DE2548453A1 - (+)-(2,5)-Dihydro-(4)-methyl-(5)-oxo-furan-(2)-acetic acid prepn. - by microbial oxidation of (3)-methyl-pyrocatechol or (3)-methyl-benzoic acid - Google Patents

Abstract

(+)-2,5-Dihydro-4-methyl-5-oxo-furan-2-acetic acid (I) is a new cpd. (I) is prepd. by biological co-oxidation of 3-methyl-pyrocatechol (II) or 3-methylbenzoic acid (III) using bacteria of the strain Pseudomonas B 13 after cultivation on 3-chlorobenzoic acid. (I) is used as a starting material for the chemical synthesis of organic cpds. (esp. 4-hydroxy-2-pentene-2,5-dicarboxylic acid). (I) is also used as a component in odourant preparations.

Description

(+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2)

The invention relates to the compound (+) - 2,5-dihydro-4-methyl-5-oxo-furanessigsAure- (2) and a method for their production.

The compound is produced through biological co-oxidation of 3-methylpyrocatechol using bacteria of the Pseudomonas B 13 strain (registration number DSM 624) after growing on 3-chlorobenzoic acid. The procedure is marked by the process steps a) cultivation of a biologically active cell suspension of Pseudomonas strain B 13 on a mineral medium with 3-chlorobenzoate as the only one Carbon and energy source.

b) continuous co-oxidation of a 1 molar aqueous solution (PH = approx. 7.5) of 3-methylpyrocatechol as structurally analogous, non-isoelectronic Substrate for the cell- Suspension, with a Exceeding toxic concentrations of this compound is avoided, c) extraction the (+) -2, 5-dihydro-4-methyl-5-oxo-furanacetic acid formed by biological co-oxidation according to b) (2) by separating the bacterial cells from the culture fluid, concentrating the Supernatant liquid to-about one-tenth of its volume, acidifying the concentrate Cooling to approx. pH2 in an ice bath, the majority of the product crystallizing out and is separated from the concentrate in a known manner, shaking out the remaining amount of the product with a solvent and working up the solution in a known manner.

Biologically active suspension is intended to mean here that those in the suspension The bacteria contained in it still have certain catabolic activities other process is the compound (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) by biological co-oxidation of 3-methylbenzoic acid using bacteria of the Pseudomonas B 13 strain (registration number DSM 624) after cultivation on 3-chlorobenzoic acid manufactured. The process steps of this process are a) cultivation of a biological active cell suspension - by growing Pseudomonas strain B 13 on a mineral medium with 3-chlorobenzoate as the only source of carbon and energy1 b) continuous or discontinuous addition of a 1 molar aqueous solution (pH = approx. 7.5) of 3-methylbenzoic acid as a structurally analogous, non-isoelectronic substrate to the suspension mentioned under a), with the concentration falling below a minimum of 1 mM of this compound is avoided, c) Obtaining the through biological co-oxidation resulting (+) 2p 5-dihydro-4-methyl-5-oxo-furanacetic acid (2) by separating the bacterial cells from the culture fluid, concentrating the supernatant liquid to about a tenth of its volume, acidifying the concentrate Cool to approx. pH2 in an ice bath, with the majority of the product crystallizing out and is separated from the concentrate in a known manner, shaking out the remaining amount of the product with a solvent and working up the solution in a known manner.

Under structurally analogous-non-isoelectronic connections here those are to be understood which admittedly fill the same space as the utilizable Substrate of growth (i.e.

3-chlorobenzoate), but different polarities (= different electronic Character).

The compound according to the invention is in a simple manner in the pure state and can be produced with a relatively high yield. Relatively high yield means here that a yield of more than 50% of that expected from the stoichiometry is obtained. In the pure state means that the compound according to the invention is not arises in the form of a racemate and that it is not mixed with other oxidation products, which occur as products of possible side reactions, as is the case with chemical reactions is often the case in organic chemistry.

For example, in the oxidation of 4-methyl-pyrocatecin with peroxyacetic acid a mixture with cis-, trans-ß-methyl-muconic acid, 2,5-dihydro-3-methyl-5-oxo-furanacetic acid- (2) 2,5-dihydro-2-methyl-5-oxo-furanacetic acid (2) and 2,8-dihydro-2-hydroxy-3-methyl-5-oxo-furanacetic acid (2) obtained [J.C. Farrand, D.C. Johnson: Journal of Organic Chemistry, Vol. 36, o. 23 (1971) pp. 3606 to 3612].

From the publication by JC Farrand et al. it can also be seen that the compound 2,5-dihydro-2-methyl-5-oxo-furanacetic acid- (2) in the oxidation with peroxyacetic acid from different starting compounds in each case in only a low yield as a racemate and always in a mixture with other oxidation products: from 4-methylpyrocatechol (= VII) 4% from 4-methylveratrole cJ: 1 from p-cresol : 3, from -p-methyl anisole 1 1 from 2-methoxy-p-cresol : 2% from 4-methyl-o-benzoquinone : 4% The biological co-oxidation of 3-methyl-pyrocatechol results in the compound of the formula according to the invention according to the reaction sequence with a yield of almost 90%.

If the compound (I) according to the invention is prepared from 3-methylbenzoic acid which is structurally analogous to that of 3-chlorobenzoic acid - non-isoelectronic 3-methylbenzoic acid, the sequence of reactions found for this is shown that in addition to the compound (r) according to the invention, an isomeric compound which can be easily separated from I, namely (+) - 2,5-dihydro-2-methyl-5-oxo-furanacetic acid- (2) is obtained in a relatively low yield, about 9%, which is obtained in the co-oxidation of 4-methylpyrocatechol in 93% yield. While the reaction sequence for 3-methylbenzoic acid terminates in products I and II, it is not yet complete for 3-chlorobenzoic acid because halide is split off and total degradation occurs with substrate utilization. Both structurally analogous but non-isoelectronic compounds, for example 3-chlorobenzoate and 3-methylbenzoate, go through the same enzymatic steps as long as steric factors determine the conversion.

At the stage of halide cleavage, methyl cannot be isoelectronic (i.e. as CH O) are removed, leading to the accumulation and excretion of the corresponding Metabolite leads.

In this reaction ema is the consequence of biological co-oxidation contain 3-methylpyrocatechol.

The compound (I) according to the invention is already in this case to Beginning of process step c) already in over 60% yield in pure form won. The final yield is more than 80Y. .

The preparation according to the invention is illustrated in the following examples the compound (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) explained in more detail, Example 1: Preparation of (+) -2, 5-dihydro-4-methyl-5-oxofuranacetic acid- (2) by co-oxidation of 3-methylbenzoate For the production of a biologically active bacteria suspension the following stock solutions were initially created: Stock solution 1: 70 g Na2HPO4 # 12H2O 10 g KH2PO4 in 1000 ml water Stock solution 2: 100 g (NH4) 2 SO4 5.0 g Ca (NO3) 2 # 4H2O 1.0 g Fe (III) ammonium citrate 20 g MgSO4 7 1120 100 ml trace element solution after N. Pfennig and K.D. Lippert [Arch.

Microbiol., Vol. 55 (1966), pages 245 to 256]. but without iron salts and ethylenediaminetetraacetic acid (EDTA) in 1000 ml of aqueous solution.

Stock solution 3: 156.6 g -3-chlorobenzoic acid- in 1000 ml aqueous solution, adjusted to pH 7.5 with NaOH.

A mineral salt chlorobenzoate medium was used to cultivate Pseudomonas strain B 13 used that in 1 liter 100 ml of the stock solution 1 and 10 ml each of the stock solutions 2 and 3 included.

The bacteria of strain B 13 were grown in a 5 liter fermenter. 3 l of chlorobenzoate mineral salt medium were inoculated with 60 ml of a preculture, which in a 1000 ml baffle flask on the rotary shaker (turbidity E546 = 1.7-) 8).

This fermenter culture was at 30 under RLihren (600 rpm) with 3 1 / min. ventilated After about 10 hours of incubation, a peristaltic pump was used Simultaneously through three separate channels equal amounts of the stock solutions 2 and 3 as well as 1 -M NaOII solution fed in. The ones required for the exponentially growing culture Delivery volumes were determined with the help of empirical values from the ratio of turbidity determined for the existing chlorobenzoate concentration. The latter was made with the help of the Liquid chromatography determines what happens from the time the sample is taken around the 15th time Min.

required. There were initially 0.1 and last 1.2 ml of medium per min.

pumped in, the 3-chlorobenzoate concentration between 1.0 and 5 mM fluctuated. With a turbidity of E546> -6, the air supply was set to 8 1 / min. increased and foam formation by adding approx. 0.02 ml / min. n-Octanol pushed back.

Continuous replenishment of 3-chlorobenzoate resulted in over 20 Nearly exponential growth was observed over hours.

With 90 mmol chlorobenzoate / l, a cell density finally became corresponding a turbidity of E546 nm = 12.5 is achieved.

Co-oxidation of 3-methylbenzoic acid was made because the cells were absent of substrate rapidly lose activity before the 3-chlorobenzoate is completely consumed 3-methylbenzoic acid as 1 M aqueous sodium salt solution, pH 7.5, in portions of 25 mmol of the fermenter culture added. There was no separation and washing of the cells required and would only have resulted in a loss of activity. As with the cultivation of the Cells, the substrate concentration was continuously determined by means of liquid chromatography controlled. The further consumption of 3-chlorobenzoates was in the presence of 3-methylbenzoate slowed down so much that after the last addition of methylbenzoate Chlorobenzoate was still detectable for 4.5 hours.

To obtain the lactone compounds I and II, the cells were through Centrifugation separated and the culture liquid in the rotary evaporator to about 300 ml concentrated. On acidification with hydrochloric acid to Pg 2.0 and cooling in an ice bath crystallized most of the lactones. The products still remaining in the mother liquor were shaken out with 1000 ml of ether. After drying over MgS04 and evaporation the ether remaining partially oily, light brown colored The residue was treated with warm methyl ethyl ketone.

extracted. On cooling (approx. -20 ° C) the lactones crystallized.

With the aid of preparative layer chromatography on silica gel in a solvent system Diisopropyl ether-formic acid-water, 200: 7: 3 (v / v / v) and extraction of the in transmitted light The bands that appear transparent could also be those remaining in the methyl ethyl ketone Extract leftover lactones.

In the co-oxidation of 3-methylb-enzoaL, I was obtained by acid precipitation alone and subsequent crystallization from hot methyl ethyl ketone in 64% strength Yield obtained in pure form.

The total yield of (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) (I) - was 82% of the theoretically possible, based on the amount of 3-methylbenzoate used , the yield of dihydro-2-methyl-5-oxo-furanacetic acid- (2) (II) in this case 9% of the theoretically possible (a total of 91% of the 3-methylbenzoate used converted into products I and II).

The maximum transfer rate for methyl benzoate was with the existing cell density (E546 nm = 12.5) at a substrate concentration> 4 mM achieved. 3-methylbenzoate was fed in in portions, thereby reducing the rate of conversion was measurable by liquid chromatography. The concentration of the substrate was if possible kept above the saturation concentration (approx. # 4 mM). It it was found that the activity of the cells up to the termination of the reaction (4.5 Hours) after conversion of 70 mmol of methyl benzoate per liter was unchanged. There were about 11 g / l lactones (I and II) were produced. An inhibition of the methyl benzoate conversion by excretion products could not be observed.

Example 2: Preparation of (+) -2,5-dihydro-4-methyl-5-oxofuranacetic acid- (2) (I) by co-oxidation of 3-methylbenzcatechol.

The procedure of Example 1 was repeated with the exception that instead of 3-methyl-pyrocatechol was co-oxidized by 3-methyl-benzoate. Because of toxicity of methyl catechol became the substrate. fed in continuously, the critical concentration of 0.5 mM was not exceeded.

The methyl catechol was converted at such a high rate that that in samples which were taken 1 min. after adding the substrate, the substrate was included Was only barely detectable with the help of liquid chromatography. Opposite to the co-oxidation of 3-methylbenzoate tended to have a significantly lower cell density (E546 = 3.6).

One observed temporarily in the liquid chromatographic-nV-spectrometric Control a very intense signal indicating the excretion of 2-methylmuconic acid indicated (hmax 269 nm when recorded in situ). A cell suspension with the turbidity E546 nm = 3.6 sets 2.5 g (20.2 mmol) of 3-methylcatechol per liter completely in 150 minutes around. (+) - 2,5-Dihydro-4-methyl-5-oxo-furanacetic acid- (2) (I) is formed as a product in 89% of the theoretically possible yield, (+) -2, 5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) (I) is the main product of the co-oxidation of 3-methylbenzoate and was made with the Almost quantitatively formed product identified by 3-methylpyrocatechol Melting point, UV and IR spectrum as well as chromatographic behavior (liquid chromatography; Thin-layer chromatography: brown-violet spot with the nitroprusside reagent, Rf = 0.25). The compound crystallizes from methyl ethyl ketone in colorless compacts Crystals of m.p. 154.5-1550C. Initially, the compound could be made from diisopropyl ether in needles, sctnmp. 77-78 ° C. This crystal shape could later be found after the high-melting form has appeared, only briefly when evaporating Observe the solutions on the slide. The elemental analysis correlates with the Gross Formula C7H804: C 53.85%; H 5.16%, found: C 53.86%; H 5.50%.

The compound of the invention can be used as a starting compound for syntheses of organic compounds that are difficult to access chemically, those of the same Base body how the output connection can be derived can be used, for example for the preparation of 4-hydroxy-pentene- (2) -dicarboxylic acid- (2,5) or their salts.

The compound according to the invention can also be used as a component in fragrance preparations Find application.

Claims (8)

Claims (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2). 2. Process for the preparation of (+) -2, 5-dihydro-4-methyl-5-oxofuranacetic acid (2), characterized by using biological co-oxidation of 3-methyl-catechol of bacteria of the Pseudomonas B 13 strain (registration number DSM 624) after cultivation on 3-chlorobenzoic acid. 3. The method according to claim 2, characterized by the method steps a) Cultivation of a biologically active cell suspension by cultivation of Pseudomonas Strain B 13 on a mineral medium with 3-chlorobenzoate as the only carbon and energy source7 b) continuous co-oxidation of a 1 molar solution (PH = approx. 7.5) of 3-methylpyrocatechol as a structurally analogous, non-isoelectronic substrate to the cell suspension mentioned under a), with an excess of toxic concentrations this compound is avoided, c) extraction by biological co-oxidation (+) -2, 5-Dihydro-4-methyl-5-oxo-furanacetic acid (2) formed after b) by separating the bacterial cells from the culture liquid, concentrating the supernatant Liquid to about a tenth of its volume, acidification of the concentrate to approx. PH², cooling in an ice bath, the majority of the product crystallizing out and out the concentrate is separated in a known manner, shaking out the remainder of the Product with a solvent and working up the solution in a known manner. 4. Process for the preparation of (+) - 2,5-dihydro-4-methyl-5-oxofuranacetic acid (2), characterized by using biological co-oxidation of 3-methylbenzoic acid of bacteria of the Pseudomonas B 13 strain (registration number DSM 624) after cultivation to 3-chlorobenzoic acid. 5. The method according to claim 4, characterized by the method steps a) Cultivation of a biologically active cell suspension by cultivation of Pseudomonas Strain B 13 on a mineral medium with 3-chlorobenzoate as the only carbon and energy source, b) continuous or discontinuous addition of a 1 molar Solution (pH = approx. 7.5) of 3-methylbenzoic acid as a structurally analogous, non-isoelectronic one Substrate to the suspension mentioned under a), with a fall below a Minimum concentration of 1 mM of this compound is avoided, c) obtaining the (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) formed by biological co-oxidation by separating the bacterial cells from the culture liquid, concentrating the supernatant Liquid to about a tenth of its volume, acidification of the concentrate to approx. PH2, cooling in the bath, whereby the main part of the product crystallizes out and is separated from the concentrate in a known manner, shaking out the remaining amount of the product with a solvent and working up the solution in a known manner. 6. Use of (+) - 2, 5-dihydro-4-methyl-5-oxo-furanacetic acid- (2) as a starting compound for syntheses of chemically difficult to obtain organic compounds that have the same basic structure as the starting compound are derivable. 7. Use of (+) - 2,5-dihydro-4-methyl-5-oxo-furanacetic acid (2) for the preparation of 4-hydroxy-pentene- (2) -dicarboxylic acid- (2,5) or their salts. 8. Use of (+) - 2, 5-dihydro-4-methyl-5-oxo-furanacetic acid (2) as a component in fragrance preparations.