ES2357989T3 - LUMINISCENT COMPOUNDS. - Google Patents

Abstract

Water-soluble squaric or croconic acid derivative fluorescent labeling dyes (I) are new. Fluorescent dyes of formula (I) are new. E = direct bond or -C(=B)-; A, B, D = O, S or N-R; or at least one may be replaced by CRxRy; R = aliphatic or aromatic group, or a reactive derivative such as (CH2)nCOOH or (CH2)nNH2; Rx, Ry = COOH, COCl, CONH2 or CO-NHS; NHS = N-hydroxy-succinimide; X, Y = O, S, Se, NR or C(Me)2; at least one of R1-R10 = reactive group suitable for covalent bonding to a substrate molecule, selected from (i) NCO, NCS, mono- or dichlorotriazine, aziridine, sulfonyl halide, N-hydroxysuccinimide ester, imido ester, glyxoal and CHO for bonding to amino or hydroxy functions or (ii) maleimide or iodoacetamide for bonding to thiol functions; the reactive group optionally being bonded to the chromophore via a spacer of formula (CH2)n; n = 1-18; at least one of R1-R10 = ionizable substituent providing hydrophilicity, such as SO3<->, COO<-> or N(R)3<+>; R3-R10 may also = groups forming further fused aromatic or heterocyclic rings.

Description

Field of the Invention

The invention relates to luminescent compounds based on squaric acid.

Background of the invention

A luminescent compound, or luminophore, is a compound that emits light. In turn, a luminescence procedure is a procedure that involves detecting light emitted by a luminophore, and that uses properties of that light to understand properties of the luminophore and its surroundings. Luminescence procedures may be based on chemiluminescence and / or photoluminescence, among others, and may be used in spectroscopy, microscopy, immunoassays, and other fields.

Photoluminescence is a particular type of luminescence that involves the absorption and subsequent remission of light. In the photoluminescence, a luminófer is excited from a base state of low energy to an excited state of higher energy by absorption of a photon of light. The energy associated with this transition is subsequently lost through one or more mechanisms, including the production of a photon by fluorescence or phosphorescence.

The photoluminescence can be characterized by several parameters, including the extinction coefficient, the excitation and emission spectrum, the Stokes shift, the luminescence life time and the quantum yield. An extinction coefficient is a measure dependent on the wavelength of the absorber force of a luminopher. An excitation spectrum is the dependence of the emission intensity after the excitation wavelength, measured at a constant individual emission wavelength. An emission spectrum is the distribution of the emission wavelength measured after excitation with a constant individual emission wavelength. A Stokes shift is the difference in wavelengths between the maximum of the emission spectrum and the maximum of the absorption spectrum. A luminescence life time is the average time in which a luminophore is in excited state before returning to the base state. A quantum yield is the ratio of the number of photons emitted to the number of photons absorbed by a luminophore.

Luminescence procedures can be influenced by extinction coefficient, excitation and emission spectra, Stokes shift and quantum performance, among others, and may involve characterization of fluorescence intensity, fluorescence polarization (FP) , fluorescence resonance energy transfer (FRET), fluorescence lifetime time (FLT), total internal reflection fluorescence (TIRF), fluorescence correlation spectroscopy (FCS), recovery of fluorescence after photobleaching (FRAP) and its fluorescence analogs, among others.

Luminescence procedures have several significant potential characteristics. First, luminescence procedures can be very sensitive because modern detectors, such as photomultiplier tubes (PMT) and charge-coupled devices (CCD), can detect very low levels of light. Second, the luminescence procedures can be very selective because the luminescence signal can come almost exclusively from the luminophore.

Despite these potential capabilities, the luminescence procedures suffer from several drawbacks, of which at least some relate to the luminophore. For example, the luminophore may have an extinction coefficient and / or a quantum efficiency that is too low to allow the detection of an adequate amount of light. The luminophore may also have a Stokes offset too low to detect the emission light without significant excitation light detection. The luminophores can also have an excitation spectrum that does not allow to be excited by limited wavelength light sources, such as common lasers and arc lamps. The luminophore can also be unstable, so it easily bleaches and becomes non-luminescent. The luminophore may also have an excitation and / or emission spectrum that overlaps with the well-known autoluminescence of biological and other samples; such autoluminescence is particularly significant at wavelengths below 600 nm. The luminophore can also be expensive, especially if it is difficult to manufacture.

Terpetschnig and others, Anal. Biochem 217, 197-204 (q1987) disclose the synthesis and fluorescence characteristics of a squaraine-N-hydroxysuccinimide ester and a sulfobutyl quaraine-N-hydroxysuccinimide ester:

WO 96/41144 describes marker components comprising a fluorophore moiety coupled to one or more polyoxyhydrocarbyl moieties. Escuarilio dyes are mentioned as possible fluorophores and a squarilio compound is exemplified in Fig. 7v.

The following publications are referred to the reader JOSEPH R. LAKOWICZ, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (1983), RICHARS J. LEWIS SR, HAWLEY’S CONDENSED CHEMICAL DICTIONARY (12th Ed., 1993).

Summary of the invention

The invention provides photoluminescent compounds and methods of synthesis and use of photoluminescent compounds, among others.

Generally, the compounds refer to the following structure:

image 1

in which:

(to)

each of B and C is selected from the group consisting of W1 and W2, W1 and W2 being:

(b)

one of B and C is W1 and the other of B and C is W2;

(C)

n is independently selected for each of B and C from the group consisting of 0, 1 and 2;

(d)

Y is independently selected for each of B and C from the group consisting of O, S, Se, Te, N-Rh and C (Ri) (Rj), Rh being selected from the group consisting of H, aliphatic groups, alicyclic groups and aromatic groups, and each of Ri and Rj being selected from the aliphatic groups;

(and)

R is independently selected for each of B and C from the group consisting of H, aliphatic groups, alicyclic groups and aromatic groups, and

(F)

each of X1, X2, X3 and X4 is independently selected for each B and C from the group consisting of N, O, S and C-Rk, Rk being selected from the group consisting of H, F, Cl, Br, I, aliphatic groups, alicyclic groups, aromatic groups and parts of a substituted condensed aromatic or heterocyclic cycle;

image2

the photoluminescent compound being covalently or non-covalently associated with a vehicle that is a pearl.

In some embodiments, the compound includes at least one S. In other embodiments, the compound includes at least one heteroatom in X1 through X4 of W1 or W2. In yet other embodiments, the compound includes a vehicle. Generally, the procedures refer to the use of photoluminescent compounds, described above.

The nature of the invention will be more readily understood after considering the drawings, chemical structures and detailed description of the invention set forth below.

Brief description of the drawings

Figure 1 is a graph showing the excitation (dotted line) and emission (continuous line) spectra for (13) -HSA in buffered saline (PBS).

Abbreviations In this application, the following abbreviations may be used, among others: Detailed description of the invention

BSA

bovine serum albumin

Bu

butyl

 DCC

dicyclohexylcarbodiimide

DMF

 dimethylformamide

D / P

 dye to protein ratio

Et

ethyl

 g

grams

h

hours

hSA

human serum albumin

HCG

human chorionic gonadotropin

l

liters

m

 mili (10-3)

M

 cool

I

 methyl

mol

moles

 m.p.

 melting point

n

 nano (10-9)

NHS

N-hydroxysuccinimide

NlR

near infrared region

PBS

 buffered saline

Prop

propyl

 TMS

tetramethylsilane

TSTU

 N, N, N ’, N’-tetramethyl (succinimido) uronium tetrafluoroborate



 micro (10-6)

The invention generally relates to photoluminescent compounds and methods of using such compounds. These photoluminescent compounds can be useful, both in free or condensed form, as probes, markers and / or indicators. This utility may partly reflect the intensification of one or more of the following characteristics: quantum yield, Stokes shift, extinction coefficients, and photostability. This utility may also reflect excitation and emission spectra in relatively inaccessible regions of the spectrum, including the red and near infrared regions.

The rest of the discussion includes (1) a perspective of the structures, (2) a perspective of the synthesis procedures and (3) a series of illustrative examples.

Structure Perspective

Photoluminescent compounds and their synthetic precursors generally comprise the following structure:

image2

The various symbols have the meanings previously indicated.

 Photoluminescent compounds. In the photoluminescent compounds, B and C are chosen between W1 and W2, one of B and C is W1 and one of B and C is W2.

Other characteristics of the compounds have been previously described.

 Vehicle groups The photoluminescent compounds of the invention are covalently or non-covalently associated with a vehicle that is a pearl. Covalent association can be accomplished by several mechanisms, including a reactive group, and may involve a spacer to separate the photoluminescent compound or the precursor from the vehicle. The non-covalent association can also be performed by various mechanisms, including the incorporation of the photoluminescent compound or the precursor into or into a matrix, such as a bead, or by non-specific interactions such as hydrogen bonding, ionic bonding or hydrophobic interactions.

Alicyclic groups include groups of organic compounds characterized by the arrangement of carbon atoms in closed ring structures that sometimes resemble boats, chairs or even bird cages. These compounds have properties that resemble those of aliphatic compounds and should not be confused with aromatic compounds that have the hexagonal ring of benzene. The alicyclic compounds comprise three subgroups: (1) cycloparaffins (saturated), (2) cycloolefins (unsaturated with two or more double bonds) and (3) cycloacetylenes (cyclin) with a triple bond. The best known cycloparaffins (sometimes referred to as naphthenes) are cyclopropane, cyclohexane and cyclopentene; of the cycloolefins are typical cyclopentadiene and cyclooctatetraen. Most alicyclic compounds are derived from petroleum or coal tar, and many can be synthesized by various procedures.

Aliphatic groups include groups of organic compounds characterized by a linear or branched chain arrangement of the constituent carbon atoms. Aliphatic hydrocarbons comprise three subgroups: (1) paraffins (alkanes), which are saturated and comparatively non-reactive, (2) olefins (alkenes or alkanes), which are unsaturated and quite reactive and (3) acetylenes (alkynes), which contain a triple bond and they are very reactive. In complex structures, the chains can be branched or crosslinked.

Aromatic groups include groups of unsaturated cyclic hydrocarbons containing one or more rings. A typical aromatic group is benzene, which has a 6-carbon ring that contains three double bonds. Most aromatic groups are very reactive and chemically versatile. Most aromatics are derived from petroleum and coal tar. Some 5-member cyclic compounds, such as the furan (heterocyclic) group, are analogs of aromatic compounds.

Heterocyclic rings include closed ring structures, usually of 5 or 6 members, in which one or more of the ring atoms is a non-carbon element, for example, sulfur, nitrogen, etc. Examples include pyridine, pyrrole, furan, thiophene and purine.

Perspective of the synthesis and characterization

Typically, the synthesis of the photoluminescent compounds according to the present invention is carried out by multistage reaction from the synthesis of a methylene base. Synthesis of suitable bases can be performed based on literature procedures or new procedures. Generally, the spectral properties of the photoluminescent compounds, including the excitation and emission wavelength, depend greatly on the type of the methylene base used. Typical starting materials include benzoindoles, benzoselenzoles, benzooxazoles, benzoimidazoles, etc., and squaric acid. Squaric acid is a dibasic acid that undergoes a series of nucleophilic substitution reactions with various reagents, including amines, phenols, and CH-acid compounds such as 1,2,3,3-tetramethyl-benzoindole. The squaraine bridge in the resulting compounds stabilizes the conjugate chain and shifts the excitation and emission wavelength of these dyes to red compared to cyanine-based dyes. In particular, the exchange of oxygen in the rest of squaraine with a methylene functionality (= CR2) is seen here that it is the route to new dyes of the squaraine group with useful fluorescence properties.

In the examples that follow this section, the synthesis and spectral characterization of several long-wavelength fluorescent markers based on squaraine dyes, including some reactive versions, are presented. These dyes may include a cyanine-type chromophore and a squared bridge. To achieve water solubility, sulfonic acid or other groups can be introduced into heterocyclic ring systems and, thus allowing covalent protein binding, Nhydroxysuccinimide ester (NHS ester) or other forms can be synthesized. To modify the spectral properties of the dyes, dicyomethylene substituted versions of the squaraines were synthesized and tested for their potential use to label biopolymers. Squaraine-based markers have lower quantum yields in water ( = 0.15) and very high quantum yields ( = 0.5-0.7) when bound to biomolecules. The absorption and emission wavelengths can be tuned by replacement of the squarin ring or by introduction of heteroatoms in the heterocyclic moiety. Thus, indolenin-squaraines absorb around 635 to 840 nm in water and approximately 645 to 650 nm when bound to proteins. The absorption and emission spectra of benzothiazolium and benzoselenzolium squaraines, on the other hand, are shifted towards longer wavelengths. Typical emission wavelengths for such squaraine dyes are around 680 to 690 nm for benzothiazole dyes and more than 700 nm for benzoselenzole derivatives. It is important that Stokes shift increases in these dyes that emit longer wavelengths, which ultimately increases the sensitivity of a fluorescent measurement.

The resulting dyes have absorption and emission maximums at more than 600 nm and their wavelength can be tuned by changing the heterocyclic moiety and / or the substitution in the annular system of the squaraine. Stokes 'displacement of methylene derivatives of symmetric squaraines (15) increases more than 2.5 times with respect to Stokes' displacement of analogous squaraines containing oxygen (8) and (3b). In addition, the replacement of C = O with C = C in example (15) results in a batochromic shift of both the absorption and emission properties of these dyes. A greater increase in Stokes displacement can be achieved by introducing asymmetry into the molecule. Thus, asymmetric versions (15) are expected to have even greater Stokes offsets.

The substitution of an oxygen in the central square bridge with CH-acid reagents, for example, dicyanomethane, HOOC- (CH2) -COOH or ROOC- (CH2) -CN leads to the group of luminescent methylene quarantine derivatives. Compared to basic squaring, these compounds have excitation and emission properties shifted to red and greater Stokes shifts. The absorption and emission maxima of a representative reactive dye (15) (example 7) were found to be 667 nm and 685 nm in PBS, respectively.

Example 8 describes synthetic routes to asymmetric dyes of thioescuaraine and methylene quarantine. The squaraine compounds of this example do not form part of the invention as claimed. The key intermediates for this subgroup of squaraine are monosubstituted thioescuaraine and methylene quarantine derivatives, which are synthesized by reacting the disodium salt of 1,3-dithioescuaric acid (2c) or 4-cyanomethylene-2,3-dibutyl (2d) squarate with 1 equivalent of indolenin (1a). The intermediate is subsequently reacted with an equivalent of a different methylene base. The synthesis of asymmetric squaraine dyes allows access to monofunctional reactive squaraine dyes (Scheme 1). Such dyes show improved marker behavior because of reduced protein crosslinking.

Example 9 shows colorant structures based on thioescuaraine and methylene quarantine which further demonstrate the variety of structures that can be synthesized from this invention. Only the dicyanomethylene quarantine compounds of this example form part of the invention as claimed. Most of the representative structures are based on water-soluble reactive dyes that have different substitution configurations in the central bridge. Two of the structures contain phosphoramidite bonds for direct coupling used in the synthesis of DNA or RNA on solid support. For the synthesis of phosphoramidites, sulfonated and non-sulfonated versions of squaraine dyes can be used. The phosphoramidite binding can be incorporated into the heterocyclic bases or can be attached to the square central ring by activating a carboxycyanomethylene function to an NHS ester and reacting with an aminoalcohol spacer group. The hydroxylic function introduced is then converted into a phosphoramidite by standard procedures.

EXAMPLE 1

Synthesis of precursors

This section describes the synthesis of several precursors. Using literature procedures, p-hydrazinobenzenesulfonic acid (Illy et al., J. Org. Chem. 33, 4283-4285 (1968)), potassium salt of 1- (-carboxypentyl) -2,3,3 acid was synthesized. , timethylindolenium-5-sulfonic acid (1a), potassium salt of 2,3,3-trimethylindole-5-sulfonic acid (Mujumdar et al., Bioconj. Chem. 4, 105-111 (1993)) (1b) and 1,2 , 3,3-tetramethylindoleninium-5-sulfonate (1c). The disodium salt of 1,3-dithiosquaric acid (2c) and dicyanomethylene dimethyl quarate (2d) were synthesized according to

G. Seitz et al., Chem. Ber. 112, 990-999 (1979) and B. Gerecht et al., Chem. Ber. 117, 27142729 (1984), respectively.

Preparation of the potassium salt of 1- (-carboxypentyl) -2,3,3-trimethylindolenium-5-sulfonic acid (1a)

P-Hydrazinobenzenesulfonic acid

33 g of sodium carbonate were added to a suspension of 104 g (0.6 mol) of paminobenzenesulfonic acid in 400 ml of hot water. The solution was cooled to 5 ° C in an ice bath and 70 g of concentrated sulfuric acid were added slowly under rapid stirring. Then a solution of 42 g of sodium nitrite in 100 ml of water was added with cooling. A light yellow precipitate of diazo compound formed, which was filtered, washed with water but not dried.

The wet diazo compound was added under stirring and cooling (5 ° C) to a solution of 170 g of sodium sulphite in 500 ml of water. The solution, which took an orange color, was stirred under cooling for 1 h and then heated to reflux. Finally 400 ml of conc. Hydrochloric acid was added. The solution took a yellow color and the product precipitated as a white solid. For complete bleaching, 1-2 g of zinc powder was added. The reaction mixture was cooled overnight and the precipitate was filtered off, washed with water and dried in an oven at 100 ° C.

Yield: 96 g (85%), white powder, m.p. = 286 ° C (bibliography 285 ° C); Rf 0.95 (RP-18, water: MeOH 2: 1)

Preparation of the potassium salt of 2,3,3-trimethylindole-5-sulfonic acid (1b)

18.2 g (0.12 mol) of p-hydrazinobenzenesulfonic acid and 14.8 g (0.17 mol) of isopropylmethyl ketone were stirred for one hour at room temperature in 100 ml. The mixture was then refluxed for 4 h. The mixture was cooled to room temperature and the resulting pink solid precipitate was filtered off and washed with ether.

The precipitate was dissolved in methanol and a concentrated solution of potassium hydroxide in 2-propanol was added until a yellow solid precipitated completely. The precipitate was filtered off, washed with ether and dried in desiccator over P2O5.

Yield: 20.4 g (71%), off-white powder, m.p. 275 ° C, Rf 0.40 (silica gel, isopropanol: water: ammonia 9: 0.5: 1).

Potassium salt of 1- (-carboxypentyl) -2,3,3-trimethylindolene-5-sulfonic acid (1a)

15.9 g (57 mmol) of the potassium salt of 2,3,3-timethylindolene-5-sulphonic acid and 100 ml of 1,2-dichlorobenzene were refluxed under nitrogen atmosphere for 12 h

12.9 g (66 mmol) of 6-bromohexanoic acid. The solution was cooled to room temperature and the pink precipitate was filtered off, washed with chloroform and dried. Yield: 15.8 g (58%), pink powder, Rf 0.75 (RP-18, MeOH: water 2: 1).

5 Synthesis of 1,2,3,3-tetramethylindoleninium-5-sulfonate (1c)

1.1 g of 2,3,3-trimethylindoleninium-5-sulfonate was suspended in 30 ml of methyl iodide. The reaction mixture was boiled for 25 h in a closed tube. After cooling the mixture, the excess methyl iodide was decanted and the residue was suspended in 50 ml of acetone. The solution was filtered and the residue dried in desiccator

10 over CaCl2. The resulting light yellow powder was used without further purification. Yield: 90%, light yellow powder.

image2

image2

EXAMPLE 2

5 Synthesis of 2,4-bis [N- (butoxycarbonylpentyl) -3,3-dimethyl-5-sulfo-2-indolinylidene methyl cyclobutenediyl-3-dicyanomethylene-1-oxolate (15) 3-dicyanomethylene-2,4-dibutyl -scuarate (2d)

10 3-Dicyano-2,4-dibutyl-squarate (2d) was prepared according to Gerecht et al. (1984)

image2

2.16 ml (10 mmol) of squaric acid dibutyl ester were dissolved in 40 ml of THF.

15 660 mg (10 mmol) of malonodinitrile was added with stirring. A solution of 1.64 ml of triethylamine in 3 ml of THF was then added and the mixture was stirred at room temperature for 15 h. The solvent was removed under reduced pressure and a yellow-brown oil remained. The crude product was purified by multiplex liquid chromatography (MPLC) using silica gel as stationary phase and methanol as eluent.

Yield: 173 mg (63%) of (2d).

image 1

472 mg of the potassium salt of 1- (carboxypentyl) -2,3,3-trimethylindolene-5-sulfonic acid (1a) and 137 mg of the dibutyl ester of malononitrile escarric acid (2d) were maintained at reflux for 4 hours. ml of butanol: toluene (1: 1, v / v) using a Dean trap

10 Stark After the mixture was cooled to room temperature, the solvents were removed in vacuo and the crude product was triturated with ether and dried. The crude product was purified by preparative thin layer chromatography on RP-18 glass plates using a mixture of methanol: water (2: 1 v / v) as eluent. The blue-green band was collected with an Rf of 0.55. Yield: 32%. FAB-EM-FAB calculated for C41H44N4O11S2K2 (M2-) 832.9, found

15 633.2. IR (KBr): 2100 cm-1 (CN). 1H NMR (D2O):  8.00 (2H, s), 7.90 (2H, d), 7.75 (2H, d), -CH = is exchanged, 4.45 (4H, t), 2 , 10 (4H, t), 1.85 (4H, m), 1.55 (4H, m), 1.45 (12H, s), 1.35 (4H, m); max (abs) = 667 nm (PBS), max (em) = 685 nm (PBS) (4%), max (abs) = 687 nm (PBS + HSA), max (em) = 704 nm (PBS + HSA), (8%),  = 110,000 l / mol.cm (H2 =).

EXAMPLE 3 The thioscuaraine compounds of this example do not form part of the invention as claimed, but are retained as useful base information.

Thioescuaraine and asymmetric dicyanomethylene quarantines

Synthesis of monosubstituted thioescuaraine and dicyanomethylene quarantine derivatives The following compounds are important intermediates for dye synthesis

5 asymmetric squaraine and can be synthesized using starting materials derived from squaric acid or squaric ester. A general procedure is then given for the synthesis of two classes of asymmetric analogs of squaraine.

It was dissolved in 10 ml of ethanol containing 100 µl of triethylamine 1 mmol of 1- (carboxypentanil) -2,3,3-trimethylindolene-5-sulfonate. The mixture was heated to 40 ° C and 1.1 mmol of dicyanomethylene tearic acid dibutyl ester (2d) or disodium salt of 1,3-dithiosquaric acid (2c) were added in small portions. The reaction mixture was then heated to 60 ° C and stirred for 4 h. After the mixture was cooled to room temperature, the solvent was removed under reduced pressure. The residue was redissolved in ethanol, 5 ml of 0.1 M HCl was added and the mixture was refluxed for 10 min. After cooling, they were removed

The solvents and the residue were washed several times with ether or chloroform.

image2

Esquaraine derivative

R R1

Diciano-methylene Ditio

C (CN) 2 S OH SH

General synthesis procedure for asymmetric dyes of thioescuaraine and methylene quarantine The above intermediates were heated to reflux with 1 mmol of 1,2,3,3-tetramethylindoleninium-5-sulphonate in a mixture of butanol: toluene (1: 1 v / v) using a trap

5 Dean-Stark. After cooling the solvents were removed under reduced pressure. The product can be purified by preparative thin layer chromatography (RP-18F254S) using as eluent methanol: water (2: 1 v / v).

Yield: 25-30% asymmetric compound.

10 Synthesis scheme for an asymmetric thioescuaraine dye EXAMPLE 4

image2

Only part of the invention is claimed as claimed by the compounds of

dicyanomethylene quaraine of this example. The remaining compounds are preserved as

Useful background information.

Additional structures of thioescuaraine and methylene quarantine dyes

image3

The last example shows a central square ring (X = O-, S-) converted so that it contains a reactive end group for covalent binding to vehicle molecules. To introduce an amino function into a ring of squaraine (X = O-, S-), the squaraine or squaraine

The substituted one becomes a monoester version (X = OMe, OBu) in which Me is methyl and Bu is butyl. The monoester is then reacted with the amine or an amino acid derivative to result in an amino derivative. This mechanism offers a convenient route for converting a non-reactive dichlorine dye into a reactive analog.

EXAMPLE 5

General procedures for marking proteins and determining dye to protein ratios

Protein labeling reactions were performed using 50 mM bicarbonate buffer (pH 9.1). A stock solution of 1 mg of dye in 100 µl of anhydrous DMF was prepared. 10 mg of protein was dissolved in 1 ml of 100 mM bicarbonate buffer (pH 9.1). Coloring of the stock solution was added and the mixture was stirred for 24 h at room temperature.

Unconjugated dye was separated from the labeled proteins using gel penetration chromatography with Sephadex G50 column (0.5 cm x 20 cm column) and as eluent phosphate buffer solution 22 mM (pH 7.3). The first colored band contained the dye-protein conjugate. A subsequent blue band with a much higher retention time contained the separated free dye. A series of labeling reactions were prepared as described above to obtain different dye to protein ratios. Compared to the free forms, the protein-bound forms of the dyes have definite changes in their spectral properties.

Protein concentrations were determined using the Pierce BCA Protein Assay Reagent kit (Rockford, IL). The ratio of dye to protein (D / P) gives the number of dye molecules covalently bound to the protein.

Covalent ester bonding of NHS (14) to polyclonal anti-HSA

385 µl (5.2 mg / ml) of anti-HSA was dissolved in 750 µl of bicarbonate buffer (0.1 M, pH 9.0). 1 mg of NHS ester (14) was dissolved in 50 µl of DMF and slowly added with stirring to the protein solution prepared above. After 20 h of stirring, the protein conjugate was separated from the free dye using a Sephadex G50 column and phosphate buffer (20 mM, pH 7.2). The first blue band that was isolated contained the labeled conjugate.

Conjugation of (14) to HSA

0.5 mg of (14) in 50 µl of DMF was slowly added to a stirring solution of 5 mg of HSA in 750 µl of bicarbonate buffer (0.1 M, pH 9.0). The mixture was stirred for another 6 h at room temperature. The mixture was dialyzed against a phosphate buffer (22 mM; pH 7.2) using a dialysis membrane (1500 FT, Union Carbide) cut to

10,000 Analysis: max (abs) = 642 nm (PBS); max (em) = 654 nM (PBS).

Similar reactions were performed using the other reactive dyes.

 Extinction times of fluorescence of different dyes and their conjugates

The following table shows the fluorescence extinction times of various dyes and their conjugates. Experimental conditions included (1) excitation at 600 nm using rhodamine B dye laser, (2) emission observed at 660 nm using an interference filter with 10 nm bandpass, and (3) at a temperature of 20 ° C.

Sample

 Extinction time, ns Amplitude  Fractional intensity Half-life, ns Chi squared

3rd

0.21 0.752 0.496 0.43 3.7

0.65

0.248 0.504

4

0.20 0.558 0.286 0.51 3.8

0.64

0.442 0.714

3b-HSA

0.18 0.676 0.142

0.96

0.089 0.097 2.26 1.7

3.81

0.253 0.761

13-HSA

0.011 0.865 0.036

0.768

0.068 0.201 2.44 5.22

2.99

0.067 0.764

Cy5

1.02 one one 1.01 2.1

Cy5-hCG

0.16 0.408 0.071 1.33 2.8

1.41

0.592 0.929

10 Spectral properties and protein-to-protein dye ratios for various reactive dyes of squaraine and its conjugates

The spectral properties and ratios of dye to protein were determined for several reactive dyes of squaraine and its conjugates. Figure 1 shows the absorption (excitation) and emission spectra for (13) -HSA in PBS. The following table summarizes data for (13) -HSA and several other reactive dyes of squaraine and its conjugates in PBS.

Squaraine

max (abs), nm maz (em), nm , l (mol.cm) Yield c,% D / P, mol / mol

3b 3b-HSA 6a 7 7-HSA 13 13-HSA 15 15-HSA

635 642 627 634 635 630 642 667 685 642 653 647 646 660 649 654 685 704 180,000 -100,000 120,000 -66,000 -110,000 - 13 60-70 3 13 50 5 60ñ-70 4 n.d. -1 -0.5 -0.8 -n.d.

image 1

Description of applications of the invention In addition to the compounds, the invention provides several methods for using these compounds in various assay formats. 10 The assay can be a competitive trial that includes a recognition moiety, a binding participant and an analyte. Binding participants and analytes can be selected from the group consisting of biomolecules, drugs and polymers. In some competitive assay formats, a compound or several are marked with photoluminescent compounds according to the invention. For example, the binding partner can be labeled with one of such photoluminescent compounds and, by the appearance of fluorescence in a liquid phase of the

assay, the displacement of the compound from an immobilized recognition moiety can be detected. In other competitive assay formats, an immobilized emzyme can be used to form a complex with the fluorophore-conjugate substrate.

The binding of antagonists to a receptor can be tested by a competitive binding procedure in so-called ligand / receptor assays. In such assays, a labeled antagonist competes with an unlabeled ligand for the receptor binding site. One of the union participants can be immobilized, but not necessarily immobilized. Such tests can also be performed on microplates. Immobilization can be achieved by covalent bonding to the wall of the well or to the surface of the beads.

Other preferred assay formats are immunological assays. There are several types of immunological tests. Competitive binding assays, in which labeled and unlabeled antigens compete for binding sites on the surface of an antibody (binding material). Typically, there are incubation times required so that there is sufficient time to balance. Such tests can be performed heterogeneously or homogeneously.

Sandwich assays use secondary antibodies and analyte access binding material is removed by a washing step.

Other types of reactions include binding between avidin and protein, protein A and immunoglobulins, lectins and sugars (for example, concanavalin A and glucose).

The photoluminescent compounds described herein can also be used to sequence nucleic acids and peptides. For example, fluorescence labeled oligonucleotides can be used to detect DNA fragments.

Other applications of labeled DNA primers, in addition to DNA sequencing, are in the process of fluorescent in situ hybridization (FISH) and for applications of individual nucleotide polymorphisms (SNPs).

Multicolored labeling experiments allow simultaneous control of different biochemical parameters. To this end, two or more fluorophores can be introduced into the biological system to inform about different biochemical functions. The technique can be applied to fluorescent in situ hybridization (FISH), DNA sequencing, fluorescence microscopy and flow cytometry. One way to achieve multicolored analysis is to label biomolecules such as nucleotides, proteins or DNA primers with different fluorescent markers and defined fluorescence properties. For multicolored marking, fluorophores with narrow emission bandwidths are preferred, because they have only a small overlap with the other dyes and, therefore, mutually separate so that there is a sufficient resolution of the signal. A suitable multicolored trio of fluorophores would include Cy3, TRICT and a photoluminescent compound as described herein, among others.

The simultaneous use of FISH (fluorescent in situ hybridization) primers in combination with different fluorophores is useful for the detection of chromosomal translocations, for mapping genes on chromosomes and for marking tumors, to name only a few applications. One way to achieve simultaneous multiple sequence detection is to use combinatorial dialing. Up to seven different DNA targets can be visualized using a combination of haptenized DNA primers (eg, biotin, digoxigenin or dinitrophenol) with three sets of distinguishable fluorophores that show emission in green (fluorescent), red (Texas Red) and blue (7-amino-4-methylcumarin-3-acid or Cascade Blue acid) (Ried et al., Proc. Natl. Acad. Sci. USA 89, 1388-1392 (1992)). Three labeled DNA primers of the three fluorescent markers can be visualized, while another four will appear as mixtures of fluorophores, for example, primer 4 (fluorescein and rhodamine); primer 5 (fluorescein and Cascade Blue); primer 6 (rhodamine and Cascade Blue) and primer 7 (fluorescein, rhodamine and Cascade Blue).

The other way is to label each nucleic acid primer with a fluorophore with defined spectral properties. Similar conjugates of this invention can be synthesized and used in a multicolored multisequential analysis approach.

The luminescent compounds of the invention can also be used for screening tests in a combinatorial library of compounds. The compounds can be explored in terms of various characteristics, including their specificity and greediness in favor of a particular recognition.

Screening tests of a library of compounds are well known. An exploration assay is used to determine compounds that bind to a target molecule and thereby create a signal change that generates a labeled ligand bound to the target molecule. Such assays allow to explore compounds that act as agonists or antagonists of a receptor or that disrupt the protein-protein interaction. They can also be used to detect hybridization or binding of DNA and / or RNA.

Another way to perform screening assays is based on compounds that influence enzyme activity. To this end, non-fluorescent enzyme substrates of the invention could be used to detect interaction with the substrate.

It is also possible to test compounds that interfere with proteins that inhibit enzyme activity using an electrophoretic assay. In such assays, the most active compounds prevent enzyme inhibition, which results in more enzymatic catalysis.

In addition to non-fluorescent enzyme substrates, fluorescent substrates can be used in such assays. The cleavage of the fluorescent substrate leads to a change in spectral properties such as maximum excitation and emission, which makes it possible to distinguish between free and bound fluorophore.

Analytes

The invention can be used to detect an analyte that interacts with a recognition moiety in a detectable manner. As such, the invention can be attached to a recognition moiety that is known to those skilled in the art. Such recognition moieties allow the detection of specific analytes. Examples are pH or potassium sensitive molecules, for example, synthesized by introduction of potassium chelators such as corona ethers (azacorones, thiacorones, etc.). To detect intracellular ionic concentrations, calcium sensors based on BAPTA (1,2-bis (2-aminophenoxy) ethan-N, N, N ’, N’-tetra-acetic acid) are frequently used as chelating species. The combination of a compound of the invention and the BAPTA, calcium binding moiety, can lead to new absorbent Ca sensors and long wavelength emitters that could be used for the determination of intracellular and extracellular calcium concentrations (Akkaya et al. , Tetrahedron Lett. 38, 4513-4516 (1997)).

Fluorescence procedures

These compounds of the new invention can be detected by applying commonly used intensity-based fluorescent procedures. It is known that squaraine dyes have life times in the range of hundreds of ps to a few ns (see table). The nanosecond life time and the long wavelength of absorption and emission of these dyes when bound to proteins would allow them to be measured with cheap instrumentation using laser diodes for excitation and avalanche photodiodes for detection. Typical tests are based on the measurement of the fluorescence lifetime as a parameter of the FRET tests (fluorescence resonance energy transfer). The union between a species labeled fluorescent donor (typically an antigen) and a species labeled fluorescent acceptor is accompanied by a change in the intensity and lifetime of the fluorescence. Life time can be measured using procedures based on intensity or phase modulation (J.R. LAKOWITZ, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (1983)).

Esquaraine dyes have a high intrinsic polarization in the absence of rotary motion, which makes them useful as markers in fluorescence polarization (FP) assays. Fluorescence polarization immunoassays (IPF) are widely used to quantify low molecular weight antigens. The assays are based on measures of polarization of antigens with fluorescent probes. The requirement of polarization probes used in FPI is that the emission of unbound labeled antigen can be depolarized and increased after binding to the antibody. In such binding assays low molecular weight species labeled with the compounds of the invention can be used, and the unknown concentration of the analyte can be determined by changing the polarized emission of the fluorescent marker molecule.

The materials, procedures and applications of the invention are exemplified by the following description of the synthesis and spectral properties of the compounds.

The synthesis and spectral characterization of a squaraine derivative for covalent binding to biomolecules has already been indicated. Squaraines, which are 1,3-substituted derivatives of squaric acid, have high absorption coefficients (> 200,000 l mol-1 cm-1) in the red region, have high photostability and allow the introduction of reactive functional groups such as NSH esters. These dyes have good quantum yields in protein binding. The hydrophilic character of the dyes can be improved by introducing sulfonic acid groups.

The advantages of fluorescence detection with high wavelength excitation are the reduced autofluorescence of cells and tissues and the use of cheap laser light sources operating at 635, 645 and 650 nm. Autofluorescence of biological samples decreases with increasing wavelength, in particular beyond 600 nm. There are only a few probes that absorb in the region of red or near infrared (NIR) and even fewer are available reactively. Functionalities such as isocyanate and N-hydrosuccinimide esters and thiol-reactive iodoacetamide and maleiimide groups can be used to covalently bind marker molecules to drugs, DNA, antibodies and synthetic polymers.

Although the invention has been described in preferred forms, its specific embodiments as disclosed and illustrated herein are not to be considered in a limiting sense because numerous variations are possible.

Claims (5)

1. A composition of matter comprising a photoluminescent compound, a photoluminescent compound having the structure: image 1 in which:

(to)

each of B and C is selected from the group consisting of W1 and W2, W1 and W2 being

(b)

one of B and C is W1 and the other of B and C is W2,

(C)

n is independently selected for each of B and C from the group consisting of 0, 1 and 2;

(d)

Y is independently selected for each of B and C from the group consisting of O, S, Se, Te, N-Rh and C (Ri) (Rj), Rh being selected from the group consisting of H, aliphatic groups, alicyclic groups and aromatic groups, and each of Ri and Rj being selected from the aliphatic groups;

(and)

R is independently selected for each of B and C from the group consisting of H, aliphatic groups, alicyclic groups and aromatic groups, and

(F)

each of X1, X2, X3 and X4 is independently selected for each B and C from the group consisting of N, O, S and C-Rk, Rk being selected from the group consisting of H, F, Cl, Br, I, aliphatic groups, alicyclic groups, aromatic groups and parts of a substituted condensed aromatic or heterocyclic cycle;

image 1 the photoluminescent compound being covalently or non-covalently associated with a vehicle that is a pearl.

2.

The composition of claim 1, wherein the photoluminescent compound is symmetric.

3.

The composition of claim 1, wherein the photoluminescent compound is asymmetric.

Four.

The composition of claim 1, wherein n is 1.

5.

A procedure to perform a photoluminescent test, a procedure that

comprises: exciting a photoluminescent compound as defined in any of claims 1 to 4 with excitation light, and detecting the emission light emitted by the photoluminescent compound.