RU2506293C2 - Diad compounds containing azo groups and ferrocene nucleus in molecule, and use thereof as fluorescence extinguishers - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing novel diad compounds (I) having two different chromophoric fragments that are not coupled to each other and contain azo groups and ferrocene residues, and use thereof to extinguish fluorescence of fluorophores (I)

where Fc is ferrocenyl; R₁-H or Fc;R₂ is H or ortho- or para-hydroxy-; R₃ is ortho- or meta-, or para-nitro-, or ortho- or meta-, or para-nitrophenylazo-, or para-N,N-dimethylamino-, or para-carboxy-; L is para-carbamoyl vinylidene acetophenone or para-carboxamidovinylidene acetophenone, or para-N-(2-carbamoylethyl)-carboxamidovinylidene acetophenone, or para-(4-[methylamino] butoxy)-vinylidene acetophenone, or N,N-di[4{1-(para-vinylidene acetophenylamino)-methyl-1,2,3-triazolyl}butyl]amino group. The method of producing (I) involves aldol-crotonic condensation of ferrocene aldehyde with para-substituted acetophenone and reacting the obtained ferrocenylidene acetophenone (2) with an azo compound, or adding to (2) reactive groups and azo coupling with a diazo salt. Effectiveness of (I) in extinguishing fluorophores in a solution and in a composition of nucleic acids in a wide spectral range is shown, which enables to use (I) to label biological macromolecules and design oligonucleotide hybridisation probes for molecular diagnosis methods.

EFFECT: diad compounds (I) have high molar extinction coefficients, a wide absorption spectrum and electroactivity; also described is a method of extinguishing fluorescence of fluorophores in double-stranded structures of nucleic acids using (I).

2 cl, 8 dwg, 2 tbl, 8 ex

Description

The invention relates to a method for the preparation and use as fluorescence quenchers of complex dyad substances having two functional parts in their molecular structure, of the general formula (I):

Where

Fc is a ferrocenyl radical of the following structure:

R ₁ is a hydrogen atom, or Fc,

R ₂ is a hydrogen atom, or ortho or para-hydroxyl,

R ₃ is an ortho or meta or para-nitro group, or an ortho or meta or para-nitrophenyl azo group, or a para-N, N-dimethylamino group, or a para-carboxy group,

L is a para-carbamoyl vinylidene acetophenone group, or a para-carboxamidinyl vinylidene acetophenone group, or a para-N- (2-carbamoylethyl) -carboxamidinyl vinylidene-acetophenone group, or a para- (4- [methylamino] butoxy) -vinylidene acetophenone group, or {1- (para-vinylideneacetophenylamino) -butyl-1,2,3-triazolyl} methyl] amino group.

The combination of two functional parts in the molecules: azo groups (one or more) and ferrocene residues (one or several), which are chromophores, provides unique photophysical and photochemical properties of these substances: high molar extinction coefficients, a wide absorption spectrum, which is the algebraic sum of absorption spectra components (azo dye and ferrocene), electroactivity. The proposed compounds are capable of quenching the fluorescence of a large number of fluorophores in a wide spectral range and can be used for labeling various molecules, including biological ones, giving them unusual properties and allowing the analysis of the same labeled sample using different physicochemical methods of analysis simultaneously, for example photometric and electrochemical.

Description of drawings

Figure 1. The synthesis scheme of the dyad FcD1.

Figure 2. The synthesis scheme of the FcD2 dyad.

Figure 3. The synthesis scheme of the FcD3 dyad.

Figure 4. The synthesis scheme of the FcD4 dyad.

Figure 5. The synthesis scheme of the FcD5 dyad.

Table 1. Absorption bands and dynamic quenching quenching constants of dyads.

6. Scheme for the synthesis of labeled nucleic acids.

Table 2. Used oligonucleotides.

7. The structure of oligonucleotide duplexes.

Fig. 8. Melting profiles of oligonucleotide duplex structures.

Description of the invention

Fluorescence analysis methods are widely used in scientific research and in practice. At present, supersensitive methods have been proposed that make it possible to operate with single molecules by trapping individual quanta of light radiation. Their modern versions are very flexible and allow real-time analysis using special molecular systems that provide switching of the fluorescence intensity and give a fluorescence signal due to the processes of intra- and intermolecular energy transfer. Several mechanisms of fluorescence energy transfer are known, but molecular systems with resonant fluorescence energy transfer (FRET) are mainly used [1, 2]. FRET is based on the transfer of excitation energy between two molecules or photoactive groups, which act as a donor and acceptor (quencher) of energy, respectively.

The most used quenchers in bioanalytical applications are azo compounds (azo dyes): Dabcyl, groups BHQ [3, 4], RTQ [5], LQ [6], QSY [7] and several others [8]. In recent years, compound quenchers ("super quenchers") have been created to interact with a single fluorophore, which leads to greater quenching efficiency and increases the sensitivity and specificity of the analysis. For example, the quenching efficiency of the FAM fluorophore in molecular bicones was compared with one, two, and three Dabcyl quenchers at the 5'-end [9]. Ferrocene can also be used to quench luminescence [10]. It is known that it actively quenches the luminescence of fluorophores due to the ability to deactivate both singlet [11, 12] and triplet [13] states. However, at present, ferrocene in the study of biological objects is used exclusively as an electroactive label [14–20]. There is practically no data on the applicability of ferrocene as a fluorescence quencher for dyes routinely used for labeling nucleic acids, although ferrocene derivatives are widely used in luminescent systems for a number of applications due to their high stability in the visible spectral range [21–29]. There is no information in the literature on the use of complex compound compounds: dyads, triads, etc., containing both ferrocene and azo groups in the molecule.

The difference of our approach to quenching of fluorescence is the use of complex dyad substances as quenchers, which have two functional parts in their molecular structure and are chromophores with high molar extinction coefficients. Using the methods of organic and bioorganic chemistry, we synthesized dyads FcD1-FcD5 based on derivatives of ferrocene and azo dyes according to the schemes given in the "Drawings" section (Figs. 1-5).

FcD1-FcD5 is characterized by the presence of an extended system of conjugated double bonds in the azo fragment, which provides effective delocalization of the electron density of π bonds. The azo group causes the appearance of an intense band π → π ^* transition in the region of 280-320 nm. The introduction of an electron-donating substituent in the position conjugated with the azo group shifts the π → π ^* transition band to the visible region of the spectrum; the introduction of an electron-withdrawing substituent in the position of the second aromatic residue conjugated with the azo group further enhances this shift [30]. The presence of electron-withdrawing substituents in the dyes obtained: nitro, carboxy groups and electron-donating dimethylamine groups, leads to an effective shift in the electron density of bonds in the molecules, which ensures the presence of an absorption band in the visible region of the spectrum and a high molar extinction coefficient. The ferrocene fragment is also a chromophore; in addition, it has a conjugation of the density of the nucleus with a multiple bond and a keto group. Ferrocene derivatives themselves are generally inferior in terms of molar extinction coefficient and quenching efficiency to azo dyes. Both azo dyes and ferrocene derivatives have characteristic absorption bands, which makes them capable of quenching only certain fluorophores. The combination of two chromophore systems in one molecule should theoretically provide a wider absorption band for the conjugate and high values of molar extinction coefficient and fluorescence quenching efficiency, the magnitude of which will depend on the characteristics of the starting materials.

The structure of the FcD1-FcD5 compounds is such that both chromophore fragments, firstly, do not have their own fluorescence, and secondly, they do not have conjugation. This suggests that there is no interaction and energy transfer between them, and the absorption spectra of these substances will be a simple algebraic sum of the absorption spectra of the constituents. To confirm this, the spectral characteristics were studied and the fluorescence quenching constants of the FcD1-FcD5 compounds were found (Table 1). It turned out that all compounds are characterized by a broadened absorption band in the electronic spectrum in the range 470–560 nm. For FcD1 and FcD3, the absorption band has two peaks.

Extinguishing can be static or dynamic; in the case of ferrocene, it can occur either due to the resonant fluorescence energy transfer (FRET), or due to electron transfer. For effective quenching, the maximum emission of the fluorophore and the maximum absorption of the quencher should partially or completely overlap. We have studied the fluorescence quenching dyads 5-carboxyfluorescein (FAM, λ = 520 nm _es) and X-rhodamine (ROX, λ = 605 nm _es) most commonly used as reporter groups in the study of nucleic acids. For these dyes, fluorescence intensities were obtained in the presence of different amounts of quencher. Based on the data obtained, the fluorescence quenching constants were found on the basis of the Stern-Volmer equation (Table 1). The highest fluorescence quenching constant from synthesized dyads has FcD5. For this compound, an activated FcD5-NHS ester and its labeled IQ oligonucleotide were obtained (Fig. 6).

To study the possibility of quenching fluorescence by dyads in the composition of nucleic acids, oligonucleotide duplex structures A-D were monitored (Fig. 7). In them, FcD5 and fluorescent dye (FAM or ROX) are located as close as possible on one side of the duplex on different chains. For these structures, melting profiles were obtained (Fig. 8). The presence of a single nucleotide substitution destabilizes the secondary structure, which leads to a decrease in temperature (duplexes B and D).

Thus, the proposed dyads can act as effective quenchers of fluorescence, including as part of biomacromolecules: proteins, nucleic acids, etc. In addition, the presence of a ferrocene core in dyad molecules determines their electroactivity, therefore, these compounds are promising precursors of a new type of reporter groups, which combine both electroactive properties and the ability to quench fluorescence. This makes it possible to use simultaneously different analytical methods for studying the same biological samples labeled with only one dyad.

The proposed methods for the synthesis of dyads and their use as quenchers for fluorescence are illustrated by the following examples.

Example 1. Synthesis of (2, or 3, or 4) -nitro-2'-hydroxy-5 '- [N- (para-ferrocenylideneacetophenyl) -carboxamido] azobenzene (FcD1).

9 ml of dichloromethane, 0 5 ml of pyridine and a solution of 0.6 g of n-aminoacetophenone in 6 ml of dichloromethane were added to 1.0 g of 4,4-dimethoxytrityl chloride. The reaction mixture was stirred for 4.5 hours, after which the solution was evaporated. 1.1 g of N-DMTr-n-aminoacetophenone (1) were obtained as an oil. Ferrocenaldehyde in an amount of 0.46 g and 0.98 g (1) was dissolved in 4 ml of ethanol, 200 μl of piperidine was added and refluxed for 24 hours. Then 0.3 g of trichloroacetic acid was added to the reaction mixture and stirred vigorously for 1 h, 5 ml of water was added and the organic was separated off. phase. The aqueous layer was extracted with dichloromethane (2 × 5 ml), the organic phases were combined, washed with water (2 × 5 ml), the aqueous phase was removed. The organic layer was evaporated, 5 ml of ethyl acetate was added to the residue and divided on a column (Al ₂ O ₃ adsorbent, ethyl acetate: hexane 1: 1 eluent). Product (2) is a dark purple oil, R _f = 0.38 (silufol, 1: 1 hexane ethyl acetate). ¹ H NMR Spectrum (CDCl ₃ , δ, ppm): 4.15 (s, 5H, C6); 4.45 (t, 2H, C3.4); 4.75 (d, 2H, C2.5); 6.65 (d, 2H, C _Ar 2.6); 7.4 (d, 1H, C8, J 15.3); 7.6 (d, 1H, C7, J 145: 3); 7.75 (d, 2H, C _Ar 3.5).

To a solution cooled to 0 ° C, 3 g of nitroanilines in 36 ml of 10% HCl and 30 ml of H ₂ O was gradually poured a solution of 1.5 g of NaNO ₂ in 10 ml of H ₂ O. It was kept at room temperature for 40 minutes and poured onto the solutions under cooling 3 g of 4-hydroxybenzoic acid in 3.5 ml of 20% NaOH and 10 ml of H ₂ O. It was kept under cooling for 40 minutes, the precipitates were filtered off and dried in air. The resulting azo compounds (3) were isolated by column chromatography (hexane: ethyl acetate 1: 2). 0.16 g (2) and 0.13 g (3) were dissolved in 15 ml of anhydrous THF, 0.1 g of DCC was added. The reaction was carried out at room temperature for 5 hours, then the solvent was evaporated. The resulting product was divided on a column of silica gel, hexane-ethyl acetate 3: 1 → 1: 2. Mass spectrum: m / z 599 [M-H] ⁺ .

Example 2. Synthesis of 4- (2 ', or 3', or 4 ') - nitrophenylazo-4' - (para-ferrocenylideneaceto-phenylcarboxamido) azobenzene (FcD2).

0.64 ferrocenaldehyde and 0.5 g of 4-acetylbenzoic acid were dissolved in 20 ml of toluene, 400 μl of piperidine and molecular sieves (3A) were added. The reaction mixture was heated for 3 hours, after which 2 ml of 10% HCl solution was added and the precipitate was filtered on a Schott filter, washing several times with water. The violet crystals (4) obtained were R _f = 0.3 (ethanol: 1: 1 chloroform). ¹ H NMR Spectrum (DMSO-d ₆ , δ, ppm, J / Hz): 4.2 (s, 5H, C ₅ H ₅ ); 4.6 (s, 2H, C3.4); 4.8 (s, 2H, C2.5); 7.4 (d, 1H, C1H, J 15.3); 7.6 (d, 1H, C2H, J 15.3); 8.1-8.3 (m, 4H, 4C _Ar H). Compound (6) was obtained by double azo coupling of aniline with nitroanilines according to Example 1 with the formation of an intermediate ion (5). Further, FcD2 was obtained by condensation in the presence of DCC, by analogy with example 1. Mass spectrum: m / z 686 [M-H] ⁺ .

Example 3. Synthesis of 4-N, N-dimethidamino-4 '- [N- {2- (para-ferrocenylideneacetophenyl-carboxamido) ethyl} carbamoyl] azobenzene (FcD3).

Compounds (7), (8) and FcD3 were obtained by analogy with example 1. Mass spectrum: m / z 652 [M-H] ⁺ . Example 4. Synthesis of 4-carboxy-4'-N-methyl-N- [4-butoxy- (para-ferrocenylideneacetophenyl)] - azobenzene (FcD4).

Compound (9) was obtained by aldol-croton condensation by analogy with Example 2. A waxy substance of dark violet color was obtained. R _f = 0.58 (dichloromethane: 3: 1 isopropanol). ¹ H NMR spectrum (DMSO-d ₆ , δ, ppm, J / Hz) 4.25 (s, 5H, C ₅ H ₅ ), 4.45 (s, 2H, C3.4); 4.7 (s, 2H, C2.5); 7.35 (d, 1H, C ₁ H, J 13.2); 7.6 (d, 1H, C ₂ H, J 13.2), 8.1-8 3 (m, 4H, 4C _Ar H).

0.3 g (9) and 200 μl of 1,4-diiodobutane were dissolved in toluene, 0.12 g of K ₂ CO ₃ was added and boiled for 32 hours. Target product (10) was isolated by column chromatography (hexane: ethyl acetate 1: 1). Received needle-shaped crystals of copper color R _f = 0.65 (hexane: ethyl acetate 2: 1). Compound (10) was introduced into interaction with N-methylaniline in an equimolar ratio in toluene during boiling for 18 h in the presence of potash. The hot reaction mass was decanted, the solvent was evaporated. The residue was purified by silica gel column chromatography (hexane: ethyl acetate 1: 1). Received a substance (11) of brick color. R _f = 0.45 (hexane: ethyl acetate 2: 1). Compound (11) was introduced into the azo coupling reaction with the diazonium salt of n-aminobenzoic acid. The target product was isolated by flash chromatography (hexane: chloroform 1: 3). Mass spectrum: m / z 640 [M-H] ⁺ .

Example 5. Synthesis of 4-carboxy-4'-N, N-di [4- {1- (para-ferrocenylideneacetophenylamino) -butyl-1,2,3-triazolyl} methyl] aminoazobenzene (FcD5).

Compound (13) was obtained by analogy with example 4 in the form of a waxy substance of dark purple color. Based on it, the azide derivative (14) was obtained by interaction with NaN ₃ in an aqueous-organic medium. Complete alkylation of aniline with propargyl bromide synthesized (15), which then leads to the adduct (16) by the reaction of the [3 + 2] dipolar cycloaddition catalyzed by copper (I) salts. For this, 15 ml of DMSO, 30 ml of aqueous triethylammonium acetate (0.2 M, pH 7) were mixed, 20 ml of a 0.1 M solution of (14) in DMSO and 12.6 ml of an 8 mM aqueous solution of copper (II) sulfate pentahydrate, 8.0 ml of 12 was added. 5 mm aqueous solution of ascorbic acid and 20 ml of 0.1 M solution (15) in DMSO. The mixture was purged with nitrogen and kept at room temperature for 24 hours, after which the mixture was poured into 100 ml of water, 10 ml of saturated sodium bicarbonate solution was added. The reaction product was extracted with ethyl acetate (3 × 20 ml), the organic phases were dried over MgSO ₄ , and the solvent was evaporated. The dry residue was chromatographed on silica gel, 1: 1 isopropanol-dichloromethane eluent. Subsequent azo coupling, by analogy with Example 4, gives FcD5. Mass spectrum: m / z 1088 [M-H] ⁺ .

Example 6. Electronic absorption spectra of compounds FcD1-FcD5 and the study of the efficiency of quenching by them fluorescence of dyes in solution.

Electronic spectra of FcD1-FcD5 compounds were recorded on a UV-3600 UVI spectrometer (Shimadzu, Japan) in the range of 190–700 nm. Braley solutions thereof in absolute ethanol, the concentration of 1 x 10 ^-4 M. For dyes FAM (λ = 520 nm _es) and ROX (λ _isp = 605 nm) was obtained fluorescence intensity in the presence of various amounts of the compounds FcD1-FcD5. 2 × 10 ^-6 M solutions in ethanol were used. The fluorescence intensity was measured on an LS 55 luminescent spectrometer (PerkinElmer, USA). Based on the data obtained, quenching curves were constructed in the coordinates of the Stern-Volmer equation and the values of quenching constants

Example 7. Synthesis of labeled nucleic acids.

To a solution of 1 mmol FcD5 in 5 ml of dry THF (water content less than 0.003%) was added a solution of 115 mg of N-hydroxysuccinimide (NHS, 1 mmol) and 206 mg of dicyclohexylcarbodiimide (DCC, 1 mmol) in 5 ml of THF. The reaction mixture was stirred at room temperature for 5 hours. N, N'-Dicyclohexylurea was planted with absolutely dry diethyl ether. The mixture was kept for another 18 hours at 0 ° C; the precipitated crystals of FcD5-NHS ether were filtered off, washed with 1 ml of diethyl ether and dried in a desiccator over CaCl ₂ . Yield 65%. Received maroon crystals

To obtain labeled IQ oligonucleotide, a solution of 1 mg FcD5-NHS in 10 μl DMSO was added to a solution of 5.5 pmol of amino-oligonucleotide I in 20 μd of 0.5 M carbonate buffer (NaHCO ₃ / Na ₂ CO ₃ , pH 9). The reaction mixture was stirred for 18 hours at room temperature. Then, 1 ml of alcohol was added and kept at -20 ° C for 2 h, after which it was centrifuged (14000 rpm, 15 min). The alcohol phase was taken, the precipitate was dried, resuspended in 100 μl of water of the highest quality category. Labeled oligonucleotide was isolated using LC-20AT HPLC system (Shimadzu, Japan).

Example 8. The study of the effectiveness of quenching of fluorescence dyad FcD5 duplexes

The fluorescence quenching of FAM and ROX dyes by the FcD5 dyad in duplex structures was studied by melting them in the iCycler iQ DNA amplifier (BioRad, USA). 30 μl samples contained 0.15 pu of IQ oligonucleotide and 0.03 pu of one of the fluorescently labeled oligonigides of IF II-F, IR or II-R in 1 ^x SSC buffer. The step of temperature change was set equal to 0.5 ° C with an interval of 10 s, the range of temperature change was from 10 to 95 ° C.

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Claims (2)

1. The method of obtaining compounds dyads of General formula (I):

where
Fc is a ferrocenyl radical of the following structure:

,
R ₁ is a hydrogen atom or Fc,
R ₂ is a hydrogen atom, or ortho or para-hydroxyl,
R ₃ is an ortho or meta or para-nitro group, or an ortho or meta or para-nitrophenyl azo group, or a para-N, N-dimethylamino group, or a para-carboxy group,
L is a para-carbamoyl vinylidene-acetophenone group, or a para-carboxamidinyl vinylidene-acetophenone group, or a para-N- (2-carbamoylethyl) -carboxamidinyl vinylidene-acetophenone group, or a para- (4- [methylamino] butoxy) -vinylidene acetophenone group, or {1- (para-vinylideneacetophenylamino) methyl-1,2,3-triazolyl} butyl] amino group,
characterized in that for their preparation ferrocenaldehyde is used as the starting compound, which is involved in the aldol-croton condensation reaction with para-substituted acetophenone followed by the interaction of the obtained ferrocenylidene acetophenone with the azo compound in the condensation reaction of the amino and carboxy groups, respectively, or with the subsequent introduction of ferroenocenoceneenide into the molecule groups and the interaction of this group with the diazosol by the azo coupling reaction, leading to the creation of molecules with two different, non-conjugated, chromophore fragments, the combination of which in one molecule provides high molar extinction coefficients, a wide absorption spectrum and the electroactivity of the resulting substances. 2. The method of quenching the fluorescence of fluorophores in double-stranded structures of nucleic acids, characterized in that quenchers use dyad compounds based on ferrocene and azo dyes obtained according to claim 1.

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