WO2014094312A1

WO2014094312A1 - Method for synthesizing nucleoside amino acid derivatives through enzyme catalysis

Info

Publication number: WO2014094312A1
Application number: PCT/CN2012/087204
Authority: WO
Inventors: 李金亮; 张仙; 赵楠; 金圣芳
Original assignee: 上海迪赛诺化学制药有限公司
Priority date: 2012-12-21
Filing date: 2012-12-21
Publication date: 2014-06-26

Abstract

Disclosed is a method for synthesizing nucleoside amino acid derivatives through enzyme catalysis, which comprises the step of: under the catalysis of protease or lipase, nucleoside compounds and L-amino acid ester reacting to obtain nucleoside amino acid derivatives. This method does not require other solvents.

Description

Method for catalyzing the synthesis of nucleoside amino acid derivatives by enzyme

Technical field

The invention belongs to the technical field of biocatalysis synthesis, and particularly relates to a method for enzymatically synthesizing a nucleoside amino acid derivative. Background technique

In recent years, nucleoside drugs have received extensive attention for the treatment of viral diseases. However, clinically used nucleoside drugs have problems such as toxic side effects and drug resistance. Nucleoside prodrugs can improve the oral bioavailability and pharmacokinetics of nucleoside drugs, target drugs to specific lesions, prolong the action time, reduce toxic side effects, improve antiviral effect, expand antiviral spectrum, and modify nucleosides. An important direction of drug-like structure. For example, valacyclovir is acyclovir L-valine methyl ester, which can improve the malabsorption of acyclovir by oral administration. It can be well absorbed in the gastrointestinal tract after oral administration and completely hydrolyzed by enzyme. Conversion to acyclovir, which can increase the bioavailability of acyclovir by 3 to 5 times, reaching 65%. The antiviral activity and anti-HIV activity of the amino acid ester prodrug derivatives of tenofovir are significantly higher than that of tenofovir. Cytarabine is mainly used in the treatment of acute leukemia, including lung cancer, digestive tract cancer, head and neck cancer and malignant lymphoma. It is an anti-metabolic drug, which is improved by improving the molecular structure of the drug. An important way.

Modification of the molecular structure of nucleoside drugs by traditional chemical synthesis requires complex steps such as acylation and catalytic hydrogenolysis, and requires the use of special chemical catalysts, usually with low yield, poor selectivity, and contamination. And various disadvantages such as toxicity. Enzymatic synthesis has the characteristics of mild conditions and good selectivity, and is widely used in the derivatization of multi-functional drugs, especially nucleoside drugs. In recent years, many biocatalytic methods have been used to structurally modify nucleoside drugs. Reports such as Riva et al. (J Am Chem Soc, 1988, 110: 584) first reported the selective esterification of adenosine and uridine primary hydroxyl groups in DMF with subtilisin, which has a long reaction period (requires 7~) 8 days), the reaction selectivity is poor, and there are many products. Hanson et al. (Bioorg. Med. Chem., 2000, 8: 2681) reported the preparation of lobe in acetone and DMF using ChiroCLECTM-BL and immobilized lipase derived from Pseudomonas cepacii {Pseudomonas ce ^ci^ A proline prodrug of Lobucavir (BMS 180194), which is used to treat herpes virus and hepatitis B. However, the concentration of the reaction substrate is low (10 g/l), the reaction by-products are more, the selectivity is lower, and the post-treatment is complicated. Tamarez et al. (Org Process Res Dev, 2003, 7: 951) discloses a method for the catalytic synthesis of ribavirin amino acid derivatives by CAL lipase in anhydrous tetrahydrofuran, which requires a large amount of anhydrous tetrahydrofuran as a solvent. It is not conducive to environmental protection, and post-processing is cumbersome. Kathleen McClean et al. (Tetrahedron Letters, 2011, 52: 215) report the use of immobilized enzymes of commercial subtilisin (ChiroCLEC-BL) catalyzes the transesterification of L_proline methyl ester with acyclovir to synthesize valacyclovir, and the reaction is small in scale, and the enzyme source ChiroCLEC-BL cannot be obtained by simple commercial means. The difficulty of industrial application of this process. Lin et al. (Bioorg Med Chem Lett, 2005, 15 : 4064) reported the catalytic synthesis of cytarabine prodrugs in an organic medium using Bacillus subtilis alkaline protease, which is poorly selective and produces a polymerizable 5'-0- Various products such as acyl cytarabine derivatives and 4-N-acyl cytarabine derivatives; catalyzed synthesis of cytarabine in organic medium by using Candida antarctica lipase B (CALB) as a catalyst The prodrug only selectively esterifies the 5'-O-hydroxyl position of cytarabine, however the conversion of this reaction is very low.

Existing enzyme-catalyzed methods for synthesizing nucleoside prodrugs generally have the problem of requiring a large amount of organic solvent, low selectivity or the need to select a specific protease, resulting in environmentally unfriendly, low conversion rate, complicated post-treatment, and process. It is cumbersome, raw materials or reagents are not easy to obtain, and economic costs are high. Therefore, the development of a high-conversion, simple, and environmentally friendly enzyme-catalyzed synthesis of nucleoside prodrugs is an important direction for antiviral drug research. Summary of the invention

In order to solve the defects of the prior art, the present invention provides a method for synthesizing a nucleoside amino acid derivative by a protease or a lipase which is simple in operation, high in selectivity, mild in temperature, and low in solvent and low in pollution.

The first aspect of the present invention provides a method for enzymatically synthesizing a nucleoside amino acid derivative, comprising the steps of: reacting a nucleoside compound and an L-amino acid ester under a catalysis of a protease to obtain a nucleoside amino acid derivative;

The nucleoside compound is acyclovir, ganciclovir, inosine, guanosine, adenosine or cytarabine; the L-amino acid ester is a liquid L-serine ester, a liquid L- Alanine ester, liquid L-cysteine ester or liquid L-phenylalanine ester;

The nucleoside amino acid derivative is acyclovir-L-serine ester, acyclovir-L-alanine ester, acyclovir-L-cysteine ester, acyclovir-L -phenylalaninate, ganciclovir-L-serine ester, ganciclovir-L-alaninate, ganciclovir-L-cysteine, ganciclovir-L-benzene Alanine ester, inosine-L-serine ester, inosine-L-alaninate, inosine-L-cysteine, inosine-L-phenylalaninate, guanosine-L- Serine ester, guanosine-L-alanine ester, guanosine-L-cysteine ester, guanosine-L-phenylalanine ester, adenosine-L-serine ester, adenosine-L-alanine Acid ester, adenosine-L-cysteine ester, adenosine-L-phenylalanine ester, cytarabine-L-serine ester, cytarabine-L-alanine ester, arsenic Glycosyl-L-cysteine ester or cytarabine-L-phenylalanine ester;

The proteinase is Bacillus subtilis alkaline protease, Bacillus licheniformis alkaline protease, alkaline protease 3. 0T, alkaline protease 2. 4L FG, Saiwei proteinase 8. 0T, Saiwei proteinase 16. 0 L, Yi Rui Protease 8. 0 L, Iverase 16. 0 L or thermolysin.

In another preferred embodiment, the protease is alkaline protease 3. 0T.

In another preferred embodiment, the L-serine ester is L-serine d- ₄ ester; and/or the L-alanine ester is L-alanine d- ₄ ester; and/or The L-cysteine ester is L-cysteine (and ₄ ); and/or the L-phenylalanine ester is L-phenylalanine d- ₄ ester.

In another preferred embodiment, the L-serine ester is L-serine methyl ester, L-serine ethyl ester, L-serine n-propyl ester or L-serine methyl ester.

In another preferred embodiment, the L-alanine ester is L-alanine methyl ester.

In another preferred embodiment, the L-cysteine ester is L-cysteine methyl ester.

In another preferred embodiment, the L-phenylalanine ester is L-phenylalanine methyl ester.

The second aspect of the present invention provides a method for enzymatically synthesizing a nucleoside amino acid derivative, comprising the steps of: reacting a nucleoside compound and an L-amino acid ester under the catalysis of a lipase to obtain a nucleoside amino acid derivative; ,

The nucleoside compound is acyclovir, ganciclovir, inosine, guanosine or adenosine;

The L-amino acid ester is a liquid L-valine ester, a liquid L-serine ester, a liquid L-alanine ester, a liquid L-cysteine ester or a liquid L-phenylalanine. Acid ester

The nucleoside amino acid derivatives are acyclovir-L-valine ester, acyclovir-L-serine ester, acyclovir-L-alaninate, acyclovir-L- Cysteine ester, acyclovir-L-phenylalanine ester, ganciclovir-L-valine ester, ganciclovir-L-serine ester, ganciclovir-L-alanine Acid ester, ganciclovir-L-cysteine ester, ganciclovir-L-phenylalanine ester, inosine-L-valine ester, inosine-L-serine ester, inosine- L-alanine ester, inosine-L-cysteine ester, inosine-L-phenylalanine ester, guanosine-L-valine ester, guanosine-L-serine ester, guanosine- L-alanine ester, guanosine-L-cysteine ester, guanosine-L-phenylalanine ester, adenosine-L-valine ester, adenosine-L-serine ester, adenosine- L-alanine ester, adenosine-L-cysteine ester or adenosine-L-phenylalanine ester;

The lipase is Libo lipase 100T, Νονο435 lipase, lipase PS Amano SD, lipase AS Amano, lipase AK Amano, lipase G, lipase AYS Amano, Candida antarctica lipase B (CALB) ) or alkaline lipase Greasex 50L.

In another preferred embodiment, the L-valine ester is an L-valine C1-4 ester; and/or the L-serine ester is an L-serine d- ₄ ester; and/or The L-alanine ester is L-alanine d- ₄ ester; and/or the L-cysteine ester is L-cysteine CH ester; and/or the L- The phenylalanine ester is L-phenylalanine d- ₄ ester.

In another preferred embodiment, the L-valine ester is L-valine methyl ester or L-valine ethyl ester. In another preferred embodiment, the L-serine ester is L-serine methyl ester, L-serine ethyl ester, L-serine n-propyl ester or L-serine methyl ester.

In another preferred embodiment, the L-alanine ester is L-alanine methyl ester.

In another preferred embodiment, the L-amino acid ester is L-valine methyl ester.

In another preferred embodiment, the lipase is Candida antartica lipase B.

In another preferred embodiment, the nucleoside compound is acyclovir or ganciclovir.

In another preferred embodiment, the concentration of the nucleoside compound in the reaction system is 10 to 50 g/L. In another preferred embodiment, the concentration of the nucleoside compound in the reaction system is 20 to 30. g/L o In another preferred embodiment, the protease or lipase is present in the reaction system at a concentration of 8 to 65 g/L. In another preferred embodiment, the concentration of the protease or lipase in the reaction system is 15 to 30 g/L. In another preferred embodiment, the reaction is carried out at 20 to 70 ° C; and / Or the reaction described is carried out 24~

120 hours.

In another preferred embodiment, the reaction is carried out at 30 to 50 °C.

In another preferred embodiment, the reaction is carried out for 72 to 96 hours.

In another preferred embodiment, the reaction is carried out at a relative vacuum of from -100 to -500 mbar.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. Specific embodiment

The inventors have extensively and intensively studied and unexpectedly discovered that a nucleoside amino acid derivative is prepared by a solventless reaction of a nucleoside compound with an L-amino acid ester which is liquid at a reaction temperature by using a protease or a lipase as a catalyst. Economical, environmentally friendly, mild reaction conditions, high conversion rate, simple product purification and high purity. On this basis, the inventors have completed the present invention. the term

As used herein, the L-valine d- ₄ ester is L-valine decyl ester, such as L-valine methyl ester, L-valine ethyl ester, L-valine n-propyl Ester, L-valine isopropyl ester, L-valine n-butyl ester, L-valine isobutyl ester, L-valine t-butyl ester, or the like.

The L-serine d- ₄ ester is L-serine d- ₄ decyl ester, such as L-serine methyl ester, L-serine Ethyl ester, n-propyl L-serine, isopropyl L-serine, n-butyl L-serine, isobutyl L-serine, tert-butyl L-serine, or the like.

The L-alanine d- ₄ ester is L-alanine CH-ester, such as L-alanine methyl ester, L-alanine ethyl ester, L-alanine n-propyl ester, L - isopropyl alaninate, n-butyl L-alanine, isobutyl L-alanine, tert-butyl L-alanine, or the like.

The L-cysteine d- ₄ ester is L-cysteine CH-ester, such as L-cysteine methyl ester, L-cysteine ethyl ester, L-cysteine N-propyl ester, L-cysteine isopropyl ester, L-cysteine n-butyl ester, L-cysteine isobutyl ester, L-cysteine tert-butyl ester, or the like.

The L-phenylalanine d- ₄ ester is L-phenylalanine d- ₄ decyl ester, such as L-phenylalanine methyl ester, L-phenylalanine ethyl ester, L-phenyl propyl N-propyl propyl ester, isopropyl L-phenylalanine, n-butyl L-phenylalanine, isobutyl L-phenylalanine, tert-butyl L-phenylalanine, or the like .

As used herein, the "nucleoside amino acid derivative" is a compound shown in Table 1.

Cytarabine-L-serine ester

Cytarabine-L-alanine ester

Cytarabine-L-cysteine ester

Cytarabine-L-phenylalanine ester

Enzyme catalyzed reaction

The present invention provides a preferred method for enzymatically synthesizing a nucleoside amino acid derivative, which comprises the steps of: reacting a nucleoside compound with an L-amino acid ester under the catalysis of a protease to obtain a nucleoside amino acid derivative.

The nucleoside compound is preferably acyclovir, ganciclovir, inosine, guanosine, adenosine or cytarabine. The L-amino acid ester is preferably a liquid L-serine ester, a liquid L-alanine ester, a liquid L-cysteine ester or a liquid L-phenylalanine ester. Preferably, the L-serine ester is L-serine d- ₄ ester; the L-alanine ester is L-alanine CH ester; and the L-cysteine ester is L- Cysteine d- ₄ ester; the L-phenylalanine ester is L-phenylalanine (^ ₄ ester).

The protease is preferably Bacillus subtilis alkaline protease, Bacillus licheniformis alkaline protease, Alcalase 3. 0T, Alcalase 2. 4L FG, Savinase protease 8. 0T , Savinase 16. 0 L, Esperase 8. 0 L, Eversase 16. 0 L or thermolys in; preferably, said Egg The white enzyme is alkaline protease (Alcalase) 3. 0T, which has better catalytic effect and higher catalytic efficiency and selectivity. The nucleoside amino acid derivatives are: acyclovir-L-serine ester, acyclovir-L-alanine ester, acyclovir-L-cysteine ester, acyclovir- L-phenylalanine ester, ganciclovir-L-serine ester, ganciclovir-L-alaninate, ganciclovir-L-cysteine, ganciclovir-L- Phenylalanine ester, inosine-L-serine ester, inosine-L-alanine ester, inosine-L-cysteine ester, inosine-L-phenylalanine ester, guanosine-L - Serine ester, guanosine-L-alanine ester, guanosine-L-cysteine ester, guanosine-L-phenylalanine ester, adenosine-L-serine ester, adenosine-L-propyl Acid ester, adenosine-L-cysteine ester, adenosine-L-phenylalanine ester, cytarabine-L-serine ester, cytarabine-L-alanine ester, arabinose Cytidine-L-cysteine ester or cytarabine-L-phenylalanine ester.

The present invention provides another preferred method for the enzyme-catalyzed synthesis of a nucleoside amino acid derivative, which comprises the steps of: reacting a nucleoside compound with an L-amino acid ester under the catalysis of a lipase to obtain a nucleoside amino acid derivative.

The nucleoside compound is preferably acyclovir, ganciclovir, inosine, guanosine or adenosine; the L-amino acid ester is preferably a liquid L-valine ester, a liquid L-serine Ester, liquid L-alanine ester, liquid L-cysteine ester or liquid L-phenylalanine ester. Preferably, the L-amino acid ester is preferably a liquid L-valine ester, a liquid L-serine ester, a liquid L-alanine ester, a liquid L-cysteine ester or a liquid L. - phenylalanine ester. Preferably, the L-valine ester is L-valine d- ₄ ester, the L-serine ester is L-serine d- ₄ ester; and the L-alanine ester is L - alanine d- ₄ ester; the L-cysteine ester is L-cysteine ester; and the L-phenylalanine ester is L-phenylalanine d- ₄ ester.

The lipase is Lipolas e 100T, Novo435 lipase, lipase PS Amano SD, Lipase AS Amano, Lipase AK Amano ), Lipase G, lipase AYS L ipase AYS Amano, Candida antarctica lipase B (CALB) or alkaline lipase Greasex 50L. Preferably, the lipase is Candida antarctica lipase B, which has better catalytic effect and higher catalytic efficiency and selectivity.

The nucleoside amino acid derivatives are: acyclovir-L-valine ester, acyclovir-L-serine ester, acyclovir-L-alaninate, acyclovir-L -cysteine ester, acyclovir-L-phenylalanine ester, ganciclovir-L-valine ester, ganciclovir-L-serine ester, ganciclovir-L-propyl Acid ester, ganciclovir-L-cysteine ester, ganciclovir-L-phenylalanine ester, inosine-L-valine ester, inosine-L-serine ester, inosine -L-alanine ester, inosine-L-cysteine ester, inosine-L-phenylalanine ester, guanosine-L-valine ester, guanosine-L-serine ester, guanosine -L-alanine ester, guanosine-L-cysteine ester, guanosine-L-phenylalanine ester, adenosine-L-valine ester, adenosine-L-serine ester, adenosine -L-alanine ester, adenosine-L-cysteine or adenosine -L-phenylalanine ester.

The above two methods of the present invention employ an L-amino acid ester (preferably L-amino acid- ₄ ester) which is liquid at the reaction temperature, and can be enzymatically reacted without adding other solvents by utilizing its liquid state. The liquid L-amino acid ester serves as both a reactant and a solvent, and an increase in the amount of the L-amino acid ester can increase the conversion efficiency of the product, and the excess L-amino acid ester can be recycled and reused. Therefore, the method has good selectivity, high conversion rate, high yield (yield exceeds 65%, preferably 65-95% or 70-90%), good quality, low cost, less environmental pollution, It is easy to operate and is suitable for industrialization.

In the method of the present invention, the reaction system is composed of an L-amino acid ester and a nucleoside compound, and a protease or a lipase is added, and the enzyme is catalyzed at a certain temperature (for example, 20 to 70 ° C; preferably 30 to 50). The reaction is carried out for a period of time (e.g., 24 to 120 hours; preferably 72 to 96 hours) to synthesize a series of nucleoside amino acid derivatives. The reaction time refers to the time required until the conversion of the catalytic reaction is substantially constant, and the so-called "time at which the conversion rate is substantially constant" means a time when the conversion rate changes by 5% every 24 hours. The concentration of the nucleoside compound in the reaction system is 10 to 50 g/L, preferably 20 to 30 g/Lo.

Increasing the amount of protease or lipase can increase the catalytic effect, but the use of too much enzyme easily affects the dispersion of the enzyme in the reaction system, which is not conducive to the entry and exit of the substrate at the enzyme catalytic site, thereby hindering the reaction. Therefore, the present invention selects an appropriate amount of protease or lipase to facilitate the reaction. The protease or lipase has a concentration in the reaction system of 8 to 65 g/L, preferably 15 to 30 g/L. Preferably, the protease or lipase is anhydrous or substantially anhydrous. Compared with the prior art, the method of enzymatically synthesizing nucleoside amino acid compounds is mainly used in the present invention.

1. The enzyme used in the method is convenient in source, and can be used as a catalyst to catalyze the reaction of a nucleoside compound and an L-amino acid ester without a special treatment procedure, thereby producing a product with high optical purity and high yield. The enzyme of the present invention has the characteristics of good selectivity and high conversion rate.

2. The L-amino acid ester in the method is a reactant and a solvent, and the reaction is carried out without adding other solvents, the reaction substrate concentration is high, the product conversion efficiency is high, the quality is good, and the excess L-amino acid ester can be recovered. Reuse, which reduces costs and reduces environmental pollution.

3. The method has mild reaction conditions, simple operation, simple post-treatment and wide industrial application prospects, and has important application value in the preparation and research of nucleoside prodrugs. The invention will be further elucidated below in conjunction with specific implementations. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples that do not specify the specific conditions are usually According to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Raw materials or reagents such as acyclovir, ganciclovir, inosine, guanosine, adenosine, cytarabine, L-amino acid ester hydrochloride, etc., which are used in the present invention, are commercially available products unless otherwise specified. Example 1

1. Preparation of 1 L-valine methyl ester

Add L-valine methyl ester hydrochloride (30 g, 178.9 mmol) to a 500 ml stoppered flask, add water (50 ml) and dichloromethane (30 ml), and dissolve the L_valine methyl ester salt with magnetic stirring. salt, wait until completely dissolved, sodium hydrogen carbonate (18g, 214. 2 bandit _01, 1.2 equivalents) was added to the flask, reaction was rapidly stirred until no bubble generation. The mixture was transferred to a separatory funnel, and the methylene chloride layer was separated. The aqueous layer was extracted with methylene chloride (30 ml). The organic layer was combined and dried over anhydrous sodium sulfate. Liquid (L-valine methyl ester 22g, 93.7%) o

The preparation of the other L-amino acid esters is the same as this, except that different L-amino acid ester hydrochlorides are used instead of L-valine methyl ester hydrochloride.

1. 2 Preparation of Bacillus subtilis and Bacillus licheniformis alkaline protease

1) Fermentation culture of Bacillus subtilis SO Bacillus licheniformis S02

Bacillus subtilis S01, Bacillus licheniformis S02 was purchased from the China General Microorganisms Collection and Management Center (CGMCC).

Bacillus subtilis S01 fermentation medium (g/L): yeast extract 20, sucrose 10, Tween-80 5, magnesium sulfate 0·2, pH 9.

2, Sodium citrate 0. 3, Sodium citrate 0. 02, Manganese sulphate 0. 02, Sodium sulphate 50. , pH 8. 7.

The above two kinds of medium were sterilized at 121 °C for 15 min, cooled, sterilized, inoculated with Bacillus subtilis S01 or Bacillus licheniformis S02, inoculated in 4%, fermented for 36 h at 37 ° C, 200 r/min. The supernatant of the fermentation broth was collected by centrifugation at 7500 rcf for 10 min.

2) Alkaline protease extraction from Bacillus subtilis, Bacillus licheniformis

Take 100 mL of the fermentation broth supernatant, slowly add 2 volumes of frozen acetone under ice bath conditions, continue to stir for 1~2 h, and then centrifuge at 10 °C for 7 min at 4 °C to obtain protein precipitate. The precipitate will be at room temperature. The cells were dried under air, and the obtained proteins were Bacillus subtilis alkaline protease and Bacillus licheniformis alkaline protease, respectively. Used after Continued in the examples. Example 2 Enzyme catalytic reaction

To the reaction system, add acyclovir 40 mg, alkaline protease Alcalase 3. 0T 20 mg, then add L-alanine methyl ester 2 ml, mix and add 4A molecular sieve 0. 1 g, put in 50 ° C The reaction was carried out in a constant temperature shaking incubator for 24 h at a speed of 250 rpm/min. The conversion of the above reaction was determined by liquid chromatography to evaluate the catalytic activity of the enzyme catalyst (see Table 2).

Conversion rate (%) = product peak area / (product peak area + substrate peak area) * 100% content determination method - acyclovir and product acyclovir-L-alanine ester content using high-performance liquid phase The chromatographic conditions were as follows: C18-XDB column (250 X 4. 6 mm, Agi lent), mobile phase methanol/water (volume ratio 20/80), column temperature 30 ° C; flow rate 0. 8 Ml/min, detection wavelength 254 nm, injection volume 5 μ 1 . The peak time of acyclovir standard was 3. 4 min, and the peak time of acyclovir-L-alanine standard was 5. 3 min. Example 3-10 Enzymatic Reaction

The experimental method was the same as in Example 2, except that the reaction system was carried out under the conditions shown in Table 2, respectively.

9 Bacillus subtilis alkaline protease according to Example 1. Step 1. 2 Preparation 65 +

10 Bacillus licheniformis alkaline protease according to the procedure of Example 1. 1. Preparation of 65 + + Note: When the conversion rate is less than 5%, the catalytic activity is marked as "+"; when the conversion rate is 5~10%, the catalytic activity Marked as "++"; when the conversion rate is 10~15%, the catalytic activity is marked as "+++"; when the conversion rate is 15~20%, the catalytic activity is labeled "++++".

The results showed that alkaline protease (Alcalase 3. 0T and Alcalase 2. 4L FG) was selected as the catalyst to catalyze the highest catalytic activity of acyclovir for the synthesis of acyclovir-L-alanine ester, and lipase such as CALB. It exhibited a catalytic effect comparable to that of Alcalase 3. 0T and Alcalase 2. 4L FG. Example 11 Enzyme catalytic reaction

To the reaction system, 40 mg of inosine was added, and then 2 ml of L-alanine methyl ester was added to form a reaction system having a substrate concentration of 20 g/L, and Savinase 8. 0 T 40 mg and 4A molecular sieves were added. The reaction was carried out in a 50 ° C constant temperature shaking incubator for 48 h at a speed of 250 rpm. The conversion rate of the above reaction was measured by liquid chromatography for 48 hours, and the catalytic activity of the enzyme catalyst was evaluated, wherein inosine and L-alanine were evaluated. The reaction activity of the acid ester at the end of the transesterification reaction was determined to be 100%. The results are shown in Table 3. Example 12-21 Enzymatic Reaction

The experimental procedure was the same as that of Example 11, except that the conversion of the above reaction was measured by liquid chromatography using a raw material or an enzyme catalyst as shown in Table 3, and the catalytic activity of the enzyme catalyst was evaluated.

The results are shown in Table 3.

Table 3 Commercialization of enzymes to synthesize several nucleoside amino acid derivatives

The results show that the choice of alkaline protease or lipase as a catalyst can catalyze the selection of various nucleoside compounds. The transesterification reaction can be used to structurally modify the polyfunctional nucleoside compound. The lipase CALB is used to modify the acyclovir with L-valine methyl ester. The catalytic activity is the highest, which is cytarabine 5 . Times around. Example 22

Add acyclovir 200 mg (0.89 mmol) to a 50 ml round bottom flask, and add 20 ml of L-serine methyl ester to form a reaction system with a substrate concentration of 10 g/L, and add thermolysin. 8度。 The reaction was carried out at 70 ° C under a magnetic stirring condition (vacuum degree -300 mbar) for 72 h, the conversion of the reaction was determined by liquid chromatography to be 45.3%, the reaction was continued for 24 h, and the conversion of the reaction was 68. 2%。 3%, the reaction was continued for 24h, the conversion rate of the determination was 78.2%. 9%。 The conversion was determined to be 81.9%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to obtain acyclovir-L-serine ester hydrochloride 240. 7 mg (0. 69 leg ol, molar yield 77.5%), optical purity 99. 2%. Example 23

Add acyclovir 200 mg (0.89 mmol) to a 50 ml round bottom flask, and add 10 ml of L-cysteine methyl ester to form a reaction system with a substrate concentration of 20 g/L. Add alkaline protease. (3), Continued reaction. The reaction was determined by liquid chromatography. The conversion was 43.3%, and the reaction was continued at 50 ° C under a magnetic stirring condition (vacuum degree - 700 mbar). 6%。 The conversion rate of the reaction was determined to be 61. 6%, and the conversion of the reaction was continued for 24 hours, the conversion of the reaction was determined to be 76. 5%, and the reaction was continued for another 24 hours, the conversion of the reaction was determined to be 79.6%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to obtain acyclovir-L-cysteine ester hydrochloride 251.7 mg (0. 69 mmol, molar yield 77.5%), optical 1%。 Purity 99.1%. Example 24

Add 50 mg (0. 39 mmol) of ganciclovir to a 50 ml round bottom flask, add 10 ml of L-serine methyl ester to form a reaction system with a substrate concentration of 10 g/L, and add alkaline protease (Alcalase). The reaction conversion rate is 46.2%, the reaction is continued for 24 hours, and the reaction is determined. The reaction is carried out at a temperature of 50 ° C under a magnetic stirring condition (vacuum degree - 500 mbar) for 72 h. The conversion rate was 86.1%. The conversion rate of the reaction was 86.1%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to obtain ganciclovir-L-serine ester hydrochloride 149. Omg (0. 32 leg ol, molar yield 82.0%), optical purity 99. 5%. Example 25

Inosine 200 mg (0.74 mmol) was added to a 50 ml round bottom flask, followed by L-alanine methyl ester 10 ml. A reaction system having a substrate concentration of 20 g/L was formed, and Savinase 16. OL O. 5 g was added, and the reaction was carried out under reduced pressure (vacuum degree - 700 mbar) at 60 ° C for 72 h. The reaction rate of the reaction was determined to be 41.2%, and the reaction was continued for 24 hours. The conversion of the reaction was determined to be 59.8%, and the reaction was continued for 24 hours. The conversion of the reaction was 74.5%, and the reaction was continued for 24 hours. 8%。 The conversion rate of the determination was 78.8%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to give inosine-L-alanine ester hydrochloride 214. 2 mg (0. 57 mmol, molar yield 77.0%), optical purity 99. 6%. Example 26

Add cytarabine 200 mg (0. 82 mmol) to a 50 ml round bottom flask, and add 10 ml of L-phenylalanine methyl ester to form a reaction system with a substrate concentration of 20 g/L. Add ivermase. (1), Continue the reaction. The conversion of the reaction was determined by liquid chromatography. The reaction was determined to be 40.1% by continuous liquid reaction. 2%。 The reaction conversion rate was 78.2%. The conversion of the reaction was 78.2%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to give cytarabine-L-phenylalanine hydrochloride 269. Omg (0. 61 leg ol, molar yield 74.4%) 2质量。 Optical purity 99.2%. Example 27

To a 50 ml round bottom flask, guanosine 200 mg (0.70 mmol) was added, and then 10 ml of L-serine methyl ester was added to form a reaction system having a substrate concentration of 20 g/L. The lichens spore prepared in Example 1 was added. 8%, continued reaction for 24 hours, the reaction was carried out by liquid chromatography at a pressure of 30 ° C under a magnetic stirring condition (vacuum degree of -300 mbar) for 48 h. 7%。 The conversion of the reaction was determined to be 51. 7%, and the reaction was continued for another 24h, the conversion of the reaction was determined to be 64.7%. The reaction rate was 74.3%. The reaction conversion rate was 84.5%. The conversion rate was determined to be 84.5%. After the end of the reaction, the acid is acidified, filtered, concentrated, and recrystallized from ethanol to give guanosine-L-serine ester hydrochloride 227. 8 mg (0. 56 leg ol, molar yield 80. 0%), optical purity 99.7% . Example 28

Add 50 mg (0. 89 mmol) of acyclovir to a 50 ml round bottom flask, add 10 ml of L-valine methyl ester to form a reaction system with a substrate concentration of 20 g/L, and add Candida antarctica. The reaction rate was determined to be 38.1%, and the reaction was continued for 24 hours. The reaction was carried out by liquid chromatography at a temperature of 50 ° C under a magnetic stirring condition (vacuum degree of -300 mbar) for 48 h. The conversion rate of the reaction was determined to be 79.8%, and the reaction was continued for another 24 hours. The conversion of the reaction was 86.6%, and the reaction was continued for another 24 hours. 91. 3%, the end of the reaction, acidified by hydrochloric acid, filtered, concentrated, 50% ethanol recrystallization to obtain valacyclovir hydrochloride 285. Omg (0. 79mmol, molar yield 88.8%), optical purity of 99. 4%. Example 29

Add acyclovir 700 mg (3.11 mmol) to a 50 ml round bottom flask, and add 20 ml of L_valine methyl ester to form a reaction system with a substrate concentration of 35 g/L. Add Libo fat. 8%, Continue reaction 24h, the reaction was determined by liquid chromatography. The reaction was determined by liquid chromatography to determine the conversion rate of 40. 8%, continue to react for 24 hours. The reaction was carried out at a temperature of 55 ° C under a magnetic stirring condition (vacuum degree of -300 mbar) for 48 h. 7%。 The conversion of the reaction was determined to be 78. 6%, the reaction was continued for 24h, the conversion of the reaction was determined to be 89.6%, and the reaction was continued for another 24h, the conversion of the reaction was determined to be 90.7%. 6%。 The reaction was completed, acidified by hydrochloric acid, filtered, concentrated, 50% ethanol recrystallized to give valacyclovir hydrochloride 970. 6mg ( 2. 69 leg ol, molar yield 86. 5%), optical purity 99.6%. Example 30

5,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The reaction was converted to a reaction rate of 75. 8 and the reaction was determined to be 75. 6%。 The conversion of the reaction was determined to be 87.6%. The reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from ethanol to obtain ganciclovir-L-valine ester hydrochloride 488. 0 mg (0. 98 mmol, molar yield 83.7%), optical purity 98. 5%. Example 31

Inosine 200 mg (0.74 mmol) was added to a 50 ml round bottom flask, and 10 ml of L-serine methyl ester was added to form a reaction system having a substrate concentration of 20 g/L, and lipase PS Amano SD 0. 2 was added. g, the reaction was carried out under reduced pressure (vacuum degree -300 mbar) at 50 ° C for 48 h, and the conversion of the reaction was determined by liquid chromatography to be 41. 5%, the reaction was continued for 24 h, and the conversion of the reaction was determined to be 73. 9%。 The reaction rate was determined to be 85.9%. 0%。 The end of the reaction, the acidification of hydrochloric acid, filtration, concentration, ethanol recrystallization to obtain inosine-L-serine ester hydrochloride 239. Omg (0. 61 leg ol, molar yield 82.4%), optical purity of 99.0% . Example 32

Add guanosine 200 mg (0.70 mmol) to a 50 ml round bottom flask, and add 10 ml of L-cysteine methyl ester to form a reaction system with a substrate concentration of 20 g/L. Add lipase AK Amano 0 4 g, reacted under reduced pressure (vacuum degree -300 mbar) at 30 ° C for 48 h, and the reaction was measured by liquid chromatography. 6%。 The conversion rate was 35.1%, the reaction was continued for 24h, the conversion of the reaction was determined to be 51.3%, and the reaction was continued for another 24h, the conversion rate of the determination was 65.6%. The reaction conversion rate was 73.6%. 9%。 The conversion of the reaction was continued for 24h, the conversion of the reaction was determined to be 79. 6%, and the reaction was continued for another 24h, the conversion rate of the reaction was determined to be 82.9%. After the end of the reaction, the acid is acidified, filtered, concentrated, and recrystallized from ethanol to give guanosine-L-cysteine ester hydrochloride 232. 6 mg (0. 55 mmol, molar yield 78.6%), optical purity 99. 5 %. Example 33

Add adenosine 200 mg (0.75 mmol) to a 50 ml round bottom flask, and add 10 ml of L-alanine methyl ester to form a reaction system with a substrate concentration of 20 g/L. Add alkaline lipase Greasex 50L. 0%。 After the reaction under reduced pressure (vacuum degree -300 mbar) for 40h, the conversion rate of the reaction was determined by liquid chromatography to be 41.1%, the reaction was continued for 24h, the conversion rate of the reaction was determined. 78%。 After the reaction was continued for 24h, the conversion of the reaction was determined to be 85.9%, and the reaction was continued for another 24h, the conversion of the reaction was determined to be 90.1%, the reaction was completed, acidified by hydrochloric acid, filtered, concentrated, 50% 4%。 The ethanol was recrystallized to give adenosine-L-alanine ester hydrochloride 175. 3mg (0. 65mmol, a molar yield of 86.7%), optical purity of 99.4%. Example 34 (Comparative Example 1)

Add acyclovir 400 mg (1.78 mmol) to a 50 ml round bottom flask, and add 10 ml of L_valine methyl ester and 10 ml of dimethyl sulfoxide (DMSO) to form a substrate concentration of 20 g/ The reaction system of L was added with Candida antarctica lipase B (CALB) 0.3 g, and the reaction was carried out under reduced pressure (vacuum degree -300 mbar) at 50 ° C for 48 h. The conversion of the reaction was determined by liquid chromatography. The rate of conversion was 31. 2%, and the reaction was continued for 24 hours. The conversion of the reaction was determined to be 55.8%. The reaction was further continued for 24 hours. The conversion of the reaction was determined to be 63.6%. The reaction was continued for another 24 hours. 7%, the reaction was completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to give valzallovir hydrochloride 389. 7 mg (1. 08 mmol, molar yield 60.7%). Example 35 (Comparative Example 2)

Add acyclovir 400 mg (1.78 mmol) to a 50 ml round bottom flask and add L_valine methyl ester ^! ^ and deionized water? !^, a reaction system having a substrate concentration of 33. 3 g / L, adding Candida antarctica lipase B (CALB) 0. 3g, decompression at 50 ° C, magnetic stirring conditions (vacuum degree -300 7%。 The reaction rate was determined by the liquid chromatography, the reaction was determined to be 16.7%, the reaction was continued for 24h, the conversion of the reaction was determined to be 25.6%, and the reaction was continued for 24h, the conversion rate of the reaction was 29.7% And the reaction is carried out for 24 hours, the conversion of the reaction is determined to be 34.3%, and the reaction is completed, acidified by hydrochloric acid, filtered, concentrated, and recrystallized from 50% ethanol to obtain valacyclovir hydrochloride 182. 5 mg (0. 51 mmol, mol Yield 28.7%). The experimental results of Examples 34 and 35 indicate that the conversion rate is poor after the addition of an organic solvent such as dimethyl sulfoxide or water. Experiments have shown that organic solvents or water have a greater influence on the activity of the enzyme in the reaction system, which is not conducive to the continuation of the reaction, and will have a greater impact on the post-treatment. It can be seen that in the preparation method of the present invention, the protease or lipase used is commercially available or can be obtained by a simple fermentation method without going through some special treatment process. This method can obtain a product of high purity without additional solvent addition. Under the catalysis of the protease or lipase of the present invention, the method has high conversion rate, high selectivity, few by-products, and simple post-treatment. Therefore, the method is simpler, more economical and more environmentally friendly, and is very suitable for industrialization. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art after the above-described teachings of the present invention.

Claims

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A method for catalyzing the synthesis of a nucleoside amino acid derivative by an enzyme, comprising the steps of: reacting a nucleoside compound and an L-amino acid ester under a catalysis of a protease to obtain a nucleoside amino acid derivative;

The proteinase is Bacillus subtilis alkaline protease, Bacillus licheniformis alkaline protease, alkaline protease 3. 0T, alkaline protease 2. 4L FG, Saiwei proteinase 8. 0T, Saiwei proteinase 16. 0 L, Yi瑞酶 8. 0 L, avidin 16. 0 L or thermolysin.

2. The method of claim 1 wherein

The L-serine ester is L-serine d- ₄ ester; and/or

The L-alanine ester is L-alanine d- ₄ ester; and/or

The L-cysteine ester is L-cysteine (^ ₄ ester; and/or

The L-phenylalanine ester is L-phenylalanine CH ester.

A method for catalyzing the synthesis of a nucleoside amino acid derivative by an enzyme, comprising the steps of: reacting a nucleoside compound and an L-amino acid ester under a catalysis of a lipase to obtain a nucleoside amino acid derivative;

The nucleoside amino acid derivatives are acyclovir-L-valine ester, acyclovir-L-serine ester, acyclovir-L-alaninate, acyclovir-L- Cysteine ester, acyclovir-L-phenylalanine, ganciclovir Wei-L-valine ester, ganciclovir-L-serine ester, ganciclovir-L-alaninate, ganciclovir-L-cysteine, ganciclovir-L -phenylalanine ester, inosine-L-valine ester, inosine-L-serine ester, inosine-L-alanine ester, inosine-L-cysteine ester, inosine-L -phenylalanine ester, guanosine-L-valine ester, guanosine-L-serine ester, guanosine-L-alanine ester, guanosine-L-cysteine ester, guanosine-L -phenylalanine ester, adenosine-L-valine ester, adenosine-L-serine ester, adenosine-L-alanine ester, adenosine-L-cysteine ester or adenosine-L - phenylalanine ester;

The lipase is Libo lipase 100T, Νονο435 lipase, lipase PS Amano SD, lipase AS Amano, lipase AK Amano, lipase G, lipase AYS Amano, Candida antarctica lipase B or alkali Sex lipase Greasex 50L.

4. The method of claim 3, wherein

The L-valine ester is L-valine C1-4 ester; and/or

The L-serine ester is L-serine d- ₄ ester; and/or

The L-alanine ester is L-alanine d- ₄ ester; and/or

The L-cysteine ester is L-cysteine (^ ₄ ester; and/or

The L-phenylalanine ester is L-phenylalanine CH ester.

The method according to claim 3, wherein the lipase is Candida antarctica lipase B.

6. The method according to claim 1 or 3, wherein the nucleoside compound is acyclovir or ganciclovir.

The method according to claim 1 or 3, wherein the concentration of the nucleoside compound in the reaction system is 10 to 50 g/L.

The method according to claim 1 or 3, wherein the protease or lipase has a concentration in the reaction system of 8 to 65 g/L.

The method according to claim 1 or 3, wherein the reaction is carried out at 20 to 70 ° C; and / or the reaction is carried out for 24 to 120 hours.

10. Process according to claim 1 or 3, characterized in that the reaction is carried out at a relative vacuum of from -100 to -500 mbar.