RU2790836C2 - Europium complex of monosodium salt of 2,2',2",2'"-(2,2'-((4-(4-aminophenyl)-2,2'-bipyridine-6-yl)methylazadiyl)bis-(ethane-2,1-diyl))-bis(azatriyl)tetraacetic acid - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of luminescent europium complex compounds, namely to a europium complex of monosodium salt of 2,2',2",2'"-(2,2'-((4-(4-aminophenyl)-2,2'-bipyridine-6-yl)methylazadiyl)bis-(ethane-2,1-diyl))-bis(azatriyl)tetraacetic acid of the formula 1.

EFFECT: invention can be used as a luminescent mark for immune analysis; it can be used in medicine.

1 cl, 4 dwg, 2 tbl

Description

1. The field of technology to which the invention relates.

The invention relates to the field of luminescent complex compounds of europium with a heterocyclic 2,2'-bipyridine ligand 1 with a chelating fragment based on diethylenediaminotetraacetic acid (DTTA) and an amino group suitable for creating a peptide bioconjugation bridge (Fig. 1). The invention can be used as a luminescent label for immunoanalysis and can be used in medicine, as well as in research laboratories.

2. State of the art.

Pyridine derivatives and their annelated analogs are fairly well known ligands for complexation with various metal cations. Their complexes with lanthanides such as Sm ³⁺ , Dy ³⁺ , Pr ³⁺ , Ho ³⁺ , Yb ³⁺ , Nd ³⁺ , Er ³⁺ have acquired the greatest practical importance. Such compounds exhibit interesting magnetic properties (GM Davies, SJA Pope, H. Adams, S. Faulkner, MD Ward, Inorg. Chem. 44 (2005) 4656. DOI:10.1021/ic050512k) and are also capable of luminescence in the IR range. (S. Quici, M. Cavazzini, G. Marzanni, G. Accorsi, N. Armaroli, B. Ventura, F. Barigelletti, Inorg. Chem. 44 (2005) 529. DOI:10.1021/ic0486466) and in the visible part of the spectrum (L. Armelao, S. Quici, F. Barigelletti, G. Accorsi, G. Bottaro, M. Cavazzini, E. Tondello, Coord. Chem. Rev. 254 (2009) 487. DOI:10.1016/j.ccr.2009.07 .025), which makes their use in magnetic resonance therapy possible (S. Aime, SG Crich, E. Gianolio, GB Giovenzana, L. Tei, E. Terreno, Coord. Chem. Rev. 250 (2006) 1562. DOI :10.1016/S1002-0721(13)60022-8), as well as in the field of telecommunications (Y. Oshishi, T. Kanamori, T. Kitagawa, S. Takashashi, E. Snitzer, GH Jr. Sigel, Opt. Lett. 16 (1991) 1747. DOI:10.1364/OL.16.001747) and as immunoassay labels (J. Yan, G. Wang, Trends Anal. Chem. 25 (2006) 4 90. DOI:10.1016/j.trac.2005.11.013). In the latter case, successful application requires the presence in the composition of such compounds of a linker for binding to biological molecules of the body, which can be such functional groups as carboxyl (SP Martsev, VA Preygerzon, YI Mel'nikova, ZI Kravchuk, GV Ponomarev, VE Lunev, A. P. Savitsky, J. Immunol Methods 186 (1995) 293. DOI:10.1016/0022-1759(95)00154-3), aldehyde (S. Poupart, C. Boudon, P. Peixoto, M. Massonneau, P.-Y. Renard, A. Romieu, Org. Biomol. Chem. 4 (2006) 4165. DOI:10.1039/B612805J), sulfochloride (A. Chan, EP Diamandis, M. Krajden, Anal. Chem. 2 (1993 ) 158. DOI:10.1021/ac00050a012), and some others, such as the amino group (N. Meltola, P. Jauria, P. Saviranta, H. Mikola, Bioconjugate Chem. 10 (1999) 325. DOI:10.1021/bc980013q). If the latter is present in the composition of the ligand, it is possible both its direct binding to the carboxyl group of the biomolecule and its transformation into isothiocyanate by reaction with thiophosgene, which makes it possible to obtain a more convenient linker in use, which usually smoothly and selectively reacts with the primary amino groups of biological molecules to form thiourea derivatives (H. Takalo, VM Mukkala, H. Mikola, P. Liitti, I. Hemmila, Bioconjugate Chem. 5 (1994) 278. DOI:10.1021/bc00027a015).

In addition, in order to avoid dissipation of the energy of the excited state of lanthanide by transferring it to solvent molecules, it is necessary to saturate its coordination sphere. Since the coordination number of the lanthanides is nine, the ligand must be nine-dentate. This is achieved by introducing additional chelating groups into its structure, which are used as residues of polyaminoacetic acids, such as IDA, DO3A and DTTA (A. M. Prokhorov, V. N. Kozhevnikov, D. S. Kopchuk, H. Bernard, N. Le Bris, R. Tripier, H Handel, B. Koenig, D. N. Kozhevnikov, Tetrahedron 67 (2011) 597. DOI:10.1016/j.tet.2010.11.058).

The ligands presented in the literature that meet all the above requirements are based on the use of 2,2':6',2''-terpyridines as a chromophore (Z. Wang, J. Yan, K. Matsumoto, Luminescence 20 (2005) 347. DOI :10.1002/bio.843), as well as pyridine functionalized at the C4 position (H. Takalo, P. Pasanen, J. Kankare, Acta Chem. Scand. B 42 (1988) 373. DOI:10.3891/acta.chem.scand .42b-0373), or conjugated with five-membered nitrogen-containing heterocycles (J. Yuan, G. Wang, K. Majima, K. Matsumoto, Anal. Chem. 73 (2001) 1869. DOI:10.1021/ac0013305).

Ligands based on the use of 2,2′-bipyridines as a chromophore are much less common. To date, only two examples of such ligands have been presented in the literature (ligands2 And3, Fig. 2) suitable for protein bioconjugation (A. M. Prokhorov, V. N. Kozhevnikov, D. S. Kopchuk, H. Bernard, N. Le Bris, R. Tripier, H. Handel, B. Koenig, D. N. Kozhevnikov, Tetrahedron 67 (2011) 597. DOI :10.1016/j.tet.2010.11.058). The structure of only one of them, the ligand2, ensures saturation of the europium coordination sphere, which is ensured by the presence of a DTTA residue in the α-position to the nitrogen atom. Its europium complex showed a rather high excited state lifetime (0.60 ms) with a low photoluminescence quantum yield (0.6%). ligand2 (Fig. 2), as a connection with the most matching features was chosen by us as a prototype.

3. The essence of the invention.

The essence of the invention is a water-soluble europium complex 1 of the ligand of the 2,2'-bipyridine series 4 (Fig. 1), the structure of which ensures saturation of the europium coordination sphere and can be suitable for bioconjugation with a protein (H. Takalo, VM Mukkala, H. Mikola, P Liitti, I. Hemmila, Bioconjugate Chem 5 (1994) 278. DOI:10.1021/bc00027a015), characterized by a twofold increase in quantum yield, the position of the amino group in the aminophenyl substituent ( para - instead of meta -), the position of the aminophenyl substituent (there is no in the 5′, and in the 4 position of 2,2′-bipyridine), an increased lifetime of the excited state.

The present invention demonstrates improved photophysical properties compared to the prototype, namely: a twofold increase in the quantum yield (from 0.6% to 1.2%) while increasing the excited state lifetime (from 0.6 ms to 1.06 ms), which makes it more suitable for practical applications .

4. Information confirming the possibility of carrying out the invention.

4.1. To confirm the possibility of carrying out the invention, a method for obtaining compound 1 is given.

The synthesis scheme is shown graphically in Fig. 3.

Synthesis of tetraether 5.

Dissolved tetraether6(255 mg, 0.3 mmol) synthesized according to the described method (A. P. Krinochkin, D. S. Kopchuk, G. A. Kim, I. N. Ganebnykh, I. S. Kovalev, S. Santra, G. V. Zyryanov, A. Majee, V. L. Rusinov, O. N. Chupakhin, ChemistrySelect 4 (2019) 6377) in methanol (50 ml), catalytic palladium (10%) on carbon (25 mg), reduced with hydrogen, for 8 hours at room temperature. The reaction mass was evaporated in vacuum. The resulting residue was purified by column chromatography, eluent - methylene chloride, then - ethyl acetate. The eluates containing the product were evaporated in vacuo.

Tetra- tert -butyl 2,2',2'',2'''-(((((4-(4-aminophenyl)-[2,2'-bipyridin]-6-yl)methyl)azadiyl)bis( ethane-2,1-diyl)) bis(azatriyl)) tetraacetate("tetraether5"). Dark yellow oil. Yield 146 mg (0.18 mol, 59%).NMR ¹ H (CDCl₃, δ, ppm): 1.39 (s, 36H, Bu^t), 3.31-3.50 (m, 12H, NCH ₂, CH ₂COOBu^t), 3.70-3.83 (m, 4H, NCH₂), 5.17 (s, 2H, PyCH ₂), 6.78 (m, 2H, CH_aroma), 7.34 (m, 1H, H-5'(Py)), 7.70 (m, 2H, CH_aroma), 7.81 (t, 1H,³ J 7.6 Hz, H-4′(Py)), 7.89 (d, 1H,⁴ J 1.2 Hz CH_aroma(Py)), 8.58 (d, 1H,³ J 7.2 Hz, H-3′(Py)), 8.67 (d, 1H,⁴ J 1.2 Hz CH_aroma(Py)), 8.70 (d, 1H,³ J4.8 Hz, H-6'(Py)).ESI-MS,m/z: found 819.51, calculated 819.50 (M+H)⁺.

Obtaining a ligand 4.

Tetraester 5 (164 mg, 0.2 mmol) was dissolved in a mixture of 5 ml of 11N hydrochloric acid and 5 ml of water and kept at room temperature for 8 hours. The reaction mass was evaporated in a vacuum, 10 ml of 11N hydrochloric acid was added to the residue, the mixture was kept at room temperature for 2 hours, evaporated in a vacuum, the residue was treated with dry acetonitrile (25 ml), the resulting mixture was kept at room temperature for 8 hours. The precipitate was filtered off, dried in a vacuum.

Hydrochloride (2,2',2'',2'''-(2,2'-((4-(4-Aminophenyl)-2,2'-bipyridin-6-yl)methyl-azadiyl)bis-( ethane-2,1-diyl)) bis(azatriyl)tetraacetic acid("ligand4"). Yield 134 mg (0.16 mmol, 78%). Found, %: C 40.57, H 5.20, N 9.73, Cl 22.95. Calculated for C₂₉H₃₄N₆O₈•11/2HCl•7/2H₂O: C 40.59, H 5.46, N 9.79, Cl 22.72.

Obtaining the europium complex 1.

Ligand 4 (43 mg, 0.05 mmol) was dissolved in 10 mL of water, and the calculated amount of sodium hydroxide (19 mg, 0.48 mmol) was added to the solution. Then EuCl ₃ •6H ₂ O (18 mg, 0.05 mmol) was added to the solution and stirred for 2 h at room temperature. The solvent was distilled off in vacuo, the residue was extracted with hot methanol (3×20 ml), the extract was evaporated in vacuo.

1 ("complex"). Yield 26 mg (0.030 mmol, 61%). Found, %: C 40.60, H 4.79, N 9.94. Calculated for C ₂₉ H ₃₀ EuN ₆ NaO ₈ •5H2O: C 40.71, H 4.71, N 9.82. ESI-MS , m/z : found 743.13, calculated 743.12 [M-Na] ^-- .

The claimed compound is a colorless powdery substance, soluble in methanol and water, insoluble in chloroform, 1,2-dichloroethane and toluene.

4.2. Confirmation of the saturation of the europium coordination sphere.

Table 1. Coordination properties of the europium complex 1 .

τ _H2O , ms τ _D2O , ms q 1.06 1.0 -0.29

τ -the lifetime of the excited state;q is the parameter corresponding to the number of water molecules in the europium coordination sphere,q = 1.2*(1/τ_H2O – 1/τ_D2O – 0.25)

To measure the lifetime of europium photoluminescence, a Varian Cary Eclipse spectrofluorometer with a xenon flash lamp was used, the lamp flash time was 0.001 ms. 5 parallels of 50 measurements were obtained, the results were averaged. Measurements in H ₂ O and D ₂ O were carried out under the same conditions.

As follows from the results of measuring the lifetimes of the excited state of europium in water and heavy water, given in Table. 1 and the corresponding calculations made in accordance with the formula (A. Beeby, IM Clarkson, RS Dickins, S. Faulkner, D. Parker, L. Royle, AS De Sousa, JAG Williams, M. Woods, J. Chem. Soc ., Perkin Trans. 2 3 (1999) 493. DOI:10.1039/A808692C) in the coordination sphere of europium chelated with 4 ligand, there are no water molecules (the denticity of the ligand is 9), i.e., the structure of the latter excludes the nonradiative relaxation of the energy of the excited state of europium.

4.3. Measurement of the relative quantum yield.

Table 2. Photophysical properties of the europium complex 1 .

Absorption, λ _max , nm (ε, 10 ^-3 M ^-1 cm ^-1 ) _ΦEM 245, 314 (8.70) 1.2

Ф - quantum yield of photoluminescence

Electronic absorption spectra were recorded using the standard Shimadzu Scan program on a two-beam UV-2600 spectrophotometer (Shimadzu, Japan) in the range of 190–700 nm with a wavelength accuracy of ±0.1 nm.

Luminescence spectra were recorded on a Varian Cary Eclipse spectrofluorimeter with mutually perpendicular beams, the wavelength accuracy was 0.5 nm. The measurements were carried out in the range from 190 to 800 nm in SUPRASIL 111-QS 10 quartz cuvettes (Hellma, Germany), the width of the zone around the stationary point of excitation/emission is 10 nm. The wavelength of the stationary point of excitation was assigned each time to the maximum in the absorption and emission spectra, the wavelength of the stationary point of emission was set to the maximum in the excitation spectra. The PMT voltage was chosen based on the luminescent response during test registration. The spectra took into account the luminescence of solvents.

The relative quantum yields of compound solutions were measured at 22+1°C using the method available at www.jyhoriba.co.uk (Jobin Yvon Ltd. 2 Dalston Gardens, Stanmore, Middlesex HA7 1BQ UK). The reference sample was an aqueous solution of the ruthenium complex [Ru(bpy) ₃ ]Cl ₂ ·3H ₂ O. To measure the quantum yield, at least 4 parallels were used with averaging the results, R ² ≥0.99. Absorption at the point of excitation did not exceed 0.05 Abs to exclude reabsorption.

As follows from the data (Fig. 4 and Table 2), the absorption maximum of the europium complex is in the region of 314 nm, the compound exhibits a characteristic europium photoluminescence with a maximum emission wavelength of 613 nm (red color). The quantum yield of europium photoluminescence is 1.2%.

Claims (2)

Europium complex of monosodium salt (2,2',2'',2'''-(2,2'-((4-(4-aminophenyl)-2,2'-bipyridin-6-yl)methylazadiyl)bis (ethane-2,1-diyl))-bis(azatriyl)tetraacetic acid of formula 1 - luminescent dye:

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