Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P33/00—Preparation of steroids
  • C12P33/06—Hydroxylating

Definitions

• the present invention relates to the provision of an enzymatic method for the preparation of 1 ⁇ , 3a,7a,12a-tetrahydroxy bile acid (Formula I).
• Bile acids are steroid acids found predominantly in the bile of mammals and other vertebrates. Different molecular forms of bile acids can be synthesized in the liver by different species.
• Forma I 1 ⁇ , 3a,7a,12a-tetrahydroxy bile acid (Formula I) belongs to the family of bile acids. It has been isolated from the urine of patients with hepatobiliary disorders, in the urine of women in late pregnancy, in newborn infants and the human meconium (Tohma et al, Chem. Pharm. Bull. 1985, 33(7), p.3071).
• the complex methodology of Tohma is used to create a double bond between carbon atoms 1 and 2 and apply Tamao-Fleming oxidation methodology to introduce a hydroxyl group at position 1 of the steroid ring system (Scheme 1).
• the hydroxyl group of position one, with its natural (and desired) stereochemistry is converted to a carbonyl group, which later needs to be reconverted to hydroxyl again.
• the patent application discloses alternative preparation, shown in Scheme 2.
• the introduction of the hydroxyl group in position 1 is realized by eliminating one of the already present hydroxyl groups (with the desired stereochemistry) again and utilizing a complex sequence of oxidative reactions, similar to that of Scheme 1, which creates an epoxide ring and also inserts bromine atoms in the ring system. Not only is it required to reduce again the carbonyl group created in position 3, it is further required to open the epoxide ring stereo selectively and remove the bromine atoms under catalytic conditions.
• the present invention discloses an enzymatic method for the preparation of compound of formula I, starting from readily available cholic acid.
• the method disclosed herein comprises of one step, catalyzed by an enzyme, which selectively introduces a hydroxyl group at position 1 of the steroid ring system of cholic acid.
• enzyme process or "enzymatic method” as used herein denotes a process or method employing an enzyme or microorganism.
• Compound of formula I formally is ip,3a,7a,12a-tetrahydroxy bile acid.
• Bile acids further provide conjugated bile acids, bile acid salts and bile acid derivatives.
• the present invention therefore, provides a method for the preparation of compound of formula I, further comprising conversion of the latter to a bile acid conjugate, or bile acid salt or a bile acid derivative.
• conjugated bile acids (compounds of formula II) usually refers to the product of condensation of a bile acid with glycine or taurine (see for example Jose JG Marin, World Journal of Gastrenterology 2009, 15(7), 804).
• Bile acid salts are salts of the conjugated bile acids, wherein the bile acid conjugate is in the form of an anion and the cation is sodium or potassium cation or any other cation of elements belonging to the group of alkali metals or alkaline earth metals. Preferable are sodium and potassium salts.
• the provision of conjugated bile acids and bile acid salts (Formula II) from compound of Formula I, is also encompassed in the present invention.
• the conversion of compound of Formula I to compound of Formula II is based on the formation of an amide bond, a chemical transformation well-known to the skilled person and exemplified in common text books of chemistry (see for example March's Advanced
• Genetic engineering also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. Genetic engineering is a process that alters the genetic make-up of an organism by either removing or introducing DNA. DNA can be introduced directly into the host organism or into a cell that is then fused or hybridized with the host. This relies on recombinant nucleic acid techniques that are known in the field of molecular biology to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro- injection or micro-encapsulation.
• the term "genetically engineered cytochrome P450 enzymes” or “genetically engineered heme-depended oxygenase enzymes” includes genetic variations created in vitro in various organisms or cell lines and with various molecular biology techniques.
• Non-limiting examples are heterologously expressed enzymes, commercially available engineered custom enzymes and microorganisms, sometimes referred to as biocatalysts, available through companies such as Codexis Inc., Cypex Ltd, etc.
• the enzymes employed in the present invention may be derived from the respective organisms. They may also be synthetically or otherwise prepared. For example, they may derive from genetically engineered host cells.
• the use of the genetically engineered host cells themselves, or cells which have otherwise been modified, is also contemplated where such cells are capable of producing enzymes having the structure of enzymes derived from the above recited genera of organisms.
• the present invention discloses an enzymatic method for the preparation of compound of formula I.
• Cytochrome P450 enzymes known to perform hydroxylations onto their substrates, have been disclosed in the prior art. However, the insertion of a hydroxyl group at position 1 of the steroid ring system is not a usual transformation within the prior art (see for example Donova et al, Appl. Microbiol. Biotechnol. 2012, 94, table 1). Enzymes produced from the cytochrome P450 genes are involved in the synthesis and metabolism of various molecules and chemicals within cells. Cytochrome P450 enzymes (CYPs) play a role in the synthesis of many molecules including steroid hormones. Additional cytochrome P450 enzymes metabolize external substances, such as medications, and internal substances, such as toxins that are formed within cells.
• CYP proteins There are approximately 60 CYP genes in humans.
• the CYP proteins contain a heme as a cofactor and are therefore hemoproteins. For the same reason those enzymes are considered heme-depended oxygenases, to underline the specific structural characteristic which is responsible for their behavior as powerful oxyfunctionalisation catalysts.
• the proteins are in general the terminal oxidase enzymes in electron transfer chains, broadly categorized as P450-containing systems. Cytochrome P450 enzymes are primarily found in liver cells but are also located in cells throughout the body. Within cells, cytochrome P450 enzymes are located in the endoplasmic reticulum; a structure involved in protein processing and transport and the energy-producing centers of cells, the mitochondria. The enzymes found in mitochondria are generally involved in the synthesis and metabolism of internal substances, while enzymes in the endoplasmic reticulum usually metabolize external substances, primarily medications and environmental pollutants.
• Cytochrome P450 enzymes account for 70 percent to 80 percent of enzymes involved in drug metabolism.
• Each cytochrome P450 gene is named with CYP, indicating that it is part of the cytochrome P450 gene family.
• the gene is also given a number associated with a specific group within the gene family, a letter representing the gene's subfamily, and a number assigned to the specific gene within the subfamily.
• the cytochrome P450 gene that is in group 27, subfamily A, gene 1 is written as CYP27A1.
• the cytochrome P450 system is an ubiquitous superfamily of monooxygenases that is present in plants, animals, and prokaryotes.
• the human genome encodes more than 50 members of the family, whereas the genome of the plant Arabidopsis encodes more than 250 members.
• Microbial cytochromes P450 are often soluble enzymes and are involved in diverse metabolic processes. In bacteria the distribution of P450s is very variable with many bacteria having no identified P450s (e.g. E.coli). Some bacteria, predominantly actinomycetes, have numerous P450s. Due to the solubility of bacterial P450 enzymes, they are generally regarded as easier to work with than the predominantly membrane bound eukaryotic P450s. This, combined with the remarkable chemistry they catalyse, has led to many studies using the heterologously expressed proteins in vitro.
• CYP enzymes have been disclosed to perform hydroxylation reactions in various positions of the steroid ring system.
• cholic acid has been reported not to undergo metabolism by means of hydroxylation by enzymes of the CYP family, in sharp contrast to other bile acids (Bodin et al, Biochimica et Biophysica Acta, 2005, 1687, 84).
• the inventors have surprisingly found that it is possible to obtain compound of formula I from cholic acid (Formula III) in one step, by performing a hydroxylation in position 1 catalyzed by a cytochrome P450 enzyme.
• the method disclosed herein overcomes the lengthy synthetic processes disclosed in prior art, wherein at least 10 chemical reactions are required to achieve this transformation.
• the heme-dependend oxygenase enzyme is a monooxygenase cytochrome P450 enzyme. It is therefore another object of the present invention to provide an enzymatic method for the preparation of compound of formula I, comprising the selective hydroxylation of cholic acid in one step in the presence of a monooxygenase cytochrome P450 enzyme, wherein the monooxygenase cytochrome P450 enzyme is genetically engineered.
• the cytochrome P450 enzyme may belong to any family of the P450 enzymes, which is suitable of carrying out the above described transformation.
• Preferable enzymes are CYP102A1, CYP102A7, CYP3A4, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1. More preferable is CYP102A1 enzyme. Even more preferable are the following enzymes commercially available from Codexis Inc.
• CYP102A1 enzymes are also microbial CYP enzymes. More preferable is CYP102A1 enzyme. Even more preferable are the following enzymes commercially available from Codexis Inc. (Codex® MicroCyp® kit): MCYP0002, MCYP0005, MCYP0009, MCYP0013, MCYP0014, MCYP0015, MCYP0016, MCYP0027, MCYP0029, MCYP0030, MCYP0032, MCYP0034, MCYP0035, MCYP0052, MCYP0057, MCYP-P1.2-A05, MCYP-P1.2-A07, MCYP-P1.2-A12, MCYP-P1.2-B10, MCYP-P1.2-B11, MCYP-P1.2-B12, MCYP-P1.2-D07, MCYP-P1.2-D09.
• the cytochrome P450 enzyme may be used with any suitable cofactor.
• cofactors are NAPH or NADPH.
• the cofactor regeneration system may be based on dehydrogenase substrate system.
• Dehydrogenase may be d/l-isocitrate dehydrogenase (IDH), glycerol dehydrogenase (GlyDH), formate dehydrogenase (FDH), alcohol dehydrogenase (ADH), glucose dehydrogenase (GDH), or glucose-6-phosphate dehydrogenase (G-6P-DH).
• IDH d/l-isocitrate dehydrogenase
• GlyDH glycerol dehydrogenase
• FDH formate dehydrogenase
• ADH alcohol dehydrogenase
• GDH glucose dehydrogenase
• G-6P-DH glucose-6-phosphate dehydrogenase
• the co-factor may be provided separately. Alternatively, the cofactor may be co-expressed with the enzyme.
• Sources of enzymes suitable for use in the present invention may be commercially available enzymes, available from sources such as Codexis Inc. USA. Alternatively, other suitable enzymes may be identified by methods well-known in the art. The enzyme may be used in the disclosed method according to techniques well known to the skilled person. They may be used as part of the cells producing them (whole cell catalysis) or in vitro, where the enzyme is available and is employed in the reaction media under appropriate reaction conditions.
• the method of the present invention may be conducted in an aqueous medium.
• the aqueous medium is water, preferably deionized water, or a suitable aqueous buffer solution, preferably a phosphate buffer solution.
• the reaction of the present method may also be conducted in a medium which is a mixture of an organic medium and an aqueous medium.
• the organic medium may be an organic solvent miscible or immiscible with water.
• Preferable organic solvents are alkanols, such as methyl, ethyl or isopropyl alcohol, dimethylsulfoxide, acetonitrile or acetone. Other solvents may include ionic liquids and deep eutectic solvents.
• the amount of enzyme employed, where employed in the present process, is preferably ranging from about 0.1 to about 100 ⁇ per 1 mol of the compound of the starting material compound of formula III.
• the amount of cofactor employed is preferably between 0.8 and 1.2 mM. It is preferable to employ a cofactor regeneration system based on dehydrogenase in the range between 0.3 and 1 mg per milliliter of liquid medium, and the appropriate dehydrogenase substrate in the range of 0.15 and 0.5 mM in the reaction liquid medium.
• the method of the present invention may be employed by means of whole cell catalysis.
• the reaction medium temperature may be such that the enzyme retains its enzymatic activity. It may be adjusted according to the restrictions of the enzyme. It is preferably maintained between 25 and 35° C, and most preferably between 28 and 32° C.
• the reaction medium is preferably shaken between 180 rpm and 250 rpm.
• the pH may be such that the enzyme retains its enzymatic activity. It may be adjusted according to the restrictions of the enzyme. Preferably, the pH is between 6.0 and 8.0.
• the reaction time can be varied depending upon the amount of enzyme and its specific activity. It may further be adjusted by the temperature or other conditions of the enzymatic reactions, which the skilled person is familiar with. Typical reaction times are ranging between 1 hour and 72 hours.
• the starting material is first dissolved, for example, in potassium phosphate buffer solution or acetonitrile or dimethylsulfoxide or an alcohol, and then is added to the reaction medium.
• the phosphate buffer is preferably between 50 mM and 150 mM and the pH between 7.5 and 8.5.
• the reaction medium preferably contains between about 0.1 to about 5 g of a compound of the formula III (cholic acid) as starting material per liter of liquid medium.
• the reaction medium is phosphate buffer, preferably potassium, between 50 mM and 150 mM, and pH between 7.5 and 8.5.
• the hydroxylation reaction of the present invention may require the addition of water or an organic alcohol, for example, an alkanol such as methyl, ethyl or isopropyl alcohol, or DMSO or acetonitrile or acetone.
• these materials are employed in an amount providing a molar excess, or a large molar excess, based on the starting material compound of formula III.
• a UHPLC MS method was developed and implemented for the determination of 1 ⁇ ,3 ⁇ ,7 ⁇ ,12 ⁇ - tetrahydroxy bile acid (Formula I) and cholic acid (Formula III). The analysis was performed on an UHPLC Nexera X2 - MS 2010EV system (Shimadzu Corp).
• Mobile phases A and B consisted of ultrapure water and Acetonitrile, respectively, wherein Mobile phase A (ultrapure water) contains 0.1% formic acid. Initially, a linear gradient of A/B changing from 90: 10 to 30:70 in 4 min, was employed after being held at the initial solvent composition for 2 min. Mobile phase composition changed linearly to A/B 10:90 over one minute and was held at A/B 10:90 for 2 min. The flow rate was set at 0.5 niL/min and the injection volume was 3 ⁇ L ⁇ .
• ESI mass spectrometric parameters was performed by using the reference standards of compound of formula I and cholic acid (compound of formula III).
• LC-MS determination was performed in selected ion monitoring (SIM) mode. Both modes positive and negative were applied, using rapid polarity switching.
• SIM selected ion monitoring
• MS scan was used to detect possible matrix interferences and unexpected co-elutions.
• a MicroCyp® 24-well plate (available from Codexis Inc.) is thawed at room temperature for 20 - 30 minutes.
• the MCYP-RXN buffer 1.65 g, included in the screening kit, containing potassium phosphate salts, NADP+, glucose and glucose dehydrogenase is dissolved in 46.6 mL DM water assisted by gentle shaking.
• the pH is adjusted to 8 by using 1M KOH aqueous solution.
• Cholic acid 4.23 mg are dissolved in 1.4 mL potassium phosphate buffer 100 mM, pH 8 and the resulting solution is added in the MCYP-RXN buffer solution bringing the final volume to 48 mL.
• the solution is homogenized assisted by gentle shaking.
• IPTG Isopropyl ⁇ -D-l-thiogalactopyranoside
• ALA 5-Aminolevulinic acid hydrochloride
• the cells were harvested at 8,000xrpm for 15min at 4°C.
• the cell pellet was resuspended in 5ml potassium phosphate buffer 50 mM pH 7.5. Cells were disrupted by sonication in ice water (2x 5min, intensity 50% (200watts), pulser 30%, OMNI Sonic Ruptor 400, Ultra Sonic Homogenizer), followed by centrifugation at 8,000xrpm, 4°C for 30min. The supernatant was stored at -20°C.
• E.coli C41 and 43 cells with no vector- gene for cytochrome P450 were used (this samples were treated in the same way as the rest of the samples).
• Stock solution B [Potassium phosphate buffer 50 mM pH 7.5]: A 100 mL volumetric flask is charged with 0.112 g potassium phosphate monobasic and 0.953 g potassium phosphate dibasic trihydrate. The volumetric flask is filled up to 100 mL with MQ-water and the solution is gently shaken for homogenization.
• Stock solution C [Stock solution of NADPH in potassium phosphate buffer 50 mM pH 7.5]: A 1.5 mL plastic tube is charged with 12.5 mg NADPH and 0.5 mL Stock solution B. The solution is homogenized.
• a 1.5 mL plastic tube is charged with 3 mg of enzyme, 0.980 mL from Stock solution B and 10 from the Stock solution A. The mixture is homogenized by shaking gently. Then, 10 of the Stock solution C are added and the plastic tube is place in a thermostated shaker at 200 rpm at 30 °C with cap off. Stock C 3.5 are added in the reaction mass every 30 minutes up to 6 h. Samples of 100 are withdrawn at 3 h, 4.5 h, 6 h and 22h and submitted to LC-MS analysis, as per example 1, after adjustment of the samples pH to 4-5 and centrifuging them at 4000 rpm for 15 minutes.

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