MXPA00001608A - Microbial conversion of 2-methylquinoxaline - Google Patents

Abstract

The present invention relates to methods for the microbial oxidation of 2-methyl-quinoxaline to 2-quinoxaline-carboxylic acid comprising contacting 2-methyl-quinoxaline with a microorganism, or a suitable setete mutant, and incubating the mixture resulting in conditions sufficient to provide an amount of 2-quinoxaline-carboxylic acid, the present methods optionally further comprising the isolation and purification of 2-quinoxaline-carboxylic acid

Description

CONVERTING MICROBIAL PE 2-METHYL-QUINOXAL1NA FIELD OF THE INVENTION The present invention relates to novel methods for preparing 2-quinoline-carboxylic acid and relates, more specifically, to the microbial oxidation of 2-methyl-quinoxaline to form 2-quinoxaline-carboxylic acid.

BACKGROUND OF THE INVENTION Methods for the microbial oxidation of certain aromatic heterocycles are known in the art and, in particular, for the microbial oxidation of methyl groups existing in certain aromatic heterocycles, such as, for example, those described in the following two articles: " Gene Order of the TOL Catabolic Plasmid Upper Pathway Operating and Oxidation of Both Toluene and Benzyl Alcohol by the xy / IA Product ", by S. Harayama et al, J. Bacteriol. 167 (2): 455-461 (1986) and "Enzymatic Oxidation of Methyl Groups on Aromatic Heterocycles: A Versatile Method for the Preparation of Heteroaromatic Carboxylic Acids", by A. Keiner, Angew. Chem. International Edition in English, 31 (6): 774-775 (1992).

The patent of the U.S.A. do not. No. 4,859,592 describes a microbial process for the production of picolinic acid, which can then be converted into pyridine products by chemical means. The patent of the U.S.A. do not. 5,104,798; 5,213,973; and 5,236,832 describe a microbial process for the oxidation of methyl groups in certain aromatic heterocycles with 5 or 6 membered rings, to give the corresponding carboxylic acids, which is made by a bacterium of the Pseudomonas species using toluene, xylene or cymene as inducer. As described therein, it is known in the art that the oxidation of the methyl group of toluene to benzoic acid by the strain Pseudomonas putida ATCC No. 33015 comprises three operations catalyzed by toluene-monooxygenase, alcohol dehydrogenase and aldehyde dehydrogenase, respectively . As described above with reference to the aforementioned article by Harayama et al., The plasmid pWWO of TOL of P.putida mt-2 is a transmissible extrachromosomal element that encodes all the enzymes required for the oxidative catabolism of various aromatic hydrocarbons, including toluene, m-xylene and p-xylene. Bacteria that are carriers of TOL plasmids, eg P. putida ATCC No. 33015, can convert certain aromatic hydrocarbons to their corresponding aromatic carboxylic acids; both the xyl operon encoding enzymes for the degradation of xylene and the genes that are responsible for the regulation of the xyl gene are located in the plasmid pWWO of TOL. The genes located in the plasmid pWWO of TOL, which encode the enzymes required for the above oxidations, must be induced to produce said enzymes. Therefore, the description of said induction is applied in the aforementioned US patents. do not. 5,104,798; 5,213,973 and 5,236,832. As described in an article by Gaucher et al. In Dev. Ind. Microbio /., 22: 219-232 (1981), the fungus Penicillium gríseofulvum contains three enzymes for the conversion of m-cresol to m-hydroxybenzoic acid; m-cresol-methyl-hydroxylase, m-hydroxybenzyl alcohol dehydrogenase and m-hydroxybenzaldehyde hydroxylase. Reiterating, as is known in the art, certain fungi and bacteria contain enzymes for the oxidation of methyl groups existing in certain aromatic rings to give their corresponding carboxylic acids. While it is known that methyl groups existing in said heteroaromatic rings can be oxidized to their corresponding carboxylic acids using microorganisms, as would be appreciated by those skilled in the art, the chemical and optical performances of said microbial oxidations generally vary substantially depending on, for example, , of the particular microorganism chosen, the concentration of the substrate, the structure of the substrate, and similar issues. It has now been found that a range of microorganisms, including fungi and bacteria, substantially oxidize 2-methyl-quinoxaline to give 2-quinoxaline-carboxylic acid. In addition, the present process allows an appropriate recovery of 2-quinoxaline-carboxylic acid. The provisional patent application of the US. No. 60 / 073,801 (hereinafter referred to as "the application '801") filed on February 5, 1998, now international PCT application no. PCT / IB99 / 00067 filed January 18, 1999, describes the use of 2-quinoxaine carboxylic acid as an intermediate in the synthesis of new dihydroxy-hexanoic acids which are useful for treating, eg, inflammation and other immune disorders. The 2-quinoxaline-carboxylic acid provided by the novel methods of the present invention can be used to synthesize said dihydroxy-hexanoic acid. All documents cited here, including the preceding ones, are incorporated by reference to the present in its entirety.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a microbiological process for preparing 2-quinoxaline-carboxylic acid from 2-methyl-quinoxaline. More particularly, the present invention relates to microbiological processes for preparing the compound of formula I contacting the compound of formula II with a microorganism capable of performing the oxidation of the methyl group of the compound of formula II to give the carboxyl group of the compound of formula I, and incubating the resulting mixture under appropriate conditions to provide an amount of the compound of formula I. Correspondingly, the present invention provides methods for carrying out the microbial oxidation of the compound of formula II, 2-methyl-quinoxaline, which comprises: contacting the compound of formula II with a microorganism, or a mutant thereof which is known or otherwise obtainable by those skilled in the relevant technology and which is capable, in spite of said mutation, of carrying out the oxidation in question (hereinafter referred to as "an appropriate ester mutant"), and incubating the resulting mixture under conditions sufficient to provide an amount of the compound of 2-quinoxaline-carboxylic acid, wherein said microorganism is selected from the group consists of Absidia glauca ATCC No. 22752, Absidia glauca ATCC No. 74480, Absidia pseudocylindrospora ATCC No. 24169, Absidi repens ATCC No. 14849, Absidia repens ATCC No. 74481, Actinomucor elegans ATCC No. 6476, Alternaria sotani ATCCN No. 11078, Aspergillus tamarii ATCC No. 16865, Coniophora puteana ATCC No. 12675 Cunninghamella echinulata ATTC No. 8688a, Cunninghamella echinulata ATCC No. 8688b, Cunninghamella echinulata ATCC No. 8983, Cunninghamella echinulata ATCC No. 9244, Cunningamella echinulata ATCC No. 9245, Cunninghamella echinulata ATCC No. 10028b, Cunninghamella echinulata ATCC No. 26269, Cunninghamella echinulata ATCC No. 36190, Cuninghamella echinulata ATCC No. 36112, Cunninghamella homothallica ATCC No. 16161, Cylindrocarpon destructans ATCC No. 66963, Diplodia gossypina ATCC No. 20575, Epicoccum neglectum ATCC No. 12723, Glomerella lagenaria ATCC No. 14724, Penicillium claviforme ATCC No. 10426, Penicillium duclauxii ATCC No. 10440, Penicillium glabrum ATCC No. 11080, Pseudocochliobolus ATCC No. 24155, Psedomonas putida ATCC No. 33015, Pseudomonas putida ATCC No. 202190, Rhodococcus rhodochrous ATCC No. 19067 and Thamnostylum piriforme ATCC No. 8686; and appropriate mutants thereof; with the proviso that when said microorganism is said Pseudomonas putida ATCC No. 33015 or said Pseudomonas putida ATCC No. 202190, said Pseudomonas putida ATCC No. 33015 or said Pseudomonas putida ATCC No. 202190 is induced by interaction with an inducer before said contacting said Pseudomonas putida ATCC No. 33015 or said Pseudomonas putida ATCC No. 202190 with said 2-methyl-quinoxaline. The methods in question optionally further comprise the isolation of the desired product, 2-quinoxaline-carboxylic acid, by any appropriate method. For example, the reaction mixture can be extracted with an organic solvent, preferably ethyl acetate, and then the extracted material can be chromatographed. Alternatively, the 2-quinoxaline carboxylic acid can be adsorbed from the reaction mixture onto a resin, preferably a polymeric absorbent resin, eluted therefrom using n organic solvent, preferably ethyl acetate, and crystallized from the eluted material using an organic solvent, or a combination of organic solvents, preferably ethyl acetate and methanol. Moreover, the 2-quinoxaline carboxylic acid produced by the present processes can be treated with an appropriate base, e.g., sodium hydroxide, resulting in the formation of a salt, e.g., the sodium salt. , of 2-quinoxaline-carboxylic acid. The alkali metal salt of 2-quinoxaline carboxylic acid can then be isolated from the bioconversion medium by removal of the cells from the medium by filtration or centrifugation, followed by concentration of the cell-free medium, eg, by evaporation. The microorganism in question is preferably an intact microorganism.

In a preferred embodiment of the present invention in which the microorganism is a fungus, the fungus is selected from the group consisting of the genera Absidia, Aspergillus, Alternaria, Penicillium, Diplodia and Cunninghamella. In a particularly preferred embodiment of the present invention in which the microorganism is a fungus, the fungus is of the genus Absidia. In an especially preferred embodiment of the present invention wherein the microorganism is a fungus of the genus Absidia, the microorganism is A. glauca ATCC No. 22752 or A. glauca ATCC No. 74480, or an appropriate mutant thereof, or, more still, any deposit of A. glauca ATCC No. 22752, or an appropriate mutant thereof, made to comply with the terms of the Budapest treaty. In another especially preferred embodiment of the present invention wherein the microorganism is a fungus of the genus Absidia, the microorganism is A. repens ATCC No. 14849 or A. repens ATCC No. 74481, or an appropriate mutant thereof, or, more even, any deposit of A. repens ATCC No. 14849, or an appropriate mutant thereof made to comply with the terms of the Budapest Treaty. A preferred cell density for the fungal cultures of the present invention is from about 10 to about 30 g of dry weight of cells / l. In another preferred embodiment of the present invention, the microorganism is a bacterium.

In a preferred embodiment of the present invention in which the microorganism is a bacterium, the bacterium is selected from the group consisting of the genera Pseudomonas and Rhodococcus. In a partially preferred embodiment of the present invention in which the microorganism is a bacterium, the bacterium is of the genus Pseudomonas. In an especially preferred embodiment of the present invention wherein the microorganism is a bacterium of the genus Pseudomonas, the microorganism is P. putida ATCC No. 33015 or P. putida ATCC No. 202190, or an appropriate mutant thereof, or more so , any deposit of P. putida ATCC No. 33015, or an appropriate mutant thereof, made to comply with the terms of the Budapest Treaty. A preferred cell density for the bacterial cultures of the present invention is a density that provides an optical density of from about 10 to about 30 to 650 nm. As discussed above, in embodiments of the present invention in which the microorganism is P. putida ATCC No. 33015 or P. putida ATCC No. 202190, or an appropriate mutant thereof, the microorganism, or an appropriate mutant thereof, it is induced before or during the contacting. It is preferred that the contacting takes place after the termination of the induction of the microorganism. Preferred inductors include p-xylene. A partially preferred inducer is p-xylene.

In a preferred embodiment of the present invention, wherein the microorganism is P. putida ATCC No. 33015 or P. putida ATCC No. 202190, or appropriate mutant thereof, and the microorganism is cultured in a growth medium within a flask, the inducer is added to said medium before contacting the microorganism with 2-methyl-quinoxaline and is incubated in said growth medium for a period of time sufficient for the substantial completion of said induction. The cells of the induced microorganism are collected by centrifuging the contents of the flask, removing, eg, decanted, the spent growth medium (and therefore the inducer in question), washing the cell pellet and resuspending the pellet in a aqueous medium, such as DPBS (Biowhittaker), before contacting said 2-methyl-quinoxaline with said microorganism. In another preferred embodiment of the present invention wherein the microorganism is P. putida ATCC No. 33015 or P. putida ATCC No. 202190, or an appropriate mutant thereof, and the microorganism in question is cultured in a growth medium within of a fermentor, the inducer is added continuously or constantly to said growth medium before the contact in question of the microorganism with 2-methyl-quinoxaline and is incubated in said growth medium for a period of time surface for substantial termination of said induction, and then it is interrupted before said 2-methyl-quinoxaline is contacted with said microorganism.

In a further preferred embodiment of the present invention the contacting in question is achieved by adding 2-methyl-quinoxaline to a growth medium comprising the microorganism in question when the microorganism is a fungus. In a preferred embodiment of the present invention in which the contacting in question is achieved by adding 2-methyl-quinoxaline to a growth medium comprising the fungus in question, the growth medium is a medium of solid maceration materials. corn. A preferred medium of maceration corn solids comprises from about 20 to about 40 g / liter (I) of maceration corn solids and about 20 g / l dextrose, which has a pH of about pH 4.85. Another preferred growth medium comprises about 20 g / l of Pharmamedia® (Traders Protein) and about 20 g / l of dextrose, which has a pH of about pH 7.2. In still another preferred embodiment of the present invention, contacting is accomplished by adding the compound of Formula II adsorbed to a resin. See, for example, the article by J.T. Vicenzi et al., "Large-scale stereoselective enzymatic ketone reduction with in situ product removal via polymeric adsorbent resins", Enzyme and Microbial Techonology, 20: 494-499 (1997). In yet another preferred embodiment of the present invention, contacting is accomplished by adding 2-methyl-quinoxaline to an aqueous medium comprising washed cells of the microorganism.

In still another preferred embodiment of the present invention, the microorganism is washed before contacting the microorganism with 2-methyl-quinoxaline. In a preferred embodiment of the present invention in which the microorganism is washed before contacting the microorganism with 2-methyl-quinoxaline, the washed microorganism is immobilized before contacting. In another preferred embodiment of the present invention, the microorganism is grown in a medium of macerating corn solids for a period of time from about seventy-two hours prior to contacting which is accomplished by adding 2-methyl- quinoxaline to this one. The methods of the present invention optionally further comprise the isolation or separation of 2-quinoxaline-carboxylic acid, which is carried out eg by extraction with an organic solvent, adsorption on a resin, crystallization, or as discussed above, when The metal salt is provided with an alkali metal of 2-quinoxaline-carboxylic acid, by concentration by evaporation of a cell-free medium, or the like. The present invention includes the use of 2-quinoxaline-carboxylic acid in the synthesis of the novel dihydroxy-hexanoic acids described in the aforementioned '801 application following any of the methods described in that' 801 application or using any other methods appropriate therefor. .

Those skilled in the art will fully understand the terms used herein to describe the present invention; however, the following terms used here are as described immediately below. The term "intact microorganism" means that the cells of the microorganism possess substantially their inherent mechanical, physical and biochemical integrities (and / or induced, as the case may be). The term "microbial oxidation" means the oxidation of the present invention, as has been done by the intact microorganism, or by any preparation thereof, and the like. The term "microorganism" includes any intact microorganism or appropriate preparation thereof, includes, for example, a washed microorganism to be exempt from, eg, the fermentation medium, the growth medium, the culture broth, and the like. , as may be the case; and the microorganism immobilized, eg, in a column, fixed to globules, and the like.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise stated, throughout the specification and the appended claims: ° C is degrees Centigrade; % is so much percent; ACN is acetonitrile; DMSO is dimethyl sulfoxide; DPBS is Dulbecco's phosphate buffered saline solution; EtOAc is ethyl acetate; EtOH is ethanol; g is gram (s); HPLC is high performance liquid chromatography; I is liter (s); MeOH is methanol; mg is milligarm (s); 10 min is minute or minutes; mm is millimeter (s); mmol is mylimole (s); my is milliliter (s); A77-xylene is meta-xylene; 15 N is (concentration) normal; nM is nanomolar (concentration); PBS is phosphate buffered saline; p-xylene is para-xylene rpm is revolutions per minute; TFA is trifluoroacetic acid; β is microliter (s); v / v is volume by volume; American National Can is domiciled in Menasha, Wisconsin, USA; Becton Disckinson® Labware is domiciled in Franklin Lakes, New Jersey, USA; Becton Dickinson® Microbiology Systems is domiciled in Sparks, Maryland, USA; Biowhittaker® is domiciled in Walkersville, Maryland, USA; Column Engineering®, Inc. is domiciled in Ontario, California, USA; IEC® Centrifuge is domiciled in Needham Heights Massachusetts, USA; Rohm and Haas® is domiciled in Philadelphia, Pennsylvania, USA; and Traders Proteins® is domiciled in Memphis, Tennessee, USA: In addition, ATCC is the American Type Culture Collection that is domiciled at 10801 University Boulevard, Manassas, Virginia, 20110-2209, USA. Table 1 below lists the microorganisms described here and their depositor (s) (see, www.ATCC.com). 1NRRL is Northern Regional Research Laboratories (Peoria, Illinois). 2 A. glauca, 22752, deposited within the terms of the Budapest Treaty on January 13, 1999. 3 A. repens, 14849, deposited within the terms of the Budapest Treaty of January 13, 1999. 4 P. putida , 33015, deposited within the terms of the Budapest Treaty on January 13, 1999.

As discussed above, the present invention relates to microbiological processes for preparing the compound of formula I contacting the compound of formula II with a microorganism capable of carrying out the oxidation of the methyl group of the formula II, 2-methyl-quinoxaline, to give the carboxyl group of the formula I, 2-quinoline-carboxylic acid, and incubating the resulting mixture under appropriate conditions to provide the 2-quinoxaline-carboxylic acid. The methods of the present invention are carried out with ease. The microorganism is cultured, with induction when necessary, eg, when the microorganism is eg P. putida ATCC No. 33015 or P. putida ATCC No. 202190, or an appropriate mutant thereof, and then contact with 2-methyl-quinoxylin to oxidize the methyl group of 2-quinoxaline to give the -COOH group of 2-quinoxaline-carboxylic acid. The 2-quinoxaline carboxylic acid can then be reacted, e.g., additionally by methods described in the aforementioned '801 application to finally provide the novel dihydroxy-hexanoic acids described in the' 801 application which are useful for treating a inflammation and other immune disorders. The activity, the methods for assaying the activities, the dosages, the dosage forms, the administration methods and the background information concerning the new dihydroxy-hexanoic acids described in the '801 application are set forth herein. As noted above, any suitable microorganism or an appropriate mutant thereof can be used in the methods of the present invention. As will be understood by those skilled in the art in light of the present disclosure, the conditions of the methods in question would be chosen depending, e.g., on the class of the microorganism and the particular preparation thereof. For example, the pH, the temperature, the concentrations of components, and the like, eg, of the fermentation medium and the organic solvent, as well as the concentrations of 2-methyl-quinoxaline and the inducer (when employed) will be chosen for provide the particular desired result using the selected microorganism. Preferred fungi include the members of the genera Absidia, Actinomucor, Alternaria, Aspergillus, Coniophora, Cunninghamella, Cylindrocarpon, Diplodia, Epicoccum, Fusarium, Glomerella, Penicillium, Pseudocochliobolus, Thamnostylum and Verticillium, but the species of these are not particularly limiting, with the condition that the microorganisms, or mutants thereof, are capable of performing the oxidation in question.

Particularly preferred fungi belong to the genera Absidia, Alternaria, Aspergillus, Cunninghamella, Diplodia and Penicillium. Especially preferred fungi belong to the genus Absidia. More particularly, preferred fungi include A. glauca ATCC No. 22752, A. glauca ATCC No. 74480, A. pseudocylindrospora ATCC No. 24169, A. repens ATCC No. 14849, A. repens ATCC No. 74481, A. elegans ATCC No. 6476, A. solani ATCC No. 11078, A. tamarii ATCC No. 16865, C. puteana ATCC No. 12675, C. echinulata ATCC No. 8688a. C. echinulata ATCC No. 8688b, C. echinulata ATCC No. 8983, C. echinulata ATCC No. 9244, C. echinulata ATCC No. 9245, C. echinulata ATCC No. 10028b, C. echinulata ATCC No. 26269, C. echinulata ATCC No. 36190, C. echinulata ATCC No. 36112, C. homothallica ATCC No. 16161, C. destructans ATCC No. 66963, D. gossypina ATCC No. 20575, E. neglectum ATCC No. 12723, G. lagenaria ATCC No. 14724, P. claviform ATCC No. 10426, P. duclauxii ATCC No. 10440, P. glabrum ATCC No. 11080, P. / t / pafo / s ATCC No. 24155 and T. piriforme ATCC No. 8686; and appropriate mutants thereof. Most preferred fungi A. glauca ATCC No. 22752, A. glauca ATCC No. 74480, ATCC Repens No. 14849, ATCC Repens No. 74481, ATLAN No. 11078, ATCC No. 16865, ATCC No. 8983, ATCC No. 20575 and P. Gossypina. ATCC Glabrum No. 11080; and appropriate mutants thereof.

Particularly preferred fungi include A. glauca ATCC No. 22752, A glauco ATCC No. 74480, A. repens ATCC No. 14849 and A. repens ATCC No. 74481; and appropriate mutants thereof. Especially preferred fungi include A. repens ATCC No. 14849 and A. repens ATCC No. 74481; and appropriate mutants thereof. Preferred bacteria include those belonging to the genera: Bacillus, Brevibacterium, Micrococcus, Pseudomonas and Rhodococcus, but its species are not particularly limiting with the proviso that the microorganisms, or their mutants, are capable of carrying out the oxidation in question. Particularly preferred bacteria include those belonging to the genera Pseudomonas and Rhodococcus. Especially preferred bacteria include those belonging to the genus Pseudomonas. More particularly, preferred bacteria include P. putida ATCC No. 33015, P. putida ATCC No. 202190 and R. rhodochrous ATCC No.; and appropriate mutants of these. Especially preferred bacteria are P. putida ATCC No. 33015 or P. putida ATCC No. 202190; and appropriate mutants of these. As discussed above, the present invention includes the use of any appropriate mutants of any of the appropriate microorganisms. In addition, a group of mutants with more desirable properties, eg, capable of oxidizing greater amounts of substrate, compared to the parent strain, can also be used in the method in question, and these new strains can be produced using known methods , including, for example, classical mutagenesis and selection techniques, and even recombination methods, for example site-directed mutagenesis. Classical mutagenesis methods include chemical mutagenesis with N-methyl-N'-nitroso-guanidine (Delic et al. (1970), Mutat.Res.9: 167), nitrous acid (Crueger and Crueger (1984), Biotechnologv: A Textbook of Industrial Microbioloqy, page 15, Sinauer Associates, Inc., Sunderland, MA, USA) and irradiation with ultraviolet light (Thrum (1984), in Biotechnology of Industrial Antibiotics (Vandame, editing coordinator), Marcel Dekker , New York, pages 373-374). Selection techniques include simple reisolation of the strain by selection of an isolated colony, selection of specific morphologies of the colony and selection as to resistance to compounds analogous to known components or that are considered to be in the biosynthesis pathway of the compound of formula I (Crueger and Crueger (1984), Biotechnologv: A Textbook of industrial Microbiology, page 24-25, Sínauer Associates, Inc., Sunderland, MA, USA). These new strains are used in the present methods since, for example, they have improved properties relative to their respective progenitor strains, eg they produce more amount of 2-quinoxaline-carboxylic acid, they exhibit less undesired intrinsic degradative activity of 2- methyl-quinoxaline and / or 2-quinoxaline-carboxylic acid and intermediates that can be generated in the process of the present invention depending, for example, on the particular microorganism chosen. In addition, when the mutant is used since it is used it results in more 2-quinoxaline-carboxylic acid, it is necessary to grow less volume of the culture to obtain the necessary material in order to generate a quantity of 2-quinoxaline- carboxylic acid according to the present process which can result in substantial cost savings. As described above any suitable preparation of the microorganism can be used in the methods of the present invention such as, for example, a microorganism in a growth medium, a microorganism washed until free of eg fermentation medium, culture broth, and the like, or immobilized microorganism, eg in a column, fixed to globules, and the like. Those skilled in the art will understand from the description provided herein how an appropriate immobilized intact microorganism is to be prepared as described, for example, by Bauer et al. In the article "Polyvinyl alcohol-immobilized whole-cell preparations biotransformation of nitriles "published in Biotechnology Letters, 18 (3): 343-348 (1996). The preferred intact microorganisms will be those which substantially oxidize 2-methyl-quinoxaline to give the product, specifically 2-quinoxaline-carboxylic acid, while leaving the product substantially unchanged, for example free of intrinsic activity which can degrade or otherwise have a negative impact for the desired product at any stage of the procedures in question. Suitable microorganisms for use in the microbial oxidation in question can be prepared by any suitable method known to those skilled in the relevant technology. An example of an appropriate method for the preparation of a microorganism from a commercially available reserve material is provided below. Based on the present disclosure which includes the methods provided below, those skilled in the art will understand how to modify any part of these methods, for example, a method for contacting 2-quinoxaline-carboxylic acid with the microorganism.; the components and conditions of the growth medium, for example, temperature, pH and the like; the respective concentrations of 2-methyl-quinoxaline, of the inducer (when used); or the incubation conditions; to achieve the desired result using any appropriate microorganism. In embodiments of the present invention in which the microorganism is a fungus, a preferred range of 2-methyl-quinoxaline concentrations is from about 0.01 g / L to about 2.5 g / L, and particularly preferred range is from about 0.1 g. / the approximately 2.0 g / l. In embodiments of the present invention wherein the microorganism is a fungus selected from the group consisting of A. repens ATCC No. 14849, A. repens ATCC No. 74481, A glauca ATCC No. 22752, A glauca ATCC No. 74480 and appropriate mutants thereof, a preferred range of 2-methyl-quinoxaline concentrations is from about 0.1 g / L to about 2.0 g / L. In embodiments of the present invention in which the microorganism is a bacterium, a preferred range of 2-methyl-quinoxaline concentrations is from about 0.01 g / l to about 1.5 g / l and a particularly preferred range is from about 0.1 g / l. the approximately 1.0 g / l. In embodiments of the present invention wherein the bacterium is selected from the group consisting of P. Putida ATCC No. 33015, P. Putida ATCC No. 202190 and appropriate mutants thereof, a preferred range of 2-methyl- Quinoxaline is approximately 0.1 g / L to approximately 1.0 g / L. In addition, and as discussed above, a bacterium carrying a TOL plasmid, for example, P. Putida ATCC No. 33015 or P. Putida ATCC No. 202190 in a medium within a fermenter, the inducer, preferably p -xylene is added at a preferred addition rate of about 4.5 mmol / hour to about 6.5 mmol / hour, and a particularly preferred rate of addition is from about 4.9 mmol / L / hour to about 6.1 mm / L / hour. In embodiments of the present invention in which P. Putida ATCC No. 33015 or P. Putida ATCC No. 202190 is in a medium inside a flask, the inducer preferably p-xylene is added continuously in gaseous form to the medium. As would be understood by those skilled in the art from the present disclosure and from the articles and patents mentioned above (e.g., U.S. Patent No. 5,236,832), the concentration of the inductor is usually selected. so that it is lower than the minimum inhibitory concentration of the enzymes responsible for oxidation. See also the citation of Claus and Walker, J. Gen. Microbio /., 36: 107-122 (1964). Any suitable method for contacting the substrate, 2-methyl-quinoxaline, with the microorganism can be used in the present invention. The substrate can be contacted with the microorganism in any appropriate order. For example, 2-methyl-quinoxaline can be added to a medium, such as a culture broth, comprising the microorganism, free or immobilized, or some combination thereof; or the medium can comprise 2-methyl-quinoxaline and the microorganism can then be added to said medium; or 2-methyl-quinoxaline and the organism can be added together to said medium; either the 2-methyl-quinoxaline or the microorganism can be added to an appropriate solvent comprising the other; or the 2-methyl-quinoxaline can be absorbed in a resin; and similar. Those skilled in the art will understand from the description provided herein how any part of the procedures in question may be modified, as desired.

As discussed above, it is preferred in the present invention that the microorganism be A. glauca ATCC No. 22752. As also discussed above, a lyophilized sample of A. glauca ATCC No. 22752 was deposited in the ATCC collection within the terms of the Budapest Treaty of January 13, 1999. This newly deposited culture was given the new deposit number of ATCC No. 74480. Therefore, it is also preferred in the present invention that the microorganism be A glauca ATCC No. 74480. All restrictions regarding the availability to the public of the microorganism culture thus deposited will be irrevocably eliminated when a patent is granted from the specification of the present invention. As also described above, it is especially preferred in the present invention that the microorganism be A. repens ATCC No. 14849. A lyophilized sample of A repens ATCC No. 14849 was deposited with the ATCC within the terms of the Budapest Treaty of the January 13, 1999. This newly deposited culture was given the new deposit number of ATCC No. 74481. Therefore, it is also preferred in the present invention that the microorganism be A. repens ATCC No. 74481. All the Restrictions as to the availability to the public of the microorganism culture thus deposited will be irrevocably eliminated when a patent is granted from the specification of the present invention.

Cultures of A. repens ATCC No. 14849 (or A. repens ATCC No. 74481, ATCC glauca No. 22752, or ATCC glauca No. 74480) can be obtained from the ATCC, and an example of a Appropriate method for the preparation from said available reserve is provided immediately below. The stock cultures can be prepared from rice cultures such as, for example, indicated below: Erlenmeyer flasks (250 ml capacity) containing approximately 50 g of brown rice and approximately 20 ml of distilled water are treated in autoclave at approximately 121 ° C for approximately 30 min, a suspension of A. repens ATCC No. 14849 (or A. repens ATCC No. 74481, A glauca ATCC No. 22752, or A glauca ATCC No. 74480), vegetative cells, or spores, is prepared by adding either aliquot part of a liquid culture or a rag from an inclined culture that has grown on a sterile distilled water agar medium. Each flask with rice is inoculated with approximately 5 ml of the spore suspension or cells and incubated for approximately 10 days at approximately 28 ° C, at which time the spore reserve is prepared by washing the rice culture with approximately 0.5 solution. % Tween 80 in distilled water, decanting the spore suspension out of the rice, and adding from about 10% to about 20% glycerol. The spore reserve is stored at approximately -70 ° C. As will be understood by those skilled in the art for any selected fungus, and as specifically provided hereinbelow in the examples for the preferred A. glauca ATCC No. 22752 or ATCC No. 74480 and the especially preferred A repens ATCC N No. 14849 or ATCC No. 74481, an appropriate method for preparing the selected fungus is as follows: the fungus is inoculated from the stock culture of frozen vegetative cells or spores as described above, into a flask or tube glass with a metal closure containing a growth medium (containing an aliquot from a sterile solution including Tween 80, glycerol and distilled water) whose composition is described below in greater detail. The fermentation is carried out at temperatures ranging from about 22 ° C to about 32 ° C, and preferably at about 29 ° C, with proper shaking, preferably from about 200 rpm to about 220 rpm and, most preferably, at around 210 rpm. When so desired, the pH of the growth medium can be maintained by the use of appropriate buffers incorporated in the fermentation medium and / or adjusted periodically by addition of either a base or an acid, as required. A preferred pH range is from about pH 6 to about pH 7. Any appropriate duration of growth of the microorganism (ie, fungus or bacteria), contacting the microorganism with 2-methyl-quinoxaline, and 2-methyl- Quinoxaline with the microorganism can be used in the present invention. An appropriate growth of the microorganism can be achieved, for example, in the course of about 24 hours, in which period of time it can be added to the culture either (a) the 2-methyl-quinoxaline itself, (b) an aliquot part A suitable solution of 2-methyl-quinoxaline is an appropriate solvent, for example, it does not undesirably affect the growth or function of the microorganism, preferably EtOH or (c) 2-methyl-quinoxaline absorbed in a resin. Then, the incubation may be continued for, for example, a period of time from about two to twenty-four days, depending, for example, on the vessel in which the bioconversion is performed, the medium and the incubation conditions, for example, the temperature, pH and agitation. The incubation broth can then be extracted using any suitable extraction method, for example, (a) in which an appropriate solvent such as, for example, EtOAc, methyl isobutyl ketone, methyl ethyl ketone, methylene chloride , and the like, preferably, EtOAc, removes the organic components from the incubation broth or (b) by adsorbing the product, 2-quinoxaline-carboxylic acid, onto an appropriate resin, preferably a polymeric adsorbent resin, more preferably a selected resin among those of the trademark Amberlite® (Rohm and Haas), most preferably XAD4 (of the Amberlite resins). After extraction of the incubation broth with an appropriate organic solvent and separation of the organic and aqueous phases, the compounds comprising the organic residue can be determined using any suitable method, such as, for example, chromatography. Alternatively, after extraction of 2-quinoxaline-carboxylic acid from the incubation broth using a resin, the 2-quinoxaline-carboxylic broth can be eluted therefrom using an appropriate solvent, preferably EtOAc or MeOH, and then crystallized from from, for example, EtOAc, using for example EtOAc and MeOH. Any suitable growth medium can be used in the process of the present invention, and the appropriate growth medium will contain a source or several sources of assimilable carbon, assimilable nitrogen and inorganic salts containing essential minerals. In general, many carbohydrates such as, for example, glucose, maltose, mannose, sucrose, starch, glycerol, millet jelly, molasses, soybean and the like, can be used as assimilable carbon sources. Sources of assimilable nitrogen include, for example, materials such as yeast and yeast hydrolysates and casein, primary yeast, yeast extracts, cottonseed meal, soybean solid materials, wheat germ, meat extracts, peptone, liquid of maceration of corn, solid materials of corn maceration, and ammonium salts. Nutrients of inorganic salts suitable for use in the culture medium of the present invention include, for example, customary salts containing sodium, iron, magnesium, potassium, cobalt, phosphate, and the like. More particularly, the components of growth media suitable for use in the present invention when the microorganism is a fungus include, for example, corn steep liquor, corn steep solids, Pharmamedia® and a malt extract. The maceration liquid of corn is prepared with approximately 40 g / l of maceration liquid of corn and approximately 20 g / l of dextrose, and is adjusted to approximately pH 4.85 before sterilization. The corn maceration solid material medium is prepared with about 20 g / l to about 40 g / l of maceration corn solids and about 20 g / l of dextrose, and is adjusted to approximately pH 4.85 before sterilization . Another suitable medium for use in the methods of the present invention is prepared with approximately 20 g / l of Pharmamedia® and approximately 20 g / l of dextrose, and adjusted to approximately pH 7.2 before sterilization. The malt extract medium is prepared with approximately 10 g / l of malt extract, approximately 10 g / l of dextrose, approximately 5 g / l of peptone, and approximately 2 g / l of yeast extract, and adjusts to approximately pH 7 before sterilization. Another means suitable for use in the methods of the present invention is prepared with approximately 20 g / l of dextrose, approximately 5 g / l of nutrisoja flour, approximately 5 g / l of yeast extract, approximately 5 g / l of NaCl and about 5 g / l of K2HP04, the pH being adjusted to around pH 7.0 with H2SO4 before sterilization. A particularly preferred growth medium for the fungi suitable for the present process is the aforementioned medium of corn maceration solids.

As discussed above, it is particularly preferred in the present invention that the microorganism be P. putida ATCC No. 33015. As also discussed above, a lyophilized sample of P. putida ATCC No. 33015 was deposited with the ATCC within the terms of the Budapest Treaty on January 13, 1999. This newly deposited culture was given the new deposit number of ATCC No. 202190. Therefore, it is also preferred in the present invention that the microorganism be P. putida ATCC No. 202190. All restrictions on the availability to the public of the culture of microorganisms thus deposited will be irrevocably eliminated upon granting a patent from the specification of the present invention. In addition, growth media suitable for use in the present invention when the microorganism is a bacterium include any known means, for example Nutrient Broth (approximately 32 g / l, from Becton Dickinson Microbiology Systems) and glycerol (approximately 5 g / l). As will be understood by those skilled in the art from any selected bacteria, and as specifically provided below in the examples for P. putida ATCC No. 33015, an appropriate method for preparing the selected bacteria is as follows: the bacterium is inoculated to Starting from a frozen stock culture prepared as known in the art (a stock with approximately 17% glycerol) inside a flask or a glass tube with a metal closure or a fermentor containing a growth medium (which contains an aliquot of a sterile solution that includes Tween 80, glycerol and distilled water) whose composition is described below in greater detail. The fermentation is carried out at temperatures ranging from about 20 ° C to about 40 ° C, and preferably at temperatures ranging from about 25 ° C to about 32 ° C, with appropriate agitation, preferably from about 200 rpm to about 220 ° C. rpm and, most preferably, at about 210 rpm. When so desired, the pH of the growth medium may be maintained by the use of appropriate buffers incorporated in the fermentation medium and / or may be adjusted periodically by addition of either a base or an acid as required. A preferred inoculum is from about 1% to about 20% v / v (from inoculum to medium). A preferred pH range is from about pH 6 to about pH 8. It should be noted that reference to particular buffers, media, reagents, conditions of contact or culture, amount of substrate, amount of inducer is not intended. when used, and the like, in any part of the present specification is limiting, but should be read to include all related materials that those persons having ordinary experience in the technology will recognize as possessing interest or value in the particular context in the one that presents the present debate. For example, it is often possible to replace one buffering system or culture medium with another, so that a different but known way is used to achieve the same goals as those to which the use of a method, material or composition is directed. Suggested In addition, it should be understood that the present invention includes scaling up the process in question for commercial purposes. The microbial oxidation in question optionally further comprises the isolation of the desired product, 2-quinoxaline-carboxylic acid. 2-Quinoxaline-carboxylic acid can be isolated as described below from the medium in which the new microbial oxidation process was carried out and, more specifically, from any intermediate compounds that may have been produced but have not been completely converted to 2-quinoxaline-carboxylic acid, depending for example on the selected microorganism and the incubation conditions. Any suitable methods for isolating and / or purifying any of the intermediates or the desired product of the process in question, can be used in the present invention, including filtration, extraction, crystallization, column chromatography, thin layer chromatography, liquid chromatography. at low preparative pressure, HPLC, adsorption to resins, or any suitable combination of such methods. The detailed examples given below show that a range of microorganisms, specifically fungi and bacteria, oxidize 2-methyl-quinoxaline to provide 2-quinoxaline-carboxylic acid, which can then be separated from any 2-methyl undesired unqualified quinofaline, or any intermediates, and can be further reacted according to methods well known in the art to provide, for example, the compounds of the '801 application. Although the present disclosure is directed primarily to the use of intact microorganisms in the methods in question, those skilled in the art will understand that the microbial methods in question can be achieved by appropriate preparations thereof, for example crushed and dehydrated cell preparations, extracted materials that they comprise the microbial enzymes capable of achieving the oxidations in question, or the enzymes themselves, together with any necessary cofactors, and the like. The present invention is illustrated by the following examples. The preceding and following descriptions of the present invention and the various embodiments are not intended to be limiting of the invention, but are instead illustrative thereof. Therefore, it will be understood that the invention is not limited to the specific details of these examples.

EXAMPLE 1 Oxidation of 2-methyl-quinoxaline in culture tubes using A. repens ATCC no. 14849 A. Bioconversion using the fungus A repens ATCC no. 14849 Three "test" cultures (T1, T2 and T3) were prepared as follows: approximately 2.5 ml of a sterile growth medium (with approximately 20 g / l of dextrose, approximately 5 g / l of nutrisoja meal, approximately 5 g / l of a yeast extract, approximately 5 g / l of NaCl and approximately 5 g / l of K2HP0, the pH being adjusted to approximately pH 7.0 with H2SO4 before sterilization) were added to each of three tubes of glass of 16 x 125 mm each of which had a metal closure (T1, T2 and T3), followed by the addition of spores (approximately 1% v / v of a spore reserve culture) of A. repens ATCC No. 14849 to T1, T2 and T3. The three cultures in tubes were incubated at about 29 ° C, with shaking at about 210 rpm. After about 48 hours (for T1), 72 hours (for T2) or 96 hours (for T3), about 0.05 ml of a stock solution (with approximately 50 mg / ml in approximately 100) was added to the cultures in tubes. % EtOH, final concentration of approximately 1 mg / ml) of 2-methyl-quinoxaline. After further incubation at about 29 ° C (see Table 2 below) the fermentation broths from the cultures in tubes were adjusted to approximately pH 2 with 4 N HCl. The contents of each tube culture were extracted with an equal volume of EtOAc (pure): EtOAc was added, the tube culture was vortexed and then centrifuged at approximately 2,000 rpm (IEC Centrifuge). The EtOAc layer was removed and the aqueous layer was extracted a second time. The combined organic extracts were dried, under nitrogen, in a water bath at about 50 ° C.

B. Yield of 2-quinoxaline-carboxylic acid determined by reverse phase HPLC Each of the extracts, prepared as described above, was resuspended in about one ml of a mixture of ACN and water (1: 9, v / v) and approximately 20 μl of each resuspended extract were analyzed by injection on a HPLC column: Inertsil® C8 HPLC column (4.6 x 250 mm, Column Engineering, Inc.). The compounds contained within each injected resuspended extract were separated socratically at about 1.0 ml per minute in a mobile phase (a mixture of ACN and 0.05% aqueous TFA, 1: 4, v / v). Under these conditions, the 2-quinoxaline-carboxylic acid was eluted at approximately 8.6 min and the 2-methyl-quinoxaline eluted at about 15 min. The yields of 2-quinoxaline-carboxylic acid were determined from such analyzes by HPLC for several sets of experimental conditions (ie T1, T2 and T3), and these yields are presented in Table 2 below: TABLE 2 As illustrated by the data for T1, T2 and T3 in Table 2, HPLC analysis shows that the microbial procedure in question results in yields of 56%, 76% and 79%, respectively, of the desired 2-quinoxaline acid. -carboxylic Correspondingly, the inclusion of the intact microorganism, ie A. repens ATCC No. 14949, results in the oxidation of the 2-methyl-quinoxaline to give the 2-quinoxaline-carboxylic acid, and a substantial amount of the acid remains intact. quinoxaline-carboxylic.

EXAMPLE II Oxidation of 2-methyl-quinoxaline in tube cultures using A. repens ATCC No. 14849 in four different growth media A. Preparation of four different growth media Medium 1 was prepared with approximately 40 g / l of maceration liquid of corn and approximately 20 g / l of dextrose, and adjusted to approximately pH 4.85 before sterilization.

Medium 2 was prepared with approximately 40 g / L of maceration corn solids and approximately 20 g / L dextrose, and adjusted to approximately pH 4.85 before sterilization. Media 3 was prepared with approximately 20 g / l of Pharmamedia® and approximately 20 g / l of dextrose, and adjusted to approximately pH 7.2 before sterilization. Medium 4 was prepared with approximately 10 g / l of malt extract, approximately 10 g / l of dextrose, approximately 5 g / l of peptone, and approximately 2 g / l of yeast extract, and adjusted to around pH 7 before sterilization.

B. Bioconversion using the fungus A repens ATCC No. 14849. Eight "test" cultures (T1a, T1b, T2a, T2b, T3a, T3b, T4a and T4b) were prepared as follows: approximately 2.5 ml of a sterile culture medium (respectively medium 1, medium 2, medium 3 and medium 4) were added to each of eight glass tubes of 16 x 125 mm each of which had a metal closure (T1a, T1b , T2a, T2b, T3a, T3b, T4a and T4b), followed by the addition of spores (approximately 1% v / v of spore reserve culture) of A repens ATCC No. 14849 to all tube cultures. The eight cultures in tubes were incubated at about 29 ° C, with shaking at about 210 rpm. After either approximately 48 hours for (T1a, T2a, T3a and T4a) or approximately 72 hours for (T1b, T2b, T3b and T4b) approximately 0.05 ml of an original solution (approximately 50 mg / ml) was added to the cultures in tubes. ml in DMSO, final concentration of approximately 1 mg / ml) of 2-methyl-quinoxaline. After further incubation at about 29 ° C for 12 days, the fermentation broth of each culture was extracted and the combined organic extracts were dried as described in example 1.

C. Yield of 2-quinoxaline-carboxylic acid as determined by reverse phase HPLC Each of the extracts, prepared as described above, was then treated and analyzed by reverse phase HPLC as described in Example 1 The yields of 2-chenoxaline-carboxylic acid were determined from said HPLC analysis for various sets of experimental conditions (namely, T1a, T1b, T2a, T2b, T3a, T3b, T4a and T4b), and these yields were presented in table 3 below.

TABLE 3 As illustrated by the data in Table 3, HPLC analysis shows that the microbial procedure in question in which the microorganism is A. repens ATCC No. 14849 results in the production of 2-quinoxaline-carboxylic acid in all media tested. The data in Table 3 also indicate that, of the four media tested, medium 2 provides the highest yield in% of the desired product, 2-quinoxaline-carboxylic acid.

EXAMPLE III Oxidation of 2-methyl-quinoxaline in flask cultures using A. repens ATCC No. 14849 or A. glauca ATCN No. 22752 A. Bioconversion using the fungus A. repens ATCC No. 14849 or the fungus A glauca ATCC No. 22752 Four "test" cultures (T1a, T1b, T2a and T2b) were prepared as follows: approximately 25 ml of medium of sterile culture (approximately 20 g / l of dextrose, approximately 5 g / l of nutrisoja meal, approximately 5 g / l of a yeast extract, approximately 5 g / l of NaCl and approximately 5 g / l of K2HPO4, being adjusted the pH to approximately pH 7.0 with H2SO4 before sterilization) were added to each of the four conical flasks (300 ml), followed by the addition of spores (approximately 1% v / v of a spore reserve culture) from either A repens ATCC No. 14849 (for T1a, T1b) or A. glauca ATCC No. 22752 (for T2a, T2b). The four flask cultures were incubated at about 29 ° C, with stirring at about 210 rpm. Immediately after inoculation (for T2a), or after approximately 24 hours (for T2b), approximately 0.5 ml of a stock solution (approximately 50 mg / ml in approximately 100% EtOH, final concentration of approximately 1 mg / ml of 2-methyl-quinoxaline were added to the flask cultures of A. glauca ATCC No. 22752, and after approximately 48 hours (for T1a) or 72 hours (for T1b), approximately 0.5 ml of a stock solution ( approximately 50 mg / ml in approximately 100% EtOH, final concentration of approximately 1 mg / ml) of 2-methyl-quinoxaline were added to the flask cultures of A. repens ATCC No. 14849. After further incubation to approximately 29 ° C for 24 days (for T1a), for 16 days (for T1 b), 25 days (for T2a) or 24 days (for T2b), the fermentation broths from the flask cultures were adjusted around pH 2 with HCl 4 N. The content of each flask culture was extracted with two 25 ml aliquots of EtOAc, and the solvent was removed from the combined EtOAc extracts under reduced pressure to provide the crude products.

B. Yield of 2-quinoxaline-carboxylic acid as determined by reverse phase HPLC. Each of the extracts, prepared as described above, was resuspended in approximately 5 ml of a mixture of MeOH and ACN (3: 2, v / v) and diluted to 1: 19 with water for analysis by HPLC: These HPLC analyzes were performed as described for example 1. The yields of 2-quinoxaline-carboxylic acid were determined from such analyzes by HPLC for various sets of experimental conditions (i.e. T1a, T1b, T2a and T2b), and these returns are given in Table 4 below.

TABLE 4 As illustrated by the four 4 data, the inclusion of the intact microorganism, ie A. glauca ATCC No. 22752 or A. repens ATCC No. 14849, results in the oxidation of 2-methyl-quinoxaline to the acid 2-quinoxaline-carboxylic acid. The% of the starting material, ie 2-methyl-quinoxaline, remaining in T1a, T1b, T2a and T2b is around 7%, 7%, 6% and 6%, respectively.

EXAMPLE IV Screening for the microbial conversion of 2-methyl-quinoxaline to 2-quinoxaline-carboxylic acid Cells of various microorganisms were grown in the tubes containing 2.5 ml of the dextrose medium and nutrisoja meal, as described in example 1. Individual tubes were inoculated with spores or plant cells (approximately 1% v / v of culture of spore reserve or vegetative cells) of various microorganisms stored in the form of frozen glycerol suspensions, and incubated at about 29 ° C with shaking (210 rpm) on a rotary shaker. After about 48 hours, 0.05 ml of a 10 mg / ml solution of 2-methyl-quinoxaline in DMSO was added to each tube. After incubation for approximately 4 days, the content of each tube was extracted, and the individual extracts were analyzed by HPLC as described in example 1. The yields of 2-quinoxaline-carboxylic acid were determined by HPLC and the results they collect in table 5.

TABLE 5 EXAMPLE V Oxidation of 2-methyl-quinoxaline in flask cultures using P. putida ATCC No. 33015 P. putida ATCC No. 33015 cells were grown in the medium (Nutrient broth (approximately 32 g / l) and glycerol (approximately 5 g / l)). Six conical flasks (300 ml capacity) containing approximately 30 ml of the medium were incubated with approximately 0.10 ml of a glycerol suspension of P. putida ATCC No. 33015 cells previously stored at about -70 ° C. After adding approximately 2 ml of p-xylene contained in a 15 ml conical polypropylene centrifuge tube (Falcon®, Becton Dickinson Labware), the flasks were sealed with Parafilm® (American National Can) and shaken ( at about 225 rpm) on a rotary shaker for about 18 hours at about 29 ° C. These flask cultures had a density of approximately 1.9 measured at 650 nm. The cells were harvested from the six flasks by centrifugation, washed once with about 250 ml of DPBS, and resuspended in approximately 20 ml of PBS (Biowhittaker) in a 300 ml conical flask. The bioconversion was initiated by the addition of about 0.1 ml of a solution of approximately 100 mg / ml 2-methyl-quinoxaline DMSO, corresponding to an initial concentration of approximately 0.5 mg / l. Incubation was continued for about 4 days at about 29 ° C with shaking at about 225 rpm. Samples of the bioconversion broth were removed at various times and, after removing the cells by centrifugation and having diluted with MeOH as required, they were analyzed by HLPC. Approximately 20 pl of each of these samples were analyzed by injection on an Inertsil® HPLC C8 column (4.6 x 250 mm). Each column was eluted at about 1.0 ml / min with a mobile phase consisting of a mixture of ACN and aqueous TFA at approximately 0.05% (1: 4, v / v). The yields of 2-quinoxaline-carboxylic acid were respectively 86%, 90% and 94% after about 1, 2 and 4 days of incubation, respectively.

EXAMPLE VI Oxidation of 2-methyl-quinoxaline in a fermentor culture using P putida ATCC No. 33015 P. putida ATCC No. 33015 was grown in a fermenter with approximately 10 I of medium 5. The fermenter was inoculated with six cultures of P. putida each of which was grown in a conical flask ( 300 ml capacity) containing approximately 50 ml of medium 5.

Each flask culture was inoculated with approximately 175 μl of a spore stock of P. putida ATCC No. 33015, a polypropylene centrifuge tube of 15 ml capacity was introduced, containing approximately 2 ml of p-xylene and the flask it was hermetically sealed Parafilm®. These flask cultures were incubated at about 29 ° C for about 17 hours with shaking at about 210 rpm. After inoculation of the fermentor with the 6 flask cultures, p-xylene was added to the fermenter in approximately 2 ml aliquots at about 20 minutes for a total of 2 hours. After that, aliquots of about 2.5 ml of p-xylene were added to the fermentor for about 20 minutes for about 3.5 hours. The addition of xylene was then stopped and 2-methyl-quinoxaline was added at about 5.25 hours (about 1.95 g) and about 7.75 hours (about 7.76 g) after inoculation. Incubation was continued for about 22 hours after the final addition of 2-methyl-quinoxaline. A sample of the incubation medium was centrifuged to remove the cells, diluted with MeOH and analyzed by HPLC using the method described in Example V. This analysis revealed an approximately 81% yield of 2-quinoxaline-carboxylic acid.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A process for the microbial oxidation of 2-methyl-quinoxaline to 2-quinoxaline-carboxylic acid, comprising contacting said 2-methyl-quinoxaline with a microorganism and incubating the resulting mixture under conditions sufficient to provide an amount of said 2-quinoxaine carboxylic acid, wherein said microorganism is selected from the group consisting of: Absidia glauca ATCC No. 22752, Absidia glauca ATCC No. 74480, Absidia pseudocylindrospora ATCC No. 24169, Absidia repens ATCC No. 14849, Absidia repens ATCC No. 74481, Actinomucor elegans ATCC No. 6476, Alternaria solani ATCC No. 11078, Aspergillus tamarii ATCC No. 16865, Coniophora puteana ATCC No. 12675, Cunninghamella echinulata ATCC No. 8688a, Cunninghamella echinulata ATCC No. 8688b, Cunninghamella echinulata ATCC No. 8983, Cunninghamella echinulata ATCC No. 9244, Cunninghamella echinulata ATCC No. 9245, Cunninghamella echinulata ATCC No. 10028b, Cunninghamella echinulata ATCC No. 2 6269, Cunninghamella echinulata ATCC No. 36190, Cunninghamella echinulata ATCC No. 36112, Cunninghamella homothallica ATCC No. 16161, Cylindrocarpon destructans ATCC No. 66963, Diplodia goosypina ATCC No. 20575, Epicoccum neglectum ATCC No. 12723, Glomerella lagenaria ATCC No. 14724 , Pencillium claviforme ATCC No. 10426, Pencillium duclauxii ATCC No. 10440, Pencillium glabrum ATCC No. 11080, Pseudocochliobolus lunatus ATCC No. 24155, Pseudomonas putida ATCC No. 33015, Pseudomonas putida ATCC No. 202190, Rhodococcus rhodochorus ATCC No. 19067 and Thamnostylum piriforme ATCC No. 8686; and appropriate mutants thereof; with the proviso that when said microorganism is said Pseudomonas putida ATCC N °. 33015 or said Pseudomonas putida ATCC No. 202190, said Pseudomonas putida ATCC No. 33015 or said Pseudomonas putida ATCC No. 202190 is included by interaction with an inducer of said contacting said Pseudomonas putida ATCC No. 33015 or said Pseudomonas putida ATCC No. 202190 with said 2-methyl-quinoxaline. 2 - The method according to claim 1, further characterized in that it also comprises asylating the 2-quinoxaline-carboxylic acid. 3. The process according to claim 1, further characterized in that said isolation is carried out by extraction of said mixture with an organic solvent. 4. The process according to claim 3, further characterized in that said organic solvent is ethyl acetate. 5. The method according to claim 4, further characterized in that it also comprises subjecting said extraction to chromatography. 6. The process according to claim 2, further characterized in that said isolation is carried out by adsorption of said 2-quinoxaline-carboxylic acid adsorbed from resin with an organic solvent. 7. The process according to claim 6, further characterized in that said resin is a polymeric adsorbent resin. 8. The process according to claim 7, further characterized in that said organic solvent is ethyl acetate or methanol. 9. The process according to claim 8, further characterized in that it also comprises crystallizing said 2-quinoxaline-carboxylic acid eluted from ethyl acetate. 10. The process according to claim 8, further characterized in that it also comprises crystallizing said 2-quinoxaline-carboxylic acid eluted from ethyl acetate and methanol. 11. The method according to claim 1, further characterized in that said microorganism is an intact microorganism. 12. The method according to claim 11, further characterized in that said microorganism comprises washed cells of said microorganism. 13. The method according to claim 12, further characterized in that it also comprises immobilizing said washed cells. 14. - The method according to claim 12, further characterized in that said washed cells are in an aqueous solvent. 15. The method according to claim 14, further characterized in that said contacting is carried out by adding said 2-methyl-quinoxaline to said solvent. 16. The method according to claim 11, further characterized in that said microorganism is in a growth medium. 17. The method according to claim 16, further characterized in that said contacting is performed by adding said 2-methyl-quinoxaline to said growth medium. 18. The method according to claim 1, further characterized in that said microorganism is selected from said Absidia glauca ATCC N °. 22752, Absidia glauca ATCC N °. 74480, Absidia repens ATCC No.14849, Absidia repens ATCC No.74481, Alternaria solani ATCC No.11078, Aspergillus tamarii ATCC No.16865, Cunninghamella echinulata ATCC N °. 8983 and Diplodia gossypina ATCC N °. 20575; and said mutants of these. 19. The method according to claim 18, further characterized in that said microorganism is selected from the group consisting of said Absidia glauca ATCC N °. 22752, Absidia glauca ATCC N °. 74480, Absidia repens ATCC No.14849, Absidia repens ATCC No.74481, Alternaria solani ATCC No. 11010, Aspergillus tamarii ATCC No. 16685; and said mutants of these. 20. The method according to claim 19, further characterized in that said microorganism is selected from the group consisting of said Absidia glauca ATCC N °. 22752, Absidia glauca ATCC No. 74480, Absidia repens ATCC No.14849, Absidia repens ATCC No.74481; and said mutants of these. 21. The process according to claim 20, further characterized in that said microorganism is Absidia repens ATCC No. 14848, Absidia repens ATCC No. 74481. 22. A process for the microbial oxidation of 2-methyl-quinoxaline to give 2-quinoxaline-carboxylic acid, further characterized by comprising contacting said 2-methyl-quinoxaline with a microorganism and incubating the resulting mixture under sufficient conditions to provide an amount of said 2-quinoxaline-carboxylic acid "further characterized in that said microorganism is Absidia repens ATCC No. 14848, Absidia repens ATCC No. 74481; and appropriate mutants thereof. 23. The method according to claim 22, further characterized in that said microorganism is in a growth medium. 24. The method according to claim 23, further characterized in that said contacting is carried out by adding said 2-methyl-quinoxaline to said growth medium. 25. - The method according to claim 1, further characterized in that said microorganism is said Pseudomas putida ATCC No. 33015 or said Pseudomas putida ATCC N °. 202190. 26.- The method according to claim 25, further characterized in that said inducer is p-xylene. 27. The method according to claim 26, further characterized in that said Pseudomas putida ATCC No. 33015 or said Pseudomas putida ATCC N °. 202190 is in a growth medium. 28. The process according to claim 27, further characterized in that said p-xylene is added to said growth medium. 29. The method according to claim 28, further characterized in that said growth medium is inside a flask. 30. The method according to claim 29, further characterized in that it further comprises the operation of collecting said microorganism after the termination of said induction. 31. The process according to claim 30, further characterized in that said collection is performed by centrifuging the contents of said flask, decanting the fluid, washing the cell pellet and resuspending said pellet in a buffer. 32. - The method according to claim 31, further characterized in that said contacting is performed by adding said 2-methyl-quinoxaline to said buffer after said resuspension. + 33.- The method according to claim 28, further characterized in that said growth medium is inside a fermentor. 34. The method according to claim 33, wherein said addition of said p-xylene to said growth medium is interrupted after said induction. The method according to claim 34, further characterized in that said contacting is carried out by adding said 2-methyl-quinoxaline to said growth medium after said interruption of the addition of said p-xylene. 36.- A process for the microbial oxidation of 2-methyl-quinoxaline to give 2-quinoxaline-carboxylic acid, further characterized by comprising contacting 2-methyl-quinoxaline with a microorganism after the enzymes of said microorganism are induced by interaction with an inducer and incubating the resulting mixture under conditions sufficient to provide an amount of said 2-quinoxaline-carboxylic acid, further characterized in that said microorganism is selected from the group consisting of Pseudomas putida ATCC No. 33015 or said Pseudomas putida ATCC N °. 202190; or appropriate mutants thereof. 37. - The method according to claim 36, further characterized in that said inductor is p-xylene or m-xylene.