Technical Field

The invention relates to hydroxylation of codeine of formula 1 to 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3 by the microorganism strain Rhizobium radiobacter R89-1 CCM 7947. In hydroxylation, 14-hydroxycodeine of formula 2 is predominantly formed. The reaction provides a 100% conversion of codeine to 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3 with an initial concentration of codeine 1.5 g/1, a 90 % one with 3.0 g/1, and with an initial concentration of 6 g/1 the conversion is 47%.

These compounds are important intermediates in preparation of pharmaceutically active substances.

Background Art

Codeine, morphine and thebaine belong to natural alkaloids produced by poppy (Papaver somniferum). Introduction of the hydroxyl group to the C-14 position of the morphine alkaloid structure is utilized in preparation of oxycodone, naloxone, oxymorphone, and naltrexone, which are used in medicine as analgesics, antitussives, and medicines for overcoming the addiction to drugs and alcohol. The most widely used feedstock in the chemical production of these opioids is thebaine, a minority component of the natural opium extract. This is converted to 14-hydroxycodeinone by oxidation (with a peroxy acid) (Lopez, D., Quiooa, E., Riguera, R., 1994 Tetrahedron Lett., 31,5727-5730). Considering that codeine and morphine are much more available than thebaine, they are perspective precursors for preparation of 14-hydroxy opioids. The described chemical methods provide low yields, include accumulation of difficult-to-separate by-products, and require using undesirable heavy metals (Zhang,Q., Rich, J.O., Cotterill, C, Pantaleone, D.P., Michels, P.C. 2005 J. Am. Chem. Soc. 127,7286-7287).

The interest in obtaining more effective analgesics from natural opioids by means of biotransformations originated in the 1960s. The first works describing capability of the basidiomycete Trametes sanguinea to transform thebaine by allylic oxygenation and demethylation to 14-hydroxycodeinone and subsequent reduction to 14-hydroxycodeine were published by Iizuka et al. (Iizuka, ., Okuda, S., Aida, K., Asai, T., Tsuda, K., Yamada, M., Seki, I. 1960 I. Chem. Pharm. Bull. 8,1056-7 and Iizuka, 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, I. A.M. Symposium on Microbiology No.6, Institute of Appl. Microbiol., University of Tokyo, Tokyo, Japan) described conversion of thebaine to 14-hydroxycodeinone and 14-hydroxycodeine with Trametes sanguinea (and 120 other basidiomycete strains from 1200 tested various bacterial and fungal strains). Similar results were published in another independent study with the fungus Trametes cinnabarina (Groger, D and Schmauder, H.P. 1969, Experientia 25, 95-96). Microbial transformation of morphine alkaloids was also described for bacterial strains. Liras, P. and Umbreit, W.W. (1975, Appl. Microbiol. 30, 262-266) described conversion of morphine to 14-hydroxymorphine by resting cells of Arthrobacter sp.

Low concentration of 14-hydroxymorphine, as a product of biotransformation, was also found for Pseudomonas testosteroni. a- And β-hydroxysteroid dehydrogenases were also described for this strain, which produced morphinone and codeinone from codeine or morphine in presence of NAD ⁺ (Liras, P., Kasparian, S.S., Umbreit, W.W. 1975, Appl. Microbiol. 30, 650-656).

The transformation of morphine and codeine to 14-hydrxymorphinone and 14- hydroxycodeinone was also described for the genus Bacillus sp. (Madyastha, K.M., Reddy, G.V.B. 1994, J. Chem. Soc. Perkin Trans. 1 (8), 91 1-912).

Harder, P.A., and Kunz, D.A. (1989 United States Patent No. 4,798,792) patented bacterial hydroxylation of codeine and its water-soluble salt to 14-hydroxycodeine by the strains of the genus Streptomyces. The highest conversion was found for the strain Streptomyces griseus NRRL B8090 using a rich medium containing soya meal and initial concentration of codeine 1 mM (0.29 mg/ml) during 30-day cultivation. After 27 days, 0.2 mM (63 mg/1) of 14-OH codeine and 0.03 mM (8.55 mg/1) of norcodeine was obtained with 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 capability to hydroxylate codeine to 14-hydroxycodeine and mechanism of the reactions for Mycobacterium neoaurum (MTP650).

The bioconversion of codeine to semi-synthetic opioid derivatives (6-acetylcodeine, oxycodone, norcodeine, morphine) was described for Nostoc muscorum by Niknam et al. (Nikam, S., Faramazi, M.A., Abdi, K., Yazdi, M.T., Aminei, M., Rastegar, H., 2010, World J. Microbiol. Biotechnol. 26, 1 19-123). The 14-OH codeine derivative was not detected. Optimum concentration of codeine for biotransformation by whole cells was 0.5 - 1.0 mg/ml: the derivatives were identified after conversion lasting 5 to 10 days.

Wick, A., Wagner, M., Ternem, T.A., 201 1 , Environ. Sci. Technol. 46, 3374-3385, described transformation paths of codeine activated by sludge under aerobic conditions. Hydroxylation of codeine in the C-14 position is one of the described paths. Starting concentration of codeine was 5 mg/1.

Hydroxylation of codeine 1 to 14-hydroxycodeine 2 and 14-hydroxycodeinone 3 by microorganism strains of genus Rhizobium has not been described in the literature.

Disclosure of Invention

The invention relates to the new microorganism strain Rhizobium radiobacter R89-1 CCM 7947, which is capable to hydroxylate codeine of formula 1 in position 14

(1) to 14-hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3

(2) (3) with 100% conversion of codeine of formula 1. 14-Hydroxycodeine is predominantly formed during the hydroxylation.

Detailed Description of Invention

The strain Rhizobium radiobacter R89-1 CCM 7947 was obtained by an extensive screening of more than 2000 microorganisms of collections and isolated from nature (yeasts, moulds, and bacteria). The screening provided two bacterial strains showing the given properties. However, only the strain R89-1 converted codeine (1.5 g/1) with 100% efficiency. This microorganism was taxonomically classified and designated as Rhizobium radiobacter R89-1. Its biochemical and morphological classification has also been confirmed by sequential analysis of 16S rDNA. The described bacterial strain does not belong to taxonomically described strains with the capability to hydroxylate codeine of formula 1 to 14- hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3.

This invention provides the new microorganism strain Rhizobium radiobacter R89-1 CCM 7947, which is capable to hydroxylate codeine 1

(1)

to 14-hydroxycodeine 2 and 14-hydroxycodeinone 3

(2) (3) with 100% conversion of codeine of formula 1. 14-Hydroxycodeine of formula 2 is predominantly formed during the hydroxylation.

The said process has an advantage of 100% conversion of codeine at the initial concentration of codeine 1.5 g/1; 90% at 3.0 g/1, and 47%> at 6 g/1. The conversion with the said microorganism has the advantage of absence of other intermediates of biotransformation.

The new microorganism, which is the subject of this invention, is deposited in the Czech Collection of Microorganisms at the Masaryk University of Brno, Tvrdeho 14, under the denomination CCM 7947. Properties of the strain are shown in Table 1.

Table 1 Characteristics of the strain Rhizobium radiobacter R89-1 CCM 7947

A. Morphology (microscopy)

Shape of cells rods, individually in irregular clusters

Spores asporogenic

Gram-colouring negative

Mobility positive

B. Morphology of colonies round, smooth, glossy, slightly convex, unbroken margin,

2 - 3 mm in diameter

C. Growth conditions

Temperature grows well at 20 - 35 °C, it grows also at 37 °C, no growth at 42 °C

pH 6.0 - 8.5

Relation to oxygen aerobic

Growth in presence of 6.5 %> NaCl negative D. Physiological characteristics

Hydrolysis of starch negative

Hydrolysis of casein negative

Hydrolysis of esculin positive

Hydrolysis of tyrosine negative

Hydrolysis of o-nitrophenyl-p-D- positive

galactoside

Hydrolysis of lecithin negative

Hydrolysis of gelatine negative

Hydrolysis of Tween 80 negative

Hydrolysis of DNA negative

Decarboxylation of ornithine negative

Reduction of nitrates negative

Reduction of nitrites negative

Acid from glucose by oxidation positive

Acid from glucose by fermentation negative

Acid from xylose positive

Acid from mannitol negative

Acid from maltose negative

Acid from fructose positive

Growth on Simons citrate negative

Urease positive

Catalase positive

Oxidase positive

Arginine dihydrolase negative

Production of fluorescein negative

Composition of the cultivating medium was optimized and influence of various sources of carbon and energy, complex components of medium, as well as various physical conditions of cultivation on the culture growth and its capability to bio-transform codeine was tested.

Unless mentioned otherwise, the strain R89-1 was cultivated in shaken flasks on Luria-Bertani complex medium (LB) and on the medium supplemented with trace elements (LBTE medium), at temperatures 20 to 35 °C and initial pH 4 to 9. Composition of both media is shown in Example 2. Optimum conditions for cell growth were chosen, under which the strain was cultivated in a stirred bioreactor under conditions of a batch or fed-batch cultivation in order to show capability of codeine bioconversion. The batch cultivations were carried out without regulation of pH and concentration of dissolved oxygen. The fed-batch cultivations were conducted under regulated pH and concentration of dissolved oxygen in the medium. The fed-batch solution contained glucose or sucrose in concentration of 20 to 50% by weight, as a source of carbon and energy.

Determination of the degree of conversion was determined in the culture samples under the following conditions: the collected samples were mixed with rt-butylmethylether (TBME) and NH ₄ OH (adjustment of pH), and substances 1, 2 and 3 were extracted by shaking at temperature of 28 °C for 20 min. The conversion of substance 1 to derivatives 2 and 3 was determined by means of HPLC.

The biotransformation of morphine of formula 4 and of thebaine of formula 5 proceeded under the same conditions as that of codeine with the difference that concentrations of morphine and thebaine were 0.2 g/1. At hour 96, the conversion of both morphine and thebaine was 100%.

The properties of the strain CCM 7947, i.e. 100% conversion of codeine of formula 1 to 14- hydroxycodeine of formula 2 and 14-hydroxycodeinone of formula 3, imply a real assumption of utilizing the subject strain for hydroxylation of codeine of formula 1, of morphine of formula 4, and of thebaine of formula 5 to provide the respective 14-hydroxy-derivatives in the pharmaceutical industry.

(4) (5)

The following Examples describe in detail methods of obtaining the strain Rhizobium radiobacter R89-1, methods of its cultivation focused on biotransformation activity of codeine, and optimization of conditions of biotransformation, without being limited by said Examples. Brief Description of Drawings

Fig. 1. Time development of the concentration of biomass (X,■) and of the biotransformation activity (conversion,*) in a batch culture of the strain Rhizobium radiobacter R89-1 CCM 7947 on LBTE medium according to Example 4. Fig. 2. Time development of the concentration of biomass (X,■) and of the biotransformation activity (conversion,*) in a batch culture of the strain Rhizobium radiobacter R89-1 CCM 7947 on LBTE medium with glucose (0.5 %) in a stirred bioreactor according to Example 5.

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

Examples

Example 1 Isolation of the strain Rhizobium radiobacter R89-1

The strain Rhizobium radiobacter R89-1 was obtained from a compost soil where wastes from the poppy processing had been composted. 10 g of the soil were re-suspended in 100 ml of the physiological solution (9 g/1 NaCl ) and shaken (200 rpm) for 1 hour. The autochthonous microflora was appropriately diluted in the physiological solution and inoculated on the LB agar. Individual morphologically different colonies of isolates, grown at 28 °C for 3 days, were re-inoculated on a fresh LB agar and, after growth at 28 °C, their capability to hydroxylate codeine of formula 1 to 14-OH-codeine of formula 2 and 14-OH-codeinone of formula 3 was determined by the following method: 2 ml of the LB medium supplemented with codeine at a concentration 0.1 g/1 in 12 well culture plates (mcwp) was inoculated with the respective isolate; after 72 hours of cultivation (28 °C and 200 rpm), 0.1 ml NH ₄ OH and 0.5 ml TBME were added to 1 ml of the sample and the cultivating 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 means of HPLC under the following conditions: Column: C-18 Hibar Lichrom manu-fix Elution mixture: water + 0.1% TFA: MeOH (4: 1 V/V)

Flow rate: 1.3 ml/min

Injected volume: 0.05 ml

Detection at wavelength: 220 and 214 nm

Column temperature: 40 °C

Time of analysis: 5 min

Retention times: compound 1 codeine 2.6 min,

compound 2 14-hydroxycodeine 2.2 min

compound 3 14-hydroxycodeinone 3.6 min Out of 2000 tested isolates (collection strains and isolates from natural materials), two bacterial strains were obtained that showed capability to hydroxylate the compound of formula 1 to products of formulae 2 and 3. These strains were cultivated in 50 ml of the LB medium in 500 ml cultivating flasks by the following method: the medium was inoculated with the colonies of strains grown on the LB agar, and the flasks were incubated in a rotatory shaking apparatus (200 rpm, 28 to 30 °C) for 24 hours (first vegetative generation). 2 ml of the grown culture were used to inoculate three parallel flasks with 50 ml of the LBTE medium supplemented with codeine at a concentration of 0.1 g/1 (second vegetative generation) that were cultivated under the same conditions. After 48 hours of cultivation (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 means of HPLC (see above). Of both the strains tested, the natural isolate from the sample of the compost containing wastes from the poppy processing providing 100% conversion was selected. The strain was designated as R89- 1 and deposited in the collection of microorganisms under the number CCM7947.

Example 2

Optimization of composition of the cultivating medium for Rhizobium radiobacter R89-1 CCM 7947

Optimization of the composition of the medium for growth and conversion was carried out in 12 well mcwp plates as described in Example 1. Composition of cultivation media: Mineral medium (M9):

Na ₂ HP0 ₄ 14.6 g/1

KH ₂ P0 ₄ 3.0 g/1

NaCl 0,5 g/1

NH ₄ C1 1.0 g/1

MgS0 ₄ . 7H ₂ 0 0.2 g/1

distilled water 1000 ml

Source of carbon and energy:

glucose, sucrose, xylose, glycerol, sorbitol

in concentrations: 1 ; 5; 10; 15; and 20 g/1

Complex substrates:

casein hydrolysate (CH) 20 and 50 g/1

yeast extract (YE) 20 and 50 g/1

tryptone (TRY) 20 and 50 g/1

Luria-Bertani complex medium (LB):

tryptone 10 g/1

yeast extract 5 g/1

NaCl 10 g/1

distilled water 1000 ml

Trace elements (TE)

MgS0 ₄ .7 H ₂ 0 0.2 g/1

CaCl ₂ . 2H ₂ 0 50 mg/1

FeS0 ₄ .7H ₂ 0 lO mg/l

Table 2 Influence of the medium and complex source on growth (OD ₆ oo) of Rhizobium radiobacter R89-1 CCM 7947 and on conversion of codeine (0.2 g/1)

Type of OD ₆₀₀ Conversion 48. h

Complex substrate (g/1)

medium 24 h cultivation (%)

M9 YE 20 5.4 0

M9 YE 50 5.5 0 M9 TR 20 5.5 50

M9 TRY 50 4.1 20

M9 CH 20 3.7 10

M9 CH 50 1.8 0

LB YE 5 and TRY 10 3.2 100

LB and TE YE 5, TRY 10 and TE 4.6 100

Based on the results shown in Table 2, the culture medium LB+TE (LBTE) was selected for further optimization. Influence of the source of carbon and energy is shown in Table 3.

Influence of the source of carbon and energy on growth (OD ₆ oo) of Rhizobium radiobacter R89-1 CCM 7947 and on conversion of codeine (0.2 g/I) in LBTE medium

Source of carbon and OD ₆₀₀ Conversion 48 h energy (g/1) 24 h cultivation (%) glucose 1 5.4 100 glucose 5 6.8 100 glucose 10 7.5 99

glucose 15 7.0 30

glucose 20 6.8 12

sucrose 1 5.7 100 sucrose 5 6.2 100 sucrose 10 6.0 70

sucrose 15 6.5 15

sucrose 20 6.3 10

glycerol 1 4.8 50

glycerol 5 4.5 50

glycerol 10 4.0 30

glycerol 15 4.1 20

glycerol 20 4.2 15 Glucose and sucrose at 0.5% concentration are optimum source of carbon and energy. Xylose and sorbitol can also be used as a source of carbon and energy.

Example 3 Optimization of cultivating conditions for Rhizobium radiobacter R89-1 CCM 7947

The influence of pH on growth of cells was followed in 12 well mcwp plates at 29 °C: 2.0 ml of the LBTE media with the respective initial pH were inoculated with 40 μΐ of the culture (24 h, 29 °C, LBTE medium of corresponding pH). The influence of pH on conversion of codeine was also followed in 12 well mcwp plates at 29 °C with the difference that a 10% cell suspension prepared by the following method was used: the culture grown in LBTE (24 h, pH 7.0, 29 °C) was decanted and re-suspended in the fresh LBTE medium of the respective initial pH. After a 48-h conversion, concentration of 1, 2 and 3 was determined as described in Example 1.

Table 4 Influence of the initial pH value of LBTE medium on growth (OD ₆ oo) of the

Rhizobium radiobacter R89-1 CCM 7947 culture and on conversion of codeine (0.2 g/1)

The influence of temperature on growth of cells was followed in 12 well mcwp plates with initial pH of the medium 7.0: 2.0 ml of the LBTE medium was inoculated with 40 μΐ of the culture grown in the LBTE medium (24 h, 29 °C). The influence of temperature on conversion of codeine was followed in 12 well mcwp at initial pH of the medium 7.0 with the difference that a 10 % cell suspension prepared in the same way as in monitoring the influence of pH (this Example) was used. After a 48-h conversion at various temperatures, concentrations of 1, 2 and 3 were determined as described in Example 1.

Table 4 Influence of the temperature on growth (Οϋβοο) of the Rhizobium radiobacter

R89-1 CCM 7947 culture and on conversion of codeine (0,2 g/1)

Example 4

Batch cultivation of the strain Rhizobium radiobacter R89-1 CCM 7947 in LBTE medium in a stirred bioreactor

Batch cultivation in the LBTE medium was carried out in a stirred bioreactor Biostat MD: working volume 6 1, aeration with 6 1 of air/min, initial frequency of stirring 300 rpm, at temperature 30 °C and initial pH 7.2. The medium was inoculated with a 5% culture of the second generation (see Example 1 , with the difference that the 500 ml cultivating flasks contained 100 ml of the LBTE medium). During cultivation, pH of the medium and concentration of dissolved oxygen were not regulated. During the cultivation in the bioreactor, the culture was sampled in regular time intervals and concentration of the biomass (dry weight of cells, X) and capability of the cells to hydroxylate codeine of formula 1 to 14-OH-codeine of formula 2 and 14-OH-codeinone of formula 3 were determined (the cell pellet from 2 ml of the cultivating liquid was maintained at -30 °C; after the cultivation was complete, all samples were re-suspended in 2 ml of the LBTE medium with codeine at a concentration 0.2 g/1 and, after a 48-h conversion, concentration of the substances of formula 1, 2 and 3 were determined as described in Example 1). A maximum concentration of dry weight of biomass (1.7 g/1) was attained after 16 hours of cultivation; a maximum conversion of the substance of formula 1 (82 %) was achieved only at the end of cultivation (25 hours). The time developments of the concentration of biomass (X,■) and of biotransformation activity are shown in Fig. l .

Example 5

Batch cultivation of the strain Rhizobium radiobacter R89-1 CCM 7947 in LBTE medium with glucose in a stirred bioreactor.

Batch cultivation was carried out under the same conditions as in Example 4, with the difference that glucose at a concentration 0.5 % was added to the medium in the beginning of cultivation. A maximum concentration of dry weight of biomass (5.7 g/1) was attained after 16 hours, a maximum conversion of substance 1 (100 %) was achieved after 18 hours of cultivation. The time development of the biomass concentration (X, ■) and of biotransformation activity (conversion,♦) in the stirred bioreactor is shown in Fig. 2.

Example 6

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

The fed-batch cultivation was carried out under the same conditions as in Example 5, with the difference that the initial concentration of glucose was 1.0 % and, from hour 13 to hour 22 of the cultivation, a 40% solution of glucose was added to the reactor in order to maintain the concentration of dissolved oxygen in the medium (p0 ₂ ) within the range of 25 to 35% of the maximum saturation of the medium with oxygen. The pH value was regulated with ammonia to pH = 7.5. Fig. 3 shows time development of the concentration of biomass and of biotransformation activity. In this regime of cultivation, maximum concentration of dry weight of the biomass (11.9 g/1) and maximum conversion of the substance of formula 1 (100 %) were attained at hour 20 of cultivation. Example 7

Influence of initial concentration of codeine 1 on its biotransformation by 10 % cell suspension

The cells from the end of cultivation described in Example 6 were decanted, washed with the physiological solution, and re-suspended in the LBTE medium to obtain a concentration of cells 10% (wet weight of cells). The biotransformations were carried out in 500 ml cultivating flasks containing 50 ml of the cell suspension in a rotatory shaker at 200 rpm and 29 °C. The starting concentration of substrate 1 was 0.2 to 6.0 g/1 .

Example 8

Influence of the initial concentration of codeine 1 on its biotransformation with a 20% cell suspension

The bioconversion proceeded under the same conditions as in Example 7, with the difference that the resulting concentration of cells was 20% (wet weight of cells) and the concentration of the starting substance of formula 1 was 1.5; 3; or 6 g/1 . initial concentration conversion

of I (g 1) (%)

24 h 48 h 72 h 96 h

1.5 95 100 - -

3.0 46 80 87 90

6.0 21 36 46 47

Example 9

Biotransformation of morphine and thebaine with a 20% cell suspension The biotransformation of morphine of formula 4 and thebaine of formula 5 was carried out under the same conditions as in Example 8, with the difference that the concentrations of morphine and thebaine were 0.2 g/1. At hour 96, the conversion of morphine and thebaine was 100%.