ES2368276A1 - Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna - Google Patents

Abstract

The invention relates to compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded DNA. The invention further relates to a group of compounds having a benzamidine structure, to a method for the synthesis thereof and to the use of same as fluorogenic agents for recognizing DNA sequences.

Description

Compounds derived from bis-benzamidiniums as fluorogenic agents for signal specific sequences of double stranded DNA.

The present invention relates to a group of compounds derived from bis-benzamidiniums such as Fluorogenic agents for DNA sequence recognition.

Prior art

An important objective of current research on the border between chemistry and biology is the development of chemical agents capable of efficiently interacting with specific sequences of double stranded DNA. Over the past decades, a large number of heterocyclic agents capable of selectively binding to DNA have been described by inserting into their minor groove (cf. Lown, JW Pharmacol. Ther . 1999, 84 , 1-111). Among these molecules, probably the most studied are those based on polyamides of pyrrole and imidazole in hairpin structure, since they show excellent properties in terms of affinity and sequence specificity. Unfortunately, the synthesis of these compounds is laborious, and their usefulness in vivo is largely limited due to their intrinsic difficulties in crossing biological membranes, as well as their remarkable toxicity.

Another type of agents that also recognize the minor groove of DNA are bis-benzamidinium-type compounds, such as pentamidine (1a) or propamidine (1b). These molecules are especially attractive from the pharmacological point of view, since they are stable and have good transport properties in numerous cell lines. For example, pentamidine has been used for years against trypanosomiasis, lesmaniasis and pneumonia of P. Carinii . Among the bis-amidine derivatives, those with aromatic type connectors are also especially important; in fact, DB289, a prodrug of furamidine (3), is in clinical phase III against sleeping sickness, and is very active against other parasites (cfr. Mathis, AM, et al., Antimicrob. Agents Chemother . 2007 , 51 , 2801-2810).

one

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These properties, together with their structural simplicity, have encouraged the search for new analogues with improved recognition properties and reduced toxicities. At the moment, the major limitations for the development of this type of benzamidiniums have to do with the low versatility and practicality of existing synthetic routes and with the fact that there is no simple, fast and reliable method that allows to evaluate and quantify their affinity and selectivity for specific DNA sequences. The usual method to study the affinity of these types of molecules is based on the thermal denaturation of their complexes with DNA. However, this method only gives indirect and qualitative information on the recognition characteristics of these compounds and is also difficult to implement in high-throughput screening strategies (cf. Dardonville, C. et al., J. Med. Chem ., 2006, 49 , 3748-3752).

The donor / acceptor configuration in the units of 4-carbamidoylphenoxy characteristics of the propamidine structure could lead to properties Fluorogenic and induced emission when inserted into the groove minor DNA, as a result of environmental and polarity change that occur when these molecules are inserted into the minor groove of the DNA; however, the excitation wavelength of the structure of propamidine matches the typical DNA absorption band at 260 nm This coincidence prevents the application of techniques of fluorescence for the study of its interaction with DNA.

The authors of the present invention have observed that by substituting the 4-phenoxyamidinium units, characteristics of the classical benzamidinic derivatives, with 4-aminobenzamidiniums analogs are obtained which, in addition to selectively interacting with DNA sequences, have the additional advantage that they have a red-shifted absorption spectrum, which allows excitation at a longer wavelength (329 nm) and also experience a significant increase in their fluorescence emission intensity at 387 nm when they bind to rich sequences in A / T belonging to double stranded DNA, (O. Vázquez et al., Org. Lett ., 2010, 12 , 216-219).

Brief Description of the Invention

In the present invention, new ones are described type derivatives bis-4-aminobenzamidine which incorporate in its structure an acceptor fluorophore. These compounds have high affinity for specific DNA sequences of double chain, and also signal this interaction by issuing fluorescence.

The additional advantage of these compounds is that being present in its structure an acceptor fluorophore emit light at an even longer wavelength than in the compounds in the that these groups are not present, due to a process of intramolecular energy transfer from the system bis-4-aminobenzamidine until acceptor fluorophore group. Thanks to this transfer process it effectively extends the range of emission wavelengths to which the presence of DNA can be monitored. In a case particular Stokes shift increased by 145 nm, in instead of at 60 nm as in cases where this group does not exist acceptor fluorophore. And also, modifying the fluorophore group acceptor it is possible to design derivatives that emit light at different wavelengths that are of interest to your application such as luminescent biosensors.

Another additional advantage is that the intensity of the emission band can be increased up to 60 times, which allows to detect amounts of DNA of the order of nanograms.

In a first aspect, the present invention is refers to a compound of general formula (I)

3

where Z is a fluorophore group acceptor and n has a value of 1 a 5.

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In another aspect, the invention relates to a preparation process of a compound of general formula (I) which comprises a reaction between a compound of formula II and a acceptor fluorophore,

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where Y is selected from hydrogen and a chelating agent, and n has a value of 1 to 5.

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In another aspect, the invention relates to the use of a compound of general formula (I) as a fluorogenic agent of DNA sequence recognition.

Detailed description of the invention

In the present invention, the term "agent Fluorogenic "refers to a fluorescent molecule that has a functional group that absorbs energy of a wavelength specific and emits it in another determined one of greater wavelength (that is, with less energy), and in which the amount of energy emitted (quantum yield) depends so much on the group itself Fluorescent functional as of its chemical environment.

The term "acceptor fluorophore" refers, in the present invention, to a species that participates in a resonance energy transmission (FRET) process accepting the energy transferred by another species that is in an excited state (this transfer can take place through any photophysical process, including Förster or Dexter type mechanisms). A key parameter for the transfer of energy between two fluorophores is the combination of species with nearby energy levels. This proximity is related to the transfer theory so that the higher the value of the overlap integral between the donor and acceptor (JDA), between the emission spectrum of the donor and the absorption spectrum of the acceptor, the better the FRET . In the present invention, the theory of energy transfer is incorporated, as well as the calculation of said integral, the interpretation of its values and the detection of transfer energy by means of the following article: "Resonant Energy Transfer: Methods and Applications", Analytical Biochemistry , 1994, 218 , 1-13. In addition, these energy requirements can be easily visualized by the overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore. In a particular case of the present invention, this overlap was studied (Figure 4) between the emission spectrum of the aminobenzamidinium derivative N 1, N 3 -bis (4-aminodinophenyl) propane-1,3-diamine and the absorption spectrum of compound 6, proving that this combination of species is suitable for carrying out the invention.

The term "alkyl" refers, in the present invention, to aliphatic, linear or branched chains, having 1 to 10 carbon atoms, preferably between 1 and 5 carbon atoms. For example, but not limited to, methyl, ethyl, n -propyl, n -butyl, n-pentyl, n-hexyl, n-heptyl etc. The alkyl groups may be optionally modified by one or more substituents such as amine, halogen, hydroxyl or carboxylic acid.

In a preferred embodiment, the present invention refers to a compound of formula I where Z, the group acceptor fluorophore, is selected from a complex lanthanide ion with a chelating agent or a coumarin of general formula III.

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5

where R1 is selected from -NR4R5, -OH, -OR4, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl, Y

R3 is selected from hydrogen, halogen and I rent.

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In a more preferred embodiment, the ion Lanthanide is selected from Eu + 3 and Tb + 3.

In a preferred embodiment, the chelating agent is selected from 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 4,7,10-Tetraazacyclododecane -1,4,7,10-tetra
(methylene phosphonic acid) (DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).

In another preferred embodiment, n is 3.

In another preferred embodiment, the invention is refers to a process for obtaining the compounds of formula (I) as described above, which comprises the reaction between a compound of formula II, as described previously, with an acceptor fluorophore species, where the species acceptor fluorophore is selected from a lanthanide ion or derivatives of coumarin of formula IV,

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6

where R1 is selected from -NR4R5, -OH, -OCOR4, R4 and R5 being the same or different selected from hydrogen, I rent,

R2 is selected from hydrogen, alkyl, triflate, tosyl, succinimidyl or halide, and

R3 is selected from hydrogen, halogen and I rent.

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In a particular embodiment, the invention is refers to a process for obtaining the compounds of formula (I) as described above, which comprises the reaction between a compound of formula II, as described above and a acceptor fluorophore species, which

- when Y is hydrogen then the species acceptor fluorophore is selected from coumarin derivatives of formula IV, as defined above. When R2 is hydrogen the procedure comprises a coupling between both in the presence of an activator of carboxylic groups.

Activators of carboxylic groups are those which modify the carboxylic group by converting the hydroxyl into a better leaving group and allow to carry out the reaction of coupling faster, more effectively and in more conditions soft. For example, they are activators of carboxylic groups carbodiimide derivatives such as dicyclohexylcarbodiimide, or diisopropylcarbodiimide; triazole derivatives such as the 1-hydroxy-benzotriazole, 1-hydroxy-7-aza-benzotriazole; derivatives of phosphonium or uronium salts of a non-anion nucleophilic such as tetrafluoroborate or hexafluorophosphate, by example hexafluorophosphate O-benzotriazol-N, N, N ', N'-tetramethyl-uronium (HBTU), hexafluorophosphate 2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyl uronio matanaminio (HATU), hexafluorophosphate 7-azabenzotriazol-1-yloxy-tris- (pyrrolidino) -phosphonium (PyOAP), hexafluorophosphate benzotriazol-1-yloxy-tris- (pyrrolidino) -phosphonium  (PyBOP);

- when Y is a chelating agent then the acceptor fluorophore is selected from lanthanide ions, preferably between Eu + 3, Tb + 3, and the reaction having place is a complexation reaction with the corresponding salt of the trivalent lanthanide ion, which may be trichlorides, tribromides and triiodides, sulfates or triflates.

In a more particular embodiment, the compound of formula II where Y is a chelating group is prepared by a method comprising a coupling reaction between a compound of formula IIa

7

where is hydrogen and n se select between 1 and 5,

and a chelating agent that possesses an acid group carboxylic, such as acid 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), 4,7,10-Tetraazacyclododecane-1,4,7,10-tetra (acid methylene phosphonic) (DOTP), acid 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid  (TETA), in the presence of a carboxylic group activating agent. Preferably the chelating agent is in the form protected, that is, all acetic groups are protected except one, for example with tert-butyl groups such as acid 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert-butylacetate-10-acetic acid.

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In another particular embodiment, compound II can be deprotected by routine reactions known to those skilled in the art under usual deprotection conditions ( Greene, TW " Protective Groups in Organic Synthesis ", 3rd Ed, Wiley-Interscience, 1999 ). The deprotection of a tert-butylcarboxylate in acid medium is typical, for example, trifluoroacetic acid.

In another preferred embodiment, the present invention relates to the use of a compound as described above, in which the DNA sequence that is recognized is formed by 4, 5 or 6 pairs of A / T bases.

Preferably, the DNA sequence that is recognize is formed by 4 or 5 base pairs A / T.

In another preferred embodiment, the sequence of bases are selected between AATT and AATTT.

In a preferred embodiment, the present invention relates to the use of the compound as described previously for agent identification tests of recognition of the minor groove of the DNA by displacement of the fluorescence.

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Description of the figures

Figure 1a. Emission spectra of compound 5a in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl, which show the corresponding electronic transitions; spectra in absence of DNA (\ blacksquare); with 9 equivalents of non DNA specific GGCCC (\ medcirc); with 9 equivalents of AATTC DNA (\ boxempty); with 9 equivalents of AATTT target DNA (\ medbullet).

Figure 1b Emission spectra of compound 5b in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl, which show the corresponding electronic transitions; spectra in absence of DNA (\ blacksquare); with 9 equivalents of non DNA specific GGCCC (\ medcirc); with 9 equivalents of AATTC DNA (\ boxempty); with 9 equivalents of AATTT target DNA (\ medbullet).

Figure 2. The graph compares:

to)

Fluorescence emission of N 1, N 3 -bis (4-aminodinophenyl) propane-1,3-diamine (0.5 µM in Tris-HCl buffer) with 9 equiv. target DNA (AATTT, \ blacksquare);

b)

the fluorescence emission of compound 6 (0.5 µM in buffer Tris-HCl) (? The intensity of this spectrum is presented multiplied by 10 for clarity); Y

C)

the fluorescence emission of compound 6 (0.5 µM in buffer Tris-HCl) in the presence of 9 equiv. target DNA (AATTT, \ medbullet).

In the figure inserted the fluorescence of the cuvettes with solutions is shown of 500 nM of 6 with an excitation at 329 nm: a) no DNA was added; b) 2 equiv of AATTT DNA.

Figure 3a. Emission spectra of a solution 0.5 micromolar compound 6 increasing the amounts of DNA target (AATTT): 0, 0.4, 0.7, 1, 1.8, 2.5, 3.2 and 4 micromolar. The inserted graph shows the 472 nm titration of compound 6 in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl with DNA AATTT and demonstrates that it conforms to a 1: 1 union model, being the dissociation constant (814 ± 90) nM.

Figure 3b Emission spectra of a solution 0.5 micromolar compound 6 increasing the amounts of DNA truncated (AAT): 0, 0.4, 0.7, 1, 1.8, 2.5, 3.2 and 4 micromolar. The inserted graph shows the 472 nm titration of compound 6 in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl with DNA AATGC and demonstrates that it conforms to a 1: 1 union model, being the dissociation constant (2.38 ± 0.08) µM.

Figure 4. Absorption band of the aza-benzamidinium unit: dotted line a). Coumarin absorption band: dotted line b). Fluorescence emission band of N 1, N 3 -bis (4-aminodinophenyl) propane-1,3-diamine, solid line (med). Fluorescence emission band of compound 6, solid line (?). The spectra were normalized for comparison.

Figure 5a. Excitation spectrum at 472 nm of 0.5 micromolar compound 6 in Tris-HCl 20 buffer mM pH 7.5, 100 mM NaCl (dotted line); and with 9 equiv. from AATTT ADN (continuous line).

Figure 5b Emission spectrum of compound 6 in the absence of DNA (\ blacksquare); emission spectrum of compound 6 with 9 equiv. of AATTT DNA and wavelength of 432 nm excitation (med); emission spectrum with 9 equiv. of AATTT DNA and 329 nm excitation wavelength (\ medbullet).

The following examples and drawings are provided by way of illustration, and are not intended to be Limitations of the present invention.

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Examples Compound Preparation

Reagents and solvents were purchased in commercial houses, and specifically acid 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert-butylacetate-10-acetic acid It was supplied by Macrocyclics and coumarins can be purchased to Sigma-Aldrich.

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Example 1 Preparation of compound 3

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A dispersion of 60% sodium hydride (68 mg, 476 µmol, 4 equiv) was added to a solution of compound 2 (66 mg, 119 µmol, 1 equiv) in dry DMSO (1.5 mL). After 30 min, l-iodo- tert- butyl-2-aminopropylcarbamate (19 mg, 239 µmol, 2 equiv) was added portionwise. The reaction mixture was stirred under argon at room temperature for 6 h. The reaction crude was purified by reverse phase chromatography ( Büchi Sepacore ) (A: methanol; B: 0.1% water trifluoroacetic acid; gradient: 15% B, 5 min; 15%? 95% B, 30 min.).

The isolated compound was dissolved in CH2Cl2 (1 mL) and cooled to 0 ° C, trifluoroacetic acid (1 mL) was added and the mixture was stirred at 0 ° C for 1 h. The solvent was removed to dryness and the crude was purified by reverse phase chromatography (Büchi Sepacore) (gradient: 0% B, 5 min; 0% → 50% B, 30 min.) To obtain compound 3 (13 mg , 39%). 1 H-NMR δ (DMSO- d 6) : 1.72-1.82 (m, 2H), 2.81-2.91 (m, 2H), 3.11-3.16 m, 2H), 3.27-3.40 (m, 4H), 3.78-3.90 (m, 1H), 6.75 (d, J = 8.8 Hz, 4H), 7.65 (d, J = 8.8 Hz, 4H), 8.66 (s, 4H), 8.80 (s, 4H). 13 C-NMR δ (DMSO- d 6) : 27.6 (CH 2), 36.5 (CH 2), 43.8 (CH 2), 66.4 (CH 2), 76.4 (CH), 111.2 (CH), 112.4 (C), 129.6 (CH), 153.5 (C), 158.4 (C, TFA), 164.1 (C).

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Example 2 Preparation of compounds 5a and 5b

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A solution of 1,4,7,10-tetraazacyclododecane-1,4,7-tris- tert- butyl acetate-10-acetic acid (4,52 mg, 92 µmol, 4 equiv) in 0.2M dimethylformamide / diisopropylethylamine ( 460 µL, 92 µm 4 equiv) was activated with HATU (35 mg, 92 µmol, 4 equiv) for 5 min, added over 3 (9 mg, 23 µmol, 1 equiv) in dimethylformamide / diisopropylethyl-
0.2M amine (230 µL, 46 µm 2 equiv) and stirred for 2 h. The solvent was removed and the crude was redissolved in CH2Cl2 (500 µL), cooled and trifluoroacetic acid (500 µL) was added. It was concentrated to dryness and the crude was purified by reverse phase chromatography ( Büchi Sepacore ) (gradient: 8% B, 5 min; 8% → 40% B, 30 min.), Obtaining the intermediate compound (19 mg, 85 %). 1 H-NMR δ (DMSO- d 6 / , CD 2 Cl 2) : 1.72-1.82 (m, 2H), 2.68 (s, 3H), 2.72 (s, 3H) , 2.84 (dd, J = 13.1, J = 6.61 Hz, 2H), 2.88 (s, 4H), 3.05 (s, 2H), 3.11-3.16 (m, 4H), 3.27-3.36 (m, 4H), 3.39 (s, 2H), 3.58-3.64 (m, 8H), 3.80-4.50 (b, 1H overlays by water signal), 6.75 (d, J = 8.9, 4H) 7.65 (d, J = 8.9, 4H), 8.73 (s, 4H), 8.80 (s, 4H). 13 C-NMR δ (DMSO- d 6 / CD 2 Cl 2) : 27.6 (CH 2), 28.6 (CH 2), 28.8 (CH 2) , 30.7 (CH2), 36.6 (CH2), 41.7 (CH2), 43.8 (CH2), 44.9 (CH2), 45.0 (CH2), 47.5 (CH 2), 66.5 (CH 2), 76.5 (CH), 111.1 (CH), 112.4 (C), 129.7 (CH), 153.5 (C), 158.0 (C, TFA), 164.0 (C ), 169.5 (C).

Chelation with the lanthanide was carried out in the same way for both cases. Intermediate dissolved obtained in a buffer solution 600 µL HEPES buffer 10 mM, pH 7.5, 100 mM NaCl and the corresponding lanthanum chloride was added (50 mM in 1 mM HC1). The mixture was stirred for 1 h and purified by chromatography (gradient: 95/5 → 25/75 30 min, water / acetonitrile, 0.1% trifluoroacetic acid).

5th: MALDI / TOF-MS : [M + H] + calcd. for C_ {36} H_ {56} Eu N_ {O} {8} = 920.3 found 920.1.

5b: MALDI / TOF-MS : [M + H] + calcd. for C_ {36} H_ {56} N_ {O} {8} Tb = 926.3 found 926.3.

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Example 3 Preparation of compound 6

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A solution of 7-diethylaminocumarin-3-carboxylic acid (24 mg, 92 µmol, 4 equiv) in 0.2 M dimethylformamide / diisopropylethylamine (460 µL, 92 µm 4 equiv) was activated with PyAOP (48 mg, 92 µmol , 4 equiv), was added over 3 (9 mg, 23 µmol, 1 equiv) in 0.2 M dimethylformamide / diisopropylethylamine (230 µL, 46 µm 2 equiv) and stirred for 2 h. The residue was purified by reverse phase chromatography (Büchi Sepacore) (gradient: 15% B, 5 min; 15%? 95% B, 30 min.), To obtain the product (12 mg, 62%). 1 H-NMR δ (DMSO- d 6) : 1.14 (t, J = 6.9 Hz, 6H), 1.87 (q, J = 6.5 Hz, 2H), 3.10-3.14 (m, 2H) , 3.22-3.32 (m, 2H), 3.44-3.52 (m, 4H), 3.82-3.86 (b, 1H), 5.23 (s, 1H) 6.62 (s, 1H), 6.73 (d, J = 7.4 Hz, 4H), 6.81 (d, J = 9.0 Hz, 1H), 7.59 (dd, J _ {2} = 46.4, J {1} = 8.8 Hz, 4H), 7.68 (d, J = 9.0 Hz, 1H) , 8.47 (s, 1H), 8.65 (s, 1H), 8.78 (s, 4H), 8.95 (s, 1H), 9.18 (s, 1H). 13 C-NMR δ (DMSO- d 6) : 12.3 (CH 3), 27.7 (CH 2), 36.4 (CH 2), 44.1 (CH 2), 44.3 (CH 2), 46.6 (CH 2), 67.6 (CH), 95.5 (CH), 107.6 (C), 109.3 (CH), 110.1 (CH), 111.9 (C), 113.5 (C) , 129.7 (CH), 131.6 (CH), 147.7 (CH), 152.4 (C), 153.7 (C), 157.2 (C), 158.2 (C, TFA), 162.1 (C), 162.5 (C), 164 ( C).

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Fluorescence assays

The fluorescence experiments were led to Jobin-Yvon fluorimeter out Fluoromax-3 (DataMax 2.20), coupled to a system of Wavelength Electronics temperature control LFI-3751.

The measurements for compound 5a were brought to carried out with the following acquisition parameters: 1 nm increment; integration time: 0.2 s; excitation bandwidth: 6 nm; emission bandwidth: 6 nm; excitation wavelength: 329 nm. The emission spectra were recorded at 20 ° C between 550 and 750 nm.

The measurements for compound 5b were brought to carried out with the following acquisition parameters: 1 nm increment; integration time: 0.2 s; excitation bandwidth: 3 nm; emission bandwidth: 6 nm; excitation wavelength: 329 nm The emission spectra were recorded at 20 ° C between 345 and 600 nm

The DNAs, supplied by Termo Fischer Scientific GMBH, were double helix forks with the following sequences

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12

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The emission spectra are shown in the Figure 1a and 1b. As the spectra with AATGC are very similar to those obtained with GGCCC, it is concluded that three base pairs A / T is a sequence too short to accommodate the unit of bis-aminobenzamidinium. They fit better in pairs A / T bases of 4, 5 or 6 base pairs.

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Design and fluorescence of compound 6

The emission fluorescence of compound 6 is performed with a buffer solution, and with 9 equiv. of AATTT ADN. After excitation at 329 nm, the emission fluorescence of the compound 6 in buffer is very weak and is dominated by the band of emission of coumarin at 472 nm.

The addition of AATTT DNA induces an increase in approximately 60 times the fluorescence intensity of emission of compound 6. The emission is so intense in this case that it is even possible to detect less than 3 nanograms of DNA (figure 2).

Thus, the proper conjugation of the bis- (benzamidinium) structure with the acceptor fluorophore group, of coumarin-derived structure, leads to a significant fluorescence change when bound to specific DNA sequences (AATTT), as evidenced by the increase of the displacement (145 nm) with respect to the emission fluorescence of N 1, N 3 -bis (4-aminodinophenyl) propane-1,3-diamine, a bis (benzamidino) derivative in which they are not present acceptor fluorophores groups (figure 2).

The spectra are compared in Figure 5b above with the emission spectrum of compound 6 (performed in the same conditions) with 9 equiv. of AATTT DNA and at a length 432 nm excitation wave.

The maximum excitation of compound 6 corresponds to the expected excitation band for coumarin at 432 nm. After the addition of AATTT, there is a large increase in the intensity of the excitation spectrum, and the excitation band of bis (benzamidine) at 329 nm becomes more intense, Showing the transfer energy of the two fluorophores: exciting at 329 nm bis (benzamidine) results in an increase in the emission of coumarin at 472 nm (Figures 5a and 5b).

The excitation spectra were recorded from 220 nm at 470 nm at 20 ° C with the following parameters: increase 1 nm; integration time: 0.2 s; excitation bandwidth: 3 nm; emission bandwidth: 4 nm; emission wavelength: 474 nm

The emission spectra were carried out with the following parameters: increase 1 nm; integration time: 0.2 s; excitation bandwidth: 3 nm; bandwidth of emission: 4 nm; excitation wavelength: 329 nm or 434 nm recorded from 345 to 640 nm.

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Titration of compound 6 with different DNAs

The fluorescence experiments were led to Jobin-Yvon fluorimeter out Fluoromax-3 (DataMax 2.20 software), coupled to a Wavelength Electronics temperature control system LFI-3751.

Except for the case of experiments with lanthanides, the measurements were carried out with the following acquisition parameters: increase 1 nm; integration time: 0.2 s; excitation bandwidth: 3 nm; bandwidth of emission: 6 nm; excitation wavelength: 329 nm. The emission spectra were recorded at 20 ° C between 345 and 640 nm (figure 3).

All valuations were carried out following the same procedure: about 1 mL of 0.5 solution microM of compound 6 in 20 mM Tris-HCl pH buffer 7.5, 100 mM NaCl, successive aliquots of approximately 400 microM of DNA solution and the fluorescence spectrum for each addition.

It was observed that the addition of truncated AATGC DNA induces significantly smaller increases in the emission of compound 6 that the addition of AATTT DNA at 472 nm.

The maximum emission was used in the determination of the binding constant. So, the values of the KD dissociation constant found for compound 6 conjugated to AATTT DNA, AATCG, were respectively (814 ± 90) nM, and (2.38 ± 0.08) µM (Figure 3).

Claims (7)

1. A compound of general formula (I) 13 where Z is a fluorophore group acceptor and n has a value of 1 a 5.

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2. A compound of general formula (I) according to the claim 1, wherein Z is selected from a lanthanide ion complexed with a chelating agent or a coumarin of general formula III, 14 where R1 is selected from -NR4R5, -OH, -OR4, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl, Y R3 is selected from hydrogen, halogen and I rent.

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3. A compound of general formula (I) according to the claims 1 and 2, wherein the lanthanide ion is selected from Eu + 3, Tb + 3. 4. A compound of general formula (I) according to the claims 1 and 2, wherein the chelating agent is selected from the acid 1,4,7,10-tetraazacyclodecano-l, 4,7,10-tetraacetic (DOTA), diethylenetriaminepentaacetic acid (DTPA), 4,7,10-Tetraazacyclododecane-1.4.7.10-tetra (acid methylene phosphonic) (DOTP), acid 1,4,8,11-tetraazacyclododecane-1.4.8.11-tetraacetic (TIT). 5. A compound of general formula (I) according to previous claims, where n is 3. 6. A procedure for preparing a compound of general formula (I) comprising a reaction between a compound of formula II and an acceptor fluorophore species, fifteen where Y is hydrogen or an agent chelator, and n has a value of 1 a 5.

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7. Use of a compound of general formula (I) as a fluorogenic agent for sequence recognition of DNA