ES2249986B1 - SYNTHESIS OF FLUORESCENT DERIVATIVES OF TACROLIMUS (FK506) AND ITS USE IN THE CHARACTERIZATION OF THE INTERACTION OF FK506 WITH PROTEINS UNION TO FK506. - Google Patents

Abstract

The synthesis, purification and characterization of fluorescent derivatives of tacrolimus (FK506) are described, which have application, among others, to the study of FK506 binding proteins present, among other means, in extracellular fluids to determine their favorable or unfavorable contribution in transport , cell binding and hospitalization of this drug. The analytical method is based, although not limited to it, on the determination of the binding of the fluorescent derivative of the FK506 to the protein under study, by monitoring the changes in one or more of the following fluorescence parameters of said fluorescent derivative: fluorescence emission intensity and / or steady state fluorescence polarization, spectral shifts (maximum absorption and / or emission), extinction kinetics of fluorescence intensity and / or fluorescence polarization, as well as changes in some or several of said parameters due to the existence of phenomena of resonant energy transfer (FRET) to an acceptor or from a donor.

Description

Synthesis of fluorescent derivatives of tacrolimus (FK506) and its use in the characterization of the interaction of FK506 with FK506 binding proteins.

Technical sector

The invention relates to the fields biochemical / biotechnological / biological (interaction studies of proteins with small molecules of pharmacological activity), doctor-pharmacist (study of the mechanisms of drug action), chemical-analytical (methods of quantification of protein binding to synthetic molecules) and chemical-organic (molecule synthesis methods organic with fluorescent properties).

State of the art

Tacrolimus, also known in the literature as FK506, is a macrolide of chemical name 1 R , 9 S , 12 S ,
13 R , 14 S , 17 R , 18 E , 21 S , 23 S , 24 R , 25 S , 27 R ) -17-allyl-1,14-dihydroxy-12 - [( E ) -2 - [(1 R , 3 R , 4 R ) -4-hydroxy-3-methoxycyclohexyl] -1-methylvinyl] -23.25-dimethoxy-13,19,21, 27-tetra-methyl-11,28-dioxa-4-azatricyclo [22.3.1.0 4,9] octacos-18-eno-2,3,
10, 16-tetrone (usually obtained as a hydrate), produced by Streptomyces Tsukubaensis (T. Kino et al., J. Antibiot. 1987 , 40, 1249). The structure of tacrolimus is shown in Figure 1.

Tacrolimus is a potent immunosuppressant that is used in clinical medicine in liver, kidney and lung transplants (LJ Scott et al., Drugs, 2003 , 63, 1247). In addition to its immunosuppressive effect, FK506 can act as an anti-inflammatory or antiallergic agent (J. Clardy, Proc. Natl. Acad. Sci. 1995 , 92, 56; N. Keicho et al., Cell Immunol 1991 , 132, 285; MF Sakr et al., J. HepatoL 1993, 17, 301).

The immunosuppressive activity of tacrolimus is particularly important as it has been shown to be between 50 and 100 times more potent than the immunosuppressant cyclosporin A in inhibiting the activation of T cells (T. Kino et al., J. Antibiot. 1987 , 40 , 1249), so it turns out to be an effective alternative to cyclosporine both in primary immunosuppression after solid organ transplantation, and in rescue therapies in patients presenting with rejection (AD Mayer et al., Transplantation 1997 , 64, 436) . Tacrolimus inhibits the activation of T cells by binding, once internalized in the T cell, to a cytosolic protein called FKBP ( FK506 binding protein ). The drug-protein binding complex is in turn associated with calcineurin, inhibiting the activity of this calcium-dependent enzyme, which results in the inhibition of lymphokine expression activation. (G. Wiederrecht et al., Ann. NY Acad. Sci., 1993 , 696, 9).

Tacrolimus is administered orally or intravenously. An alternative route, not yet tested, is the inhalation route, which would be especially effective in lung transplantation. FK506 is quite insoluble in water (<0.003 mg / mL at 25 ° C) (K. Hane et al., Iyakuhin Kenkyu 1992 , 23, 33), so that commercial intravenous doses of tacrolimus usually contain a solubilizing agent, such as polyoxyethylated castor oil, which is toxic. The main side effect associated with the clinical administration of tacrolimus is nephrotoxicity. It can also cause neurotoxicity, associated with headache, tremor, insomnia, pain and other symptoms.

The binding of drugs to fluid proteins such as human plasma serum albumin or pulmonary surfactant protein A (SP-A) in the alveolar fluid, can modify the properties of such drugs, in turn decreasing the amount of free drug (D Silva et al., Spectrochim Acta A Mol. Biomol. Spectrosc. 2004 , 60, 1215) and forcing to increase the administered doses to obtain the desired effect with the consequent increase in toxicity. Alternatively, the potential binding proteins present in extracellular fluids could facilitate the transport of the drug and its internalization in the target cells. The binding of FK506 to extracellular fluid proteins is unknown until now.

Most drug interaction studies with proteins are performed using techniques that require non-physiological conditions such as high concentrations of said proteins, deuterated solvents or proteins in the crystalline state. An approach to investigate the binding of tacrolimus to proteins is to prepare a derivative or analogue of tacrolimus in which a fluorescent structure is chemically covalently bound, as described for other molecules of possible or recognized pharmacological activity ( see, eg, G. Orellana et al. Patent ES 2197811; MP Lillo, et al., Biochemistry 2002 , 41, 12436; F. Amat et al, Patent ES 2105983; JA Evangelio et al. Cell Motil Cytoskeleton 1998 , 39, 73; G. Turcatti et al. J. BioL Chem. 1997 , 272, 21167; Han et al., Biochemistry 1996 , 35, 14173; J.-M. Frére et al. Biochem. J. 1994, 300, 14 and Biochem J. 1993, 291, 19;
CD Niebylski and HR Petty, Biophys. J. 1993 , 64, 701; Teng et al., Biochemistry 1991 , 30, 7894). None of these publications, however, reveals or mentions the possibility of fluorescently labeling the tacrolimus molecule.

Fluorescence, and in particular fluorescence polarization (anisotropy) techniques, are very useful tools in the study of most biomolecular and macromolecular interactions, such as DNA-protein, DNA-drug, protein-drug, protein-protein or the binding of antigens to antibodies (see, for example, WB Dandliker et al., Methods Enzymol. 1981 , 74 Part C, 3). Unlike other analytical techniques, fluorescence does not require the physical separation of bound and free species, allowing directly measure the ratio of bound and free ligand, even in the presence of free ligand. Since it is not necessary to separate the free ligand from the bound one, all fluorescence and fluorescence polarization measurements are performed directly in solution, after reaching equilibrium.
In addition, since fluorescence and fluorescence polarization measurements do not adulterate the samples, they can be recovered to elucidate the effect on the ligand-biomolecule junction of changes in parameters such as pH, temperature and saline concentration. Thus, the interaction of tacrolimus with its binding protein or with fluid proteins can be followed by changes in fluorescence emission intensity and steady state fluorescence polarization, shifts in absorption, excitation and / or emission spectra, variations in the kinetic profiles of fluorescence extinction and / or fluorescence polarization, as well as fluorescence changes induced by resonant energy transfer (FRET) to or from third parties.

The use of substituted aminonaphthalenesulfonates as fluorescent probes in proteins and other macromolecules is well known (L. Stryer, J. Mol. Biol. 1965 , 13, 482; WO McClure and GM Edelman, Biochemistry 1966 , 5, 1908; WO McClure and GM Edelman, Biochemistry 1967 , 6, 559; WO McClure and GM Edelman, Biochemistry 1967 , 6, 567; RF Chen and JC Kernochan, J. Biol. Chem . 1967, 242, 5813; JR Lakowicz, Principies of Fluorescence Spectroscopy 2nd edition, Kluwer, New York, 1999). These compounds show characteristic changes in the location of their maximum fluorescence emission and fluorescence intensity depending on the polarity of the solvent that have allowed their use to determine "polar" and "non-polar" sites when interacting with macromolecules. They are also likely to be interrogated by fluorescence polarization techniques for the study of interaction of small molecules with proteins, for fluorescent immunoassays or for the study of cell membrane microfluidity.

KC Kasper et al. ( WO 01/09190) describe the use of tacrolimus and derivatives of tacrolimus bound to high molecular weight proteins or antibodies for the development of analytical immunological methods for the determination of tacrolimus. However, although said derivatives or analogues of tacrolimus are useful for the development of said methods and / or the production of monoclonal antibodies to tacrolimus, the authors do not prepare, mention or claim any fluorescent derivative of tacrolimus for the study of their binding to proteins present in extracellular fluids or other media, nor said derivatives could be used in any way for the study of the binding of tacrolimus to proteins, which is the object of the present invention.

Likewise, Bakhtiar and Stearns have described a procedure to investigate the binding of tacrolimus to proteins ( Rapid Commun. Mass Spectrom . 1995, 9, 240). However, the procedure for this purpose described in this publication uses the technique of mass spectrometry with electrospray ionization, which is restricted to protein molecules of a certain magnitude and requires sophisticated and expensive instrumentation, which limits its applicability. On the contrary, the techniques of study of binding to proteins or other biomolecules, based on the monitoring of the fluorescence of the labeled drug, have demonstrated a versatility, cost / performance efficiency, simplicity, resolution, safety, reliability and speed hardly exceeded (see, for example, R. Kraayenhof et al . (eds.), Fluorescence Spectroscopy, Imaging and Probes , Springer, Berlin-Heidelberg, 2002; B. Valeur and J.-C. Brochon (eds.), New Trends in Fluorescence Spectroscopy: Applications to Chemical and Life Sciences , Springer, Berlin-Heidelberg, 2001; A. Prasanna de Silva et al, Proc. Natl. Acad. Sci. USA 1999, 96, 8336).

The present invention describes the possibility of fluorescently mark tacrolimus in a position that does not It affects your protein binding. The tacrolimus thus labeled has used in the study of proteins that bind tacrolimus, particularly those that are present in fluids extracellular, without being restricted thereto media.

Description of the invention Synthesis of fluorescent derivatives of tacrolimus (FK506) and its use in the characterization of the interaction of FK506 with proteins binding to FK506

The present invention relates to the synthesis, isolation and characterization of highly fluorescent derivatives of tacrolimus (FK506) that have application, without being restricted in any way to her, to the elucidation and characterization of the interaction of tacrolimus with tacrolimus binding proteins (FKBPs). Proteins can preferably be found in extracellular fluids, but in no way the methodology analytical described here is restricted to such places.

More specifically, the invention describes the preparation and use of derivatives or analogues of the drug tacrolimus whose chemical structure has introduced another molecular entity or sub-structure that emits light (fluorescence) ultraviolet or visible when excited or illuminated with ultraviolet or visible light, without affecting the ability to join to proteins of the original molecule. Fluorescent derivatives which are revealed here are obtained, in general, by procedures Chemicals comprising three stages of synthesis, from original tacrolimus and an appropriate fluorescent molecule, always that the latter contains in its molecular structure an amino group free or can be previously incorporated said functional group.

The synthesis of tacrolimus derivatives that include a fluorescent or fluorogenic molecule in their structure, such as, but not limited to, dansyl (5- (dimethylamino) -1-naphthalenesulfonyl) or fluorescein (9- (or -carboxyphenyl) -6-hydroxy-3-isoxantenone) derivatized by introduction of at least one aminoalkyl group, consists of three stages. In the first, the incorporation of a carboxyl group to tacrolimus takes place with excellent performance through the reaction of the C-22 carbonyl group of tacrolimus (marked with an arrow in Figure 1) with carboxymethoxylamine, analogously to the conventional procedure described by KC Kasper et al .
(WO 01/09190). Subsequently, the carboxylated derivatives of tacrolimus are activated, for a more effective nucleophilic attack by the amino group carrying the derivatized fluorescent molecule, with commercial N -hydroxysuccinimide or N, N- dicyclohexylcarbodiimide, among other agents described in the literature for this purpose soluble in non-aqueous media (RP Hughland, editor, Handbook of Fluorescent Probes and Research Products , 8th edition, Molecular Probes, Eugene, OR, USA, 2002).

In the third stage the union takes place or condensation between the activated carboxylic acid derivative previously obtained and the substituted aminoalkyl derivative of the chosen fluorescent molecule (commercial or previously synthesized). This stage is preferably carried out in the absence of light. in order to avoid photodegradations of the fluorogenic group and carefully controlling the rigorous absence of water in the middle reaction, in order to avoid undesirable hydrolysis of the activated tacrolimus obtained in the previous stage.

The only limitation found to these reactions is that they should not alter the molecular structure fundamental (responsible for its activity as such and its union to proteins) of the precursor tacrolimus, except as regards C-22 carbonyl group thereof.

In addition, the patent reveals the application of fluorescent derivative of tacrolimus to the interaction study thereof with tacrolimus binding proteins using, although without being in any way restricted to them, the techniques of steady state fluorescence emission, polarization of steady state fluorescence and polarization of fluorescence with temporal resolution. Also, these techniques of fluorescence can be used, without being restricted to it, to elucidate the interaction with proteins present in spaces extracellular

Brief description of the figures

The invention includes figures that with Representative character show:

Figure 1 represents the chemical structure of the Tacrolimus (FK506). The arrow indicates the carbonyl functional group a through which the union of the structures is made fluorescents that confer this property on molecules resulting.

Figure 2 shows the chemical reaction of formation of the carboxymethoxyloxime of tacrolimus in the C-22 that allows to introduce a carboxyl group reagent in tacrolimus not existing in the original molecule, in a position that does not significantly alter the structure Drug chemistry.

Figure 3 shows activation with dicyclohexylcarbodiimide (DCC) of the carboxymethoxyloxime of tacrolimus on the C-22 to make the reaction possible of the carboxyl group with the amino group that carries the molecule fluorescent to be introduced.

Figure 4 represents the covalent junction of the dansilcadaverine to the activated carboxymethoxy oxime of tacrolimus by forming a carboxamide group.

Figure 5 shows the effect of the protein SP-A of the alveolar fluid on the spectra of emission (λ exc = 340 nm) (A) and excitation (λ em = 505 nm) of 5 x 10-4 mM dansil-FK506. The solid lines represent the Dansil-FK506 spectra in 5 mM Tris buffer, 150 mM NaCl, 0.1 mM EDTA, pH 7.4. Dashed lines represent the excitation and emission spectra of dansyl-FK506 in the presence of protein.

Figure 6 shows that the protein Alveolar fluid SP-A has no effect on the emission spectra (λ exc = 340 nm) (A) and of excitation (λ em = 505 nm) of 5 x 10-4 mM dansilcadaverine (free fluorescent probe). Continuous lines (---) represent the spectra of dansilcadaverin in 5 mM buffer Tris, 150 mM NaCl, 0.1 mM EDTA, pH 7.4. Dashed lines (- - - -) represent the excitation spectra and emission of dansilcadaverin in the presence of protein.

Figure 7 shows the effect of different fluid SP-A protein concentrations alveolar (panel A) and blood plasma albumin (panel B) on fluorescence emission anisotropy of dansil-FK506 in 5 mM Tris buffer, 150 mM NaCl, 0.1 mM EDTA, pH 7.4; (\ lambda_ {exc} = 340 nm, \ lambda_ {em} = 505 nm, 25 ° C).

Embodiment of the invention

The tacrolimus molecule (FK506), although it has a large number of functional groups (Figure 1), cannot be easily derivatized or functionalized. Indeed, some of them are poorly reactive, intrinsically (such as C8 carboxamide) or by steric hindrances (such as C10 hydroxyl). Others would result in polyiderivatization (eg hydroxyl in C24 and in the cyclohexane ring, or the two alkene groups present in the drug), while certain functionalizations would probably result in the loss of their activity or conformation. Recently Kasper, et al. ( WO 01/09190) have described a suitable method for incorporating derivatives into tacrolimus in a position that does not interfere with the production of antibodies against it (C22), which led to the belief that such position could also be useful for introducing a group or fluorescent molecule that allowed to study the binding of the analogue of tacrolimus to proteins.

In essence, the process described in this patent is based on the known reaction of tacrolimus with carboxymethoxylamine to give rise to the corresponding carboxymethoxyloxime of the drug in C-22 carbonyl. In this way, a carboxyl group slightly separated from the base structure (a methylene) is incorporated into the FK506, which can be conveniently activated for its reaction with nucleophilic groups (typically amine ) present in small fluorescent or fluorogenic molecules (Figures 2-4) . The amide resulting from the binding of fluorophore (fluorescent molecule or substructure) to tacrolimus is stable enough so that the conjugate can be used, in a large number of experimental conditions and solvent media, to study its binding to pro
Theines

In order to illustrate the FK506 drug labeling procedure, the dansyl fluorophore was selected as the label, given the affordability and low cost thereof, the extensive literature available as a biomolecule marker, as well as its sensitivity to the microenvironment , which allows the "reporter" to report its location when it is in heterogeneous (micro) media. Once the FK506 labeling procedure has been completed, at least one amino group could be introduced, without further limitation than having its chemical structure and not alter the fundamental structure of tacrolimus, other fluorescent probes such as those derived from lucifer yellow ( lucifer yellow ), dipyrrometene or fluorescein, among others, conveniently functionalized and commercially available (for example, molecular probes reactive with carboxyl groups manufactured and marketed by Molecular Probes, Eugene, OR, United States, whose chemical structures can be found in RP Hughland's book, editor, Handbook of Fluorescent Probes and Research Products , 8th edition, Molecular Probes, Eugene, OR, USA, 2002).

By way of illustration and without being considered as a limitation on the scope of the present invention, examples of obtaining a fluorescent derivative of tacrolimus (Figures 1-4), as well as the use thereof for the study of their binding are explained below. to proteins by fluorescence techniques (Figures 6-7). Tacrolimus is manufactured by Fujisawa pharmaceutical laboratories (Japan). The human SP-A protein is purified, from the pulmonary surfactant isolated from bronchoalveolar lavage, by sequential extractions with n- octylglucoside and butanol (C. Casals et al., Biochem. J. 1993 , 296, 585; García-Verdugo et al., Biochemistry 2002 , 41, 14041; García-Verdugo et al., Biochemistry 2003 , 42, 9532). Protein purity is checked using one-dimensional SDS-PAGE electrophoresis in a gel with 12% polyacrylamide under reducing conditions (50 mM dithiothreitol). The quantification of SP-A is performed by amino acid analysis on a Beckman System 6300 high resolution analyzer. Both tacrolimus binding protein (FKBP) and human serum albumin (HSA) are obtained from Sigma (St. Louis, MO). The chemical reagents and solvents mentioned in the examples are commercially available from one or more manufacturers among which are, for example, Sigma-Aldrich, Fluka, Merck, Avocado, Molecular Probes, etc.

Example 1 Formation of the carboxymethoxyloxime of tacrolimus in the C22

To a solution of 0.061 mmol of FK506 (Fujisawa) in 10 mL of anhydrous MeOH (HPLC grade, SDS) are added 0.34 mmol of sodium acetate (anhydrous, PANREAC) and 0.31 mmol of carboxymethoxylamine hydrochloride (98%, ALDRICH). The system is placed under an inert atmosphere (argon) and kept stirring at room temperature for 16 hours. The reaction evolution is followed by thin layer chromatography (TLC) using as a eluent a quaternary mixture CH 2 Cl 2 -AcOEt-MeOH-HOAc (30: 70: 11: 0.6, v / v / v / v) and as a developer iodine vapor. After that time, the MeOH is rotated and evaporated, chloroform is added, observing the partial dissolution of the reaction crude. The insoluble residue is constituted by a mixture of carboxymethoxylamine hydrochloride (used in excess) and sodium acetate, while in the liquid phase the desired oxime is found, in the form of two isomers ( anti and sin ) as demonstrated by the CCF and 1 H NMR (200 MHz). Once the oxime has been quantitatively extracted by the chloroform, the solvent is removed under reduced pressure, a white solid (65 mg) appearing that is identified as the target oxime (Figure 2) by its 1 H-NMR spectrum and its Purity is confirmed by CCF (absence of other secondary products).

Example 2 Activation of carboxymethoxyloxime with N, N'- dicyclohexylcarbodiimide (DCC)

The oxime obtained in the previous stage (0.074 mmol) is dissolved in 5 mL of anhydrous DMF (99.5%, FLUKA) and placed in a two-mouth flask. Then 0.12 mmol of DCC (Aldrich) are added and the system is left under inert atmosphere and with stirring for 1.5 h. The reaction evolution is followed by TLC using as a eluent a quaternary mixture CH 2 Cl 2 -AcOEt-MeOH-HOAc (30: 70: 11: 0.6, v / v / v / v), observing that the two spots that are observed after the first stage (oxime isomers) practically disappear and, instead, a new product is detected whose isolation was not attempted and whose nature corresponds to the carbamate resulting from the activation of the carboxyl group introduced in the stage above (Figure 3) as elucidated by spectroscopy
infrared

Example 3 Union of the dansyl cadaverine to the activated oxime of the tacrolimus

On a solution of 0.108 mmol (36.25 mg) of dansyl cadaverine (Molecular Probes) in 2 mL of anhydrous DMF under an inert atmosphere, the solution containing the activated oxime (0.074 mmol) is added by cannula, keeping the system under argon and with agitation. The evolution of the reaction is monitored by TLC using as eluent a ternary mixture n -PrOH-AcOH-H2O (7: 3: 4, v / v / v). It is left at room temperature for 72 hours and, not observing any transformation, the reaction mixture is heated to 90 ° C. After 18 h from the beginning of the heating, evolution by CCF is no longer observed and it stops. It is allowed to reach room temperature and the DMF is rotated evaporated. The resulting residue is dissolved in 50 mL of CHCl3 and extracted with 10% HCl (5 x 20 mL) and washed with H2O (3 x 20 mL). The combined organic extracts are dried over anhydrous MgSO4 and then the solvent is removed under reduced pressure. A yellow solid (52 mg) is thus obtained which, after analysis by UV-VIS absorption spectroscopy (MeOH; λ max 333 nm), 1 H-NMR (200 MHz), 13 C -RMN (50 MHz) and mass (ESI-MS, positive ion detection), is identified as the product sought (dansil-FK506). Finally, the product is dried under vacuum (0.1 Torr).

Example 4 Binding of dansyl-FK506 to proteins detected by changes in fluorescence intensity and displacements spectral

The interaction of the fluorescent derivative of tacrolimus, DNS-FK506, with proteins can be studied from changes in the intensity and displacement of the maximum fluorescence emission of the fluorescent ligand. The fluorescence intensity is measured at 90 ° from the direction of the excitation beam, selecting the emission wavelength. The emission spectra of the tacrolimus fluorescent derivative are obtained by measuring the fluorescence intensity, for an excitation wavelength of 340 nm, between 420 and 650 nm. The sample compartment is thermostatized with a bath, such as that sold by Julabo under the reference F30-C. Quartz cuvettes are used, such as those of the Hellma (111.057-QS) rectangular 0.5 cm optical path. In order to study the variation of the fluorescence intensity at a given wavelength as well as the position of the maximum emission spectrum as a function of the fluorescent tacrolimus / protein molar ratio, it is necessary to first determine the value of said parameters in the absence of protein. To this end, 2 x 10-3 mL of DNS-FK506 0.125 mM dissolved in MeOH for Merck spectroscopy is added (so that the final amount of alcohol in the solution 0.4% (v / v) does not affect the proteins ) to 0.5 mL of a physiological buffer such as PBS or in 5 mM Tris-HCl buffer, 150 mM NaCl, (Merck, Germany) pH 7.4. The fluorescence emission spectrum of the drug labeled with dansyl is collected and then the fluorescence spectra of samples in which the molar ratio tacrolimus / protein varies, setting the drug concentration and changing the protein concentration or vice versa. The contribution of the emission from the impurities of the solvent and the diffused light due to the proteins are corrected by subtracting the emission of the solvent and the different protein concentrations measured under the same conditions as the sample. To eliminate the effect of self-absorption of the emission in the fluorescence intensity measurements, we work with optimally diluted protein solutions (absorbance at the excitation wavelength <0.01 ua, 0.5 cm of optical path). To correct the deformation of the spectra due to the intrinsic characteristics of the measurement system, the experimental intensity or apparent spectrum is divided by a correction function that includes the different sensitivity of the emission monochromator system.
Photomultiplier

Figure 5 shows the study of the interaction of the SP-A alveolar fluid protein with a dansy analogue of tacrolimus (abbreviated dansyl-FK506 or DNS-FK506). Such interaction can be determined by changes in fluorescence intensity and the maximum emission of DNS-FK506. SP-A protein binding
(7.5 x 10-4 mM) to DNS-FK506 (5 x 10-4 mM) results in an increase in the fluorescence intensity of DNS-FK506, accompanied by a shift in the maximum emission towards shorter wavelengths, from 545 to 500 nm (Figure 5A). This indicates that the polarity of the medium around the fluorescent group decreases after the DNS-FK506 / SP-A interaction. Figure 5B indicates that the binding of the SP-A protein to DNS-FK506 results in a redshift of the excitation spectrum, confirming a different environment from the fluorophore in the presence of the protein. Figure 6 (panels A and B), shows that the changes observed in the emission and excitation spectra of DNS-FK506 after its interaction with the SP-A protein are not due to a non-specific interaction of the SP-A protein with the fluorescent probe (dansilcadaverine). Figure 6 clearly shows that the emission spectrum (panel A) and the excitation (panel B) fluorescence of dansylcadaverin is not modified in the presence of the SP-A protein.

Example 5 Determination of the binding of dansil-FK506 to proteins by fluorescence anisotropy

Another possible way to study the interaction of tacrolimus with proteins is to follow the changes in fluorescence anisotropy of the fluorescently labeled drug using polarizers. For this, the samples, prepared as described previously, are excited with linearly polarized light (vertical) and the components of the polarized fluorescence intensity in the directions parallel and perpendicular to the plane of polarization of the excitation light are measured alternately . For certain conditions of excitation wavelength
(340 nm) and emission (505 nm), anisotropy is calculated according to the expression (A. Jablonski, Acta Phis. Polon., 1957 , 26, 471):

\ bar {r} = \ frac {I_ {ll} - I _ {\ perp} G} {I_ {ll} + 2I _ {\ perp} G}

where G is a correction factor which takes into account the different sensitivity of the system vertical polarized light detection and horizontally.

Figure 7 (panel A) shows that anisotropy fluorescence of the fluorescent drug dansil-FK506 increases after its interaction with increasing concentrations of the SP-A protein. This experiment indicates that protein binding SP-A to DNS-FK506 cause mechanical restrictions on group rotational mobility dansyl, resulting in a significant increase in emission of anisotropy of dansil-FK506 that increases with the increase in the concentration of SP-A. In parallel, control experiments are carried out with dansilcadaverine (free fluorescent probe) in absence and presence of SP-A. The fluorescence anisotropy of the dansilcadaverine in buffer at 25 ° C is 0.002 ± 0.001 (λ ex = 340 nm; λ em = 505 nm) and said value does not change significantly in the presence of concentrations increasing amounts of SP-A protein, which indicates that the effect of SP-A protein on the anisotropy of DNS-FK506 fluorescence is due to the union of the protein to the drug FK506. Figure 7 (panel B) shows a similar experiment performed with human serum albumin.

Example 6 Determination of the binding of dansil-FK506 to proteins by means of extinction kinetics of intensity and / or fluorescence polarization

A third way to study the interaction DNS-FK506 with proteins is to follow the evolution Temporary fluorescence (intensity decay of fluorescence). The measurement of the excitation pulse profile, L (t), is determined by recording the diffuse radiation produced with purified water (Millipore MilliQ) to the length of excitation wave, consecutively picking up the decay Fluorescence and laser profile. The decays of fluorescence thus obtained are analyzed with a method of iterative convolution based on the least squares adjustment no linear, obtaining half-life distributions corresponding to each sample.

If the interaction DNS-FK506 / protein is wanted to follow from fluorescence anisotropy decays, the sample fluorescent is excited with pulses of polarized light picoseconds vertically It is selected first with the emission polarizer the fraction of polarized fluorescence intensity parallel to the direction of the excitation beam, intensity of vertically polarized fluorescence, i_ {ll} (t), and then the horizontally polarized fluorescence fraction, i {perp} (t). Fluorescence intensities polarized measurements, i_ {ll} (t) and i _ {\ perp} (t), are accumulate alternately and during the same time along with the instrumental function, L (t). The decays of fluorescence depolarization thus obtained are analyzed with a iterative convolution method / nonlinear least squares for get the rotational correlation times, \ phi_ {i}, and the limit anisotropy values, r \ infty.

Example 7 Determination of the binding of dansil-FK506 to proteins by resonant energy transfer (FRET)

Another way to study the binding of DNS-FK506 with proteins is to use the Förster-type resonant energy transfer (FRET) between the protein tryptophan and the dansyl molecule (SJ Nannepaga et al., Biochemistry 2004 , 43, 550; JK Davies et al., J. Biol. Chem. 2002 , 277, 48395). The increase in fluorescence intensity of DNS-FK506 due to the energy transfer of tryptophan is still measured by the emission between 490 and
650 nm with excitation at 285 nm.

Claims (8)

1. Tacrolimus derivatives (FK506) characterized in that they contain in their molecule a fluorogenic or fluorescent group. 2. Tacrolimus derivatives (FK506) according to claim 1, characterized in that they emit ultraviolet or visible fluorescence when illuminated by light of shorter wavelength than that emitted. 3. Procedure for obtaining tacrolimus derivatives (FK506) that contains in its structure a fluorogenic or fluorescent group characterized in that the synthesis has three stages: (1) introduction of a carboxylic acid into the tacrolimus molecule, (2) activation of the group carboxyl and (3) binding of the fluorescent molecule carrying at least one amino group reactive to activated tacrolimus. 4. Procedure for obtaining tacrolimus derivatives (FK506) containing in its molecule a fluorogenic or fluorescent group, according to claim 3, characterized in that the fluorogenic or fluorescent group can be any that contains in its molecular structure a free amino group. 5. Process for obtaining tacrolimus derivatives (FK506) containing in its molecule a fluorogenic or fluorescent group, according to claim 4, characterized in that the fluorogenic group is dansyl or fluorescein. 6. Use of tacrolimus derivatives (FK506), according to claims 1 and 2, for the elucidation or study of the binding of said derivatives to proteins using techniques of fluorescence. 7. Use of tacrolimus derivatives (FK506), according to claim 6, wherein the proteins are proteins present in the extracellular space. 8. Use of tacrolimus derivatives (FK506), according to claim 6, characterized in that said techniques are based on monitoring changes in one or more of the following fluorescence parameters of said fluorescent derivative: the emission intensity of fluorescence and / or steady state fluorescence polarization, spectral shifts (maximum absorption and / or emission), extinction kinetics of fluorescence intensity and / or fluorescence polarization, as well as changes in any or several of said parameters for existing phenomena of resonant energy transfer (FRET) to an acceptor or from a donor.