RU2680090C1 - 2-(4,4-diftor-1,3,5,7-tetramethyl-2,6-disulfo-4-boro-3a,4a-diaza-s-indacen-8-yl)-benzoic acid and its derivatives, method of their production and their application for fluorescent blinding of protein molecules - Google Patents

Abstract

FIELD: chemical industry.

SUBSTANCE: invention relates to salts of the compound of formula I with alkali metals, replacing hydrogen atoms in both sulfo groups

,

where R means the N-oxysuccinimidyl group

Also proposed a method of obtaining salts and their use.

EFFECT: salts of the compounds of formula I are fluorescent dyes with better characteristics than their analogues – water solubility, quantum yield, and extinction coefficient – and can be used as fluorescent labels of protein molecules.

3 cl, 4 dwg, 1 ex, 5 tbl

Description

FIELD OF THE INVENTION

The invention relates to the field of organic chemistry, in particular, a sulfonated derivative of bordipyrromethene, methods for their preparation, to said compounds for use as fluorescent labels of protein molecules, including in the production of labeled antibodies used in immunofluorescence analysis for the diagnosis of infectious diseases.

State of the art

In the literature there is a description of derivatives of bordipyrometers [1] and methods for their preparation.

A known method of obtaining various derivatives of bordehyrromethene of the formula (I) by the reaction of substituted pyrroles with acyl chlorides or anhydrides. First, substituted pyrroles are condensed with an acyl chloride or anhydride in an organic solvent, then, without isolating the resulting product, it is treated with ethylamine and a complex of boron trifluoride with diethyl ether [1], after which the product is isolated in the form of molecular crystalline or amorphous compounds containing one a boron atom and being zwitterions (a positively charged nitrogen atom and a negatively charged boron atom) (I).

IR ₁ - R ₈ - H, alkyl, aryl and other substituents

A known method of producing derivatives of bordehyrromethene of the formula (I) by the reaction of substituted pyrroles with aldehydes. First, substituted pyrroles are condensed with an aldehyde in an organic solvent, then the mixture is treated with an oxidizing agent, and then, without isolating the resulting product, it is treated with ethylamine and a complex of boron trifluoride with diethyl ether [1]. The product is isolated in the form of molecular crystalline or amorphous compounds having one boron atom and being zwitterions (a positively charged boron atom and a negatively charged nitrogen atom) (I).

A known method of producing disulfonated borodipyrometers (II) by sulfonation of compounds of formula (I), where R ₂ , R ₆ = H, while the composition of compound (I) contains no more than 1 boron atom, chlorosulfonic acid and subsequent neutralization of the reaction mixture with a base [2 -9].

II

The known method for sulfonation of compounds of formula (I) does not allow to obtain compounds (III) or its alkali metal salts by sulfo groups if R is a hydroxyl group (IIIa)

III

IIIa, 2- (4,4-difluoro-1,3,5,7-tetramethyl-2,6-disulfo-4-boro-3a, 4a-diaza-s-indacen-8-yl) benzoic acid

A patent is known for chemically reactive compounds of the formula I, where R is H, Hal, alkyl, aryl, cycloalkyl, alkylaryl, acyl and sulfo group [10]. The patent does not mention compound IIIa or IIIa, moreover, the patent claims describe substituents other than H. as substituents at the 8th position. Thus, the patent [10] does not describe compound IIIa or its precursor IV, which does not have sulfo groups.

Claim 2 of the patent [10] mentions a carboxyl group as one of the active groups capable of forming a chemical bond with the ligand, and clause 1 of the patent [10] mentions that any of the active groups according to the dependent claims should be obtained by chemical modification of the dyes from .1 patent claims [10] already having a finished bordipyrromethene core.

The proposed method for producing dye introduces a carboxyl group into the composition of the substance immediately at the stage of synthesis of the bordepyrromethene nucleus at position 8, so the patent [10] does not describe a method for producing compound IIIa.

A known method of obtaining the compounds of formula IV, where the carboxyl group is introduced into the composition of the substance at the stage of synthesis of bordipyrromethene core [11]. Sulfonation obtained in a known manner, compound IV does not allow to obtain compound IIIa.

IV

The real substances synthesized with sulfo groups in position 2 and 6 are small [1]; this indicates the fact that not every product with sulfonic substituents in position 2 and 6 can be obtained by sulfonation of bordepyrromethenes. Thus, the use of substances bearing activated groups, where R = SO ₃ H, known in the patent [10], in fluorescence applications does not automatically include the use of compound IIIa with an activated group, the reliable existence of which was not known at the time the patent was filed.

Disclosure of the invention

The objective of the invention is to obtain 2- (4,4-difluoro-1,3,5,7-tetramethyl-2,6-disulfo-4-boro-3a, 4a-diaza-s-indacen-8-yl) -benzoic acid (compound IIIa), as well as its derivatives with N-oxysuccimidyl groups, characterized by the possibility of using for fluorescent labeling of proteins (fluorescein channel, 490-520 nm), characterized by solubility in water, higher quantum yield, higher extinction coefficient, compared to fluorescein and with known dyes of the formula (I), for example BODIPY FL, used for the fluorescein channel.

The technical result is the possibility of obtaining a water-soluble fluorescent dye IIIa with a higher yield and purity than is possible using methods known from the prior art, the achievement of this fluorescent dye better solubility in water, higher quantum yield, and higher extinction coefficient compared with analogues, as well as the use of its derivatives with N-oxysuccimidyl groups as brighter than known from the prior art, and therefore require less consumption ode, fluorescent labels of protein molecules.

A specific feature of the preparation of sulfonated derivatives of bordipyrromethene from chlorosulfonic acid and the original bordipyrromethene is that, in comparison with the large volume of literature on derivatives of bordipyrromethene, there are few such reactions. In [1], it is noted that the number of such reactions is small. That is, the method for producing sulfonated derivatives of boripyrromethene is known from the prior art, however, there is only a few data on derivatives of bordepyrromethene that are really succesful to sulfonation, therefore, the inventive task is both the selection of the reaction sequences leading to the reaction product and the selection of the starting compound, which in principle, succesfully sulfonated. For each specific derivative of bordipyrromethene, it is not possible to predict which conditions will be optimal for sulfonation and whether it will lead to the target product.

.New compounds of formula III are provided, preferably R is hydroxyl (compound IIIa) or N-oxisucinmidyl or

.

Alkali metal salts of the compounds of formula III are proposed for sulfo groups, alkali metals preferably Na and K.

A method for producing a compound of formula IIIa and its alkali metal salts is proposed, characterized in that 2- (4,4-difluoro-1,3,5,7-tetramethyl-4-boro-3a, 4a- diaz-s-indacen-8-yl) -benzoyloxytrifluoroborate; triethylammonium salt is a compound of formula (V).

V

It is proposed the use of compounds of formula III as fluorescent dyes with water solubility, quantum yield, and extinction coefficient higher than is known from the prior art, for use as protein fluorescence labels.

The essence of the method for producing the compound of formula IIIa is that in the first step, an ionic compound of formula V is obtained from o-phthalic anhydride, 2,4-dimethylpyrrole, triethylamine and boron trifluoride etherate.

In the known method for the synthesis of sulfonated borodipyrrmethenes [2-9], chlorosulfonic acid and compounds, which are molecular crystalline or amorphous substances having one boron atom and are zwitterions, are introduced into the sulfonation reaction. In the proposed method, an ionic compound of formula V (salt with triethylamine) containing two boron atoms is introduced into the sulfonation reaction.

An advantage of the described method is that sulfonation with chlorosulfonic acid, by analogy with sulfonation methods described in the literature, leads to the resinification of the product.

The implementation of the invention

Compound V

Acetonitrile was purged with argon for 10 minutes in a round bottom flask, then 2,4-dimethylpyrrole (3.8 g; 40 mmol), phthalic anhydride (2.96 g; 20 mmol) and boron trifluoride etherate (6.3) were added under argon atmosphere. g; 44 mmol). The mixture was stirred under an argon atmosphere for 6 hours, then cooled ^to 0 ° C and boron trifluoride etherate (22.7 g; 160 mmol). The mixture was stirred for 10 minutes at this temperature, then triethylamine (22 g; 220 mmol) was added and the mixture was allowed to stir at room temperature overnight. The mixture was then poured into water (400 ml), stirred for 2 hours and diluted with dichloromethane (200 ml). The organic phase was separated and water was extracted with dichloromethane (3 × 50 ml). The organic phase was dried over sodium sulfate, the solvents were evaporated in vacuo. The product was purified by column chromatography (eluent dichloromethane-ethyl acetate-triethylamine 300: 100: 1) to obtain a dark oil, which crystallized upon stirring in a mixture of ethyl acetate (3 ml) and methyl tert-butyl ether (5 ml). The product yield in the form of an orange powder was 1.6 g (15%). Product structure (Figure 1) was confirmed by NMR spectroscopy, X-ray diffraction analysis.

Picture 1

Table 1

table 2

Table 3

Table 4

Table 5

Data on the crystal structure and structural detail of compound V:

Crystal data for compound V, C ₂₆ H ₃₄ B ₂ F ₅ N ₃ O ₂ , (M = 537.18): monoclinic, space group P2 ₁ / n. Unit cell sizes: a = 10.8302 (8) Å, b = 38.269 (3) Å, c = 13.8846 (11) Å, β = 108.844 (2), V (Volume) = 5446.1 (7) Å ³ , Z = 8, T (temperature) = 120 K, μ (MoKα) (absorption coefficient) = 0.105 mm ^-1 , Dcalc (calculated density) = 1.310 g / cm ³ , Collected / single reflections: 53766/11398, Theta angle range for data collection: 2.128 ≤ 2Θ ≤ 52.744 .. Final indicators R: R ₁ = 0.0541 (I> 2σ (I)), wR ₂ = 0.1053.

¹ H NMR spectrum of compound V (d6-DMSO, δ, ppm, J, Hz): 1.24 (9H, t, CH3CH2); 1.45 (6H, s, 1.7-CH3); 2.48 (6H, s, 3,5-CH3); 3.05 (6H, CH3CH2NH +); 6.11 (2H, 2.6-CH =), 7.44-8.02 (4H, m, -CH- benzene ring)

Synthesis of compound IIIa, sodium salt

A solution of compound V (0.2 g; 0.426 mmol) in dichloromethane (3 ml) was cooled to -50 ° C. Then, a solution of chlorosulfonic acid in dichloromethane was added over 1 minute, the mixture was stirred at this temperature for 20 minutes and the mixture was allowed to warm to room temperature for 2 hours. In this case, a precipitate formed. The liquid from the precipitate was removed by decantation, the precipitate was washed with dichloromethane (2 × 3 ml), then sodium bicarbonate (1 g; 11.3 mmol) was added, followed by water (5 ml). The mixture was stirred for 20 minutes and evaporated to dryness; ethanol and toluene were added before evaporation to avoid foam formation. An orange residue was obtained, which was chromatographed on a silica gel column using a dichloromethane-methanol mixture (1: 1) as an eluent; the residue was dissolved in eluent and the organic salts were filtered. The target product was dried, resulting in an orange powder. The substance was mixed with a mixture of tetrahydrofuran and ethyl acetate in a 1: 1 ratio of 3 ml. The yield was 115 mg (47%). The structure of the product is confirmed by NMR spectroscopy.

¹ H NMR spectrum of compound II (d6-DMSO, δ, ppm, J, Hz): 1.52 (6H, s, 1.7-CH3); 2.65 (6H, s, 3.5-CH3); 7.2-8.1 (4H, m, -CH- benzene ring)

The resulting compound IIIa, the sodium salt has the following properties:

- the compound is a fluorescent dye.

- the compound is characterized by a water solubility of 27 mg / ml. This characteristic is of high importance for use, given that compound I, as a fluorescent dye, is intended for the labeling of protein objects in an aqueous medium.

- the fluorescence spectrum of the compound is in the same range as the spectrum of fluorescein widely used in laboratory practice, due to which the substance can be operated on devices having fixed excitation / radiation wavelengths calculated for fluorescein dyes (Figures 2 and 3).

- the compound has a high quantum yield in water (0.96), which allows you to reliably cut off the background signal of the exciting radiation. This value is higher than the quantum yields for known compounds that are closest in composition and functionality, the maximum quantum yield in any solvent for which is 0.73 for compound IV [11], 0.49 for compound VI [9].

Figure 2

Figure 3

VI

- the extinction coefficient of a substance in water was 8.6 × 10 ⁴ l × mol ^-1 × cm ^-1 , which is approximately 7% higher than the extinction coefficient of compounds IV and VI, known from the prior art [9, 11]. Thus, the characteristics of compound IIIa are extremely precisely aligned with the needs of research in biochemistry and molecular biology.

Example

The use of compound III

as a fluorescent label of protein molecules

Substance IIIa was converted by reaction with O- (N-succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate into the N-hydroxysuccinimide derivative VII, and this derivative was reacted with bovine albumin.

VII

¹ H NMR spectrum of compound VII (d6-DMSO, δ, ppm, J, Hz): 1.42 (6H, s, 1.7-CH3); 2.51 (6H, s, 3.5-CH3); 2.83 (4H, t, -CH2-CH2-), 7.62-8.41 (4H, m, -CH- benzene rings)

Proteins contain amino groups, respectively, their modification is often carried out through activated esters. The speed and completeness of the reaction of activated esters with amino groups substantially depends on the pH of the medium. At low pH values, protonation of amino groups occurs, as a result of which both thermodynamics and reaction kinetics are unsatisfactory. At high values, the reaction is already affected by the hydrolysis of the activated ester. Therefore, the optimal pH in this case is 8.3–8.5.

As a solvent for the modification of bovine albumin, water was used in a mixture with dimethyl sulfoxide (DMSO).

The activated ester for modification was taken in an 8-fold excess relative to the modified amino groups. This is an experimentally selected number to achieve labeling on average for one group per macromolecule.

The protein concentration during labeling was 1 mg per ml. Activated ether was dissolved in pure DMSO (1/10 volume of the reaction mixture).

Bovine albumin was dissolved in pH 8.3 buffer (volume - 9/10 of the reaction mixture). To prepare the buffer, a 0.1 M solution of sodium bicarbonate (NaHCO ₃ ) was used.

A solution of activated ether in DMSO was added to a protein solution in buffer and thoroughly mixed, after which the reaction mixture was kept at 0 ° C for 4 hours. The conjugates were purified by reprecipitation with acetone.

The efficiency of the conjugation of dyes with bovine albumin was evaluated as follows. Since a large number of amino groups is present in the protein, conjugation proceeds randomly, and it is not possible to isolate an individual substance after conjugation. Therefore, the conjugation efficiency is evaluated by comparing the absorption of the dye at 500 nm (the absorption wavelength has shifted compared to the dye before conjugation) and protein at 280 nm, given that the extinction coefficients of the protein and dye at this wavelength are known (about 8.6 × 10 ⁴ l × mol ^-1 × cm ^-1 and 4.3 × 10 ⁴ l × mol ^-1 × cm ^-1, respectively). An example of such a spectrum is shown in Figure 4. Analysis of the spectra for dyes showed that the modification efficiency was 1-1.2 dye molecules on average per protein molecule.

Bibliography

1. Loudet A., Burgess K. Chem. Rev. 2007, 107, 4891-4932

2. Shah, M .; Thangaraj, K .; Soong, M.-L .; Wolford, L. T .; Boyer, J. H .; Politzer, I. R .; Pavlopoulos, T. G. Heteroat. Chem. 1990, 1, 389.

3. Wories, H. J .; Koek, J. H .; Lodder, G .; Lugtenburg, J .; Fokkens, R .; Driessen, O .; Mohn, G. R. Recl. TraV. Chim. Pays-Bas 1985, 104, 288.

4. Morgan, L. R .; Boyer, J. H. Boron difluoride compounds useful in photodynamic therapy and production of laser light. U.S. Patent 5446157, 1995.

5. Morgan, L. R .; Boyer, J. H. Heterocyclic compounds and their use in photodynamic therapy. WO Patent 9419355, February 18, 1994.

6. Urano, T .; Nagasaka, H .; Tsuchiyama, M .; Ide, H. Photopolymerizable composition. U.S. Patent 5498641, April 7, 1994.

7. Boyer, J. H .; Morgan, L. R. Preparation of difluoroboradiaza-s-indacene compounds and methods for using them. U.S. Patent 5189029, 1993.

8. Boyer, J. H .; Morgan, L. R. Fluorescent chemical compositions useful as laser dyes and photodynamic therapy agents, and methods for their use. U.S. Patent 361936, 1990

9. Li L., Han J., Nguyen B., and Burgess K. J. Org. Chem. 2008, 73, 1963-1970

10. Haugland R.P., Kang H.C., Chemically reactive dipyrrometheneboron difluoride dyes, US Patent 4,774,339 A, 1988

11. Wang D., Fan J., Gao X., Wang B., Sun S., Peng X., J. Org. Chem. 2009, 74, 7675–7683

Table 1. Fractional atomic coordinates (× 10 ⁴ ) and equivalent isotropic atomic displacement parameters (Å ² × 10 ³ ) for the compound V, U _eq , defined as 1/3 of the trace of the orthogonalized tensor U _IJ . Atom x y z U (eq) F1 5030.5 (15) 4658.9 (4) 4325.1 (11) 30.3 (4) F2 4358.0 (15) 4101.0 (4) 3944.1 (12) 30.0 (4) F4 9,768.1 (15) 5442.0 (4) 3205.1 (12) 32.2 (4) F7 8570.4 (18) 5667.7 (4) 1681.1 (13) 44.4 (5) F10 10,702.2 (18) 5524.6 (5) 1976.3 (14) 51.4 (5) O1 9220.1 (18) 5079,2 (5) 1736.3 (14) 27.8 (5) O2 7534.6 (17) 4993.8 (5) 2321.0 (14) 24.7 (5) N1 4514 (2) 4472.0 (5) 2579.7 (17) 20.8 (5) N2 6505 (2) 4259.9 (5) 3912.8 (16) 20.3 (5) C1 7049 (3) 4155.4 (8) 5777 (2) 32.2 (8) C2 3297 (3) 4,590.3 (7) 2088 (2) 25.8 (7) C3 3148 (3) 4637.5 (7) 1057 (2) 27.6 (7) C4 4298 (3) 4545.0 (7) 896 (2) 22.1 (7) C5 5175 (3) 4442.5 (7) 1863 (2) 19.9 (6) C6 6471 (3) 4,336.7 (6) 2167 (2) 18.4 (6) C7 7139 (3) 4243.3 (6) 3176 (2) 18.1 (6) C8 8428 (3) 4122.5 (7) 3658 (2) 22.2 (7) C9 8548 (3) 4070.7 (7) 4668 (2) 24.9 (7) C10 7366 (3) 4156.9 (7) 4812 (2) 24.9 (7) C12 2318 (3) 4654.9 (8) 2616 (2) 38.3 (8) C13 4525 (3) 4552.6 (7) -113 (2) 26.4 (7) C14 7150 (3) 4,308.0 (7) 1388 (2) 19.4 (6) C15 7992 (3) 4566.0 (7) 1234 (2) 19.3 (7) C16 8566 (3) 4515.3 (7) 483 (2) 27.2 (7) C17 8334 (3) 4217.6 (8) -109 (2) 32.4 (8) C18 7514 (3) 3962.1 (8) 48 (2) 30.4 (8) C19 6936 (3) 4008.3 (7) 792 (2) 24.3 (7) C20 8224 (3) 4901.7 (7) 1818 (2) 19.9 (7) C21 9486 (3) 4052.5 (8) 3204 (2) 32.3 (8) B1 5079 (3) 4,372.7 (8) 3717 (3) 23.2 (8) B2 9568 (4) 5438.4 (9) 2167 (3) 28.3 (9) N3 7191 (2) 5516.7 (6) 3662.2 (17) 21.7 (6) C23 7338 (3) 5892.6 (7) 3394 (2) 27.5 (7) C24 6991 (3) 6154.8 (7) 4084 (2) 40.1 (9) C25 5816 (3) 5414.5 (8) 3573 (2) 28.1 (7) C26 4859 (3) 5470.4 (8) 2526 (2) 33.2 (8) C27 8129 (3) 5428.4 (7) 4701 (2) 27.2 (7) C28 8235 (3) 5039.4 (7) 4911 (2) 34.8 (8)

Table 2. Anisotropic bias parameters (Å ² × 10 ³ ) for compound V. The formula for the anisotropic bias factor is as follows: -2π ² [h ² a * ² U ₁₁ + 2hka * b * U ₁₂ + ...]. Atom U ₁₁ U ₂₂ U ₃₃ U ₂₃ U ₁₃ U ₁₂ F1 37.7 (11) 28.9 (10) 25.7 (10) -3.2 (8) 12.3 (8) 2.1 (8) F2 29.3 (10) 27.7 (9) 36.7 (10) 4.3 (8) 15.8 (8) -3.9 (8) F4 34.4 (10) 35.4 (10) 24.4 (10) 0.3 (8) 6.1 (8) -3.4 (8) F7 59.9 (13) 23.1 (10) 38.1 (11) 6.3 (8) -0.8 (10) 1.2 (9) F10 56.6 (13) 50.6 (12) 59.1 (13) -15 (1) 35.6 (11) -29.2 (10) O1 26.6 (12) 23.6 (11) 37.6 (13) -2.6 (10) 16.5 (10) -5.4 (10) O2 27.8 (12) 23.1 (11) 26.5 (12) -3.4 (9) 13.2 (10) -1.3 (9) N1 19.9 (13) 21.5 (13) 22.5 (14) 1.3 (11) 9.2 (11) 0.7 (11) N2 24.3 (14) 19.4 (13) 16.8 (13) 1.4 (10) 6.0 (12) -2.0 (11) C1 37.0 (19) 36.9 (19) 23.4 (18) 5.4 (15) 10.8 (16) -0.8 (16) C2 24.2 (18) 23.4 (17) 30.1 (19) 0.4 (14) 9.3 (15) 2.1 (14) C3 24.7 (18) 26.5 (17) 28.2 (19) 5.4 (14) 3.9 (15) 2.3 (14) C4 24.7 (17) 14.9 (15) 26.1 (18) -0.7 (13) 7.5 (14) -2.2 (13) C5 23.7 (17) 18.6 (15) 17.5 (16) -2.6 (13) 6.9 (14) -2.3 (13) C6 25.4 (17) 9.4 (14) 20.1 (17) -4.2 (12) 7.0 (14) -6.3 (13) C7 22.0 (16) 15.0 (15) 16.0 (16) -0.7 (12) 4.4 (14) -2.8 (13) C8 22.3 (17) 17.0 (15) 27.0 (18) 0.4 (13) 7.7 (14) -4.2 (13) C9 22.4 (17) 22.2 (16) 24.9 (18) 5.8 (13) 0.2 (14) -0.7 (14) C10 30.0 (18) 21.8 (17) 20.9 (18) 2.0 (13) 5.5 (15) -5.8 (14) C12 31.9 (19) 46 (2) 40 (2) 3.6 (17) 16.5 (17) 6.7 (16) C13 27.8 (18) 31.2 (18) 18.0 (17) 0.6 (14) 4.5 (14) 0.8 (14) C14 19.6 (16) 18.9 (15) 18.0 (16) 2.0 (13) 3.5 (13) 4.1 (13) C15 19.6 (16) 19.1 (15) 19.1 (16) 2.1 (13) 6.2 (13) 4.0 (13) C16 31.6 (19) 22.1 (17) 31.5 (19) 3.3 (14) 15.5 (16) 1.2 (14) C17 42 (2) 32.2 (19) 31 (2) -3.3 (15) 22.8 (17) 4.4 (16) C18 40 (2) 26.6 (18) 25.3 (18) -5.0 (14) 12.0 (16) 0.5 (15) C19 27.2 (17) 23.4 (17) 22.1 (17) -1.1 (13) 7.9 (14) -2.1 (14) C20 21.9 (17) 19.3 (16) 17.3 (17) 5.7 (13) 4.5 (14) 2.9 (13) C21 25.4 (18) 36.6 (19) 31.6 (19) 3.3 (15) 4.8 (15) 4.3 (15) B1 30 (2) 19.2 (18) 23 (2) 2.2 (15) 11.4 (17) 0.2 (16) B2 36 (2) 23 (2) 29 (2) 0.3 (17) 13.7 (19) -7.6 (18) N3 27.1 (15) 18.9 (13) 20.1 (14) 0.4 (11) 9.1 (12) -0.5 (11) C23 35.2 (19) 17.7 (16) 29.5 (18) 3.7 (14) 10.1 (15) -3.0 (14) C24 61 (2) 23.0 (18) 32 (2) -2.5 (15) 9.2 (18) 3.7 (17) C25 27.1 (18) 29.5 (18) 27.9 (18) 1.0 (14) 9.2 (15) -4.1 (14) C26 31.2 (19) 32.8 (19) 33 (2) 0.9 (15) 6.9 (16) -0.5 (15) C27 30.6 (18) 28.9 (17) 19.4 (17) 3.5 (14) 4.4 (14) -0.5 (15) C28 40 (2) 27.1 (18) 31 (2) 6.6 (15) 3.9 (16) 6.1 (16)

Table 3. The bond lengths in compound V Atom Atom Length / Å F1 B1 1,394 (3) F2 B1 1,396 (3) F4 B2 1,386 (4) F7 B2 1,387 (4) F10 B2 1,377 (4) O1 C20 1,310 (3) O1 B2 1,498 (4) O2 C20 1,227 (3) N1 C2 1.352 (3) N1 C5 1,406 (3) N1 B1 1,545 (4) N2 C7 1,405 (3) N2 C10 1.353 (3) N2 B1 1,541 (4) C1 C10 1,487 (4) C2 C3 1,400 (4) C2 C12 1,492 (4) C3 C4 1.381 (4) C4 C5 1,427 (4) C4 C13 1,498 (4) C5 C6 1.389 (4) C6 C7 1,399 (3) C6 C14 1,495 (4) C7 C8 1,417 (4) C8 C9 1.381 (4) C8 C21 1,500 (4) C9 C10 1,396 (4) C14 C15 1,407 (4) C14 C19 1.389 (4) C15 C16 1,390 (4) C15 C20 1.497 (4) C16 C17 1,379 (4) C17 C18 1.385 (4) C18 C19 1.381 (4)

Table 4. The bond angles in compound V Atom Atom Atom Angle C20 O1 B2 123.3 (2) C2 N1 C5 107.8 (2) C2 N1 B1 126.7 (2) C5 N1 B1 125.4 (2) C7 N2 B1 125.6 (2) C10 N2 C7 107.7 (2) C10 N2 B1 126.7 (2) N1 C2 C3 109.3 (2) N1 C2 C12 122.7 (3) C3 C2 C12 128.0 (3) C4 C3 C2 108.7 (3) C3 C4 C5 106.2 (2) C3 C4 C13 125.1 (3) C5 C4 C13 128.6 (3) N1 C5 C4 107.9 (2) C6 C5 N1 120.2 (2) C6 C5 C4 131.8 (3) C5 C6 C7 121.6 (2) C5 C6 C14 119.1 (2) C7 C6 C14 119.3 (2) N2 C7 C8 108.0 (2) C6 C7 N2 120.1 (2) C6 C7 C8 131.9 (3) C7 C8 C21 129.1 (3) C9 C8 C7 106.3 (2) C9 C8 C21 124.6 (3) C8 C9 C10 108.7 (2) N2 C10 C1 122.7 (3) N2 C10 C9 109.1 (2) C9 C10 C1 128.1 (3) C15 C14 C6 123.6 (2) C19 C14 C6 117.7 (2) C19 C14 C15 118.7 (3) C14 C15 C20 121.6 (2) C16 C15 C14 119.1 (2) C16 C15 C20 119.3 (2) C17 C16 C15 121.5 (3) C16 C17 C18 119.5 (3) C19 C18 C17 119.7 (3) C18 C19 C14 121.5 (3) O1 C20 C15 113.4 (2) O2 C20 O1 124.7 (2) O2 C20 C15 121.9 (2) F1 B1 F2 108.4 (2) F1 B1 N1 110.2 (2) F1 B1 N2 110.2 (2) F2 B1 N1 110.4 (2) F2 B1 N2 110.7 (2) N2 B1 N1 107.0 (2) F4 B2 F7 109.6 (3) F4 B2 O1 111.0 (2) F7 B2 O1 109.3 (3) F10 B2 F4 110.4 (3) F10 B2 F7 111.0 (3) F10 B2 O1 105.5 (2)

Table 5. Torsion angles for compound V A B C D Angle N1 C2 C3 C4 0.3 (3) N1 C5 C6 C7 2.6 (4) N1 C5 C6 C14 -179.9 (2) N2 C7 C8 C9 -0.3 (3) N2 C7 C8 C21 178.5 (3) C2 N1 C5 C4 -0.7 (3) C2 N1 C5 C6 177.9 (2) C2 N1 B1 F1 -59.2 (4) C2 N1 B1 F2 60.5 (4) C2 N1 B1 N2 -179.0 (2) C2 C3 C4 C5 -0.7 (3) C2 C3 C4 C13 178.8 (3) C3 C4 C5 N1 0.8 (3) C3 C4 C5 C6 -177.5 (3) C4 C5 C6 C7 -179.3 (3) C4 C5 C6 C14 -1.8 (4) C5 N1 C2 C3 0.3 (3) C5 N1 C2 C12 -179.7 (3) C5 N1 B1 F1 123.2 (3) C5 N1 B1 F2 -117.1 (3) C5 N1 B1 N2 3.4 (4) C5 C6 C7 N2 -0.9 (4) C5 C6 C7 C8 178.5 (3) C5 C6 C14 C15 99.6 (3) C5 C6 C14 C19 -80.8 (3) C6 C7 C8 C9 -179.8 (3) C6 C7 C8 C21 -0.9 (5) C6 C14 C15 C16 -179.5 (3) C6 C14 C15 C20 -2.9 (4) C6 C14 C19 C18 179.4 (3) C7 N2 C10 C1 177.4 (2) C7 N2 C10 C9 -0.5 (3) C7 N2 B1 F1 -121.5 (3) C7 N2 B1 F2 118.7 (3) C7 N2 B1 N1 -1.6 (3) C7 C6 C14 C15 -82.9 (3) C7 C6 C14 C19 96.7 (3) C7 C8 C9 C10 0,0 (3) C8 C9 C10 N2 0.3 (3) C8 C9 C10 C1 -177.5 (3) C10 N2 C7 C6 -179.9 (2) C10 N2 C7 C8 0.5 (3) C10 N2 B1 F1 59.1 (3) C10 N2 B1 F2 -60.7 (4) C10 N2 B1 N1 179.0 (2) C12 C2 C3 C4 -179.8 (3) C13 C4 C5 N1 -178.7 (2) C13 C4 C5 C6 3.0 (5) C14 C6 C7 N2 -178.3 (2) C14 C6 C7 C8 1.1 (4) C14 C15 C16 C17 -0.3 (4) C14 C15 C20 O1 166.9 (2) C14 C15 C20 O2 -12.9 (4) C15 C14 C19 C18 -1.0 (4) C15 C16 C17 C18 -0.2 (4) C16 C15 C20 O1 -16.5 (4) C16 C15 C20 O2 163.7 (3) C16 C17 C18 C19 0.1 (4) C17 C18 C19 C14 0.5 (4) C19 C14 C15 C16 0.9 (4) C19 C14 C15 C20 177.5 (2) C20 O1 B2 F4 56.9 (4) C20 O1 B2 F7 -64.1 (3) C20 O1 B2 F10 176.5 (2) C20 C15 C16 C17 -177.0 (3) C21 C8 C9 C10 -178.9 (3) B1 N1 C2 C3 -177.7 (2) B1 N1 C2 C12 2.4 (4) B1 N1 C5 C4 177.3 (2) B1 N1 C5 C6 -4.2 (4) B1 N2 C7 C6 0.5 (4) B1 N2 C7 C8 -179.0 (2) B1 N2 C10 C1 -3.1 (4) B1 N2 C10 C9 179.0 (2) B2 O1 C20 O2 -6.3 (4) B2 O1 C20 C15 173.9 (2)

Claims (6)

1. Salts of the compounds of formula I with alkali metals replacing hydrogen atoms in both sulfo groups

, where R is an N-oxysuccinimidyl group

2. The method of producing salts according to claim 1, including the preparation of 2- (4,4-difluoro-1,3,5,7-tetramethyl- (from o-phthalic anhydride, 2,4-dimethylpyrrole, triethylamine and boron trifluoride etherate) 4-boro-3a, 4a-diaza-s-indacen-8-yl) benzoyloxytrifluoroborate, a triethylammonium salt, which is then introduced into the sulfonation reaction followed by the addition of an alkali metal bicarbonate, followed by a reaction with O- (N-succinimidyl) - 1,1,3,3-tetramethyluronium tetrafluoroborate. 3. The use of salts according to claim 1 for fluorescent labeling of protein molecules.

Images