WO2011135142A1 - Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna - Google Patents

Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna Download PDF

Info

Publication number
WO2011135142A1
WO2011135142A1 PCT/ES2011/070297 ES2011070297W WO2011135142A1 WO 2011135142 A1 WO2011135142 A1 WO 2011135142A1 ES 2011070297 W ES2011070297 W ES 2011070297W WO 2011135142 A1 WO2011135142 A1 WO 2011135142A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
dna
general formula
emission
bis
Prior art date
Application number
PCT/ES2011/070297
Other languages
Spanish (es)
French (fr)
Inventor
Olalla Vazquez Vazquez
Mateo Isidro SÁNCHEZ LÓPEZ
José Luis MASCAREÑAS CID
Marcos Eugenio Vazquez Sentis
Original Assignee
Universidade De Santiago De Compostela
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidade De Santiago De Compostela filed Critical Universidade De Santiago De Compostela
Publication of WO2011135142A1 publication Critical patent/WO2011135142A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C257/00—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines
    • C07C257/10—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines
    • C07C257/18—Compounds containing carboxyl groups, the doubly-bound oxygen atom of a carboxyl group being replaced by a doubly-bound nitrogen atom, this nitrogen atom not being further bound to an oxygen atom, e.g. imino-ethers, amidines with replacement of the other oxygen atom of the carboxyl group by nitrogen atoms, e.g. amidines having carbon atoms of amidino groups bound to carbon atoms of six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/18—Metal complexes
    • C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide

Definitions

  • the present invention relates to a group of compounds derived from bisbenzamidiniums as fluorogenic agents for DNA sequence recognition.
  • Another type of agents that also recognize the minor groove of DNA are bis-benzamidinium-type compounds, such as pentamidine (la) or propamidine (Ib). These molecules are especially attractive from the pharmacological point of view, since they are stable and have good transport properties in numerous cell lines.
  • pentamidine has been used for years against trypanosomiasis, lesmaniasis and pneumonia of P. Carinii.
  • 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 , 57, 2801-2810).
  • the donor / acceptor configuration in the 4-carbamidoylphenoxy units characteristic of the propamidine structure could give rise to fluorogenic and induced emission properties when inserted into the minor groove of the DNA, as a consequence of the environmental and polarity change that take place at insert these molecules into the minor groove of the DNA; however, the excitation wavelength of the propamidine structure coincides with the typical DNA absorption band at 260 nm. This coincidence prevents the application of fluorescence techniques for the study of their interaction with DNA.
  • the additional advantage of these compounds is that when an acceptor fluorophore is present in their structure they emit light at an even higher wavelength than in the compounds in which these groups are not present, due to an intramolecular energy transfer process from the bis-4-aminobenzamidine system to the acceptor fluorophore group. Thanks to this transfer process, the range of emission wavelengths at which the presence of DNA can be monitored is effectively extended. In a particular case, Stokes' displacement increased by 145 nm, instead of 60 nm, as in cases where this acceptor fluorophore group does not exist. And in addition, by modifying the acceptor fluorophore group it is possible to design derivatives that emit light at different wavelengths that are of interest for application 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.
  • the present invention relates to a compound of general formula (I)
  • the invention relates to a process for preparing a compound of general formula (I) comprising a reaction between a compound of formula II and an acceptor fluorophore,
  • Y is selected from hydrogen and a chelating agent, and n has a value of 1 to 5.
  • the invention relates to the use of a compound of general formula (I) as a fluorogenic agent for DNA sequence recognition.
  • fluorescent agent refers to a fluorescent molecule that has a functional group that absorbs energy of a specific wavelength and emits it in a given one of greater wavelength (ie, with less energy) , and in which the amount of energy emitted (quantum yield) depends on both the fluorescent functional group itself and its chemical environment.
  • 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 Forster or Dexter type mechanisms).
  • FRET resonance energy transmission
  • 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 giver and acceptor (JDA), between the emission spectrum of the donor and the absorption spectrum of the acceptor, the better the FRET.
  • 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, "-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.
  • the present invention relates to a compound of formula I wherein Z, the acceptor fluorophore group, is selected from a lanthanide ion complexed with a chelating agent or a coumarin of general formula III.
  • Rl is selected from -NR4R5, -OH, -OR4, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl, and
  • R3 is selected from hydrogen, halogen and alkyl.
  • the lanthanide ion is selected from Eu 3 and Tb
  • the chelating agent is selected from 1,4,7,10-tetraazacyclodecano-1, 4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTP A), 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic acid) (DOTP), 1,4,8,1-tetraazacyclo-dodecane-1,4, 8,11-tetraacetic acid (TETA).
  • DOTA diethylenetriaminepentaacetic acid
  • DTP A diethylenetriaminepentaacetic acid
  • DTP A 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic acid)
  • TETA 1,4,8,1-tetraazacyclo-dodecane-1,4, 8,11-tetraacetic acid
  • n 3.
  • the invention relates 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, with an acceptor fluorophore species, wherein the fluorophore acceptor species is selected from a lanthanide ion or coumarin derivatives of formula IV,
  • Rl is selected from -NR4R5, -OH, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl,
  • R2 is selected from hydrogen, alkyl, triflate, tosyl, succinimidyl or halide, and R3 is selected from hydrogen, halogen and alkyl.
  • the invention relates 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 an acceptor fluorophore species, which
  • the acceptor fluorophore species is selected from coumarin derivatives of formula IV, as defined above.
  • the process comprises a coupling between the two in the presence of a carboxylic group activator.
  • Carboxylic group activators are those that modify the carboxylic group by converting the hydroxyl into a better leaving group and allow Perform the coupling reaction faster, more efficiently and in milder conditions.
  • carbodiimide derivatives such as dicyclohexylcarbodiimide, or diisopropylcarbodiimide are activators of carboxylic groups; triazole derivatives such as 1-hydroxybenzotriazole, l-hydroxy-7-aza-benzotriazole; derivatives of phosphonium or uranium salts of a non-nucleophilic anion such as tetrafluoroborate or hexafluorophosphate, for example 0-benzotriazol-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU), 2- (lH- hexafluorophosphate) 7-azabenzotriazol-l-yl) -l, l, 3,3-tetramethyl ura
  • the acceptor fluorophore is selected from lanthanide ions, preferably from Eu 3 , Tb 3 , and the reaction that takes place is a complexation reaction with the corresponding salt of the trivalent lanthanide ion, which may be trichlorides, tribromides and triiodides, sulfates or triflates.
  • the compound of formula II wherein Y is a chelating group is prepared by a process comprising a coupling reaction between a compound of formula Ha
  • n is selected between 1 and 5
  • a chelating agent having a carboxylic acid group such as, for example, l, 4,7,10-tetraazacyclododecane-l, 4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic acid) (DOTP), 1,4,8,11-tetraazacyclo-dodecane- 1,4,8,11-tetraacetic acid (TETA), in the presence of a carboxylic group activating agent.
  • a carboxylic acid group such as, for example, l, 4,7,10-tetraazacyclododecane-l, 4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic
  • the chelating agent is in the protected form, that is, all acetic groups are protected except one, for example with tere-butyl groups such as 1,4,7,10-tetraazacyclocyclodedecane-1,4,7- tris-tert-butylacetate-10-acetic.
  • compound II can be deprotected by routine reactions known to those skilled in the art under usual deprotection conditions (Greene, T. W. "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.
  • 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 base pairs A / T.
  • the DNA sequence that is recognized is formed by 4 or 5 base pairs A / T.
  • the base sequence is selected from AATT and AATTT.
  • the present invention relates to the use of the compound as described above for identification tests of minor groove DNA recognition agents by fluorescence shift.
  • Figure Ib Emission spectra of compound 5b in 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl, showing the corresponding electronic transitions; spectra in the absence of DNA ( ⁇ ); with 9 equivalents of non-specific GGCCC (o) DNA; with 9 AATTC () DNA equivalents; with 9 equivalents of AATTT ( ⁇ ) target DNA.
  • the inserted figure shows the fluorescence of the cuvettes with solutions of 500 nM of 6 with an excitation at 329 nm: a) no DNA was added; b) 2 equiv of AATTT DNA.
  • FIG. 3a Emission spectra of a 0.5 micromolar solution of compound 6 increasing the amounts of target DNA (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 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl with AATTT DNA and demonstrates that it conforms to a 1: 1 binding model, the dissociation constant being (814 + 90 ) nM.
  • Figure 3b Emission spectra of a 0.5 micromolar solution of compound 6 increasing the amounts of truncated DNA (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 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl with AATGC DNA and demonstrates that it conforms to a 1: 1 binding model, the dissociation constant being (2.38 ⁇ 0.08 ) ⁇ .
  • Figure 4 Absorption band of the aza-benzamidinium unit: dotted line a). Coumarin absorption band: dotted line b). Fluorescence emission band of N1, N3-bis (4-aminodinophenyl) propane-1, 3-diamine, solid line (o). 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 of compound 6 in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl (dotted line); and with 9 equiv. of AATTT ADN (continuous line).
  • Figure 5b Emission spectrum of compound 6 in the absence of DNA (B); emission spectrum of compound 6 with 9 equiv. of AATTT DNA and excitation wavelength of 432 nm (o); emission spectrum with 9 equiv. of AATTT DNA and excitation wavelength of 329 nm ( ⁇ ).
  • the reagents and solvents were purchased in commercial houses, and in particular 1, 4,7, 10-tetraazacyclododecane-1, 4,7-tris-tert-butylacetate-10-acetic acid was supplied by Macrocyclics and coumarins can be purchased at Sigma-Aldrich.
  • Fluorescence experiments were carried out on a Jobin-Yvon Fluoromax-3 fluorimeter (DataMax 2.20), coupled to a Wavelength Electronics LFI-3751 temperature control system.
  • Measurements for compound 5a were carried out with the following acquisition parameters: 1 nm increase; 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.
  • Measurements for compound 5b were carried out with the following acquisition parameters: 1 nm increase; 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.
  • GGCCC GGCA GGCCC AGC TTTTT GCT GGGCC TGCC
  • the emission fluorescence of compound 6 was performed with a buffer solution, and with 9 equiv. of AATTT ADN. After excitation at 329 nm, the emission fluorescence of compound 6 in buffer is very weak and is dominated by the band of emission of coumarin at 472 nm.
  • AATTT DNA induces an approximately 60-fold increase in the emission fluorescence intensity 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).
  • AATTT 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 displacement (145 nm) with respect to the emission fluorescence of N ; , N 3 -bis (4-aminodinophenyl) propane-1, 3-diamine, a bis (benzamidino) derivative in which fluorinated acceptor forums are not present ( Figure 2).
  • the maximum excitation of compound 6 corresponds to the expected excitation band for coumarin at 432 nm.
  • AATTT a large increase in the intensity of the excitation spectrum is observed, and the excitation band of bis (benzamidine) at 329 nm becomes more intense, showing the transfer energy of the two fluorinated forums: exciting at 329 nm, bis (benzamidine) results in an increase in the emission of coumarin at 472 nm ( Figures 5a and 5b).
  • excitation spectra were recorded from 220 nm to 470 nm at 20 ° C with the following parameters: 1 nm increment; 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: 1 nm increment; integration time: 0.2 s; excitation bandwidth: 3 nm; emission bandwidth: 4 nm; excitation wavelength: 329 nm or 434 nm recorded from 345 to 640 nm.
  • the measurements were carried out with the following acquisition parameters: 1 nm increase; 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 640 nm ( Figure 3).

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

Compuestos derivados de bis-benzamidinios como agentes fluorogénicos para señalizar secuencias específicas de ADN de doble cadena.  Compounds derived from bis-benzamidiniums as fluorogenic agents to signal specific sequences of double stranded DNA.
La presente invención se refiere a un grupo de compuestos derivados de bis- benzamidinios como agentes fluorogénicos de reconocimiento de secuencias de ADN.  The present invention relates to a group of compounds derived from bisbenzamidiniums as fluorogenic agents for DNA sequence recognition.
ESTADO DE LA TECNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
Un objetivo importante de las investigaciones actuales en la frontera entre la química y la biología es el desarrollo de agentes químicos capaces de interaccionar de forma eficiente con secuencias específicas de ADN de doble cadena. A lo largo de las últimas décadas se han descrito un gran número de agentes heterocíclicos capaces de unirse selectivamente al ADN insertándose en su surco menor (cfr. Lown, J.W. Pharmacol. Ther. 1999, 84, 1-111). Entre estas moléculas, probablemente las más estudiadas sean aquellas basadas en poliamidas de pirrol e imidazol en estructura de horquilla, puesto que muestran unas propiedades excelentes en cuanto a afinidad y especificidad de secuencia. Desafortunadamente, la síntesis de estos compuestos esl laboriosa, y su utilidad in vivo está en gran medida limitada debido a sus dificultades intrínsecas para atravesar membranas biológicas, así como a su notable toxicidad.  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, J.W. 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.
Otro tipo de agentes que también reconocen el surco menor del ADN son los compuestos de tipo bis-benzamidinio, tales como la pentamidina (la) o propamidina (Ib). Estas moléculas son especialmente atractivas desde el punto de vista farmacológico, puesto que son estables y presentan buenas propiedades de transporte en numerosas líneas celulares. Así por ejemplo, la pentamidina esta siendo utilizada desde hace años contra la tripanosomiasis, la lesmaniasis y neumonía de P. Carinii. De entre los derivados de bis-amidinas, son también especialmente importantes aquellos con conectores de tipo aromático; de hecho la DB289, una prodroga de la furamidina (3), se encuentra en fase clínica III contra la enfermedad del sueño, y es muy activa contra otros parásitos (cfr. Mathis, A.M., et al., Antimicrob. Agents Chemother. 2007, 57, 2801-2810).
Figure imgf000003_0001
la
Figure imgf000004_0001
Another type of agents that also recognize the minor groove of DNA are bis-benzamidinium-type compounds, such as pentamidine (la) or propamidine (Ib). 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 , 57, 2801-2810).
Figure imgf000003_0001
the
Figure imgf000004_0001
Estas propiedades, junto con su simplicidad estructural, han animado la búsqueda de nuevos análogos con propiedades de reconocimiento mejoradas y toxicidades reducidas. En estos momentos, las mayores limitaciones para el desarrollo de este tipo de benzamidinios tienen que ver con la escasa versatilidad y practicidad de las rutas sintéticas existentes y con el hecho de que no exista un método simple, rápido y fiable que permita evaluar y cuantificar su afinidad y selectividad por secuencias específicas del ADN. El método habitual para estudiar la afinidad de este tipo de moléculas se basa en la desnaturalización térmica de sus complejos con el ADN. Sin embargo, este método sólo da información indirecta y cualitativa de las características de reconocimiento de estos compuestos y además es difícil de implementar en estrategias de high-throughput screening (cfr. Dardonville, C. et al., J. Med. Chem., 2006, 49, 3748-3752).  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).
La configuración dador/aceptor en las unidades de 4-carbamidoilfenoxi características de la estructura de propamidina podrían dar lugar a propiedades fluorogénicas y de emisión inducida cuando se insertan en el surco menor del ADN, como consecuencia del cambio ambiental y de polaridad que tienen lugar al insertarse estas moléculas en el surco menor del ADN; sin embargo, la longitud de onda de excitación de la estructura de propamidina coincide con la banda de absorción típica de ADN a 260 nm. Esta coincidencia impide la aplicación de técnicas de fluorescencia para el estudio de su interacción con el ADN. The donor / acceptor configuration in the 4-carbamidoylphenoxy units characteristic of the propamidine structure could give rise to fluorogenic and induced emission properties when inserted into the minor groove of the DNA, as a consequence of the environmental and polarity change that take place at insert these molecules into the minor groove of the DNA; however, the excitation wavelength of the propamidine structure coincides with the typical DNA absorption band at 260 nm. This coincidence prevents the application of fluorescence techniques for the study of their interaction with DNA.
Los autores de la presente invención han observado que sustituyendo las unidades de 4- fenoxiamidinio, características de los derivados clásicos de tipo benzamidínico, por 4- aminobenzamidinios se obtienen análogos que, además de interaccionar de forma selectiva con secuencias de ADN, presentan la ventaja adicional de que presentan un espectro de absorción desplazado hacia el rojo, que permite la excitación a una longitud de onda más larga (329 nm) y además experimentan un aumento significativo de su intensidad de emisión de fluorescencia a 387 nm cuando se unen a secuencias ricas en A/T pertenecientes a ADN de doble cadena, (O. Vázquez et.al., Org. Lett., 2010, 12, 216-219). DESCRIPCION BREVE DE LA INVENCION The authors of the present invention have observed that by substituting 4- phenoxyamidinium units, characteristic of the classical benzamidinic derivatives, by 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 bound to A / T rich sequences belonging to double stranded DNA, (O. Vázquez et.al., Org. Lett., 2010, 12, 216-219). BRIEF DESCRIPTION OF THE INVENTION
En la presente invención se describen nuevos derivados del tipo bis-4- aminobenzamidínico que incorporan en su estructura un fluoróforo aceptor. Estos compuestos presentan elevada afinidad por secuencias específicas de ADN de doble cadena, y además señalizan esta interacción emitiendo fluorescencia.  In the present invention, new derivatives of the bis-4-aminobenzamidine type are described which incorporate an acceptor fluorophore into their structure. These compounds have high affinity for specific double stranded DNA sequences, and also signal this interaction by emitting fluorescence.
La ventaja adicional de estos compuestos es que al estar presente en su estructura un fluoróforo aceptor emiten luz a una longitud de onda todavía más alta que en los compuestos en los que no están presentes estos grupos, debido a un proceso de transferencia de energía intramolecular desde el sistema de bis-4-aminobenzamidínico hasta el grupo fluoróforo aceptor. Gracias a este proceso de transferencia se extiende de forma efectiva el rango de longitudes de onda de emisión a las cuales se puede monitorizar la presencia de ADN. En un caso particular se incrementó el desplazamiento de Stokes en 145 nm, en lugar de en 60 nm como en los casos en los que no existe este grupo fluoróforo aceptor. Y además, modificando el grupo fluoróforo aceptor es posible diseñar derivados que emitan luz a diferentes longitudes de onda que sean de interés para su aplicación como biosensores luminiscentes. The additional advantage of these compounds is that when an acceptor fluorophore is present in their structure they emit light at an even higher wavelength than in the compounds in which these groups are not present, due to an intramolecular energy transfer process from the bis-4-aminobenzamidine system to the acceptor fluorophore group. Thanks to this transfer process, the range of emission wavelengths at which the presence of DNA can be monitored is effectively extended. In a particular case, Stokes' displacement increased by 145 nm, instead of 60 nm, as in cases where this acceptor fluorophore group does not exist. And in addition, by modifying the acceptor fluorophore group it is possible to design derivatives that emit light at different wavelengths that are of interest for application as luminescent biosensors.
Otra ventaja adicional es que la intensidad de la banda de emisión se puede incrementar hasta 60 veces, lo que permite detectar cantidades de ADN del orden de nanogramos.  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.
En un primer aspecto, la presente invención se refiere a un compuesto de fórmula general (I) In a first aspect, the present invention relates to a compound of general formula (I)
Figure imgf000005_0001
donde Z es un grupo fluoróforo aceptor y n tiene un valor de 1 a 5. En otro aspecto, la invención se refiere a un procedimiento de preparación de un compuesto de fórmula general (I) que comprende una reacción entre un compuesto de fórmula II y un fluoróforo aceptor,
Figure imgf000005_0001
where Z is an acceptor fluorophore group and n has a value of 1 to 5. In another aspect, the invention relates to a process for preparing a compound of general formula (I) comprising a reaction between a compound of formula II and an acceptor fluorophore,
Figure imgf000006_0001
donde Y se selecciona entre hidrógeno y un agente quelante, y n tiene un valor de 1 a 5.
Figure imgf000006_0001
where Y is selected from hydrogen and a chelating agent, and n has a value of 1 to 5.
En otro aspecto, la invención se refiere al uso de un compuesto de fórmula general (I) como agente fluorogénico de reconocimiento de secuencias de ADN. In another aspect, the invention relates to the use of a compound of general formula (I) as a fluorogenic agent for DNA sequence recognition.
DESCRIPCION DETALLADA DE LA INVENCION DETAILED DESCRIPTION OF THE INVENTION
En la presente invención, el término "agente fluorogénico" se refiere a una molécula fluorescente que posee un grupo funcional que absorbe energía de una longitud de onda específica y la emite en otra determinada de mayor longitud de onda (es decir, con menor energía), y en el que la cantidad de energía emitida (rendimiento cuántico) depende tanto del propio grupo funcional fluorescente como de su ambiente químico. In the present invention, the term "fluorogenic agent" refers to a fluorescent molecule that has a functional group that absorbs energy of a specific wavelength and emits it in a given one of greater wavelength (ie, with less energy) , and in which the amount of energy emitted (quantum yield) depends on both the fluorescent functional group itself and its chemical environment.
El término "fluoróforo aceptor" se refiere, en la presente invención, a una especie que participa en un proceso de transmisión de energía de resonancia (FRET) aceptando la energía transferida por otra especie que se encuentra en estado excitado (esta transferencia puede tener lugar a través de cualquier proceso fotofísico, incluyendo mecanismos de tipo Fórster o Dexter). Un parámetro clave para la transferencia de energía entre dos fluoróforos es la combinación de especies con niveles de energía próximos. Esta proximidad se relaciona con la teoría de transferencia de manera que cuanto mayor sea el valor de la integral de solapamiento entre el dador y aceptor (JDA), entre el espectro de emisión del dador y el espectro de absorción del aceptor, mejor será el FRET. En la presente invención, se incorpora la teoría de transferencia de energía, así como el cálculo de dicha integral, la interpretación de sus valores y la detección de energía de transferencia por medio del siguiente artículo: "Resonante Energy Transfer: Methods and Applications", Analytical Biochemistry, 1994, 218, 1- 13. Además, estos requerimientos energéticos pueden ser visualizados fácilmente por el solapamiento del espectro de emisión del fluoróforo dador y el espectro de excitación del fluoróforo aceptor. En un caso particular de la presente invención se estudió este solapamiento (figura 4) entre espectro de emisión del aminobenzamidinio derivado N;,N3-bis(4-aminodinofenil)propano-l,3-diamina y el espectro de absorción del compuesto 6, comprobándose que esta combinación de especies es adecuada para llevar a cabo la invención. 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 Forster 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 giver 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 easily be visualized by the overlapping 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 emission spectrum of the N-derived aminobenzamidinium ; , 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.
El término "alquilo" se refiere, en la presente invención, a cadenas alifáticas, lineales o ramificadas, que tienen de 1 a 10 átomos de carbono, preferiblemente entre 1 y 5 átomos de carbono. Por ejemplo, pero sin limitarse a, metilo, etilo, n-propilo, «-butilo, n-pentilo, n-hexilo, n-heptilo etc. Los grupos alquilo pueden estar opcionalmente modificados por uno o más sustituyentes tales como amina, halógeno, hidroxilo o ácido carboxílico. 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, "-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.
En una realización preferida, la presente invención se refiere a un compuesto de fórmula I donde Z, el grupo fluoróforo aceptor, se selecciona entre un ión lantánido complejado con un agente quelante o una cumarina de fórmula general III. In a preferred embodiment, the present invention relates to a compound of formula I wherein Z, the acceptor fluorophore group, is selected from a lanthanide ion complexed with a chelating agent or a coumarin of general formula III.
Figure imgf000007_0001
Figure imgf000007_0001
donde Rl se selecciona entre -NR4R5, -OH, -OR4, -OCOR4, siendo R4 y R5 iguales o diferentes seleccionados entre hidrógeno, alquilo, y where Rl is selected from -NR4R5, -OH, -OR4, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl, and
R3 se selecciona entre hidrógeno, halógeno y alquilo. R3 is selected from hydrogen, halogen and alkyl.
En una realización más preferida, el ión lántanido se selecciona entre Eu 3 y Tb En una realización preferida, el agente quelante se selecciona entre el ácido 1,4,7,10- tetraazaciclodecano-l,4,7,10-tetraacético (DOTA), el ácido dietilentriaminopentaacético (DTP A), 4,7,10-Tetraazaciclododecane- 1,4,7,10- tetra(ácido metilen fosfónico) (DOTP), ácido 1,4,8,1 l-tetraazaciclo-dodecano-1,4, 8,11- tetraacético (TETA). In a more preferred embodiment, the lanthanide ion is selected from Eu 3 and Tb In a preferred embodiment, the chelating agent is selected from 1,4,7,10-tetraazacyclodecano-1, 4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTP A), 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic acid) (DOTP), 1,4,8,1-tetraazacyclo-dodecane-1,4, 8,11-tetraacetic acid (TETA).
En otra realización preferida, n es 3. In another preferred embodiment, n is 3.
En otra realización preferida, la invención se refiere a un procedimiento de obtención de los compuestos de fórmula (I) como se describió anteriormente, que comprende la reacción entre un compuesto de fórmula II, como se describió anteriormente, con una especie fluoróforo aceptor, donde la especie fluoróforo aceptor se selecciona entre un ión lantánido o derivados de cumarina de fórmula IV, In another preferred embodiment, the invention relates 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, with an acceptor fluorophore species, wherein the fluorophore acceptor species is selected from a lanthanide ion or coumarin derivatives of formula IV,
Figure imgf000008_0001
iv
Figure imgf000008_0001
iv
donde Rl se selecciona entre -NR4R5, -OH, -OCOR4, siendo R4 y R5 iguales o diferentes seleccionados entre hidrógeno, alquilo, where Rl is selected from -NR4R5, -OH, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl,
R2 se selecciona entre hidrógeno, alquilo, triflato, tosilo, succinimidilo o haluro, y R3 se selecciona entre hidrógeno, halógeno y alquilo.  R2 is selected from hydrogen, alkyl, triflate, tosyl, succinimidyl or halide, and R3 is selected from hydrogen, halogen and alkyl.
En una realización particular, la invención se refiere a un procedimiento de obtención de los compuestos de fórmula (I) como se describió anteriormente, que comprende la reacción entre un compuesto de fórmula II, como se describió anteriormente y una especie fluoróforo aceptor, que In a particular embodiment, the invention relates 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 an acceptor fluorophore species, which
- cuando Y es hidrógeno entonces la especie fluoróforo aceptor se selecciona entre derivados de cumarina de fórmula IV, como se definió anteriormente. Cuando R2 es hidrógeno el procedimiento comprende un acoplamiento entre ambos en presencia de un activador de grupos carboxílicos.  - when Y is hydrogen then the acceptor fluorophore species is selected from coumarin derivatives of formula IV, as defined above. When R2 is hydrogen, the process comprises a coupling between the two in the presence of a carboxylic group activator.
Son activadores de grupos carboxílicos aquellos que modifican el grupo carboxílico convirtiendo el hidroxilo en un mejor grupo saliente y permiten llevar a cabo la reacción de acoplamiento de forma más rápida, más eficaz y en condiciones más suaves. Por ejemplo, son activadores de grupos carboxílicos los derivados de carbodiimidas como la diciclohexilcarbodiimida, o la diisopropilcarbodiimida; derivados de triazoles como por ejemplo el 1-hidroxi- benzotriazol, l-hidroxi-7-aza-benzotriazol; derivados de sales de fosfonio o de uranio de un anión no nucleofílico como tetrafluoroborato o hexafluorofosfato, por ejemplo hexafluorofosfato de 0-benzotriazol-N,N,N',N'-tetrametil-uronio (HBTU), hexafluorofosfato de 2-(lH-7-azabenzotriazol-l-il)-l,l,3,3-tetrametil uranio matanaminio (HATU), hexafluorofosfato de 7-azabenzotriazol-l-iloxi-tris- (pirro lidino)-fosfonio (PyOAP), hexafluorofosfato de benzotriazol- 1-iloxi-tris-Carboxylic group activators are those that modify the carboxylic group by converting the hydroxyl into a better leaving group and allow Perform the coupling reaction faster, more efficiently and in milder conditions. For example, carbodiimide derivatives such as dicyclohexylcarbodiimide, or diisopropylcarbodiimide are activators of carboxylic groups; triazole derivatives such as 1-hydroxybenzotriazole, l-hydroxy-7-aza-benzotriazole; derivatives of phosphonium or uranium salts of a non-nucleophilic anion such as tetrafluoroborate or hexafluorophosphate, for example 0-benzotriazol-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU), 2- (lH- hexafluorophosphate) 7-azabenzotriazol-l-yl) -l, l, 3,3-tetramethyl uranium matanaminio (HATU), 7-azabenzotriazol-l-yloxy-tris- (pyropyl) -phosphonium (PyOAP) hexafluorophosphate benzotriazol- hexafluorophosphate 1-yloxy-tris-
(pirrolidino)-fosfonio (PyBOP); (pyrrolidino) -phosphonium (PyBOP);
- cuando Y es un agente quelante entonces el fluoróforo aceptor se selecciona entre iones lantánidos, preferentemente entre Eu 3, Tb 3, y la reacción que tiene lugar es una reacción de complejación con la correspondiente sal del ión trivalente del lantánido, pudiendo ser tricloruros, tribromuros y triyoduros, sulfates o triflatos. - when Y is a chelating agent then the acceptor fluorophore is selected from lanthanide ions, preferably from Eu 3 , Tb 3 , and the reaction that takes place is a complexation reaction with the corresponding salt of the trivalent lanthanide ion, which may be trichlorides, tribromides and triiodides, sulfates or triflates.
En una realización más particular, el compuesto de fórmula II donde Y es un grupo quelante se prepara mediante un procedimiento que comprende una reacción de acoplamiento entre un compuesto de fórmula Ha In a more particular embodiment, the compound of formula II wherein Y is a chelating group is prepared by a process comprising a coupling reaction between a compound of formula Ha
Figure imgf000009_0001
Figure imgf000009_0001
donde Ya es hidrógeno y n se selecciona entre 1 y 5,  where it is already hydrogen and n is selected between 1 and 5,
y un agente quelante que posee un grupo ácido carboxílico, como por ejemplo ácido l,4,7,10-tetraazaciclododecano-l,4,7,10-tetraacético (DOTA) o ácido dietilentriaminopentaacético (DTPA), 4,7,10-Tetraazaciclododecane- 1,4,7,10- tetra(ácido metilen fosfónico) (DOTP), ácido 1,4,8,11-tetraazaciclo-dodecano- 1,4,8,11-tetraacético (TETA), en presencia de un agente activador de grupos carboxílicos. De forma preferida el agente quelante se encuentra en la forma protegida, es decir, todos los grupos acéticos están protegidos excepto uno, por ejemplo con grupos tere-butilo como ácido 1, 4,7,10-tetraazaciclododecano- 1,4,7- tris-terc-butilacetato-10-acético. and a chelating agent having a carboxylic acid group, such as, for example, l, 4,7,10-tetraazacyclododecane-l, 4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), 4,7,10- Tetraazacyclododecane- 1,4,7,10-tetra (methylene phosphonic acid) (DOTP), 1,4,8,11-tetraazacyclo-dodecane- 1,4,8,11-tetraacetic acid (TETA), in the presence of a carboxylic group activating agent. Preferably, the chelating agent is in the protected form, that is, all acetic groups are protected except one, for example with tere-butyl groups such as 1,4,7,10-tetraazacyclocyclodedecane-1,4,7- tris-tert-butylacetate-10-acetic.
En otra realización particular, el compuesto II se puede desproteger mediante reacciones rutinarias conocidas por el experto en la materia en condiciones habituales de desprotección (Greene, T. W. "Protective Groups in Organic Síntesis ", 3rd Ed, Wiley-Interscience, 1999). Es típica la desprotección de un terc-butilcarboxilato en medio ácido, por ejemplo, ácido trifluoroacético. In another particular embodiment, compound II can be deprotected by routine reactions known to those skilled in the art under usual deprotection conditions (Greene, T. W. "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.
En otra realización preferida, la presente invención se refiere al uso de un compuesto según se describió anteriormente, en el que la secuencia de ADN que se reconoce está formada por 4, 5 ó 6 pares de bases A/T. 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 base pairs A / T.
Preferiblemente, la secuencia de ADN que se reconoce está formada por 4 ó 5 pares de bases A/T.  Preferably, the DNA sequence that is recognized is formed by 4 or 5 base pairs A / T.
En otra realización preferida, la secuencia de bases se selecciona entre AATT y AATTT.  In another preferred embodiment, the base sequence is selected from AATT and AATTT.
En una realización preferida, la presente invención se refiere al uso del compuesto según se describió anteriormente para ensayos de identificación de agentes de reconocimiento del surco menor del ADN por desplazamiento de la fluorescencia. In a preferred embodiment, the present invention relates to the use of the compound as described above for identification tests of minor groove DNA recognition agents by fluorescence shift.
DESCRIPCION DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figura la. Espectros de emisión del compuesto 5a en tampón Tris-HCl 20mM pH 7.5, 100 mM NaCl, que muestra las transiciones electrónicas correspondientes; espectros en ausencia de ADN (■); con 9 equivalentes de ADN no específico GGCCC (o); con 9 equivalentes de ADN AATTC ( ); con 9 equivalentes de ADN diana AATTT (·).  Figure the. Emission spectra of compound 5a in 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl, showing the corresponding electronic transitions; spectra in the absence of DNA (■); with 9 equivalents of non-specific GGCCC (o) DNA; with 9 equivalents of AATTC () DNA; with 9 equivalents of AATTT (·) target DNA.
Figura Ib. Espectros de emisión del compuesto 5b en tampón Tris-HCl 20mM pH 7.5, 100 mM NaCl, que muestra las transiciones electrónicas correspondientes; espectros en ausencia de ADN (■); con 9 equivalentes de ADN no específico GGCCC (o); con 9 equivalentes de ADN AATTC ( ); con 9 equivalentes de ADN diana AATTT (·). Figure Ib. Emission spectra of compound 5b in 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl, showing the corresponding electronic transitions; spectra in the absence of DNA (■); with 9 equivalents of non-specific GGCCC (o) DNA; with 9 AATTC () DNA equivalents; with 9 equivalents of AATTT (·) target DNA.
Figura 2. En la gráfica se compara: Figure 2. The graph compares:
a) la emisión de fluorescencia de N;,N3-bis(4-aminodinofenil)propano-l,3-diamina (0.5 μΜ en tampón Tris-HCl) con 9 equiv. de ADN diana (AATTT,■); a) the fluorescence emission of N ; , N 3 -bis (4-aminodinophenyl) propane-l, 3-diamine (0.5 μΜ in Tris-HCl buffer) with 9 equiv. target DNA (AATTT, ■);
b) la emisión de fluorescencia del compuesto 6 (0.5 μΜ en tampón Tris-HCl) (o;  b) the fluorescence emission of compound 6 (0.5 μΜ in Tris-HCl buffer) (or;
la intensidad de este espectro se presenta multiplicado por 10 para mayor claridad); y  the intensity of this spectrum is presented multiplied by 10 for clarity); Y
c) la emisión de fluorescencia del compuesto 6 (0.5 μΜ en tampón Tris-HCl) en presencia de 9 equiv. de ADN diana (AATTT, ·).  c) the fluorescence emission of compound 6 (0.5 μΜ in Tris-HCl buffer) in the presence of 9 equiv. of target DNA (AATTT, ·).
En la figura insertada se muestra la fluorescencia de las cubetas con soluciones de 500 nM de 6 con una excitación a 329 nm: a) no se añadió ADN; b) 2 equiv de ADN AATTT. The inserted figure shows the fluorescence of the cuvettes with solutions of 500 nM of 6 with an excitation at 329 nm: a) no DNA was added; b) 2 equiv of AATTT DNA.
Figura 3a. Espectros de emisión de una solución 0.5 micromolar del compuesto 6 incrementando las cantidades de ADN diana (AATTT): 0, 0.4, 0.7, 1, 1.8, 2.5, 3.2 y 4 micromolar. La gráfica insertada muestra la valoración a 472 nm del compuesto 6 en tampón Tris-HCl 20mM pH 7.5, 100 mM NaCl con ADN AATTT y demuestra que se ajusta a un modelo de unión 1 : 1, siendo la constante de disociación (814 + 90) nM. Figure 3a. Emission spectra of a 0.5 micromolar solution of compound 6 increasing the amounts of target DNA (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 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl with AATTT DNA and demonstrates that it conforms to a 1: 1 binding model, the dissociation constant being (814 + 90 ) nM.
Figura 3b. Espectros de emisión de una solución 0.5 micromolar del compuesto 6 incrementando las cantidades de ADN truncado (AAT): 0, 0.4, 0.7, 1, 1.8, 2.5, 3.2 y 4 micromolar. La gráfica insertada muestra la valoración a 472 nm del compuesto 6 en tampón Tris-HCl 20mM pH 7.5, 100 mM NaCl con ADN AATGC y demuestra que se ajusta a un modelo de unión 1 : 1, siendo la constante de disociación (2.38 ±0.08) μΜ. Figure 3b Emission spectra of a 0.5 micromolar solution of compound 6 increasing the amounts of truncated DNA (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 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl with AATGC DNA and demonstrates that it conforms to a 1: 1 binding model, the dissociation constant being (2.38 ± 0.08 ) μΜ.
Figura 4. Banda de absorción de la unidad aza-benzamidinio: línea punteada a). Banda de absorción de la cumarina: línea punteada b). Banda de emisión de fluorescencia de Nl,N3-bis(4-aminodinofenil)propano-l,3-diamina, línea sólida (o). Banda de emisión de fluorescencia del compuesto 6, línea sólida (·). Los espectros se normalizaron para la comparación. Figura 5a. Espectro de excitación a 472 nm de 0.5 micromolar del compuesto 6 en tampón Tris-HCl 20 mM pH 7.5, 100 mM NaCl (línea punteada); y con 9 equiv. de AATTT ADN (línea continua). Figure 4. Absorption band of the aza-benzamidinium unit: dotted line a). Coumarin absorption band: dotted line b). Fluorescence emission band of N1, N3-bis (4-aminodinophenyl) propane-1, 3-diamine, solid line (o). 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 of compound 6 in 20 mM Tris-HCl buffer pH 7.5, 100 mM NaCl (dotted line); and with 9 equiv. of AATTT ADN (continuous line).
Figura 5b. Espectro de emisión del compuesto 6 en ausencia de ADN(B); espectro de emisión del compuesto 6 con 9 equiv. de AATTT ADN y longitud de onda de excitación de 432 nm (o); espectro de emisión con 9 equiv. de AATTT ADN y longitud de onda de excitación de 329 nm (·). Figure 5b Emission spectrum of compound 6 in the absence of DNA (B); emission spectrum of compound 6 with 9 equiv. of AATTT DNA and excitation wavelength of 432 nm (o); emission spectrum with 9 equiv. of AATTT DNA and excitation wavelength of 329 nm (·).
Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. EJEMPLOS The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES
Preparación de compuestos Compound Preparation
Los reactivos y disolventes se adquirieron en casas comerciales, y en concreto el ácido 1 ,4,7, 10-tetraazaciclododecano- 1 ,4,7-tris-terc-butilacetato- 10-acético fue suministrado por Macrocyclics y las cumarinas pueden adquirirse a Sigma-Aldrich.  The reagents and solvents were purchased in commercial houses, and in particular 1, 4,7, 10-tetraazacyclododecane-1, 4,7-tris-tert-butylacetate-10-acetic acid was supplied by Macrocyclics and coumarins can be purchased at Sigma-Aldrich.
Ejemplo 1. Preparación del compuesto 3. Example 1. Preparation of compound 3.
Figure imgf000012_0001
Una dispersión del 60% de hidruro sódico (68 mg, 476 μηιοΐ, 4 equiv) se añadió a una disolución del compuesto 2 (66 mg, 119 μιηοΐ, 1 equiv) en DMSO seco (1.5 mL). Después de 30 min, se añadió l-yodo-terc-butil-2-aminopropilcarbamato (19 mg, 239 μιηοΐ, 2 equiv) en porciones. La mezcla de reacción se agitó bajo argón a temperature ambiente durante 6 h. El crudo de reacción se purificó mediante cromatografía en fase reversa (Büchi Sepacore) (A: metanol; B: agua 0.1% ácido trifluoroacético; gradiente: 15% B, 5 min; 15%→ 95 % B, 30 min.).
Figure imgf000012_0001
A dispersion of 60% sodium hydride (68 mg, 476 μηιοΐ, 4 equiv) was added to a solution of compound 2 (66 mg, 119 μιηοΐ, 1 equiv) in dry DMSO (1.5 mL). After 30 min, l-iodo-tert-butyl-2-aminopropylcarbamate (19 mg, 239 μιηοΐ, 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.).
El compuesto aislado se disolvió en CH2CI2 (1 mL) y se enfrió a 0 °C, se añadió ácido trifluoroacético (1 mL) y se agitó la mezcla a 0 °C durante lh. Se eliminó el disolvente a sequedad y el crudo se purificó mediante cromatografía en fase reversa (Büchi Sepacore) (gradiente: 0%> B, 5 min; 0%>→ 50 % B, 30 min.) para obtener el compuesto 3 (13 mg, 39%). 1H-RMN δ (DMSO- ): 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). 13C-RMN δ (DMSO-í/é): 27.6 (CH2), 36.5 (CH2), 43.8 (CH2), 66.4 (CH2), 76.4 (CH), 111.2 (CH), 112.4 (C), 129.6 (CH), 153.5 (C), 158.4 (C, TFA), 164.1 (C). The isolated compound was dissolved in CH 2 CI 2 (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-): 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-í / é ): 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).
Ejemplo 2. Preparación de los compuestos 5a y 5b. Example 2. Preparation of compounds 5a and 5b.
Figure imgf000013_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000014_0001
Una disolución del ácido l ,4,7,10-tetraazaciclododecane-l ,4,7-tris-terc-butil acetato- 10-acético (4, 52 mg, 92 μηιοΐ, 4 equiv) en dimetilformamida/diisopropiletilamina 0.2M (460 μί, 92 μηιοΐ 4 equiv) sea activó con HATU (35 mg, 92 μιηοΐ, 4 equiv) durante 5 min, se añadió sobre 3 (9 mg, 23 μιηοΐ, 1 equiv) en dimetilformamida/diisopropiletilamina 0.2M (230 μί, 46 μιηοΐ 2 equiv) y se agitó durante 2 h. Se eliminó el disolvente y el crudo se redisolvió en CH2CI2 (500 μί), se enfrió y se añadió ácido trifluoroacético (500 μί). Se concentró a sequedad y el crudo se purificó mediante cromatografía en fase reversa (Büchi Sepacore) (gradiente: 8% B, 5 min; 8%→ 40 % B, 30 min.), obteniendo el compuesto intermedio (19 mg, 85 %). 1 H-RMN δ (DMSO-í é/CDzC ): 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), 3.1 1-3.16 (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). 13C-RMN δ (DMSO-
Figure imgf000014_0002
27.6 (CH2), 28.6 (CH2), 28.8 (CH2), 30.7 (CH2), 36.6 (CH2), 41.7 (CH2), 43.8 (CH2), 44.9 (CH2), 45.0 (CH2), 47.5 (CH2), 66.5 (CH2), 76.5 (CH), 1 1 1.1 (CH), 1 12.4 (C), 129.7 (CH), 153.5 (C), 158.0 (C, TFA), 164.0 (C), 169.5 (C). A solution of l, 4,7,10-tetraazacyclododecane-l, 4,7-tris-tert-butyl acetate-10-acetic acid (4,52 mg, 92 μηιοΐ, 4 equiv) in 0.2M dimethylformamide / diisopropylethylamine (460 μί, 92 μηιοΐ 4 equiv) be activated with HATU (35 mg, 92 μιηοΐ, 4 equiv) for 5 min, added over 3 (9 mg, 23 μιηοΐ, 1 equiv) in 0.2M dimethylformamide / diisopropylethylamine (230 μί, 46 μιηοΐ 2 equiv) and stirred for 2 h. The solvent was removed and the crude was redissolved in CH 2 CI 2 (500 μί), cooled and trifluoroacetic acid (500 μί) 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-í é / CDzC): 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), 3.1 1-3.16 (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-
Figure imgf000014_0002
27.6 (CH 2 ), 28.6 (CH 2 ), 28.8 (CH 2 ), 30.7 (CH 2 ), 36.6 (CH 2 ), 41.7 (CH 2 ), 43.8 (CH 2 ), 44.9 (CH 2 ), 45.0 ( CH 2 ), 47.5 (CH 2 ), 66.5 (CH 2 ), 76.5 (CH), 1 1 1.1 (CH), 1 12.4 (C), 129.7 (CH), 153.5 (C), 158.0 (C, TFA) , 164.0 (C), 169.5 (C).
La quelación con el lantánido se llevó a cabo del mismo modo para los dos casos. Se disolvió el intermedio obtenido en una disolución tampón 600 μί HEPES buffer 10 mM, pH 7.5, NaCl lOOmM y se añadió el cloruro de lantano correspondiente (50 mM en HCl lmM). La mezcla se agitó durante 1 h y se purificó mediante cromatografía (gradiente: 95/5→25/75 30 min, agua/acetonitrilo, 0.1% ácido trifluoroacético). 5a: MALDI/TOF-MS: [M+H]+calcd. para C36H56Eu Nii08 = 920.3 encontrado 920.1. 5b: M ALDI/TOF-M S : [M+H]+calcd. para C36H56Nii08Tb = 926.3 encontrado 926.3. Chelation with lanthanide was carried out in the same way for both cases. The intermediate obtained was dissolved in a 600 μί buffer solution 10 mM HEPES buffer, pH 7.5, 10 mM NaCl and the corresponding lanthanum chloride (50 mM in lmM HCl) was added. 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 3 6H 56 Eu Nii0 8 = 920.3 found 920.1. 5b: M ALDI / TOF-M S: [M + H] + calcd. for C 36 H 56 Nii0 8 Tb = 926.3 found 926.3.
Ejemplo 3. Preparación del compuesto 6. Example 3. Preparation of compound 6.
Figure imgf000015_0001
Figure imgf000015_0001
Una disolución del ácido 7-dietilaminocumarin-3-carboxílico (24 mg, 92 μιηοΐ, 4 equiv) en dimetilformamida/diisopropiletilamina 0.2 M (460 μί, 92 μιηοΐ 4 equiv) se activó con PyAOP (48 mg, 92 μιηοΐ, 4 equiv), se añadió sobre 3 (9 mg, 23 μιηοΐ, 1 equiv) en dimetilformamida/diisopropiletilamina 0.2 M (230 μΐ,, 46 μιηοΐ 2 equiv) y se agitó durante 2 h. El residuo se purificó mediante cromatografía en fase reversa (Büchi Sepacore) (gradiente: 15% B, 5 min; 15%→95 % B, 30 min.), para obtener el producto (12 mg, 62 %). 1H-RMN δ (DMSO- d6): 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, J2 = 46.4, J¡ = 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). 13C-RMN δ (DMSO- ): 12.3 (CH3), 27.7 (CH2), 36.4 (CH2), 44.1 (CH2), 44.3 (CH2), 46.6 (CH2), 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). Ensayos de fluorescencia A solution of 7-diethylaminocumarin-3-carboxylic acid (24 mg, 92 μιηοΐ, 4 equiv) in 0.2 M dimethylformamide / diisopropylethylamine (460 μί, 92 μιηοΐ 4 equiv) was activated with PyAOP (48 mg, 92 μιηοΐ, 4 equiv) , about 3 (9 mg, 23 μιηοΐ, 1 equiv) in 0.2 M dimethylformamide / diisopropylethylamine (230 μΐ ,, 46 μιηοΐ 2 equiv) was added 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¡ = 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-): 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). Fluorescence assays
Los experimentos de fluorescencia se llevaron a cabo en fluorímetro Jobin- Yvon Fluoromax-3 (DataMax 2.20), acoplado a un sistema de control de temperatura Wavelength Electronics LFI-3751. Fluorescence experiments were carried out on a Jobin-Yvon Fluoromax-3 fluorimeter (DataMax 2.20), coupled to a Wavelength Electronics LFI-3751 temperature control system.
Las medidas para el compuesto 5a se llevaron a cabo con los siguientes parámetros de adquisición: incremento 1 nm; tiempo de integración: 0.2 s; ancho de banda de excitación: 6 nm; ancho de banda de emisión: 6 nm; longitud de onda de excitación: 329 nm. Los espectros de emisión se registraron a 20°C entre 550 y 750 nm.  Measurements for compound 5a were carried out with the following acquisition parameters: 1 nm increase; 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.
Las medidas para el compuesto 5b se llevaron a cabo con los siguientes parámetros de adquisición: incremento 1 nm; tiempo de integración: 0.2 s; ancho de banda de excitación: 3 nm; ancho de banda de emisión: 6 nm; longitud de onda de excitación: 329 nm. Los espectros de emisión se registraron a 20°C entre 345 y 600 nm.  Measurements for compound 5b were carried out with the following acquisition parameters: 1 nm increase; 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.
Los ADNs, suministrados por Termo Fischer Scientific GMBH, fueron horquillas de doble hélice con las siguientes secuencias. The DNAs, supplied by Termo Fischer Scientific GMBH, were double helix forks with the following sequences.
DNA Secuencia completa (5' to 3') DNA Full sequence (5 'to 3')
AATTT GGCG AA TTT CGC TTTTT GCG AAATT CGCC AATTT GGCG AA TTT CGC TTTTT GCG AAATT CGCC
AATTC GGCG AA TTC AGC TTTTT GCT GAATT CGCCAATTC GGCG AA TTC AGC TTTTT GCT GAATT CGCC
AATGC GGCG AA TGC AGC TTTTT GCT GC ATT CGCCAATGC GGCG AA TGC AGC TTTTT GCT GC ATT CGCC
GGCCC GGCA GGCCC AGC TTTTT GCT GGGCC TGCC GGCCC GGCA GGCCC AGC TTTTT GCT GGGCC TGCC
Los espectros de emisión se muestran en la figura la y Ib. Como los espectros con AATGC son muy similares a los obtenidos con GGCCC, se concluye que tres pares de bases A/T es una secuencia demasiado corta para acomodar la unidad de bis- aminobenzamidinio. Se acomodan mejor en pares de bases A/T de 4, 5 ó 6 pares de bases. Diseño y fluorescencia del compuesto 6. The emission spectra are shown in figure la and Ib. Since the spectra with AATGC are very similar to those obtained with GGCCC, it is concluded that three base pairs A / T is too short a sequence to accommodate the bis-aminobenzamidinium unit. They are best accommodated in A / T base pairs of 4, 5 or 6 base pairs. Design and fluorescence of compound 6.
La fluorescencia de emisión del compuesto 6 se realizó con una disolución tampón, y con 9 equiv. de AATTT ADN. Después de la excitación a 329 nm, la fluorescencia de emisión del compuesto 6 en tampón es muy débil y esta dominada por la banda de emisión de cumarina a 472 nm. The emission fluorescence of compound 6 was performed with a buffer solution, and with 9 equiv. of AATTT ADN. After excitation at 329 nm, the emission fluorescence of compound 6 in buffer is very weak and is dominated by the band of emission of coumarin at 472 nm.
La adición de ADN AATTT induce un incremento de aproximadamente 60 veces la intensidad de la fluorescencia de emisión del compuesto 6. La emisión es tan intensa en este caso que incluso es posible detectar menos de 3 nanogramos de ADN (figura 2). Así, la adecuada conjugación de la estructura bis-(benzamidinio) con el grupo fluoróforo aceptor, de estructura derivada de la cumarina, conlleva a un cambio de fluorescencia significativo cuando se une a secuencias específicas de ADN (AATTT), como lo demuestra el incremento del desplazamiento (145 nm) respecto a la fluorescencia de emisión de N;,N3-bis(4-aminodinofenil)propano-l,3-diamina, un derivado bis(benzamidino) en el que no están presentes grupos fluoró foros aceptores (figura 2). The addition of AATTT DNA induces an approximately 60-fold increase in the emission fluorescence intensity 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 displacement (145 nm) with respect to the emission fluorescence of N ; , N 3 -bis (4-aminodinophenyl) propane-1, 3-diamine, a bis (benzamidino) derivative in which fluorinated acceptor forums are not present (Figure 2).
En la figura 5b se comparan los espectros anteriores con el espectro de emisión del compuesto 6 (realizado en las mismas condiciones) con 9 equiv. de ADN AATTT y a una longitud de onda de excitación de 432 nm. In Figure 5b the above spectra are compared with the emission spectrum of compound 6 (performed under the same conditions) with 9 equiv. of AATTT DNA and at an excitation wavelength of 432 nm.
El máximo de excitación del compuesto 6 corresponde con la banda de excitación esperada para cumarina a 432 nm. Tras la adición de AATTT, se observa un gran incremento en la intensidad del espectro de excitación, y la banda de excitación de la bis(benzamidina) a 329 nm se vuelve más intensa, mostrando la energía de transferencia de los dos fluoró foros: excitando a 329 nm la bis(benzamidina) resulta un incremento en la emisión de cumarina a 472 nm (figuras 5a y 5b).  The maximum excitation of compound 6 corresponds to the expected excitation band for coumarin at 432 nm. After the addition of AATTT, a large increase in the intensity of the excitation spectrum is observed, and the excitation band of bis (benzamidine) at 329 nm becomes more intense, showing the transfer energy of the two fluorinated forums: exciting at 329 nm, bis (benzamidine) results in an increase in the emission of coumarin at 472 nm (Figures 5a and 5b).
Los espectros de excitación se registraron desde 220 nm a 470 nm a 20°C con los siguientes parámetros: incremento 1 nm; tiempo de integración: 0.2 s; ancho de banda de excitación: 3 nm; ancho de banda de emisión: 4 nm; longitud de onda de emisión: 474 nm. The excitation spectra were recorded from 220 nm to 470 nm at 20 ° C with the following parameters: 1 nm increment; integration time: 0.2 s; excitation bandwidth: 3 nm; emission bandwidth: 4 nm; emission wavelength: 474 nm.
Los espectros de emisión se llevaron a cabo con los siguientes parámetros: incremento 1 nm; tiempo de integración: 0.2 s; ancho de banda de excitación: 3 nm; ancho de banda de emisión: 4 nm; longitud de onda de excitación: 329 nm o 434 nm registrado desde 345 a 640 nm.  The emission spectra were carried out with the following parameters: 1 nm increment; integration time: 0.2 s; excitation bandwidth: 3 nm; emission bandwidth: 4 nm; excitation wavelength: 329 nm or 434 nm recorded from 345 to 640 nm.
Valoración del compuesto 6 con diferentes ADNs Titration of compound 6 with different DNAs
Los experimentos de fluorescencia se llevaron a cabo en fluorímetro Jobin- Yvon Fluoromax-3 (software DataMax 2.20), acoplado a un sistema de control de temperatura Wavelength Electronics LFI-3751. Fluorescence experiments were carried out on Jobin-Yvon Fluoromax-3 fluorimeter (DataMax 2.20 software), coupled to a control system Wavelength Electronics LFI-3751 temperature.
Excepto para el caso de los experimentos con lantánidos, las medidas se llevaron a cabo con los siguientes parámetros de adquisición: incremento 1 nm; tiempo de integración: 0.2 s; ancho de banda de excitación: 3 nm; ancho de banda de emisión: 6 nm; longitud de onda de excitación: 329 nm. Los espectros de emisión se registraron a 20 °C entre 345 y 640 nm (figura 3).  Except for the case of the lanthanide experiments, the measurements were carried out with the following acquisition parameters: 1 nm increase; 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 640 nm (Figure 3).
Todas las valoraciones se llevaron a cabo siguiendo el mismo procedimiento: sobre 1 mL de disolución de 0.5 microM del compuesto 6 en tampón Tris-HCl 20mM pH 7.5, 100 mM NaCl, se añadieron sucesivas alícuotas de aproximadamente 400 microM de disolución de ADN y se registró el espectro de fluorescencia para cada adición.  All titrations were carried out following the same procedure: on 1 mL of 0.5 microM solution of compound 6 in 20mM Tris-HCl buffer pH 7.5, 100 mM NaCl, successive aliquots of approximately 400 microM DNA solution were added and recorded the fluorescence spectrum for each addition.
Se observó que la adición del ADN truncado AATGC induce incrementos significativamente más pequeños en la emisión del compuesto 6 que la adición del ADN AATTT a 472nm.  It was observed that the addition of truncated AATGC DNA induces significantly smaller increases in the emission of compound 6 than the addition of AATTT DNA at 472 nm.
El máximo de emisión se empleó en la determinación de la constante de unión. Así, los valores de la constante de disociación KD que se encontraron para el compuesto 6 conjugado con ADN AATTT, AATCG, fueron respectivamente (814 ± 90) nM, y (2.38 ±0.08) μΜ (figura 3). The maximum emission was used to determine the binding constant. Thus, the values of the dissociation constant K D found for compound 6 conjugated with AATTT DNA, AATCG, were respectively (814 ± 90) nM, and (2.38 ± 0.08) μΜ (Figure 3).

Claims

Reivindicaciones Claims
1. Un compuesto de fórmula general (I) 1. A compound of general formula (I)
Figure imgf000019_0001
Figure imgf000019_0001
donde Z es un grupo fluoróforo aceptor y n tiene un valor de 1 a 5. where Z is an acceptor fluorophore group and n has a value from 1 to 5.
2. Un compuesto de fórmula general (I) según la reivindicación 1, donde Z selecciona entre un ión lantánido complejado con un agente quelante o una cumarina fórmula general III, 2. A compound of general formula (I) according to claim 1, wherein Z selects from a lanthanide ion complexed with a chelating agent or a coumarin general formula III,
Figure imgf000019_0002
Figure imgf000019_0002
donde Rl se selecciona entre -NR4R5, -OH, -OR4, -OCOR4, siendo R4 y R5 iguales o diferentes seleccionados entre hidrógeno, alquilo, y where Rl is selected from -NR4R5, -OH, -OR4, -OCOR4, R4 and R5 being the same or different selected from hydrogen, alkyl, and
R3 se selecciona entre hidrógeno, halógeno y alquilo. R3 is selected from hydrogen, halogen and alkyl.
3. Un compuesto de fórmula general (I) según la reivindicación 1 y 2, donde el ión lántanido se selecciona entre Eu 3, Tb 3. 3. A compound of general formula (I) according to claims 1 and 2, wherein the lanthanide ion is selected from Eu 3 , Tb 3 .
4. Un compuesto de fórmula general (I) según la reivindicación 1 y 2, donde el agente quelante se selecciona entre el ácido l,4,7,10-tetraazaciclodecano-l,4,7,10-tetraacético (DOTA), el ácido dietilentriaminopentaacético (DTP A), 4,7,10-Tetraazaciclododecane-4. A compound of general formula (I) according to claim 1 and 2, wherein the chelating agent is selected from l,4,7,10-tetraazacyclodecane-l,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTP A), 4,7,10-Tetraazacyclododecane-
1.4.7.10- tetra(ácido metilen fosfónico) (DOTP), ácido 1,4,8,11 -tetraazaciclo-dodecano-1.4.7.10-tetra(methylene phosphonic acid) (DOTP), 1,4,8,11-tetraazacyclo-dodecane acid
1.4.8.11- tetraacético (TETA). 1.4.8.11- tetraacetic acid (TETA).
5. Un compuesto de fórmula general (I) según las reivindicaciones anteriores, donde es 3. 5. A compound of general formula (I) according to the preceding claims, wherein is 3.
6. Un procedimiento de preparación de un compuesto de fórmula general (I) que comprende una reacción entre un compuesto de fórmula II y una especie fluoróforo aceptor, 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,
Figure imgf000020_0001
donde Y es hidrógeno o un agente quelante, y n tiene un valor de 1 a 5.
Figure imgf000020_0001
where Y is hydrogen or a chelating agent, and n has a value from 1 to 5.
7. Uso de un compuesto de fórmula general (I) como agente fluorogénico de reconocimiento de secuencias de ADN. 7. Use of a compound of general formula (I) as a fluorogenic DNA sequence recognition agent.
PCT/ES2011/070297 2010-04-27 2011-04-26 Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna WO2011135142A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201000561A ES2368276B2 (en) 2010-04-27 2010-04-27 COMPOUNDS DERIVED FROM BIS-BENZAMIDINES AS FLUOROGENIC AGENTS TO SIGNAL SPECIFIC DNA SEQUENCES OF DOUBLE CHAIN.
ESP201000561 2010-04-27

Publications (1)

Publication Number Publication Date
WO2011135142A1 true WO2011135142A1 (en) 2011-11-03

Family

ID=44860918

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2011/070297 WO2011135142A1 (en) 2010-04-27 2011-04-26 Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna

Country Status (2)

Country Link
ES (1) ES2368276B2 (en)
WO (1) WO2011135142A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014202814A1 (en) * 2013-06-20 2014-12-24 Universidade De Santiago De Compostela Flourescent compounds

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002516A2 (en) * 2000-06-30 2002-01-10 Thomas Jefferson University Inhibitors of hiv integrase
WO2003017994A1 (en) * 2001-08-31 2003-03-06 Neurochem (International) Limited Amidine derivatives for treating amyloidosis
EP2116236A1 (en) * 2008-04-21 2009-11-11 Université de Mons-Hainaut Bisbenzamidine derivatives for use as antioxidant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002516A2 (en) * 2000-06-30 2002-01-10 Thomas Jefferson University Inhibitors of hiv integrase
WO2003017994A1 (en) * 2001-08-31 2003-03-06 Neurochem (International) Limited Amidine derivatives for treating amyloidosis
EP2116236A1 (en) * 2008-04-21 2009-11-11 Université de Mons-Hainaut Bisbenzamidine derivatives for use as antioxidant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HANNON, M.J.: "Supramolecular DNA recognition", CHEMICAL SOCIETY REVIEWS, vol. 36, 2007, pages 280 - 295 *
VAZQUEZ, O. ET AL.: "Bis-4-aminobenzamidines: Versatile, Fluorogenic A/T-Selective dsDNA Binders", ORGANIC LETTERS, vol. 12, no. 2, 2010, pages 216 - 219, XP055204419, DOI: doi:10.1021/ol902501j *
VAZQUEZ, O. ET AL.: "dsDNA-triggered energy transfer and lanthanide sensitization processes. Luminiscent probing of specific A/T sequences", CHEMICAL COMMUNICATIONS, vol. 46, no. 30, 2010, pages 5518 - 5520 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014202814A1 (en) * 2013-06-20 2014-12-24 Universidade De Santiago De Compostela Flourescent compounds

Also Published As

Publication number Publication date
ES2368276A1 (en) 2011-11-16
ES2368276B2 (en) 2012-06-28

Similar Documents

Publication Publication Date Title
ES2385268T3 (en) Compound having a structure derived from mononucleoside or mononucleotide, nucleic acid, marker substance and method and kit for nucleic acid detection
Niu et al. A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione
Bood et al. Fluorescent nucleobase analogues for base–base FRET in nucleic acids: synthesis, photophysics and applications
US8507677B2 (en) Fluorescent probe
Van Der Velde et al. Intramolecular photostabilization via triplet-state quenching: design principles to make organic fluorophores “self-healing”
EP2057279A2 (en) Bisubstrate fluorescent probe binding to protein kinases
Ekambaram et al. Benzoselenadiazole-based responsive long-lifetime photoluminescent probes for protein kinases
Deiana et al. A site-specific self-assembled light-up rotor probe for selective recognition and stabilization of c-MYC G-quadruplex DNA
Turaev et al. Benzothiazole-based cyanines as fluorescent “light-up” probes for duplex and quadruplex DNA
Guo et al. Synthesis and spectroscopic properties of fluorescent 5-benzimidazolyl-2′-deoxyuridines 5-fdU probes obtained from o-phenylenediamine derivatives
US20140080163A1 (en) Luminescent probes having a phenanthridinyl antenna, and methods of use
JP6462587B2 (en) Near-infrared quencher
WO2011135142A1 (en) Compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequences of double-stranded dna
WO2016151297A1 (en) Optical probe for thrombin
Rami et al. Synthesis of rhodamine B–benzenesulfonamide conjugates and their inhibitory activity against human α-and bacterial/fungal β-carbonic anhydrases
Pradeep et al. Molecular recognition of parallel quadruplex [d-(TTGGGGT)] 4 by mitoxantrone: binding with 1: 4 stoichiometry leads to telomerase inhibition
Fuchi et al. Luminescent europium sensors for specific detection of 8-oxo-dGTP by time-gated fluorescence
Ekambaram et al. PIM kinase-responsive microsecond-lifetime photoluminescent probes based on selenium-containing heteroaromatic tricycle
Halder et al. A flavonol that acts as a potential DNA minor groove binder as also an efficient G-quadruplex loop binder
Du et al. Synthesis and evaluation of new BODIPY-benzofuroquinoline conjugates for sensitive and selective DNA detection
WO2013144405A1 (en) Novel chlorinated derivatives of bodipy
ES2386530T3 (en) Fluorescence blocking molecules as well as the methods and uses that involve them
WO2011007038A2 (en) Use of compounds derived from bis-benzamidines as fluorogenic agents for signalling specific sequencecs of double-strand dna, and novel compounds
Nozeret et al. Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA
JP2016196608A (en) Fluorescent dye

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11774454

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11774454

Country of ref document: EP

Kind code of ref document: A1