To the mixture was added 2- (4-carboxypiperidino)-in 100 μl of acetonitrile.
4- (1,3,3-Trimethyl-2-benzindolinylidenemethyl) cyclobutenediyl-1,3-diolate NHS ester [3h] was added. The solution was stirred at room temperature, initially cloudy, but became clear with further stirring. After stirring for 5 hours, the reaction mixture was subjected to a reverse layer prep. Plate (1: 1 acetonitrile: 0.1 M TEAB (triethylammonium bicarbonate buffer)).
Purified above. The product portion was scraped, extracted with the same eluent system, filtered, evaporated, dissolved in 50 acetonitrile: water and filtered through a cotton wool plug. The solution was evaporated and the reverse layer TLC after purification (1: 1 CH ₃ CN: 0.1 M TEA)
B) showed that the product (Rf = 0.22) flows more than the starting material (0.05) and the carboxy derivative (0.14). Mass spectrometry: (ES +) 787 (MP 2 O 6), 865 (M-PO 3), 949 (M + H), 1046 (M + Et 3 NH).

2- (4-O-N-succinimidylcarbonylpiperidino) -4- (5-sulfonate-1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenedii Lium-1,3-diolate (10a) 2- (4-carboxypiperidino) -4- (5sulfonate-1,3,3-trimethyl-2-benzindolinylidenemethyl) cyclobutenediylium-1,
3-diolate [4b] (15 mg, 0.027 mmol) was added to O- (N-) in N, N-diisopropylethylamine (5 μl) in DMF (0.5 ml).
Succinimidyl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TSTU, 8.3 mg, 30 μmol). This reaction is tl
c. _{(RPC 18} methanol, 40:... water, 60 [4b], Rf = 0.48 product Rf = 0.20); and was complete after one hour. Upon addition of diethyl ether to the mixture, the product precipitated. The solvent was decanted and the product was further triturated twice with ether to give 16 mg (89%) of the NHS derivative as an orange solid (10%
a) was obtained. δ _H (300MHz, CD ₃ OD) 1.362 (3H, t), 1.90-1.95 (8H, m), 2.13- 2.19 (2H, m)
, 2.73 (1H, m), 2.82 (4H, s), 3.55-3.65 (2H, m), 4.20 (2H, broad q),
4.68 (2H, m), 5.85 (1H, s), 7.60 (1H, d), 7.93 (1H, dd), 8.01 (1H, d), 8
.30 (1H, d) and 8.46 (1H, d). MS: (ES +) 644 (M + H), 666 (M + Na).

The NHS ester derivative was then used to label a portion of a cDNA with a modified nucleotide having five aminoallyl groups incorporated into uridine. Eight labeling reactions were set up, each two consisting of duplicates of the following and an alternative Cy3 dye as a control. 1) Squaraine dye 10a labeling in a cDNA reaction containing reverse transcriptase (RT) (test) 2) Squaraine dye 10a labeling in a cDNA reaction without reverse transcriptase (negative control) 3) Including reverse transcriptase Cy3 labeling in cDNA reaction (positive control)
4) Cy3 labeling in cDNA reaction without reverse transcriptase (negative control)

Method Labeling of cDNA with dye 10a was performed as follows. Eight cDNA reactions were set up each consisting of 2 μg of human skeletal muscle mRNA, 2 μg of random primer (No Name, Amersham Pharmacia RPK0158) and 12 μg of water. 14 μg of water was added so that the reverse transcriptase did not react. The reaction solution is
Heated for 0 minutes, then changed to room temperature for 15 minutes. Add 8 μl of 5x First Stand buffer (Promega M531A), add 4 μl 0.1 M DTT, 2 μl nucleotide mix (4 mM dATP, dCTP, dGTP), 8 μl 1 mM aminoallyl-dUTP (Sigma A0410) and 2 μl reverse transcription Enzyme (Promega M368
2) was added to the relevant tube. The reaction was kept at 42 ° C. for 3 hours. To remove the mRNA, 2 μl of 5 M sodium hydroxide was added and the tube was incubated at 42 ° C. for 10 minutes. Sodium hydroxide was neutralized by adding 20 μl of 2M HEPES. Free aminoallyl-dUTP is available on a Microcon 30 column (millipo
re cat no 42410) and removed by adding 450 μl of water.
The tube was spun at 13 krpm for 8 minutes on a microfuge (MicroCentaur, MSE). 450 μl was added to the tube again and spun for 8 minutes as described above. 450 μl
Of water again and the tube was rotated as before. 13k of purified cDNA
Eluted into a new tube by spinning for 1 minute at rpm. cDNA is Speedvac
Dried in. The cDNA was resuspended in 4.5 μl of water. 3.2 × 10 ⁻⁷ moles (０．２0.25 mgs) of squalamide NHS ester and Cy3-NHS
The esters (Amersham Pharmacia PA23001) were each resuspended in 72 μl of a 0.1 M sodium carbonate solution pH9. Next, 4.5 μl of the appropriate dye solution was added to the cDNA solution. The reaction was kept at room temperature in the dark for 1.5 hours. Next,
Add 4.5 μl of 4M hydroxyamine (Sigma H2391), keep warm, add 1 more
The incubation was continued for 0 minutes. The next step is to remove the free dye from the dye incorporated into the cDNA. Next, 500 μl of buffer PB (Qiagen, Qiaquick kit)
cat28106), and the probe was placed in a Qiaquick column (Qiagen cat28106). The tube was spun for 1 minute at 13 krpm in a MicroCentaur column. 500μ
One liter of buffer PE (Qiagen cat28106) was added for washing the tube and the spinning was repeated. The washing and spinning procedure was repeated two more times. An additional 1 minute spin was performed before elution with 100 μl elution buffer (Qiagen cat28106). Next, the dye incorporated into the probe was measured using Cary UV / visible spectrophotometry.

Results

[Table 2]

By comparing the absorbance readings of the dyes 10a and Cy3 to which the reverse transcriptase labeling reaction was added with those obtained without the reverse transcriptase reaction, the dye 10a labeled the cDNA molecule. Check. The results without reverse transcriptase confirm that unincorporated dye is effectively removed in the manner used in the scheme.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] May 11, 2001 (2001.11.11)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

Embedded image (Wherein m is 1, 2 or 3), and W is a structure

Embedded image Wherein L ′ is a linker consisting of 0-3 moieties selected from a carbon atom or an arylene group, and the dotted line is a single or fused ring, aromatic or heterocyclic, unsubstituted or substituted, Containing or linking a tertiary or quaternary nitrogen atom;
Wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ are each H or a C ₁ -C ₂₀ carbon atom or group- L ⁹ is a negative charge or a group -LG, L is a linking chain of 0 to 60, one or more arylene groups, or O
Or branched or unbranched, optionally containing an N or N ⁺ or S or S ⁺ or P or P ⁺ or Se atom, and G, whereby the compound is a biological molecule, other small molecule such as A functional group or a reactive group capable of covalently bonding to a dye or a group that increases or decreases water solubility, and provides an electron donating or attracting property that changes the spectral characteristics of the compound. Homodimers and oligomers and heterodimers and oligomers.

Embedded image , And the each of ^{R 3} and ^{R 4} is H or _C 1 _{-C 20} or a group -L-G, and n is 0-3 The compound according to claim 1, wherein.

Embedded image Wherein, X is O or S or -NR ^4, or ^-CR 10 ^{R 11,} each ^{R 1 0} and ^{R 11} are _C 1 _{-C 20} hydrocarbon or a -L-G or ^{R 10} and ^{R 11,} Form a monocyclic or condensed ring aromatic, heterocyclic or cycloaliphatic).] The compound according to claim 1 or 2, wherein

Embedded image The compound according to claim 1, which is a reactive group selected from the group consisting of:

Embedded image 5 wherein A has at least one nitrogen atom and a is 2 to 6 aromatic or alicyclic groups. 6. The dimers and oligomers of the compounds according to claim 1, wherein

Embedded image The compound according to any one of claims 1 to 6, wherein

Embedded image Ii) optionally subjecting intermediate a) to hydrolysis to give intermediate b)

Embedded image Iii) Reaction of intermediate a) or intermediate b) with R ¹ R ² NH gives the final product c)

Embedded image give.

Embedded image An analytical or labeling method comprising observing dye formation by reaction with

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) C07D 277/64 C07D 277/64 4H045 401/08 401/08 4H056 401/14 401/14 413/08 413 / 08 C07H 19/10 C07H 19/10 C09B 26/02 C09B 26/02 Z C09K 11/06 C09K 11/06 G01N 33/58 G01N 33/58 A // C07K 2/00 C07K 2/00 G01N 33/68 G01N 33/68 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, (72) invention PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW William Jonathan Cummins UK, H.23.4 Jay, Har Fordshire, Tring, Thorne Tree Drive No. 5 (72) Inventor Robert James Domet Nairn United Kingdom, Ayle 4.9 Youen, Hartfordshire, St Albans, Springwood Walk No. 16 (72) Inventor Matthew Graham Bull UK, 16.0 ARB, Buckinghamshire, Great Missenden, South Heath, Wood Lane, Wick Cottage F term (reference) 2G045 DA13 DA36 FB12 4C031 BA07 4C057 BB02 BB05 DD03 LL11 LL19 LL22 4C063 AA01 BB05 BB07 CC06 CC59 DD03 DD06 EE10 4C204 BB05 CB03 CB13 DB16 EB03 FB03 GB01 4H045 AA10 BA70 EA50 4H056 CA01 CA02 CA05 CB06 CC02 CC08 CE01 CE03 CE06 DD03 FA05 FA08 .

Claims (11)

[Claims] 1. The structure W = Sq-A, wherein A has the formula —NR ¹ R ² , wherein R ¹ and R ² are the same or different, each being H or a C ₁ -C ₂₀ hydrocarbon. Or ^one of R ¹ and R ² , or ^one of R ¹ and R ² is —OR ⁵ or —NR ⁶ R ⁷ or —COR ⁷ or —NR ⁶ COR ⁷ or —N−R ⁸ or —C (O) OR ⁷ , or R ¹ and R ² together form a saturated or unsaturated, unsubstituted or substituted monocyclic or fused ring, or an azacrown or metal binding group, Sq is 1) Wherein m is 1, 2 or 3; and W is the structure Wherein L ′ is a linker consisting of 0-3 moieties selected from a carbon atom or an arylene group, and the dotted line is a single or fused ring, aromatic or heterocyclic, unsubstituted or substituted, Containing or linking a tertiary or quaternary nitrogen atom;
Wherein R ⁵ , R ⁶ , R ⁷ and R ⁸ are each H or C ₁ -C ₂₀ carbon atoms or R ⁹ is a negative charge or a group -LG, L is a linking chain of 0 to 60, one or more arylene groups, or O
Or branched or unbranched optionally containing N or N ⁺ or S or S ⁺ or P or P ⁺ or Se atoms; and G is a functional or reactive group, whereby the compound is molecule,
And other small molecules such as, for example, dyes, or which can be covalently bonded to groups that increase or decrease water solubility or provide an electron donating or attracting property that alters the spectral properties of the compound; and Homodimers and oligomers and heterodimers and oligomers of the compounds. (2) W is , And the each of ^{R 3} and ^{R 4} is H or _C 1 _{-C 20} or a group -L-G, and n is 0-3 The compound according to claim 1, wherein. 3. The dotted line is Wherein, X is O or S or -NR ^4, or ^-CR 10 ^{R 11,} each ^{R 1 0} and ^{R 11} are _C 1 _{-C 20} hydrocarbon or -L-G or ^{R 10} and ^{R 11,} Together form a monocyclic or condensed ring aromatic, heterocyclic or cycloaliphatic]. 4. The compound according to claim 1, wherein at least one group G which is a functional or reactive group is present, whereby the compound can be covalently linked to a biological molecule. 5. G is a functional group selected from NH ₂ , OH, SH, CO ₂ H, acid halide, acid anhydride, CO activated ester, NCS, O amide phosphoric acid,
NC (O) CH ₂ I and The compound according to claim 1, which is a reactive group selected from the group consisting of: 6. Structure: embedded image 5 wherein A has at least one nitrogen atom and a is 2 to 6 aromatic or alicyclic groups. 6. The dimers and oligomers of the compounds according to claim 1, wherein 7. Sq is the following: The compound according to any one of claims 1 to 6, wherein 8. A complex of a biomolecule and a 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 conjugate according to claim 8, wherein the biological molecule is an oligo or polypeptide or a nucleotide or an oligo or polynucleotide. 10. The synthesis method comprises the steps of: i) reacting the wing portion W with a dialkyl squarate or analog to form an intermediate a
) Ii) If desired, intermediate a) is subjected to hydrolysis to give intermediate b) Iii) reacting intermediate a) or intermediate b) with R ¹ R ² NH to obtain the final product c) give. 11. A sample comprising an amine and compound a) or compound b
) And amine with compound a) or b) An analytical or labeling method comprising observing dye formation by reaction with