METHOD FOR THE SYNTHESIS OF SEMI-SYNTHETIC ANTIBIOTICS IN THERMODYNAMICALLY CONTROLLED WATER-COSOLVENT ORGANIC MISCIBLE APOLAR SYSTEMS BY USING PENICILLIN G ACYLASE

Abstract: Synthesis of semi-synthetic monobactamic or β-lactamic antibiotics by using derivatives stabilized by various methods of penicillin G acylase from various microbial sources according to a thermodynamically controlled strategy in monophase water/cosolvent organic apolar systems, wherein the concentration of the cosolvent varies between 30 % and 90 %, the temperature between -10 °C and 50 °C, the pH between 4.5 and 8.5, with concentrations of the antibiotic nucleus between 0.5 and 875 mM and acyl donor between 0.2 mM and 1 M, with a relationship antibiotic ring/activated or free acyl donor, using a buffer between 0 and 1 M. Application to the pharmaceutical industry.

Description: A large number of condensation products with the final destination in food, fine chemistry or the pharmaceutical industry are normally synthesized by chemical methods, where the use of very polluting cosolvents is normally required. The need to protect the environment has caused a big increase in the search for alternative less polluting routes. In this framework, enzymatic enqineering is revealed as a possible alternative. In the case of semi-synthetic antibiotics, the use of penicillin G acylases for the synthesis thereof also has a series of advantages. Among them we can point out that there is no need to protect the carboxyl group of the antibiotic nucleus, nor the group in α of the acyl donor (such as for example in the case of phenylglycine, homopthalic acid, etc.) or the groups in any of the other positions (substituents of the ring of many thiazoles, imidazoles, etc,) that the reaction is stereospecific (important when the acyl donor is chiral such as phenylglycine or mandelic acid), that the reaction conditions are mild etc. Penicillin G acylase is the enzyme normally used by pharmaceutical industries as the catalyst of the hydrolysis reaction of penicillin G to obtain 6 aminopenicilamic acid (6APA), starting point for the synthesis of many β-lactamic antibiotics. For many years now, penicillin G anylase has been attempted to be used as a catalyst of the second part of the process, the synthesis of semi-synthetic antibiotics (M. Cole in Biochem. J. (1969)115, 757), up to now without much success. Given that the production, purification methods etc. of this enzyme from very diverse microbian sources are already known and used normally by pharmaceutical firms, the implantation of the same in other different reactions, in this case the synthesis of semi-synthetic antibiotics, seems simple. Penicillin G acylase can catalyze the synthesis of semi-synthetic antibiotics according to various strategies (Kasche in Enzyme Microb. Technol, 1986, vol 8, (4.16.)) Hereinafter we will comment on the main ones. In kinetically controlled synthesis, an activated acyl donor (in the form of an amide or, more often, an ester) is used to obtain a maximum transitory yield of product removed from balance concentrations. This is one of the more popular strategies (V. Kasche in Biotechnology Letters, 1985, Vol. 7 No. 12; T. Takahashi, K. Kato, Y. Yamazaki and M. Isono in The Japanese Journal of Antibiotics Dec. 1977,...), basically due to the mild reaction conditions used, which permit the use of unstabilized enzymes and enzymatic derivatives, and to the high activity that the enzyme shows in these conditions. A fact to be pointed out is that the maximum transitory yields obtained depend on the enzyme used. This synthesis strategy has not achieved up to the moment sufficiently good results (T.A. Savidge in Drugs and the Pharmaceuitcal Sciences, Vo. 22, 1984.) One way to improve them seems to be the use of organic cosolvents in the reaction medium, which implies some more drastic reaction conditions in which the stability of the enzyme begins to be important. Few studies have gone in this direction. (Kasche in Methods in Enzymology, 1987, Vol. 136, (26) and in Biotechnology Letters Vol. 7 No. 12 (877-882.)) The other synthesis strategy that we will consider is the thermodynamically controlled one. In it the yield obtained is given by the thermodynamic constant. We can carry out the reaction without the need of activating the acyl donor. If we use an activated subtrate, we can remarkably improve the rate of the process. This type of strategy has two main types, depending on whether the system is mono-phase or bi-phase. In this case the use of an acylase derivative or another (be it a different bacterial source of the enzyme or method of preparation of the deivative) only the rate of the reaction will vary. In the thermodynamically controlled synthesis in bi-phase systems, a water inmiscible cosolvent is used in which the antibiotic has a better solubility than in water, raising this way the yield upon removing the product from the reaction medium. In this strategy a very fine control of the water activity is required. In thermodynamically controlled synthesis in mono-phase systems miscible organic cosolvent / water mixtures are used to displace the balance in the direction of the synthesis, starting from the two unmodified substrates. We consider that this type of system is the one to choose as long as it is possible to use it. The main advantage that it has over the other two synthesis strategies is that it is much simpler, which permits a better design of the reactor and of the reaction. Thus, with regard to the kinetically controlled synthesis, we can say that thermodynamically controlled synthesis: 1.- It does not require the activation of the acyl donor, whereby at all times only the antibiotic ring, acyl donor and antibiotic are in the medium. The ester or amide of the acyl donor would have to be added tothe kinetically controlled one. 2.- In the event of using an activated acyl donor in the form of ester or amide, we can obtain a rate similar to that of the kinetically controlled systems and at the end the concentration of the acyl donor will be very low, if the activator is carefully chosen. In kinetically controlled synthesis, normally the maximum yield can still attain a large amount of acyl donor remaining in the medium. 3.- The yield obtained is stable with regard to the transitory yield that is obtained in kinetically controlled synthesis. 4.- The yield that is obtained is constant upon not depending on the state of the catalyst. The deactivation of this alone will modify the rate with which the balance is obtained. In kinetically controlled synthesis the yield can vary over time, depending on whether the enzyme is deactivated. 5.- The overall yield of the reaction can be improved by raising the concentration of the two substrates simultaneously, or the yield can be improved regarding the antibiotic nucleus increasing the concentration of the acyl donor (cheaper, stabler and, in many cases, more soluble.) In kinetically controlled synthesis it can only be improved by increasing the concentration of the antibiotic ring. 6.- The reaction conditions can vary throughout a process for the purpose of improving the rate of the reaction without lossing any yield. In kinetically controlled synthesis this would hamper even more the design of the reactor. 7.- Many antibiotics seems to be very favourable cases for this type of synthesis strategy upon not having the pK of carboxyl and of amine very distant (as we will see later on this is very important for the yields of this type of synthesis to be acceptable.) 8.- If we obtain, according to point 4, yields close to 100 % for the antibiotic nucleus, the separation of the antibiotic from the reaction mixture will be very simple: it will only be necessary to separate the antibiotic from the excess acyl donor. With regard to thermodynamically controlled synthesis in bi-phase systems, thermodynamically controlled synthesis in mono-phase systems has the following advantages: 1.- Control of the pH is simpler in mono-phase systems than in bi-phase systems. 2.- The concentration of water in mono-phase systems can be considered constant throughout the reaction, while it requires a very fine control in bi-phase systems. 3.- Synthesis in bi-phase systems must be carried out statically with vigorous stirring. Synthesis in mono-phase systems allow the continuous or static design. 4.- In the bi-phase system a large part of the volume of the reactor may not be used up, since in many cases the water/cosolvent ratio must be very high and thereforethe tank proportion where we can situate the enzyme must be very reduced. A number of processes for preparing semisynthetic antibiotics have been made. Spanish Patent ES-P-8701590 and a publication by Guisa, J.M., Blanco R.M. and Alvaro G. "Insolubilization of penicillin G acylase by multipoint covalent attachment to aldehyde agarose gels", Proceedings of the 4th European Congress on Biotechnology, vol. 2, 97-100, Elsevier Scientific Publishers; Amsterdam (1987), disclose methods for preparing very stable penicillin G acylases but fail to disclose effective methods for synthezising antibiotics using such acylases. DE-A-3507403 discloses a process for enzymatic semisynthesis of β-lactamic antibiotics, in the presence of an lower aliphatic alcohol and penicillin amidase or D-α-amino acid hydrolase, using an ester as a starting reagent and a preferred concentration of an organic cosolvent of 15-25%. FR-A-2188608 discloses a process for preparing semisynthetic penicillines using an organic cosolvent in concentrations up to 15%. Ymazaki and Kato, Chem. Abstr., 93, 645 (1980), abstr. mo. 236827, disclose a process for preparing semysynthetic caphalosporanic and penicillanic acids using an enzyme different from penicillin G acylase. However, the methods of synthesis have not resulted in totally satisfactory yields or are rather complex. Up to now, the results obtained with this synthesis strategy have been very poor (B. McDougall, P. Dunmil and M. D. Lilly in Enzyme Microb. Technol., 1982, Vol. 4, (114-115)) despite the good initial perspectives. The invention as claimed is intended to solve the aforementioned drawbacks of prior art. We consider that only a joint design of the penicillin G acylase derivatives and the synthesis reaction make it possible to work in a broad enough margin of conditions to obtain good results, as we will see throughout this introduction. The present invention as claimed relates to a method for the synthesis of a semi-synthetic antibiotic selected from aphatolines, penicillins and semisynthetic monobactamics, based on a reaction that can be expressed in the general way as: The constant of this reaction would be: Kap = (ANTIBIOTIC) . (H 2 ) (NUCLEUS) o . (DONOR) o K ter = (ANTIBIOTIC) . (H 2 O) (NUCLEUS . NH 2 ) . (DONOR . COOH) If we make F = (NUCLEUS . NH 2 ) . (DONOR . COOH) (NUCLEUS) O . (DONOR) O Therefore, we must keep in mind that only the non-ionic forms intervene in the balance. Therefore, the differaeces between the pKs of the two substrates must be as small as possible. The ideal thing would be that the pK of the amine were lower than the pK of the carboxyl. In some given conditions, two are the main options that we have to improve the yield: Reduce the concentration of the undesired product, in this case H 2 O. Have F as close to 1 as possible, in such a way that the apparent constant is as similar as possible to the actual thermodynamic constant. These two effects are obtained by adding organic cosolvents to the medium: we will reduce the water activity and we will increase the percentage of non-ionized forms, basically of the acyl donor, as a result of the increase of its pK ap due to the greater apolarity of the medium which stabilizes the non-ionic forms. The second of these effects is the main one and will be greater the greater the apolarity of the cosolvent and its concentration. In this way we can manage to raise the pK of the carboxyl above the pK of the amine, it being possible to find pH conditions in which practically all the amine is in the non-ionized form, but in which we have a very large amount of protonated carboxyls. However, when the design of a reaction with an enzyme is done, it is necessary to consider the possible deleterious effects of the cosolvent over the enzyme itself. These possible effects can be divided into two types: Those that affect the three dimensional structure of the enzyme, either immediately, causing changes in the activity of the enzyme or by varying the rate of changes in the long run, causing changes in stability. The possible uni- or multi-pointwise direct interactions with certain residues of the enzyme involved in the catalytic activity of the enzyme, causing the inhibition thereof. This effect is very important in the case of penicillin G acylase. Unfortunately, the deleterious effects of the cosolvents on penicillin G acylase go in the same direction as the positive effect on the thermodynamic constant: they become more drastic as we increase the apolarity and the concentration of the same. (G. Alvaro, doctoral thesis, "Universidad Autonoma," 1988.) Therefore, the appropriate choice of the cosolvent is a critical factor when effecting an adequate design of this synthesis reaction. The use of stabilized derivatives will protect the enzyme from part of the first effects, but only by chance can the second ones be avoided. Besides, in the case of antibiotic nuclei it is necessary to take well into account the solubility of these molecules, as well as their stability in the different reaction conditions. For this reason, we consider that a design of this reaction that is useful in practice must consider at least five points: 1.- Activity of the enzymatic derivative DESIGN OF THE 2.- Stability of the enzymatic derivative CATALYST 3.- Solubility of the antibiotic nucleus DESIGN OF THE 4.- Stability of the antibiotic nucleus and of the antibiotic REACTION 5.- Thermodynamics of the process Obviously, the greater the stability and activity of our derivatives, less important will points 1 and 2 be in the engineering of the reaction and the greater the possibilities of finding suitable conditions of points 3, 4 and 5 will be for each antibiotic. In each case, depending on the acyl donor and on the antibiotic nucleus it will be necessary to study in an integral manner the optimal conditions for the above five points: pH, T, % and nature of the cosolvent, ionic force..., reaching compromise situations, which normally will not have the optimums of any of the parameters in particular. It becomes obvious that some conditions in which the operative life of the reactor is very short should be rejected, no matter how good the results obtained are (as would happen if we introduce penicillin G acylase soluble in a mixture of 70 % apolar cosolvent / 30 % acetate, at a pH of 5 and 50º C.) The same thing will happen if in some specific conditions we have a high stability of the enzymatic derivative, but the solubilities of the substrates or yields are low (this is what happens in water or in mixtures of water / mild cosolvents.) With regard to activity, some conditions in which the inhibition of the cosolvents, products or substrates block the enzymatic activity, not even the balance conditions will be attained. If we do not use any cosolvent, the stability of the enyme is good, so is its activity, but only some acceptable yields will be obtained in some very limited pH margins (J. O'Sullivan and C.A. Aklonnis in The Journal of Antibiotics, July 1984.) The use of mild cosolvents, such as glycerine, ethyleneglycol or polyethylenglycol) hardly increases the margin of conditions in which we can move even at high concentrations (45 % of ethyleneglycol, B. McDougall, P Dunnill and M. D. Lilly in Enzyme Microb. Technol., 1982, Vol. 4), upon hardly affecting the pKs of the acyl donors whereby the obtained results are not good enough for industrial use. Nor is the use of very apolar cosolvents, but at low concentrations, sufficient to obtain satisfactory results (20 % acetone or dimethyl sulfoxide: V. Kasche in Methods in Enzymology, 1987, Vol. 136 (26.)) Therefore, we consider that despite the seriously deleterious effects of apolar cosolvents, it is essentail to work with them (methanol, butanediol, ethanol, acetonitrile, acetone, tetrahydrofuran, dimethylformamide, dioxane, dimethylsulfoxide...) at high concentrations, between 30 and 90 %. Only these drastic conditions will have a sufficient affect on the pKs of the acyl donors (R. Femández-Lafuente, G. Alvaro and J.M. Guisán, manuscripts in preparation.) Logically, only penicillin G acylase derivatives with a high stability and activity can be used on an industrial scale and even, in many cases, in the laboratory. In this way we can obtain some good yields in a broad scale of pH, T, etc. which make it possible to find practical values of solubility and stability of substrates and products. The optimum pH and T conditions for solubility and stability of substrates and products, as well as the thermodynamic constant, will be very variable depending on the antibiotic in question. Depending on the case they can vary between pH 4.5 and pH 8.5 and a temperature between -10º C and 50º C. We point out again that only with very stable and active penicillin G acylase derivatives will it be possible to prevent these factors from very drastically limiting these margins. As an indication, but not a restriction of the invention the following examples are given hereinafter: EXAMPLE 1 100 ml. of a solution 10 mM of 6 APA and 400 mM of phenylacetic acid in 50 % dioxane / 50 of acetate 50mM, is prepared at a pH of 7. At the same time, a column kept at a constant temperature of 37°C with 10ml of Kluyvera citrophila derivative prepared in a manner known per se, is prepared. 50ml are allowed to pass at a flow of 50ml/h to equilibrate the column and at that moment the reaction mixture is recirculated. The pH is maintained with the help of a pHstate. The reaction is followed by HPLC using a C-18 column, and as a mobile phase 35% methanol/0.067 M phosphate M 65% pH 4.7, measuring the absorbancy at 260 nm. It is left to recirculate until the equilibrium is attained (we consider equilibrium, a non-variation in the concentration of substrates or products for 1 hour). In these conditions yields around 90% of conversion of 6-APA (6-aminopenicillinamic acid) into penicillin G are obtained. EXAMPLE 2 A solution of 15mM of 7 ACA (7-aminocephalosporanic acid and 400mM of thienylacetic acid in 50% dioxane/50% acetate 50mM is prepared at pH 6. The column is kept at a constant temperature of 25°C. The flow and volume of the derivative are the same as in example 1. The mobile phase for HPLC is 30% methanol/70% phosphate, 0.067M at pH 4.7; the rest of the conditions are maintained like those in example 1. After attaining the equilibrium, the degree of conversion of 7-ACA into cephalotine is above 95%. EXAMPLE 3 A solution of 10mM of 6 APA (6-aminopenicillamic acid) and 120mM of tienilacetic acid in a mixture of 65% acetone/35% acetate 70mM is prepared at pH 5.5. The column is kept at a constant temperature of 4°C. The other conditions are the same as those of the above examples. The conditions for HPLC are those of example 2. In these conditions, after reaching the equilibrium, the conversion yields of 6 APA obtained are higher than 95%. EXAMPLE 4 A solution of 40mM of 6 APA (6-aminopenicillamic acid) and 200mM of phenylacetic acid in 50% butanediol/50% acetate 50 mM is prepared at pH 6.5. The column is kept at a constant temperature of at 4º C. The rest of the conditions are as in example 1. In these conditions, the degree of conversion of 6 APA 6-aminopenicillamic acid to penicillin G is higher than 85 %. EXAMPLE 5 A solution of 4 mM of 7 ADCA 7-aminodeacetoxycephalosporanic acid and 80 mM of acetic acid in 70 % ethanol / 30% acetate 85 mM is prepared at a pH of 7.5. The reaction temperature is situated at 15º C. The rest of the conditions are as in example 4. In the equilibrium, the degree of conversion of 7 ADCA is higher than 90 %. EXAMPLE 6 A solution of 110 mM of phenylacetic acid and 5.5 mM of 7 aminoacetoxycephalosporanic 2 (5-methyl-1,3,4 thiadiazol) (ADACAT) in 60 % dimethylsulfoxide / 40 % acetate 65 mM is prepared at a pH of 7.5. The temperature at which the reaction is carried out is 20º C, the rest of the conditions are like in the other examples. In the equilibrium the degree of conversion of 7 ADACAT was more than 95 %. EXAMPLE 7 A solution of 10 mM of 6 APA and 10 mM of phenylacetic acid in 50 % dimethylformamide / 50% acetate 50 mM is prepared at a pH of 5. The reaction temperature was 4º C. All the other conditions are like those in the above example. The degree of conversion of 6 APA 6-aminopenicillamic acid obtained was higher than 55 %. EXAMPLE 8 A solution of 4.5 mM of 7 ACA and 200 mM of thienyl acetic acid in 65 % dioxane / 35 % acetate 70 mM is prepared at a pH of 8. The temperature at which the reaction is carried out is 10º C. The other conditions are those of example 2. The degree of conversion of the 7 ACA 7-aminocephalosporanic acid obtained exceeds 95 %. EXAMPLE 9 A solution of 10 mM of 6 APA 6-aminopenicillamic acid and 10 mM of phenylacetatil glycine in 50 % dimethylformamide / 50% acetate 50 mM is prepared at a pH of 5. The reaction temperature was 4º C. All the other conditions are like those of the above example. The degree of conversion of 6 APA obtained was higher than 55 %.

Claims: A method for the synthesis of a semisynthetic antibiotic selected from cephatolines, penicillins, cephalosporins and semisynthetic monobactamics, using penicillin G acylase or derivatives thereof by a reaction whereby an acyl donor comprising a carboxyl group, and an antibiotic nucleus comprising an amino group are condensed, said acyl donor being selected from phenylacetic acid and homophtalic acid; characterized in that the method comprises carrying out the reaction in a thermodynamically controlled water-organic solvent monophasic system, by direct condensation of the carboxyl group of the side chain of the acyl donor and the amino group of the antibiotic nucleus, in an aqueous medium comprising 30 to 90% of an organic cosolvent and an immobilized penicillin G acylase derivative, wherein the reactants of said condensation comprise an antibiotic nucleus concentration between 0.5 and 875 mM, and an acyl donor concentration between 0.2 mM and 1 M, said concentrations being selected such that the antibiotic nucleus / acyl donor molar ratio is between 5 x 10 -4 and 1,5, said condensation being carried out at a pH between 4.5 and 8.5, at a temperature between -10°C and 50°C, and with a buffer concentration between 0 and 1 M. A method according to claim 1, characterized in that the penicillin G acylase is selected from any of the following microorganisms: Escherichia Coli , Kluyvera citrophila , Bacillus megaterium or Proteus rettgeri . A method according to claim 1, characterized in that the support used for immobilization of penicillin G acylase is selected .from agar gels, agarose gels, porous glass, alumina. A method according to claim 1, characterized in that the cosolvent is selected from compounds that at concentrations of 50%, cause increases of more than one unit of pH in the pK of phenylacetic acid. A method according to claim 4, characterized in that the cosolvent is selected from dimethylformamide, acetone, isopropyl alcohol, dioxane, tetrahydrofuran. A method according to claim 1, characterized in that the antibiotic nucleus is a β-lactamic ring. A method according to claim 6, characterized in that the antibiotic nucleus is selected from 6-aminopenicillamic acid, 7-aminocephalosporanic acid, 7-aminodeacetoxycephalosporanic acid, 7-amino 3(5 methyl-1,3,4 thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid. A method according to claim 1, characterized in that the antibiotic nucleus is a monobactamic ring. A method according to claim 8, characterized in that the antibiotic nucleus is 3-aminomonobactamic acid.

Inventors: ALVARO CAMPOS GREGORIO, BLANCO MARTIN ROSA M, FERNANDEZ-LAFUENTE ROBERTO, GUISAN SEIJAS JOSE M, MOLINA ROSELL CRISTINA, ALVARO CAMPOS, GREGORIO, BLANCO MARTIN, ROSA, M, FERNANDEZ-LAFUENTE, ROBERTO, GUISAN SEIJAS, JOSE, M., MOLINA ROSELL, CRISTINA, ALVARO CAMPOS, Gregorio Instit. de Catál. y, BLANCO MARTIN, Rosa, M Instit. de Catál. y, FERNANDEZ-LAFUENTE, Roberto Instit. de Catál. y, GUISAN SEIJAS, José, M. Inst. de Catálisis y Petr, MOLINA ROSELL, Cristina Instit. de Catál. y
Applicants: CONSEJO SUPERIOR INVESTIGACION, CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Application year: 1990
Main classification: C12P35
IPC codes: C12P37, C12P35