ES2197811B2 - SYNTHESIS OF FLUORESCENT DERIVATIVES OF BETA-LACTAMIC ANTIOBIOTICS. - Google Patents

Abstract

Synthesis of fluorescent derivatives of beta-lactam antibiotics. The synthesis, purification and characterization of highly fluorescent beta-lactam (b-lactam) antibiotics having applications, among others, for the determination of the enzymatic activity of beta-lactamases, the study of bacterial membrane proteins, the analysis is described. of microbial fermentation media or in the development of rapid and sensitive analytical methods to detect qualitatively or quantitatively antibiotics (in fluids of biological origin, in pharmaceutical preparations and formulations, in foods of animal origin, etc.). Such analytical methods may be based, although not limited to them, on chromatographic, enzymatic, immunological procedures or on the use of molecularly imprinted polymers.

Description

Synthesis of fluorescent derivatives of β-lactam antibiotics.

Technical sector

The invention relates to the fields biochemical / biotechnological / biological (activity tests enzymatic and immunoassays), medical-pharmaceutical (compounds with antibiotic and antimicrobial activity), chemical-analytical (compound determinations antibiotics and drugs, especially by liquid chromatography or HPLC) and chemical-organic (synthesis methods of organic molecules with antibiotic activity).

State of the art

Penicillins and cephalosporins (Figure 1) constitute the group of so-called "classic" beta-lactam antibiotics (several authors, Analytical Profiles of Drug Substances , vols. 1-17, Academic Press, New York, 1972-1988), since their structure contains a heterocyclic ring of beta-lactam type. However, there are many structural analogues with antibiotic properties that contain the beta-lactam ring, such as oxacefems and carbapenems, in which the heterocyclic sulfur atom of penicillins and cephalosporins is replaced by oxygen or carbon, or nocardicins and monobactams , which are monocyclic. The mechanisms of bactericidal action of these molecules are extremely complex, although the essential fundamentals are known. Beta-lactam antibiotics are lethal to bacteria but harmless to mammals because their toxicity results from the interference of these molecules with the biosynthesis of the bacterial cell wall, which mammalian cells do not possess. There is a great osmotic pressure inside a growing bacterial cell due to the increase in nutrients and, therefore, the absence of a strong cell wall causes the cell membrane to rupture with the consequent loss of cell content, which leads to The death of it.

So, antibiotics beta-lactams are chemically attacked by some of the nucleophilic groups of the enzyme that regulates biosynthesis of the cell wall. This reaction, which involves the opening of the tense ring of beta-lactam and acylation nucleophilic group of the enzyme, gives rise to products that are not able to effectively catalyze wall growth cell, causing irreversible inhibition of the enzyme. A Effective beta-lactam antibiotic should be what stable enough to reach the bacterial enzyme without decomposition, but at the same time must be able to suffer the attack nucleophilic of said enzyme.

Antibiotic design effective β-lactams is not easy due to the high susceptibility of the carbonyl group beta-lactam to undergo nucleophilic attack, which it translates, for example, into a great facility to experience hydrolysis. The reactivity of penicillins is even greater than that of simple beta-lactams due to presence of two fused rings in its fundamental skeleton.

Fluorescent derivatization of molecular probes is an area of great relevance, as it is allowing spectacular advances in medical diagnosis, imaging techniques in Physiology, biochemical research, environmental monitoring and chemical analysis in general (see, for example, RP Haughland, Handbook of Fluorescent Probes and Research Products , 9th edition, Molecular Probes, Eugene, OR, 2004 ; Prasanna de Silva et al, Proc. Natl. Acad. Sci. USA 1999 , 96 , 8336). The synthesis of fluorescent beta-lactam compounds, and more specifically of antibiotics such as penicillins, including amoxicillin or ampicillin, is an objective of maximum interest, due to the large number of applications presented by these molecules, whether for the determination of the enzymatic activity of beta-lactamases, the study of bacterial membrane proteins, the analysis of microbial fermentation media or the development of rapid and sensitive analytical methods to detect qualitatively or quantitatively antibiotics (in fluids of biological origin, in preparations and pharmaceutical formulations, in foods of animal origin, among others) (see, for example, several authors, Analytical Profiles of Drug Substances , vols. 1-17, Academic Press, New York, 1972-1988; Chen et al., US Pat . 4740459; Chen, WO 8903889 Patent; Tsien and Zlokarnik, WO 9630540 and Patent US 5741657 Patent;. Rogers et to the, J. Chromatography 1984, 297, 385 ; Medina, ACS Symp. Be . 1996 , 636 , 132; Sternesjö and Johnsson, J. Food Prot . 1998 , 61 , 808). Such analytical methods may be based, although not limited to them, on chromatographic, enzymatic, immunological procedures or on the use of molecularly imprinted polymers . These applications require or make it very desirable to have fluorescently labeled antibiotics to carry out, with high sensitivity and selectivity, the detection stage after, for example, a competitive-type assay with the unlabeled antibiotic.

The study of the bonds that take place in the PBPs ( penicillin-binding proteins or penicillin-binding proteins ) present in bacterial membranes represents the simplest way to determine the role that these proteins play in processes such as cell division (Sprat and Pardee, Nature 1975 , 254 , 516) or in the intrinsic resistance mechanisms in pathogenic bacteria of great importance such as Streptococus, Enterococus or Pseudomonas . One of the most used techniques to carry out this type of study has been that described by BG Spratt ( Eur. J. Biochem . 1977 , 72 , 341) more than 20 years ago, in which radioactively labeled penicillins are used. However, the main limitation of the method lies in the time required to carry out the tests which, in the case of the detection of PBPs, can be several days or even weeks. In addition there is an added difficulty due to the intrinsic danger of the radioactive labels of the beta-lactam antibiotic.

Therefore, other penicillin fluorescent derivatization procedures have been developed, which allow the detection of less than 10 fmol of PBPs. Thus, J.-M. Frère et al. ( Biochem. J. 1994 , 300 , 14 and Biochem. J. 1993 , 291, 19) have described and carried out the synthesis, purification and study of kinetic properties of antibiotics labeled with fluoresceins, or with boron coordination compounds , fluorine and pyrometene (Zhao et al., Antimicrobial Agents Chemother . 1999 , 43 , 1124), which have allowed the development of very sensitive new methods for the identification and quantification of PBPs. However, these authors do not mention or describe the fluorescent antibiotics object of the present patent. In addition, the intrinsic polarity of the fluorescent marker used by Frére et al. (fluorescein), an anionic molecule, is undesirable if, for example, fluorescent antibiotics soluble in non-aqueous media are required. Moreover, the intrinsic green emission of fluorescein may be undesirable or interfering in tests combined with other substrates or labeled molecules whose fluorescence appears in the same region. On the other hand, the fluorescent marker used by Zhao et al . It exhibits a small Stokes shift between the wavelength required for excitation (504 nm) and the wavelength necessary for detection (511 nm), which makes it easy to separate the two essential for the correct quantification of PBPs. The fluorescent derivatives object of the present invention have much higher Stokes shifts, greatly facilitating discrimination of the emitted light and that used to excite the fluorescent derivative.

The preparation of fluorescent derivatives of beta-lactam antibiotics with ortho- phthaldialdehyde (Rogers et al., J. Chromatography 1984 , 297 , 385), fluorescamine (Nakagawa et al., Yakugaku Zasshi 1985 , 105 , 1096), 9 has been described. -isothiocyanatoacridine (Sinseimer et al. J. Pharm. Sci . 1969 , 58 , 1041) or dansyl hydrazine (Munns et al., J. Assoc. Off. Anal. Chem . 1985 , 68 , 968) for chromatographic determination of such antibiotics. Likewise, the preparation of beta-lactam antibiotics that incorporate luminescent ruthenium (II) complexes for the quantification of beta-lactamase activity (Liang et al., J. Am. Chem. Soc . 1996) has been previously described . , 118 , 9198). However, all these documents do not mention, nor suggest the fluorescent antibiotics object of the present patent. In addition, the wavelengths of excitation and fluorescence emission of said known derivatives are not in the same region as those of the fluorescent antibiotics described here for the first time, which may be inappropriate for many applications of the mentioned above. Moreover, none of these publications describe the isolation and purification of fluorescent beta-lactam antibiotics, but their generation in situ to an unknown extent, so they could not be used in some applications of those mentioned above.

On the other hand, Cartwright; and Fink ( FEBS Lett . 1982 , 137 , 186) have published the synthesis of dansyl penicillin (G) and its use as a fluorogenic substrate for the determination of the activity of beta-lactamase enzymes. In addition to making no mention or suggestion of the possibility of preparing the fluorescent antibiotics object of the present invention, the very small quantum emission yield of the dansylated derivatives when they are in an aqueous medium, as said authors point out, makes it very difficult to monitoring its fluorescence in said medium, when they are not associated with the enzyme.

Also described the preparation and use of substrates for beta-lactamase endowed simultaneously an entity fluorescent and quencher entity fluorescence in the same molecule (Tsien and Zlokarnik, WO 9630540, Tsien and Zlokarnik, US Patent 5,955,604; Tsien and Zlokarnik, US Patent 5,741,657). However, while the substrates described by these authors have a beta-lactam ring and some of the applications listed above, the necessary simultaneous presence of the fluorogenic group and its emission deactivator limits the number and variety of beta- antibiotics. lactams that can be prepared. In addition, the authors do not reveal, describe, or suggest the use of fluorescent markers object of the present invention.

On the other hand and due to the maximum limits established by the European Union and other governments or regulatory bodies, for concentrations of antibiotics in milk (for example, EC Commission Regulation no. 2701/94 of Nov. 1994), several Chromatographic analytical procedures for the determination of penicillins and other beta-lactam antibiotics in milk (Fletouris et al., J. Agric. Food Chem . 1992 , 40 , 617; Moats and Harik-Khan, J. Assoc. Off. Anal. Chem 1995 , 78 , 49; Blanchflower et al., Analyst 1994 , 119 , 2595). Methods of detection of beta-lactam antibiotics by UV absorption spectroscopy have also been used, although absorption at wavelengths of 220 nm or 230 nm presents many problems due to interference from other species and the detection limits of the method. For this reason, fluorescence detection methods are being developed, in which the sensitivity is much higher. However, most of the antibiotics of interest lack fluorophores, so it is essential to carry out the derivatization of antibiotics using the amino functionality they present, or synthesize fluorescent analogs with the basic skeleton of 6-aminopenicillanic acid (6- APA) in its structure, both aspects that the present invention reveals.

The examples of molecules of this type included in the bibliography show the synthetic difficulties involved in the preparation of these structures since, although various antibiotic derivatization techniques have been described, in most cases the corresponding fluorescent penicillins are not They have isolated or purified. In addition, the general references mentioned above (Haughland; Prasanna de Silva et. Al .) Do not reveal or suggest in any way the fluorogenic groups or the fluorescent antibiotics object of the present patent.

The present invention reveals the synthesis, antibiotic purification and characterization fluorescent beta-lactams (derivatives of the structure of penicillins, including amoxicillin, ampicillin, cephalosporins and 6-aminopenicillanic acid, although not limited in any way to these) that have in their structure a Pyrene entity as a highly fluorogenic agent.

Description of the invention

Synthesis of fluorescent derivatives of beta-lactam antibiotics.

The present invention reveals the synthesis, purification and characterization of highly fluorescent beta-lactam antibiotics that have applications, among others but not restricted to them, for the determination of the enzymatic activity of beta-lactamases, the study of bacterial membrane proteins , the analysis of microbial fermentation media or the development of rapid and sensitive analytical methods to detect qualitatively or quantitatively antibiotics (in fluids of biological origin, in pharmaceutical preparations and formulations, in foods of animal origin, etc.). Such analytical methods may be based, although not limited to them, on chromatographic, immunological, enzymatic procedures or on the use of molecular printing polymers ( molecularly Imprinted polymers, MIPs; such as those described by Sellergren, editor, " Molecularly Imprinted Polymers " , Techniques and Instrumentation in Analytical Chemistry, Vol. 23, Elsevier, Amsterdam, 2001).

Also, the invention describes obtaining beta-lactam antibiotic derivatives (derived from the structure of penicillins and cephalosporins, including amoxicillin, ampicillin and acid 6-aminopenicillanic, although not limited in any mode to these) that have a fluorophore or group in their structure pyrogen-like fluorogenic, linked through the amino group that presents the antibiotic or its precursor beta-lactam.

More specifically, the invention describes the obtaining and using antibiotic derivatives beta-lactams in whose structure has been introduced a group of atoms or molecular sub-structure that emits ultraviolet and visible light (fluorescence) (360-470 nm) when excited or illuminated with light ultraviolet (200-370 nm), without affecting the Bioactivity of the original molecule. The derivatives here are reveal they are obtained, in general, by chemical procedures that they comprise two stages of synthesis, from the acid 6-aminopenicillanic or the antibiotic itself original, provided that the latter contains in its structure molecular a free amino group.

The synthesis of the derivatives, which include pyrene as a fluorogenic group in its structure, consists of two stages. In the first one, the activation of the corresponding carboxylic acids derived from pyrene (for example, commercial 1-pyreneacetic acid or 1-pyrenobutyric acid or any other carboxylic acid having one or more pyrene units in its structure) takes place in its form reactive against the attack of an amine group. Activated derivatives are obtained with excellent yields by reacting, by conventional procedures (for example, those described by GT Hermanson, Bioconjugate Techniques , Academic Press, New York, 1996), the carboxylic acids mentioned with N- hydroxysuccinimide or N, N commercial commercial cyclohexylcarbodiimide, among other agents described in the literature for this purpose soluble in water or non-aqueous media (RP Hughland, editor, Handbook of Fluorescent Probes and Research Products , 8th edition, Molecular Probes, Leiden, The Netherlands).

In the second stage, the bonding or condensation takes place, in basic medium, between the activated derivative of the carboxylic acid obtained previously and the antibiotic or 6-aminopenicillanic acid, analogously to the procedure described by Lakaye et al . in Biochem. J. 1994 , 300 , 141. This step is preferably carried out in the absence of light in order to avoid photodegradations of the fluorogenic group and carefully controlling the acidity of the medium, since at pH values greater than 8.5 or less than 2.5, approximately, beta-lactam antibiotics degrade significantly quickly.

Isolation and purification of antibiotics fluorescent is preferably performed by successive extractions to aqueous and organic media, since said compounds thermally decompose, so it is not convenient recrystallize them. The products obtained must be stored preferably protected from light, at -5 ° C and under an atmosphere of argon.

The only limitation found to these reactions is that they should not alter the fundamental molecular structure (responsible for its activity as such) of the antibiotic beta-lactam or acid 6-aminopenicillanne precursor, except as far as refers to the free amino group thereof.

Moreover, in a way of carrying it out but without being restricted to it, the patent reveals, the application of fluorescent beta-lactam antibiotics for the determination of beta-lactam antibiotics in a sample whose concentration is unknown a priori and, in particularly, in foods of animal origin, by means of the use of the so-called molecular printing polymers ( MIPs ).

Description of the figures

The invention includes figures that with Representative character show:

Figure 1 shows the molecular structures typical of penicillin-type beta-lactam antibiotics and cephalosporins (R is the part of the molecule that contains the group amino) and 6-aminopenicillanic acid (6-APA), in addition to the structures resulting from join them a pyrene group (R is the part of the molecule that contains the pyrogen type fluorogenic group).

Figure 2 shows with an example a scheme General Obtaining Antibiotics beta-lactams (Ab) provided with a group pyrogen type fluorogenic not activated; Key: Py = Pyrenean group (by way of example, 1-pirenyl is shown).

Embodiment of the invention

The invention related to obtaining derivatives of highly beta-lactam antibiotics fluorescents are obtained by a chemical process comprising either one or two stages (Figure 2).

First stage

Preferably, for the previous activation of the corresponding carboxylic acids derived from pyrene (for example, 1-pyreneacetic acid or acid Commercial 1-pyrenobutyric acid or any other acid carboxylic present in its structure one or more units of pyrene) to its reactive form against the attack of an amine group, it reacts 1 equivalent of carboxylic acid dissolved in 1,4-dioxane anhydrous with 1.1 equivalents of N-hydroxysuccinimide and 1.1 equivalents of N, N-dicyclohexylcarbodiimide, preferably low Argon atmosphere and in the absence of light. The reaction mixture is kept stirring at room temperature during the times which are indicated in each example. After this time, it cools at 0 ° C with ice bath and filtered through filter plate. He precipitate is washed successively with 1,4-dioxane cold (2x10 mL) and with cold diethyl ether (2x10 mL). The filtrate is stripping the solvent by distillation thereof under reduced pressure, giving rise to a solid that, attending to your data spectroscopic, is identified as the corresponding succinimidil ester.

Second stage

Once the acid activation carboxylic containing pyrene, aqueous sodium bicarbonate is added 4% (2.5 equivalents) on an acetone suspension of 1.3 6-aminopenicillanic acid equivalents (6-APA) or the antibiotic beta-lactam that has one or more amino groups free. When the antibiotic or drug has dissolved 6-APA and preferably in the absence of light, add an acetone solution of 1 equivalent of N-hydroxysuccinimidyl carboxylic acid ester that has one or more pyrene units in its structure previously synthesized, and stirred at room temperature for 24 hours. After this time, acetone is removed by distillation under reduced pressure and water is added. The mixture is extracted with diethyl ether and the aqueous phase is acidified to pH 2.5 with 10% aqueous phosphoric acid. The solid that precipitates in these Conditions are repeatedly extracted with diethyl ether. After dry over anhydrous magnesium sulfate organic extracts combined, the solvent is removed under reduced pressure, obtaining the corresponding fluorescently labeled antibiotic, which Purify by extraction to aqueous basic medium (sodium bicarbonate 5% aqueous), subsequent acidulation (10% aqueous phosphoric acid) and re-extraction with diethyl ether.

By way of illustration and without consideration as a limitation on the scope of this patent, they are illustrated by below examples of the chemical union of the groups Pyrogen fluorogenic to the antibiotic free amino group beta-lactam or acid 6-aminopenicilánico (Figure 1), as well as the use of one of the derivatives contained in the pyrene group, for analysis quantitative in a sample of an antibiotic beta-lactam (for example, Penicillin G) by the use of molecular printing polymers. Chemical reagents and solvents mentioned in the examples are available commercially from one or more manufacturers among which they are, for example, Sigma-Aldrich, Fluka, Merck, Avocado, Molecular Probes, etc.

Example 1 Preparation of succinimidyl acid ester 1-pyrenoacetic

From a solution of 103 mg (0.39 mmol) of commercial 1-pyreneacetic acid in 15 mL of anhydrous dioxane, 50.1 mg (0.43 mmol) of N- hydroxysuccinimide and 87.9 mg (0.43 mmol) of N, N- dicyclohexylcarbodiimide and following the general synthesis and purification procedure, described above, is obtained, after 2 hours of stirring at room temperature, 116 mg (85%) of a greenish-brown solid (melting point: 195-197 ° C) which is identified as the 1-pyreneacetic acid succinimidyl ester by its proton nuclear magnetic resonance spectra and carbon-13, its infrared absorption spectrum, its mass spectrum and its elemental microanalysis.

Example 2 Preparation of succinimidyl acid ester 1-pyrenebutyric

From a solution of 2.0 g (6.94 mmol) of commercial 1-pyrenobutyric acid in 69 mL of anhydrous dioxane, 879 mg (7.64 mmol) of N- hydroxysuccinimide and 1.57 g (7.64 mmol) of N, N- dicyclohexylcarbodiimide and following the typical synthesis and purification procedure, described above, is obtained, after 25 hours of stirring at room temperature, 2.66 g (98%) of a white solid (melting point: 128-129 ° C) which is identified as the succinimidilic ester of the 1-pyreneacetic acid by its proton nuclear magnetic resonance spectra and carbon-13, its infrared absorption spectrum, its mass spectrum and its elementary microanalysis.

Example 3 Acid Obtaining [2S- (2α5α6β)] - 6- (1'pyrene-acetylamino) -3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0.] Heptane-2-carboxylic

(abbreviated PAAP; see formula in the figure on the label "penicillins", where R = (1-pyrene) -CH 2 -).

On a suspension of 315 mg (1.45 mmol) of 6-APA in 2 mL of acetone, 8 mL of 4% aqueous sodium bicarbonate. When the beta-lactam antibiotic, 400 mg are added (1.12 mmol) of succinimidyl acid ester 1-pyrenea, whose synthesis is described in the Example 1, dissolved in 12 mL of acetone and, following the general procedure described above, 228 mg (44%) are obtained of a pale yellow solid (melting point: 148 ° C, decomposes), which is identified as the PAAP by its resonance spectra proton and carbon-13 nuclear magnetic, its infrared absorption spectrum, its mass spectrum and its elemental microanalysis The resulting PAAP antibiotic emits a intense fluorescence whose maximum is at 376 nm, when Illuminates with light of wavelength less than 367 nm.

Example 4 Acid Obtaining [2S- (2α5α, 6β)] - 6- [4 '- (1''pyreneyl) -1'-oxobutylamino] -3,3-dimethyl-7-oxo-4-thia-1 -azabicyclo [3.2.0.] heptane-2-carboxylic

(abbreviated PBAP; see formula in the figure on the label "penicillins", where R = (1-pirenyl) -CH2 {CH2} -CH2-).

On a suspension of 129.2 mg (0.59 mmol) of 6-APA in 3 mL of acetone, 4 mL of 4% aqueous sodium bicarbonate. When the beta-lactam antibiotic, 177 mg are added (0.46 mmol) of the dissolved pyrene-butyric acid succinimidyl ester in 7 mL of acetone and following the general procedure described more above, 71 mg (32%) of a pale yellow solid are obtained (point melting: 89 ° C, decomposes) which is identified as the PBAP by its nuclear magnetic resonance spectra of proton and of carbon-13, its infrared absorption spectrum, its mass spectrum and its elementary microanalysis. The antibiotic resulting PBAP emits an intense fluorescence whose maximum is found at 374 nm, when illuminated with wavelength light less than 367 nm.

Example 5 Acid Obtaining [2S- (2α, 5α, 6β)] - 6 - [(N- (1'-pyrenylacetyl) amino) (4'-hydroxyphenyl) acetylamino] -3,3-dimethyl-7-oxo -4-thia-1-azabicyclo [3.2.0.] Heptane-2-carboxylic

(abbreviated PAAX; see formula in the figure on the label "penicillins", where R = (1-pirenyl) -CH 2 -CONH-CH-C 6 H 4 -OH).

On a suspension of 224.7 mg (0.61 mmol) of Commercial amoxicillin in 1 mL of acetone, 3.5 mL of 4% aqueous sodium bicarbonate. When the beta-lactam antibiotic, 169 mg are added (0.47 mmol) of succinimidyl acid ester 1-pyreneacetic dissolved in 6 mL of acetone and, following the general procedure described above, they are obtained 147 mg (45%) of a pale yellow solid (melting point: 159 ° C, decomposes) that is identified as PAAX by its spectra of nuclear magnetic resonance of proton and of carbon-13, its infrared absorption spectrum, its mass spectrum and its elementary microanalysis. The antibiotic resulting PAAX emits an intense fluorescence whose maximum found at 374 nm, when illuminated with wavelength light less than 367 nm.

Example 6 Determination of the beta-lactam antibiotic penicillin G in a sample using a printing polymer molecular and beta-lactam antibiotic PAAP fluorescent

Multiple measurements of the fluorescence at 373 nm of the PAAP, obtained according to Example 3, in solution in acetonitrile-water 99: 1 v / v, at a concentration of less than 1 mmol L -1, when it is illuminated are started at 333 nm with a commercial spectrofluorimeter (for example SPEX-Fluorolog 2 that manufactures and markets the firm Jobin-Yvon-Horiba, France). The graphic representation of these measurements constitutes the calibration curve of the analytical method that allows a biunivocal correspondence to be established between the concentration of the fluorescent antibiotic and its fluorescence at 373 nm. Next, and for the prior preparation of an insoluble molecular printing polymer (MIP) specific for penicillin G, for example, the proportions of commercial monomer, crosslinker, initiator, solvent and penicillin G described by Skudar et al., Anal. Commun . 1999, 36 , 327. A known amount of the molecular impression polymer thus obtained, crushed and sieved for example at a particle size between 25-100 µm, is contacted for 12 hours with mechanical stirring, with a solution of the PAAP fluorescent beta-lactam antibiotic in acetonitrile-water 99: 1 v / v, then determining the fluorescence at 373 nm of the supernatant when it is illuminated at 333 nm with a commercial spectrofluorimeter (for example SPEX-Fluorolog 2). From the measured fluorescence value, and by comparison of the fluorescence determined under identical conditions for a PAAP solution that has not been in contact with the molecular printing polymer, the concentration is calculated from the previously determined calibration curve PAAP fluorescent antibiotic that has been bound to the polymer. Next, increasing amounts of commercial penicillin G are added, measuring the fluorescence signal of the supernatant that results in each case. Again, from this signal, the concentration of PAAP linked to the MIP is calculated in each case. The response curve for the beta-lactam antibiotic penicillin G is obtained by representing the percentage (%) of PAAP bound to the molecular impression polymer, depending on the amount of penicillin G present in the solution. From the resulting response curve the concentration of penicillin G present in any sample containing said antibiotic in unknown concentration can be obtained, provided that it is contacted with a perfectly known amount of PAAP whose optimal value depends on the concentration of Penicillin G in the sample.

Claims (7)

1. Derivatives of beta-lactam antibiotics that contain in their molecule a fluorogenic or fluorescent group, characterized in that the fluorogenic group is pyrene. 2. Derivatives of beta-lactam antibiotics 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 derivatives of beta-lactam antibiotics containing in its molecule a fluorogenic or fluorescent group, according to claim 1, characterized in that the synthesis has two stages: (1) activation of the fluorogenic group and (2) binding of the activated group to the precursor 4. Procedure for obtaining derivatives of beta-lactam antibiotics containing in its molecule a fluorogenic or fluorescent group, according to claim 3, characterized in that the precursor is 6-aminopenicillanic acid or any beta-lactam antibiotic containing in its molecular structure a free amino group. 5. Procedure for obtaining derivatives of beta-lactam antibiotics containing in its molecule a fluorogenic or fluorescent group, according to claims 3, and 4, characterized in that when the molecule containing the pyrene group is activated, the synthesis consists of a stage of binding between said molecule and the antibiotic or 6-aminopenicillanic acid. 6. Use of antibiotic derivatives beta-lactams, according to claim 1, for the determination of the enzymatic activity of beta-lactamases, for the study of the membrane bacterial, for the analysis of microbial fermentation media or in the development of rapid and sensitive analytical methods to qualitatively and quantitatively detect said antibiotics beta-lactams. 7. Use of antibiotic derivatives beta-lactams, according to claim 1, for the quantification of antibiotic residues beta-lactams by analytical procedures competitive type that use an antibody or a polymer of specific molecular impression for the antibiotic beta-lactam.