Method of producing cordycepin and its 2-halogen derivatives
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- VUAB PHARMA AS
- Filing Date
- 2025-07-21
- Publication Date
- 2026-05-15
AI Technical Summary
Current methods for producing cordycepin and its derivatives face challenges such as low yield, long cultivation cycles, strain instability, high production costs, environmental impact, and complexity in chemical synthesis, which hinder industrial scalability and demand satisfaction.
An enzymatic synthesis method using uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) to produce cordycepin and its 2-halogen derivatives through glycosylation or transglycosylation reactions, followed by alkaline precipitation and recrystallization, eliminating the need for organic solvents and chromatographic purification.
The method achieves high yields, reduced production costs, and environmental friendliness, making it suitable for both laboratory and industrial applications with simplified product isolation and scalability.
Abstract
Description
[0001] Method of producing cordycepin and its 2-halogen derivatives
[0002] Field of technology
[0003] The invention relates to a method of producing cordycepin and its 2-halogen derivatives under the catalytic action of enzymes in enzymatic synthesis.
[0004] State of the art
[0005] Cordycepin, 3 '-deoxyadenosine, whose molecule is shown in Fig. 1, is a water-insoluble structural analogue of the nucleoside adenosine. It is a secondary metabolite produced by some members of the Cordyceps genus. Cordycepin was originally isolated in 1950 from C. militaris.
[0006] Fig. 1
[0007] Based on in vitro and in vivo studies conducted to date, this compound has been confirmed to exhibit the following activities: immunostimulatory, anti-inflammatory, antiviral, antitumour, ergogenic, hypolipidemic, hypoglycaemic, and it regulates steroidogenesis and spermatogenesis.
[0008] In traditional methods, cordycepin is mainly obtained by extraction from the mycelium and fruiting bodies of C. militaris, but the amount of cordycepin produced per unit of dry weight is very small, less than 0.5%. In addition, the formation of C. militaris fruiting bodies in nature requires a specific ecological environment and host insects, which leads to its scarcity as a natural source. Given the depletion of natural sources of C. militaris and the long artificial cultivation cycle, there is a growing demand for alternative methods of cordycepin production, including microbial fermentation, chemical synthesis, and experimental biosynthesis using cordycepin synthase. However, despite undeniable progress, current production capacity is still far from meeting the actual demand for cordycepin.
[0009] Given that C. militaris can be easily cultivated on artificial media, including submerged and stationary cultures and solid media, this fungus is currently the leading candidate for the fermentation-based production of cordycepin. Therefore, considerable research efforts have been made to improve cordycepin yield, focusing on optimising extraction methods, cultivation conditions, strain improvement and biosynthesis.
[0010] Currently, increased growth and overproduction of cordycepin have been achieved using various industrial strains of C. militaris.
[0011] On the other hand, there are also many problems, such as the long growth cycle of C. militaris and the instability of the breeding strain, which often prevent the application of high-yield strains on an industrial scale.
[0012] Several semi-synthetic and total synthetic methods for the synthesis of cordycepin have also been described in the literature. The first method of chemical synthesis was described by Todd in 1960, which uses 3'-O- / ?nitrobenzenesulfonyladenosine as the starting material. McDonald then reported the first total synthesis of cordycepin based on dihydrofuranmethanol.
[0013] Aman et al., 2000, and Moreau et al., 2013, used 3-adenosine as the starting material for the chemical synthesis of cordycepin. Li et al., 2013, developed a total synthesis of cordycepin from the starting materials D-4-glucose and D-5-xylose. In 2017, Huang et al. optimised the 3- adenosine-based synthesis and increased both the product quality and the overall reaction yield.
[0014] Although the production of cordycepin using chemical synthesis processes is undergoing intensive development, it still has significant limitations because these processes often consist of multi-step reactions that involve protective and deprotective reactions starting from compounds that are expensive and / or difficult to obtain on the market, and sometimes even involve non-stereospecific reactions (i.e., reactions that lead to the production of the final product in both the a and P conformations). Furthermore, from the point of view of cordycepin production costs, it is disadvantageous that the purification process after chemical synthesis is complicated, and a large number of organic solvents are used in the chemical synthesis process, which significantly increases the ecological burden and overall environmental pollution.
[0015] Although cordycepin can be chemically synthesised, C. militaris remains its main source due to the cost of production and complexity.
[0016] An interesting alternative to chemical synthesis is the use of enzymatic synthesis of cordycepin. A procedure based on the use of PNP and cordycepin, or 3 -deoxyinosine, is available in the literature (Denisova Alexandra O. et al. The Chemoenzymatic Synthesis of 2-Chloro- and 2- Fluorocordycepins. Synthesis. Thieme, 20 July 2017, 49, Vol. 21, pp. 4853-4860, ISSN 0039- 7881).
[0017] The authors of the above article use a method based on the use of only the PNP enzyme as the sole catalyst for the enzymatic synthesis of cordycepin and its halogen derivatives, concluding thatthe use of a combination of UP and PNP enzymes is not a suitable process for the synthesis of chlorinated cordycepin derivatives.
[0018] The objective of the invention is a method of producing cordycepin and its 2-halogen derivatives which, compared to known methods of production, would be more economical, more environmentally friendly, and would at least partially satisfy the laboratory and industrial demand for cordycepin and its 2-halogen derivatives.
[0019] Essence of the invention
[0020] The task set is solved by a method of producing cordycepin and its 2-halogen derivatives according to the invention described below.
[0021] The invention provides an alternative to the above-mentioned methods of cordycepin production, introducing synthesis using enzymatic reactions such as glycosylation or transglycosylation, which are more suitable for industrial use. The advantage of enzymatic reactions is that the necessary recombinant enzymes can be prepared and produced by culturing bacterial cells.
[0022] It is also advantageous that the enzymes can be present in the reaction medium in the form of free enzymes or enzymes immobilised on suitable carriers or can be produced in situ by cells that produce them directly in the reaction medium.
[0023] The invention is supported by the fact that, to the best of the inventors' knowledge, enzymatic synthesis using transglycosidases has not yet been described in the technical literature.
[0024] The obj ective technical task of the invention is to create a completely newmethod for producing cordycepin and its 2-halogen derivatives. The invented method makes it possible to obtain cordycepin yields that are equal to or higher than the yields according to the prior art, and the invention also allows for easy isolation of the final product, including 2-halogen derivatives.
[0025] The essence of the invented method for producing cordycepin and its 2-halogen derivatives lies in the fact that the production method includes the following production steps: a) reaction of 3 -deoxyuridine with adenine to form cordycepin, or reaction of 3- deoxyuridine with 2-chloroadenine to form 2-chlorocordycepin, or reaction of 3- deoxyuridine with 2-fluoroadenine to form 2-fluorocordycepine, in the presence of uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) in an aqueous phosphate medium, b) isolation of cordycepin or its halogen derivatives, consisting in concentrating the solution and precipitating from the solution after alkalisation of the reaction medium to a pH in the range of 11.5 to 12.5, c) recrystallisation of cordycepin or its 2-halogen derivatives from a hydroalcoholic solution under reflux.
[0026] The enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) are present in the reaction medium in the form of free enzymes and may be in semi -purified or purified form. Semi-purified form means a crude protein precipitate obtained by a combination of heat precipitation followed by salting out, e.g. with ammonium sulphate. Purified form means an enzyme purified by chromatographic methods.
[0027] 3-deoxyuridine and adenine react advantageously in a molar ratio ranging from 1 : 1 to 3: l, most advantageously in a ratio of 2: 1.
[0028] The reaction between 3 -deoxyuridine and adenine is preferably carried out in an aqueous phosphate buffer solution at a pH in the range of 6.5 to 8.5, most preferably at a pH in the range of 7.3 to 7.8.
[0029] Withinthe scope of the invention, it is advantageous if the reaction is generally carried out at a temperature in the range of 40 °C to 55 °C.
[0030] In another aspect of the present invention, the reaction between 3 -deoxyuridine and adenine or 2-chloroadenine or 2-fluoroadenine is carried out by gradually adding a solution of adenine or 2-chloroadenine or 2-fluoroadenine to an aqueous phosphate medium buffer solution to a pH in the range of 6.5 to 8.5 and simultaneously containing enzymes and 3-deoxyuridine. Adenine or its halogen derivatives are added at a rate such that adenine or its halogen derivatives remain available inthe solution until they are converted to the final product, i.e. so thatno precipitation of adenine or its halogen derivatives occurs during the reaction.
[0031] The above-mentioned solution of adenine or its halogen derivatives is advantageously prepared by suspending adenine or its halogen derivatives in a concentrated hydroxide solution until complete dissolution of adenine or its halogen derivatives is achieved.
[0032] It is advantageous during the production of cordycepin or its halogen derivatives to add a strong acid solution simultaneously with the addition of the adenine or its halogen derivatives solution, in in such an amount that the pH of the reaction mixture is maintained between 6.5 and 8.5, preferably between 7.3 and 7.8.
[0033] Both mineral acids and organic acids can be used as strong acids. The synthetic method for producing cordycepin and its halogen derivatives according to the invention is fast, technologically and energetically undemanding, and environmentally friendly. The invention does not require the presence of organic solvents or chromatographic purification of pure cordycepin and is easily applicable both in laboratory operating conditions and in mass production operating conditions. At the same time, it is advantageous that the synthesis of halogenated cordycepin derivatives can be expected to significantly influence the pharmacological properties of cordycepin in the body.
[0034] When studying the possible use of a combination of UP and PNP and commonly available raw materials, the team of originators concluded that the combination of UP and PNP is a suitable, even significantly more suitable, method than the use of the method according to the above- mentioned references in the current state of the art and allows for the economically advantageous synthesis not only of chlorinated derivatives but also of cordycepin itself.
[0035] In addition, the process based on the combination of UP and PNP eliminates the need for expensive and limited resources, cordycepin or 3 -deoxyinosine, resulting in lower production costs. Therefore, the UP / PNP process is a more viable option to produce halogenated nucleosides.
[0036] The following comparison highlights the advantages of the UP / PNP process over the PNP- based process, emphasising the advantages of using inexpensive and readily available raw materials, improved yields, minimisation of by-product formation and the resulting simple purification method, which is reflected in lower production costs.
[0037] The process based on a combination of UP and PNP is superior to the PNP -based process due to its improved yield, regioselectivity and scalability to industrial scale.
[0038] Comparison of processes based on the use of PNP and a combination of UP and PNP:
[0039] Raw materials:
[0040] UP - expensive inputs - cordycepin, or 3 -deoxyinosine vs. commercially available 3- deoxyuridine and halogenated bases when using a combination of UP and PNP
[0041] Yield and regioselectivity: UP - low yields demonstrated only by analytical methods vs. improved yields and easy purification from the reaction mixture when using a combination of UP and PNP By-product formation:
[0042] UP - high level of by-products vs. minimal formation of by-products allowing easy and inexpensive purification when using a combination of UP and PNP Scale-up:
[0043] UP - difficult scale-up due to limited availability of raw materials and demanding purification vs. easy scale-up due to the use of commonly available raw materials and easy purification thanks to minimal impurity formation when using a combination of UP and PNP Costs:
[0044] UP - high production costs due to expensive raw materials and impurity production vs. lower production costs due to available raw materials and simpler synthesis and purification process when using a combination of UP and PNP
[0045] As can be seen from the above overview, the process of obtaining halogenated cordycepin derivatives based on a combination of UP and PNP is significantly more advantageous than the PNP-based process due to its improved yield, regioselectivity and transfer to industrial scale. In addition, the process based on the combination of UP and PNP allows the synthesis not only of halogenated derivatives of cordycepin, but also of cordycepin itself, which is an important raw material. Given their prices, it is difficult to imagine the use of cordycepin and 3- deoxyinosine on an industrial scale.
[0046] Example of implementation of the invention
[0047] It is understood that the specific examples of the invention described and illustrated below are presented for illustrative purposes only and do not limit the invention to the examples given. Those skilled in the art will find or be able to obtain, through routine experimentation, a greater or lesser number of equivalents to the specific embodiments of the invention described herein.
[0048] Example 1 : Process for the production of cordycepin
[0049] Dissolve 3 -deoxyuridine in 1 L of phosphate buffer solution and heat the solution to 55 °C.
[0050] While stirring constantly, add UP and PNP enzymes to the heated solution. A solution of adenine in 3% KOH is added gradually. The pH of the reaction is maintained at 7.5. The reaction is terminated after 60 minutes, when the solution is heated to 75 °C for 15 minutes. The precipitated reaction solution is filtered and concentrated on a vacuum evaporator approximately six times. The product is precipitated from the solution at pH 11.5-12.5. The product is filtered and dried at 80 °C for 2 hours. To increase purity, the product is dissolved in a hydroalcoholic solution in a ratio of 1 :5 to 1 : 15, with recrystallisation under reflux. The concentration of cordycepin produced is around 6.5 g / L (see table below).
[0051] Example 2: Procedure to produce 2-chloro cordycepin
[0052] Dissolve 3 -deoxyuridine in 1 L of phosphate buffer solution and heat the solution to 55 °C. While stirring continuously, add UP and PNP enzymes to the heated solution. Gradually add a solution of 2-chloradeninein 3% KOH. Maintain the pH of the reaction at 7.5. The reactionis terminated after 60 minutes, when the solution is heated to 75 °C for 15 minutes. The precipitated reaction solution is filtered and concentrated on a vacuum evaporator approximately six times. The product precipitates from the solution at a pH of 11.5-12.5. The product is filtered and dried at 80 °C for 2 hours. To increase purity, the product is dissolved in a hydroalcoholic solution in a ratio of 1 :5 to 1 : 15, with recrystallisation under reflux. The concentration of the 2-chlorocordycepine produced is approximately 4.5 g / L.
[0053] Example 3: Procedure for the production of 2-fluorocordycepine
[0054] Dissolve 3 -deoxyuridine in 1 L of phosphate buffer solution and heat the solution to 55 °C. While stirring continuously, add UP and PNP enzymes to the heated solution. Gradually add a solution of 2-fluoradeninein 3% KOH. Maintain the pH of the reaction at 7.5. The reactionis terminated after 60 minutes, when the solution is heated to 75 °C for 15 minutes. The precipitated reaction solution is filtered and concentrated on a vacuum evaporator approximately six times. The product precipitates from the solution at a pH of 11.5-12.5. The product is filtered and dried at 80 °C for 2 hours. To increase purity, the product is dissolved in a hydroalcoholic solution in a ratio of 1 : 5 to 1 : 15, where recrystallisation occurs under reflux. The concentration of the produced 2-fluorocordycepine is approximately 2.9 g / L.
[0055] Industrial applicability
[0056] The method of producing cordycepin and its 2-halogen derivatives according to the invention will find application in the synthesis of cordycepin as an alternative source to natural cordycepin obtained by culturing the fungus C. militaris on insects or on artificial culture media, or its 2- halogen derivatives.
Claims
PATENT CLAIMS1. Method of producing cordycepin, chemically named 3'-deoxyadenosine, and its 2-halogen derivatives, characterised in that it consists of a set of process steps: a) reaction of 3 -deoxyuridine with adenine to produce cordycepin, or reaction of 3- deoxyuridine with 2-chloroadenine to produce 2-chlorocordycepin, or reaction of 3-deoxyuridine with 2-fluoroadenine to produce 2-fluorocordycepine, in the presence of uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) in an aqueous phosphate medium, b) isolation of cordycepin or its 2-halogen derivatives by concentrating the solution and precipitating them from the solution after alkalising the reaction medium to a pH in the range of 11.5 to 12.5, c) purification of cordycepin or its 2-halogen derivatives by recrystallisation of the primary precipitate from a hydroalcoholic solution under reflux.
2. Method according to claim 2, characterised in that 3-deoxyuridine and adenine or 2- chloroadenine or 2-fluoroadenine are reacted in a molar ratio of 1 : 1 to 3 : 1 in process step a).
3. Method according to claim 1 or 2, characterised in that the reaction of process step a) is carried out in an aqueous phosphate medium which pH is in the range from 6.5 to 8.5.
4. Method according to any of claims 1 to 3, characterised in that the reaction of process step a) is carried out at a temperature in the range of 40-55 °C.
5. Method according to any of claims 1 to 4, characterised in that the reaction of process step a) is carried out by gradually adding a solution of adenine or 2-chloroadenine or 2- fluoroadenine in an alkaline medium to an aqueous phosphate reaction medium already containing the enzymes phosphorylase (UP) and purine nucleoside phosphorylase (PNP) and further 3-deoxyuridine.
6. Method according to claim 5, characterised in that the solution of adenine or 2- chloroadenine or 2-fluoroadenine is prepared by dissolving in a hydroxide solution.
7. Method according to any of claims 1 to 6, characterised in that process step a) is terminated by inactivating the enzymes used by boiling and then filtering them off.
8. Method according to any of claims 1 to 7, characterised in that, in process step b), the volume is concentrated six to seven times, the pH is adjusted using a concentrated hydroxide solution, and cordycepin or its 2-halogen derivatives are precipitated.
9. Method according to claim 8, characterised in that the resulting precipitate containing more than 90% cordycepin or its 2-halogen derivatives is separated by filtration, washed with distilled water, dried at 80 °C and recrystallised under reflux from a hydroalcoholic solution in a ratio of 1 :5 to 1 : 15.