CZ304116B6 - Codeine hydroxylation using the strain of Rhizobium radiobacter R89-1 CCM 7947 microorganism - Google Patents

Abstract

The present invention relates to a method of codeine biotransformation using the strain of Rhizobium radiobacter R89-1 CCM 7947 microorganism, which hydroxylates codeine to 14-hydroxykodeine. This compound is the only product if the biotransformation is carried out in a phosphate buffer added with a co-substrate, which is represented by sodium formate. The 14-hydroxycodeine is a significant intermediate for the preparation of pharmaceutically active substances.

Description

Technical field

The invention relates to a process for the hydroxylation of codeine of formula 1 to 14-hydroxycodeine of formula 2 and 14hydroxycodeinone of formula 3 with a strain of Rhizobium radiobacter R89-1 CCM 7947.

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This compound is an important intermediate for the preparation of pharmaceutically active substances.

BACKGROUND OF THE INVENTION

Codeine, morphine and thebaine are natural alkaloids produced by the poppy (Papaver somniferum). Introduction of the hydroxyl group into the C-14 position of the morphine alkaloid structure is used to prepare oxycodone (world production 65 t / year), naloxone (37 kg / year), oxymorphone (2.4 t / year) and naltrexone (853 kg / year) ) that are used in medicine as analgesics, antitussives, and drugs to stop drug and alcohol dependence. The most commonly used starting material for the chemical production of these opiates is codeine and thebaine from natural opium extract. Thebaine is converted by oxidation (peroxygen with line) to 14-hydroxycodeinone (López, D, Quióoa,

E., Riguera, R. 1994 Tetrahedron Lett., 31, 527-5730). Because codeine and morphine are much more accessible than thebaine, they are therefore promising precursors for the preparation of 14-hydroxyopiates. The chemical methods described show low yields, the accumulation of hardly separable by-products and the necessity of using unwanted heavy metals (Zhang, Q., Rich, JO, Cotterill, C., Pantaleone DP, Michels, PC 2005 J. Am. Chem. Soc. 127, 7286-7287).

The beginnings of interest in obtaining more effective analgesics from natural opiates by biotransformation date back to the 1960s. The first work describing the ability of Trametes sanguinea to transform thebaine by allylic oxygenation and demethylation to 14-hydroxycodeinone and subsequent reduction to 14-hydroxycodein was described by lizuka et al. (Iizuka, K., Okuda, S., Aida, K., Asai, T., Tsuda, K.,

Yamada, M., Seki, I. 1960 I. Chem. Pharm. Bull. 8,1056-7 and lizuka, K., Yamada, M., Suzuki, J., Seki, I., Aida, K., Okuda, S., Asay, T. Tsuda, K. 1962, Chem. Pharm. Bull. 10, 67-70).

Tsuda, K. (1964, IAM Symposium on Microbiology No. 6, Institute of Appl. Microbiol., University of Tokyo, Tokyo, Japan) reported a conversion of Trametes sanguinea (and another 120 basidiomycetes from 1200 different bacterial and fungal strains tested) thebaine to 14-hydroxycodeinone and 14-hydroxycodein. Similar results have been published in another independent study with Trametes cinnabarina (Groger, D and Schmauder, H.P. 1969, Experentia 25, 95-96).

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Microbial transformation of morphine alkaloids has also been described in bacterial strains. Liras, P. and Umbreit, W. W. (1975, Appl. Microbiol. 30, 262-266) described the conversion of morphine to 14-hydroxymorphine by resting Arthrobacter sp.

Low concentrations of 14-hydroxymorphine as a biotransformation product have also been found in Pseudomonas testosteronii. In this strain, α- and β-hydroxysteroid dehydrogenases have been described which produced morphinone and codeinone from codeine or morphine in the presence of NAD ⁺ (Liras, P., Kasparian, SS Umbreit, WW 1975, Appl. Microbiol. 30, 650-656) .

Also in Bacillus sp. the transformation of morphine and codeine into 14-hydroxymorphinone and 14-hydroxycodeinone has been described (Madyastha, K. M., Reddy, G.V. 1994, J. Chem. Soc. Perkin Trans. 1 (8), 911-912).

Harder, P. A. and Kunz, D. A. (1989 United States Patent No. 4,798,792) patented bacterial hydroxylation of codeine and its water-soluble salts to 14-hydroxycodeine strains of the genus Streptomyces. Streptomyces griseus NRRL B8090 showed the highest conversion. When using a rich medium containing soy flour during a 30 day culture and at an initial codeine concentration of 1 mM (0.29 mg / ml). After 27 days, 0.2 mm (63 mg / l) of 14-OH codeine and 0.03 mm (8.55 mg / l) of norodeodein were obtained in this strain.

Zhang, Q., Rich, J.O., Cotterill, I.C., Pantaleone, D.P., Michels, P.C. 2005, J. Am. Chem. Soc. 127, 7286-7287 described in Mycobacterium neoaurum (MTP650) the ability of hydroxylation of codeine to 14-hydroxycodeine and the mechanism of reactions.

Bioconversion of codeine to semi-synthetic opioid derivatives (6-acetyl codeine, oxycodone, norcodeine, morphine) has been described by Nostoc muscorum Niknam et al. (Nikam, S., Faramazi, M.A., Abdi, K., Yazdi, T. T., Amini, M., Rastegar, H. 2010, World J. Microbiol. Biotechnol. 26, 119-123). The 14-OH codeine derivative was not detected. The optimum codeine concentration for whole cell biotransformation was 0.5 - 1.0 mg / ml: derivatives were identified after conversion for 5 to 10 days.

Wick, A., Wagner, M., Ternem, T. A. 2011, Environ. Sci. Technol. 46, 3374-3385 described codeine transformation pathways by activated sludge from a sewage treatment plant under aerobic conditions. One of the described pathways is the hydroxylation of codeine at the C-14 position. The initial codeine concentration was 5 mg / L.

Hydroxylation of codeine 1 to 14-hydroxycodein 2 and 14-hydroxycodeinone 3 strains of a microorganism of the genus Rhizobium has been described in our application under the number PV2011-761 (patent 303 607).

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a novel process for the hydroxylation of codeine with the aid of a strain of Rhizobium radiobacter R89-1 CCM 7947 which hydroxylates codeine of formula 1 to preferentially 14-hydroxycodeine of formula 2,

The method is characterized in that the biotransformation is carried out in a phosphate buffer supplemented with a cosubstrate. The specific conditions of biotransformation preferentially lead to the product of formula 2.

The biological material containing Rhizobium radiobacter R89-1 CCM 7947 was suspended in 0.1 M phosphate buffer pH 7.0, the initial codeine concentration ranging from 1 to 6 g / l.

Glucose, isopropyl alcohol, glycerol and alkali metal formate were used as cosubstrate.

Where sodium formate is used as the cosubstrate, hydroxylation preferably results in 14-hydroxycodeine of formula 2. In a preferred embodiment of the invention, an aqueous solution of sodium formate at a concentration of 2.5 g / l is used.

Biotransformation is carried out, for example, in 500 ml culture flasks with 50 ml cell suspension on a rotary shaker at 200 rpm and at a temperature in the range of 28-30 ° C.

Under these conditions, in contrast to the conditions described in our application PV 2011-761, only 14-hydroxycodeine of formula 2 was obtained, with an initial codeine concentration of 1 g / l, complete (100%) conversion of codeine was observed after 24 hours. 6 g / l was then complete conversion after 72 hours.

The process according to the invention makes it possible to modify the hydroxylation of codeine to give a preferentially one product, 14-hydroxycodeine of formula 2, in an optimized embodiment even to 100% yield of the compound of formula 2.

The Rhizobium radiobacter R89-1 microorganism used in the present invention is deposited with the Czech Collection of Microorganisms at Masaryk University in Brno, Tvrdého 14, under the designation CCM 7947.

The following examples show the procedure for obtaining the microorganism of this patent and examples leading to the optimization of the hydroxylation process.

Overview of pictures

Giant. 1: Time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in a single culture of Rhizobium radiobacter strain R89-1 CCM 7947 on LBTE medium according to Example 4.

Giant. 2: Time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in a single culture of Rhizobium radiobacter strain R89-1 CCM 7947 on LBTE medium with glucose (0.5%) in a stirred bioreactor according to Example 5.

Giant. 3: Time course of biomass concentration (X,) and biotransformation activity (conversion, ♦) in fed culture of Rhizobium radiobacter strain R89-1 CCM 7947 in a stirred bioreactor at 30 ° C culture temperature in LBTE medium with glucose (1.0%) according to Example 6.

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Examples

Example 1

Isolation of Rhizobium radiobacter R89-1

The Rhizobium radiobacter R89-1 strain was obtained from compost soil where poppy residue was composted. 10 g of soil was resuspended in 100 ml saline (9 g / l NaCl) and shaken (200 rpm) for 1 h. The autochthonous microflora was appropriately diluted in saline and plated on LB agar. Individual morphologically different colonies of isolates, grown at 28 ° C for 3 days, were resuspended on fresh LB agar and, after rising at 28 ° C, were assessed for the ability to hydroxylate codeine 1 to 14-OH-codeine 2 and 14-OH-codeinone. 2 ml of LB medium supplemented with codeine at 0.1 g / L in 12 well culture plates (mcwp) were inoculated with the appropriate isolate and after 72 h of culture (28 ° C and 200 rpm), 1 ml sample added 0.1 ml NH ₄ OH and 0.5 ml TBME and the culture liquid was extracted for 20 min. After centrifugation (15 min, 10,000 rpm), the organic phase was used for quantitative determination of codeine of formula 1, 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3 by HPLC under the following conditions:

Column: C — 18 Hibar Lichrom man-fix

Elution mixture: water + 0.1% TFA: MeOH (4: 1 v / v)

Flow rate: 1.3 ml / min

Application volume: 0.05 ml

Detection at 220 and 214 nm

Column temperature: 40 ° C

Analysis time: 5 min

Retention times:

Formula 1 codeine 2.6 min,

Compound of Formula 2 14-hydroxycodeine 2.2 min

Compound of formula 3 14-hydroxycodeinone 3.6 min

From the 2000 isolates tested (collection strains and isolates from natural materials), 2 bacterial strains were obtained showing the ability to hydroxylate the compound of Formula 1 to the products of Formulas 2 and 3. These strains were cultured in 50 LB medium in 500 ml culture flasks as follows: strains grown on LB agar and flasks were incubated on a rotary shaker (200 rpm, 28-30 ° C) for 24 h (first vegetative generation). 2 ml of the grown cultures were used to inoculate three parallel cells with 50 LBTE broth with the addition of codeine at 0.1 g / l (second vegetative generation), which were cultured under the same conditions. After 48 h culture (28 ° C and 200 rpm), the culture sample was extracted with TBME (see above) and the organic phase was used for quantitative determination of bioconversion by HPLC (see above). From both strains tested, a natural isolate was selected from a sample of compost with poppy waste, which showed a conversion of 100%. The strain was designated R89-1 and deposited in the collection of microorganisms under number CCM7947.

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Example 2

Optimization of Rhizobium radiobacter R89-1 CCM 7947 culture medium composition

Optimization of media composition for both growth and conversion proceeded in 12 well mcwp as shown in Example 1.

Composition of culture media:

mineral medium (M9):

Na ₂ HPO ₄ 14.6 g / l KH ₂ PO ₄ 3.0 g / l NaCl 0.5 g / l NH4Cl 1.0 g / l MgSO ₄ .7H ₂ O 0.2 g / l Distilled water 1000 ml

carbon and energy sources:

glucose, sucrose, xylose, glycerol, sorbitol at concentrations of: 1; 5; 10; 15 and 20 g / l complex substrates:

casein hydrolyzate (CH) 20 and 50 g / l yeast extract (YE) 20 and 50 g / l trypton (TRY) 20 and 50 g / l Luria-Bertani (LB) complex medium:

trypton 10g / l yeast extract 5 g / l NaCl 10 g / l Distilled water 1000 ml trace elements (TE) MgSO ₄ .7 H ₂ O 0.2 g / l CaCl ₂ 2H ₂ O 50mg / l FeSO ₄ .7H ₂ O 10mg / l

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Tab.2 Effect of medium and complex source on the increase (OD ₆₀ o) of Rhizobium radiobacter R89-1 CCM 7947 and codeine conversion (0.2 g / l)

Media type Complex substrate (g / L) ODoo 24. h cultivation Conversions 48. h (%) M9 YE20 5.4 0 M9 YE 50 5.5 0 M9 TR20 5.5 50 M9 TRY 50 4.1 20 May M9 CH 20 3.7 10 M9 CH 50 1,8 0 LB YE5aTRY 10 3.2 100 ALIGN! LBaTE YE5, TRY 10 and TE 4.6 100 ALIGN!

Based on the results shown in Tab. 2, LB + TE (LBTE) broth was selected for further optimization. The effect of carbon and energy sources is shown in Tab. 3.

io Tab. 3 Effect of carbon source and energy on the increase (OD ₆ oo) of Rhizobium radiobacter R89-1 CCM 7947 and the conversion of codeine (0.2 g / L) in LBTE medium.

Source of carbon and energy (g / L) ODeoo 24. h cultivation Conversions 48. h (%) glucose 1 5.4 100 ALIGN! glucose 5 6.8 100 ALIGN! glucose 10 7.5 99 glucose 15 7.0 30 glucose 20 6.8 12 sucrose 1 5.7 100 ALIGN! sucrose 5 6.2 100 ALIGN! sucrose 10 6.0 70 sucrose 15 6.5 15 Dec sucrose 20 6.3 10 glycerol 1 4.8 50 glycerol5 4,5 50 glycerol 10 4.0 30 glycerol 15 4.1 20 May glycerol 20 4.2 15 Dec

Optimal sources of carbon and energy are glucose and sucrose in 0.5% concentration. Xylose and sorbitol are also a useful source of carbon and energy.

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Example 3

Optimization of culture conditions for Rhizobium radiobacter R89-1 CCM 7947

The effect of pH on cell growth was monitored in 12 well mcwp at 29 ° C: 2.0 mL of LBTE medium with appropriate initial pH was inoculated with 40 µ 40 of culture (24 h, 29 ° C, LBTE medium of appropriate pH). The effect of pH on codeine conversion was also monitored in 12 well mcwp at 29 ° C, except that a 10% cell suspension prepared as follows: LBTE grown culture (24 h, pH 7.0, 29 ° C) was centrifuged. and resuspended in fresh LBTE medium at the appropriate initial pH. After 48 h conversion, the concentrations of the compounds of Formulas 1, 2 and 3 as determined in Example 1 were determined.

Tab.4 Effect of initial pH value of LBTE medium on growth (OD ₆₀₀ ) of Rhizobium radiobacter R89-1 CCM 7947 culture and codeine conversion (0.2 g / l)

pH ODéoo 24. h cultivation Conversions 48. h (%) 3.0 0.3 0 4.0 0.5 20 May 5.0 4,5 70 6.0 4.4 100 ALIGN! 7.0 4.6 100 ALIGN! 8.0 3.7 100 ALIGN! 9.0 2.5 100 ALIGN! 10.0 0.2 0

The effect of temperature on cell growth was monitored in 12 well mcwp at an initial pH of 7.0: 2.0 mL of LBTE medium was inoculated with 40 µΐ of culture grown in LBTE medium (24h, 29 ° C). The effect of temperature on codeine conversion was monitored at 12 well mcwp at an initial pH of 7.0 with the difference that a 10% cell suspension prepared as in pH monitoring (this example) was used. After 48 h conversion at different temperatures, the concentration of the compounds of formulas 1, 2 and 3 was determined as described in Example 1.

Tab. 5 Effect of temperature on growth (OD ₆₀₀ ) of Rhizobium radiobacter R89-1 CCM 7947 culture and kon30 version of codeine (0.2 g / l)

Temperature ODéoo Conversions 48. h (° C) 24. h cultivation (%) 20 May 3.9 100 ALIGN! 25 4.3 100 ALIGN! 30 4.3 100 ALIGN! 35 3.6 100 ALIGN!

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Example 4

Single culture of Rhizobium radiobacter R89-1 CCM 7947 strain on LBTE medium in a stirred bioreactor

Single cultivation on LBTE medium was performed in a Biostat MD stirred bioreactor: working volume 6 L, aeration 6 L air / min, initial mixing frequency 300 rpm, at 30 ° C and initial pH 7.2. The medium was inoculated with 5% second generation culture (see Example 1, except that there were 100 ml of LBTE medium in 500 ml flasks). The pH of the medium and the dissolved oxygen concentration were not regulated during cultivation. During the bioreactor culture, culture samples were taken at regular time intervals and the biomass concentration (cell dry weight, X) and the ability of the cells to hydroxylate codeine of formula 1 to 14OH-codeine of formula 2 and 14-OH-codeinone of formula 3 were determined. ml of culture liquid was stored at -30 ° C). At the end of the culture, all samples were resuspended in 2 ml of LBTE medium with codeine at a concentration of 0.2 g / l and after 48 h conversion the concentrations of the compounds of formulas 1, 2 and 3 were determined as described in Example 1). The maximum dry mass concentration of biomass (1.7 g / L) was reached at 16 h of cultivation, maximum conversion of compound of formula 1 (82%) only at the end of the cultivation (25 hr). The time course of biomass concentration (X 1) and biotransformation of activity is shown in Fig. 2. 1.

Example 5

Single cultivation of Rhizobium radiobacter strain R89-1 CCM 7947 on LBTE medium with glucose in a stirred bioreactor

A single culture was performed under the same conditions as in Example 4 except that glucose was added to the medium at the beginning of the culture at a concentration of 0.5%. The maximum dry mass concentration of biomass (5.7 g / l) was reached at 4 pm, the maximum conversion of the compound of formula 1 (100%) at 18 h of culture. The time course of biomass concentration (X 1) and biotransformation activity (conversion, ♦) in the stirred bioreactor is shown in Fig. 2. 2.

Example 6

Fed-batch (40% glucose solution) culture of Rhizobium radiobacter R89-1 CCM 7947 on LBTE medium with glucose in a stirred bioreactor

The fed-batch culture was performed under the same conditions as in Example 5 except that the initial glucose concentration was 1.0% and from 1pm to 10pm a 40% glucose solution was added to the reactor to maintain dissolved oxygen concentration in the reactor. medium (pO ₂ ) in the range of 25 to 35% of the maximum oxygen saturation of the medium. The pH was adjusted to 7.5 with ammonia. Giant. 3 shows the time course of biomass concentration and biotransformation activity. Under this culture regime, both the maximum biomass dry weight concentration (11.9 g / L) and the maximum conversion of the compound of Formula 1 (100%) at 20 h of culture were achieved.

Example 7

Influence of initial codeine concentration of formula 1 on its biotransformation by 10% cell suspension in LBTE medium

The cells from the end of the culture described in Example 6 were centrifuged, washed with saline and resuspended in LBTE medium to a final cell concentration of 10% (wet cell weight). Biotransformations were performed in 500 ml culture flasks with 50 ml cell suspension on a rotary shaker at 200 rpm and 29 ° C. The substrate of formula 1 was added to the reaction mixture at time t = 0 and its initial concentration was 1.0 to 6.0 g / l.

initial concentration 1 (g / i) conversion (%) 24 h 48 h 72 h 96 h 120 h 1 46 100 ALIGN! 3 23 53 53 52 51 6 10 24 24 23 21

Example 8

Bioconversion of codeine 1 with 10% cell suspension in phosphate buffer

The bioconversion was carried out under the same conditions as in Example 7 except that the cells were resuspended in 0.1 M phosphate buffer (H 3 PO 4, pH adjusted by addition of NaOH) pH 7.0, the initial codeine concentration ranging from 1 to 6 g / L.

initial concentration (g / i) converting 1 to 3/3 (%) 24 h 48 h 72 h 1 59/10 100/0 100/0 O D 24/6 59/0 83/0 6 11/0 23/0 34/0

Example 9

Influence of cosubstrates on codeine 1 bioconversion by 10% cell suspension in phosphate buffer

The bioconversion was carried out under the same conditions as in Example 8, except that cosubstrates were added to the reaction mixture to regenerate the cofactors (glucose, isopropyl alcohol, glycerol and sodium formate. The initial concentration of codeine ranged from 1 to 24 g / l.

The highest conversion was achieved in the presence of sodium formate (2.5 g / l). Under these conditions, only 14-hydroxycodeine of formula 2 was formed.

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initial concentration of the compound of formula 1 (g / i) converting a compound of Formula 1 into a compound of formulas 2 / 3 (%) 24 h 48 h 72 h 96 h 144 h 168 h 1 100 ALIGN! 3 55 100 ALIGN! 6 29 70 100 ALIGN! 12 11 20 May 29 38 49 56 24 5 7 11 12 13 14

Example 10

Biotransformation of morphine 4 with 10% cell suspension in phosphate buffer

The biotransformation of morphine of formula 4 was carried out under the same conditions as in Example 9 except that the initial morphine concentration was 1 g / l. At 24h, the morphine conversion value was 100%.

Claims (5)

A method of biotransformation of codeine using a Rhizobium radiobacter strain R89-1 CCM 7947, which hydroxylates codeine 1 to 14-hydroxycodeine of formula 2, characterized in that the biotransformation is carried out in a phosphate buffer supplemented with a cosubstrate which is sodium formate. -10GB 304116 B6 The method of claim 1, wherein the phosphate buffer concentration is 0.1M. 3. A process according to claim 2, wherein the pH of the buffer is 7. The method of claim 3, wherein the temperature of the biotransformation is in the range of 28 to 30 ° C. Method according to claims 1 to 4, characterized in that the initial concentration of codeine is in the range of 1 to 6 g / l.