JP2001519166A - Biological conversion of colchicon compounds to the corresponding 3-glycosyl derivatives - Google Patents

Abstract

  (57) [Summary] The colchicon compound is converted to the corresponding 3-O-glycosyl derivative by the Bacillus megaterium strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the biological conversion of colchitinoid compounds to their respective 3-O-glycosyl derivatives using selected microbial strains. The method of the present invention provides a compound in which only C-3 of the aromatic ring A is glycosylated in high yield and purity, starting from the cited colchicon compound.

[0002] Compounds obtained by the bioconversion method of the present invention, in particular, thiocolchicosone (3-O
- glucosyl sheet thio Col Hikon in clogging formula (I), the a _{R 1 = -OCH 3, R 2} = -SCH a ₃₎ is mainly for the preparation of novel antitumor medicaments, significant pharmacological It is an important active element.

[0003] The highly specific glycosidation of the compounds and related colchicinoid compounds disclosed general formula of the Invention (I), in order to carry out either by chemical reaction or bioconversion, been devoted much effort Was.

[0004] Chemical pathways consist of a series of complex, non-specific reactions that non-selectively encompass different molecular sites, resulting in a mixture of glycosidated derivatives, some of which are Is inactive. Therefore, the conversion yield of the aromatic ring to the effective product specifically glycosidated at C-3 is very low.

[0005] Biological methods essentially involve the formation of the C-aromatic ring of colchitinoid compounds such as colchicine and thiocolchicine, which are not directly related to colchicon compounds.
2 and C-3 to a monoglycosidated derivative in the context of a biotransformation by a culture of Centella Asiatica, and such a transformation is not highly selective and gives sufficient yields and productivity. No (Solet, JM, et al., Phyto
chemistry 33, 4, 817-820, 1993).

[0006] Other efforts to biologically convert colchitinoid compounds include:
Only the demethylation of the methoxy group bonded to the aromatic ring (C-2 and C-3) was performed, and in any case, the yield and productivity were limited and the regioselectivity was low. Always characteristic.

For such reasons, Hufford CD, et al., (J. Pharm. Sc., 68, 10, 1239
-1242, 1979) used Streptomyces griseus and / or Streptomyces spectabilis, Bellet P. et al., (GB-923421, 1959) used a different strain of Streptomyces and other species of bacteria and fungi, colchicine And its derivatives with the corresponding 3
-Attempt to convert to demethylated derivatives. The results of these known methods confirm what has been described above in connection with the non-selectivity of the relevant microbial enzymes at alkaloid molecules such as C-2, C-3 or C-10. Furthermore, the productivity level of the catalyst system is rather low due to the low conversion yields, the low substrate concentrations that can be used, and the high frequency of tropolone ring degradation.

[0008] More recently, Poulev. Et al., (J. Ferment. Bioeng. 79, 1, 33-38, 1995).
Achieved specific demethylation using bacterial microorganisms, but its yield and productivity were still low.

[0009] Enzyme activity derived from microorganisms (Streptomyces, Bacillus, etc.) similar to those described above may cause maytansinoids (US Pat. No. 4,361,650: Izawa, M., et al., J. Antib.
iotics, 34, 12, 1587-1590, 1981). Also in this case, the catalyzed reaction consists only of demethylation,
It was characterized by low conversion yield and low productivity.

The glycosyltransferase activity of α-amylase from Bacillus megaterium strain has been described (Brumm., PJ, et al., Starch, 43, 8, 319-323, 199).
1) The receptor specificity (only glucose or glucoside) is particularly high. Cyclodextrin-glucosyltransferase produced by the same microbial source,
Starting from starch, it catalyzes the α-1,4-transglucosylation of rubusoside (13-O-β-D-glucosyl-steviol β-D-glucosyl ester). Also in this biotransformation, the acceptor of the transferase reaction is the substrate glucide fraction (Darise, M., et al., Agric. Bioel. Chem., 48, 10, 24).
83-2488, 1984). Cyclodextrin-glycosyltransferase was previously used for the preparation of cyclodextrins G6 and G8 from starch (K
Itahata, S., Okada, S., Agric. Biol. Chem., 38, 12, 2413-2417, 1974).

[0011] These examples demonstrate the high substrate specificity of glycosyltransferase activity expressed by Bacillus megaterium, which has different complex molecular structures because it involves only the glucosyl receptor. One cannot predict any reaction with secondary metabolites such as colchicon. In fact, no example is known of using the microorganism for enzymatic conversion of a colchicon compound to a 3-glycosyl derivative.

Bacillus megateriu capable of growing in the presence of high concentrations of colchicon (R ₁ −—OCH ₃ , R ₂ −—OCH ₃ ), 3-demethyl-colchicone, and their respective thio derivatives
It has now been found that strain m has a very high and very specific activity to biologically convert the substrate into a derivative glucosidated only at the C-3 of the aromatic ring. Such bioconversion occurs in a very short time and is characterized by a surprisingly high yield.

Accordingly, the present invention provides a compound of formula (I):

[0014]

Embedded image

Wherein R ₁ is a glycoside residue and R ₂ is C ₁ -C ₆ alkoxy or C ₁ -C ₆ thioalkyl. Wherein the compound wherein R ₁ is OH or methoxy is biologically converted using Bacillus megaterium.

Bacillus megaterium is a gram-positive sporulating cell having a cell diameter of 1.0 μm or more.
It is a fungus, grows aerobically on many culture media, is catalase-positive,
Hydrolyze the enzyme. The strains of Bacillus megaterium that can be used according to the invention are fully expanded, as demonstrated by growth tests and microscopic analysis.
Can be propagated, and high concentrations of colchicon and thiocolchicon (R₁= -OCH_Three, R _Two = -SCH_Three) And each of the 3-demethyl derivatives (about 2 g / l). Homologous species such as Bacillus cereus have shown difficulties in growing even at a substrate concentration of 1 g / l (10-15% absorbance of the control).

The high selectivity and efficacy of biotransformations is surprising and unusual. This is because the yield is in the range of 70 to 95%.

Furthermore, the microorganism used in the biotransformation can maintain its catalytic activity permanently even after repeated fermentation steps, thus providing a specific biotransformation in fed-batch culture and continuous processes. I do. Thus, this method provides a high level of productivity and reproducibility.

The remarkable regioselectivity ensures, in addition to the remarkable production yields, the high quality and purity of the products obtained, so that the simple downstream processing makes the products highly pure. It can be obtained at 100%.

Furthermore, the ability to reduce the steps of product purification and recovery, the economics of the process, and the affinity and safety of use are important advantages.

The sequence of operations that can be used in the method of the present invention consists of: A) A Bacillus megaterium culture that can grow in the presence of a high concentration of colchicon substrate is derived from a natural source or collection strain. To select. B) Select the isolate from A) and determine the catalytic activity of the bioconversion to the corresponding 3-O-glycosyl derivative by a bioconversion assay with the specific substrate administered at increasing concentrations. evaluate. C) Microbiological characterization of the strain selected in B). D) The target-specific selection of the bacterial population obtained from B) gradually increases the bioconversion yield. E) Optimize bioconversion by studying and optimizing critical fermentation parameters. F) Consideration and optimization of preservation methods for highly productive cultures ensure stable, uniform inoculum for application to industrial scale production. G) Scale up the process in fermenters, batch cultures, fed-batch cultures and continuous fermentations. H) Establish and optimize downstream processing and product recovery methods.

Specifically, the microorganisms that can be used in the present invention include, starting from a strain deposit center or a soil sample of various origins, or a collection culture obtained from a preselected industrial strain, Sources (peptone, yeast extract, meat extract, asparagine, etc.), various agar media containing a carbon source (glycerin, starch, maltose, glucose, etc.), pH 5-8, preferably 6-7, for selective selection. Can be. Incubation temperatures range from 20 ° to 45 ° C, preferably 28 ° to 40 ° C.

The ability of the culture to grow in the presence of a toxic concentration of the transforming colchicone substrate may vary from different agar substrates (some of which, for inhibiting the growth of most microorganisms) from 0.1 to
Evaluate by scalar dilution and parallel plating techniques on a 3 g / l concentration of a colchicon compound (for example 3-demethylthiocolchicon) which has been pre-added.

Colonies capable of growing under the above-mentioned conditions are aseptically picked, plated on different agar media, and examined for their purity and uniformity of growth.

The culture medium used for preserving the culture is a typical microorganism substrate (pH 5) containing an organic nitrogen source (eg, peptone, yeast extract, tryptone, meat extract) and a carbon source (eg, glucose, maltose, glycerin). -8, preferably 6-7). Incubation temperatures range from 20 ° to 45 ° C, preferably 28 ° to 40 ° C.

Next, the selected microorganisms are evaluated for their ability to grow in submerged culture in the presence of the colchicon compound and for their ability to convert the colchicon compound to the corresponding 3-glycosyl derivative.

This evaluation is based on one or more organic nitrogen sources (yeast extract, peptone, tryptone, casein hydrolysate, meat extract, corn step liquor, etc.) and one or more carbon sources (
Containing glucose, glycerol, starch, saccharose, etc.), inorganic phosphorus and nitrogen sources, and inorganic salts of various ions (K ⁺ , Na ⁺ , Mg ⁺⁺ , Ca ⁺⁺ , Fe ⁺⁺ , Mn ^++, etc.) Performed in 100 ml flasks containing 20 ml of liquid medium of different medium formulations.

The culture sample can optionally be subjected to a mutagenesis treatment by conventional mutagenesis techniques (such as UV irradiation) to induce a mutant having a specific bioconversion activity ( This can be evaluated by the same operation as described above).

The culture samples from each biotransformation assay were analyzed and analyzed by TLC and HLC.
Production of the 3-glycosyl derivative was assessed by PLC analysis.

The ability of the selected microorganism to convert the colchicon substrate to the respective 3-glycosyl derivative is confirmed by a bioconversion assay in a flask on a 300 ml scale in the same culture broth used in the selection process. did.

The microorganisms that showed a positive reaction were used in different culture broths on a 300 ml scale for testing for optimization of bioconversion. The main culture and fermentation parameters studied were organic nitrogen source, carbon source, mineral salts, temperature, agitation-aeration, pH, incubation time, inoculation ratio, subculture process, time and form of addition of the substrate to be converted. . The selected bacterial microorganisms capable of effecting the biological conversion of the present invention contain one or more organic nitrogen sources, preferably yeast extract, meat extract, peptone, tryptone, casein hydrolyzate, corn steep liquor, and the like. Can be grown on both solid and liquid media substrates. Carbon sources useful for growth and biotransformation
Glucose, fructose, saccharose, glycerol, malt extract and the like, preferably glucose, fructose and glycerin. The culture medium further contains an inorganic phosphorus source and salts such as K ⁺ , Na ⁺ , Mg ⁺⁺ , and NH ₄ ⁺ .

The selected microorganisms may be at a temperature of 20 to 45 ° C., preferably 28 to 40 ° C. and 5 to 8
, Preferably at a pH between 6 and 7. Under the same conditions, the considered microorganism can convert the colchicon compound to the corresponding 3-glycosyl derivative. The conversion occurs in submerged cultures as well as in flasks incubated on a rotary shaker, stirring at 150-250 rpm.

Because of the specific kinetics of this biotransformation associated with the growth of microorganisms, the optimal conditions for the purpose of biotransformation are the same conditions that are optimal for growth. . Thus, culture media useful for promoting good microbial growth, such as those based on the organic and inorganic components described above, are also useful for good activity in the biotransformation of the relevant substrate. The latter is added to the medium in the starting stage of the fermentation or in fractional aliquots starting at the start of the fermentation.

The bioconversion of the present invention is based on enzymatic conversion, which begins during the exponential growth phase and persists in parallel with growth. The maximum level of conversion to the 3-glycosyl derivative (very high; up to 95%) is reached within the first 48-72 hours, depending on the time of substrate addition. The regioselectivity of the biotransformation is absolute:
In the medium sample, the presence of the 2-glycosyl derivative was not observed at all. The products obtained are only extracellular.

The substrate to be converted can be added as an acetone or alcohol solution, an alcohol-water mixture, a dioxane solution, and the like. The biotransformation of the present invention can be scaled up to fermentor levels while maintaining the culture conditions, especially with respect to culture medium, temperature and treatment time. Proper level of agitation-aeration is important for good growth, especially 1 minute per liter of medium per minute.
A ventilation level (vvm) of ２2 liters of air, preferably 1.5-2 vvm, is required.

The product obtained from the biotransformation is extracted from the culture broth after centrifugation and recovery of the supernatant or separation of biomass from the liquid fraction by microfiltration and recovery of the permeate. From the viewpoint of optimal recovery of the product, the medium can be treated with alcohol.

Purification and recovery of the biotransformation product can be performed using chromatographic techniques for separation on an absorbing resin and elution with an alcohol, preferably methanol. The hydromethanol solution containing the product is a lipophilic organic solvent,
Preferably, it can be further purified by extraction with methylene chloride. After further treatment with a mixture of alcohol and an organic solvent, the product can be obtained in pure form by crystallization from the resulting alcohol solution. Glucose can be replaced with other sugars such as fructose or galactose without losing glycosyltransferase activity.

The following examples disclose the present invention in further detail. Example 1 An aliquot of a culture of Bacillus megaterium isolated from agricultural soil was resuspended in 20 ml of sterile saline and subjected to a scalar dilution to a dilution factor of 1: 10,000,000. Suspensions of various dilutions were plated on LB-Agar culture medium and LB-Agar supplemented with thiocolchicon or 3-demethylthiocolchicon, respectively, to a final concentration of 2 g / l (see table). Cultures were incubated at + 28 ° C for 3-4 days in the dark. Colonies that grew on selective media supplemented with colchicon compounds were isolated and plated by plating on non-selective media. The samples were incubated as described above, but for a shorter time (24 hours).

The culture was then transferred to the same agar medium in a test tube and incubated for 24 hours as described above.

Using the aliquot of the culture selected as described above, the culture medium ST (Table) 20
containing 0.4 mg of thiocolchicon or 3-demethylthiocolchicon
A 100 ml Erlenmeyer flask was inoculated to a final concentration of / ml. The culture was incubated on a rotary shaker at 200 rpm overnight at 28 ° C.

Conversion of the colchicon substrate was checked by analyzing an aliquot of the culture broth taken every 3-4 hours by TLC on silica gel with an acetone: ethyl acetate: water 5: 4: 1 elution system. .

After 4 days of incubation, aliquots of the cultures that showed obvious catalytic activity on the 3-glycosyl derivative were prepared by scalar dilution as described above for the preparation of new inoculum in vitro. Collected on plate. The bioconversion assay in the flask was repeated under the same conditions as above, except that significantly higher final concentrations of thiocolchicon and 3-demethylthiocolchicon (1 mg / ml) were used. The most active single culture (substrate conversion equal to or greater than 70%) is transferred to a cryotube (
Used for preparation of inoculum in frozen cryotubes).

[0043]

[Table 1]

Example 2 Starting from a Bacillus megaterium culture, the method described in Example 1 was used to collect the following collection strains (Deutsche Sammlung von Mikroorganismen, Braunschweig, Germa
ny) and repeated: DSM 90 DSM 322 DSM 333 DSM 1667 DSM 1670 DSM 1671

The cultures selected in Example 1 and supplemented with thiocolchicon (1 mg / ml) were incubated in liquid medium for 4 days. TLC analysis showed that conversion of the substrate to thiocolchicosone occurred with a conversion yield of 30-70%.

Example 3 An aliquot of the culture sample in a test tube, selected as described in the above example, was used to inoculate a 100 ml Erlenmeyer flask containing 20 ml of broth ST.

The broth cultures were incubated overnight at + 30 ° C. on a rotary shaker at 200 rpm. After incubation, a glycerol sterile solution was added to the culture to a final concentration of 20%. The culture was then dispersed in a 2 ml cryotube and immediately immersed in liquid nitrogen.

After several days, 10% of the culture was quickly thawed at 37 ° C. An aliquot of each cryotube was used to inoculate a 100 ml Erlenmeyer flask containing 20 ml of ST medium, which was then incubated at + 28 ° C overnight at 200 rpm (preculture). After the incubation, 2 ml of each preculture were aseptically transferred to 20 ml of fresh ST medium supplemented with 3-demethylthiocolchicon to a final concentration of 1 g / l. Biological conversion was performed and checked under the conditions described in Example 1. Analysis showed that conversion of the substrate to the 3-glycosyl derivative occurred during the quantitative period described above (70% and above), demonstrating the catalytic stability of the frozen culture. Parallel control of broth cultures plated on LBAgar immediately after thawing confirmed viability, homogeneity, and purity of the frozen culture.

Example 4 After thawing, an aliquot of the culture in a cryotube was used to prepare 50 ml of ST medium (preculture).
A 300 ml Erlenmeyer flask containing ml was inoculated. 30 ° C, 250
After overnight incubation at rpm, 5 ml of the preculture was transferred to 50 ml of the same medium to which a final concentration of 1 g / l was added by adding 3-demethyl-thiocolchicon. Cultures were incubated for 4 days under the same conditions as above. Samples are taken every 4 days to assess growth levels (absorbance measured at 600 nm), thiocorticosone production (TLC and HPLC), sterility (on LBAgar),
Morphological examination by microscopy was also performed.

Analysis by TLC was performed as described in Example 1. For HPLC analysis, 9 ml of methanol was added to the 1 ml fraction of the culture broth and centrifuged at 13,000 rpm for 2 minutes. The content of the 3-glucosyl derivative in the supernatant was analyzed by reverse phase HPLC with isocratic elution using a water: acetonitrile 80:20 elution system.

[0051] HPLC analysis demonstrated that after 72-96 hours, the biological conversion of the substrate to thiocortisone was almost complete.

The final yield of the 3-glycosyl derivative obtained by biotransformation is 70-8
It was in the range of 5%.

Example 5 The procedure described in Example 4 was repeated, but 3-demethylthiocolchicon was added to the culture in two portions: 0.25 g / l at the start and 0 after 24 hours. .74g / l
.

The growth and production reactions of the culture were similar to those obtained in Example 4, with a thiocortisone yield of about 90%.

Example 6 One liter (inoculation) of ST broth in an Ehrenmeyer flask was inoculated with a cryotube culture. The flask was incubated at + 30 ° C, 250 rpm overnight. The inoculum was aseptically transferred to a 14 liter fermentor containing 9 liters of sterile broth STL. 3-Demethylthiocolchicon was added to a final concentration of 1 g / l (25% at start, remaining after 20 hours). The fermentation was performed while maintaining agitation-aeration at an appropriate level (agitating at up to 900 rpm; aeration of 1-1.5 vvm, depending on culture growth).

Samples were taken from the culture broth every 2 hours and subjected to the following analyses:-optical density at 600 nm;-sterility and purity analysis of the strain (on LBAgar);-microscopic morphology (gram staining). Analysis of thiocorticosone content by TLC and HPLC as described in Examples 1 and 4, respectively.

After about 48 hours of fermentation, the conversion of the substrate to thiocortisone is almost complete. Final yield was about 85%.

Example 7 The procedure described in Example 6 was repeated, but after 48 hours of fermentation, 9
About 0% was recovered and the product was extracted (fraction 1). To the remaining 10%, under aseptic conditions, 9 liters of fresh sterile ST medium containing 10 g of 3-demethylthiocolchicone was added in the fermenter. Fermentation was performed as described in Example 6. 48 hours later,
9 liters of the culture broth were collected and extracted (fraction 2). To the remaining culture broth, under aseptic conditions, 9 liters or more of sterile fresh ST medium containing fresh 3-demethylthiocolchicone (10 g) was added. Fermentation was performed as described above. 48 hours later,
The culture broth was completely collected and extracted (fraction 3). The bioconversion activity of the strain remained stable in all three runs and the conversion yield was about 80%.

Example 8 The final culture broth from the fermentation (total: about 27 liters) was concentrated under vacuum to give a soft residue, which was taken up in ethanol.

After separation by filtration, the water-ethanol fraction was concentrated in vacuo to water and purified by repeated extraction with methylene chloride. The water fraction was concentrated, adjusted to pH 10 with sodium hydroxide, and extracted with a methylene chloride-ethanol mixture.

The combined organic layers were concentrated under vacuum. Ethanol was added to the obtained suspension, which was concentrated and crystallized. After the step of further redissolving the solid in a methylene chloride-ethanol mixture, a second crystallization with ethanol was performed.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission Date] March 22, 2000 (2000.3.22)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction contents]

Wherein R ₁ is a glycoside residue, especially an O-glycoside residue, and R ₂ is C ₁ -C ₆ alkoxy or C ₁ -C ₆ thioalkyl. A process for preparing a colchicon compound, wherein R ₁ is OH or methoxy,
The present invention relates to a method characterized by performing a biological conversion using acillus megaterium.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW

Claims (16)

[Claims] 1. A compound of the formula (I): Wherein R ¹ is a glycoside residue and R ² is C ₁ -C ₆ alkoxy or C ₁ -C ₆ thioalkyl. A method, wherein R ¹ is OH or methoxy, and the compound is biologically converted using Bacillus megaterium. 2. A method for preparing a compound of the formula (I), wherein R ¹ is an O-glucoside residue. 3. The method according to claim 1, wherein the Bacillus megaterium strain is selected on the basis of its ability to grow in the presence of high concentrations of the converted colchicon substrate. 4. The method according to claim 3, wherein said concentration ranges from 0.1 to 3 g / l. 5. The method according to claim 1, wherein the Bacillus megaterium is cultured in a solid or liquid medium. 6. The method of claim 5, wherein said medium contains at least one organic nitrogen source. 7. The method of claim 6, wherein said organic nitrogen source is selected from the group consisting of meat extract, peptone, tryptone, casein hydrolyzate, and corn step water. 8. The method according to claim 5, wherein the culture medium contains at least one carbon source. 9. The method of claim 8, wherein said carbon source is selected from the group consisting of glucose, fructose, glycerol. 10. The method of claim 5, wherein said medium comprises at least one source of an inorganic salt of K ⁺ , Na ⁺ , Mg ⁺⁺ , NH ₄ ⁺ . 11. The method according to claim 1, which is carried out at a pH in the range from 5 to 8. 12. The method of claim 11, wherein said pH is in the range of 6-7. 13. The method according to claim 1, which is performed at a temperature in the range of 20 ° to 45 ° C.
The method according to any of the above. 14. The method according to claim 13, wherein said temperature ranges from 28 ° to 40 ° C. 15. The method according to claim 1, wherein the method is carried out at a maximum aeration level (vvm) of 1 to 2 liters of air per minute of medium per minute. 16. The method of claim 15, wherein said level ranges from 1.5 to 2 vvm.