CN112852899A

CN112852899A - Catalytic synthesis system and catalytic synthesis method of staurosporine intermediate K252c and derivatives thereof

Info

Publication number: CN112852899A
Application number: CN202110287171.7A
Authority: CN
Inventors: 李永安; 刁刘洋; 白莉; 周亚维; 於四杰
Original assignee: Baishi Pharmaceutical Suzhou Co Ltd
Current assignee: Baishi Pharmaceutical Suzhou Co Ltd
Priority date: 2021-03-17
Filing date: 2021-03-17
Publication date: 2021-05-28

Abstract

The invention provides a catalytic synthesis system and a catalytic synthesis method of a staurosporine intermediate K252c and derivatives thereof. The catalytic synthesis system comprises: synthesizing raw materials of L-tryptophan and/or L-tryptophan derivatives, L-tryptophan oxidase, CPA synthetase, cytochrome P450 enzyme, monooxygenase, cofactor and buffer solution. In the invention, L-tryptophan and/or L-tryptophan derivatives are used as synthesis raw materials and are added into a synthetase system one by one or mixed for incubation, thus a series of staurosporine analogues with different structures can be generated. The catalytic synthesis system is suitable for constructing a staurosporine analogue library, and compounds with high selectivity and high activity can be screened from the library, so that safe and effective new drugs of kinase inhibitors can be developed, and the requirements of cancer patients are met.

Description

Catalytic synthesis system and catalytic synthesis method of staurosporine intermediate K252c and derivatives thereof

Technical Field

The invention belongs to the technical field of biosynthesis of medical intermediates, particularly relates to biosynthesis of staurosporine, and particularly relates to a catalytic synthesis system and a catalytic synthesis method of a staurosporine intermediate K252c and derivatives thereof.

Background

Cancer is a serious threat to human health, and its causative factors often stem from abnormal activation of kinases in the signaling pathway. In addition, common high-incidence chronic diseases such as autoimmune diseases and neurodegenerative diseases are also closely related to kinase abnormality. In 2001, the first kinase inhibitor drug Gleevec (Gleevec) is approved to be on the market in America, a new age of cancer targeted therapy is opened, and the curative effect is remarkably superior to that of chemotherapy. By the end of 2019, FDA has approved a total of 52 kinase inhibitors. Although the number is large, there is still a great unmet clinical need for cancer treatment due to a wide variety of cancers, a wide variety of abnormal kinases involved, and a variety of types of mutations, and drug resistance after use. In addition, these marketed drugs target only about 25 kinases, accounting for only 5% of 518 kinases in humans, and a large number of kinases have not been established. Therefore, such drugs have a huge development space.

The antineoplastic medicine is mainly from natural product analogues. Therefore, innovative techniques for the efficient synthesis of natural product analogs are critical to new drug discovery. Natural products tend to be structurally complex, containing multiple chiral centers. Compared with chemical synthesis, biosynthesis is more advantageous due to strict regio-and stereoselectivity. In particular, the increasing maturity of synthetic biology principles and technologies has made efficient synthesis of natural product analogs easy to implement. More importantly, biosynthesis can also make full use of the flexibility of chemical synthesis, providing it with structurally different synthetic raw materials, thereby producing structurally different unnatural analogues. Through constructing a non-natural analog library, the high-selectivity and high-activity compound is screened out, and then a safe and effective new kinase inhibitor medicine can be developed.

Staurosporine (Staurosporine) is a natural product from actinomycetes and has a broad-spectrum potent kinase inhibitory activity. According to incomplete statistics, staurosporine can effectively inhibit more than 200 human kinases, and the semi-inhibitory concentration (IC50) of a plurality of kinases can reach the nM level. Due to the poor selectivity, only Midostaurin (Midostaurin) of this class of compounds is currently marketed in the united states in 2017 for the treatment of acute myeloid leukemia. The development of effective synthesis technology of staurosporine analogues, the construction of analogue libraries thereof, and the screening of high-selectivity compounds are key factors for promoting the wide drug development of the compounds. Due to the complex structure, the synthesis of the staurosporine analogue library by chemical means for new drug discovery is difficult. Thus, biosynthesis provides a practical and competitive technological approach.

In 1997 staurosporine was discovered by Japanese scientists, the DNA sequence of the biosynthetic gene cluster was determined in 2002 and the natural synthetase line was initially recognized. Subsequently, the catalytic activity of several natural synthetases, such as StaO, StaD, StaP, StaC, etc., was confirmed in vitro biochemical experiments, formula I showing the biosynthetic pathway of staurosporine, wherein the R group represents ═ O or ═ NH, both reversible.

The core structural unit of staurosporine is K252c, and the synthetic raw material is L-tryptophan. K252c is glycosylated and methylated to obtain staurosporine. The staurosporine analogs such as K252c, Holorine A and the like all have remarkable kinase inhibition activity.

By genetically modifying the biosynthesis pathway of staurosporine, for example by inactivating or introducing genes encoding specific enzymes, staurosporine analogues can be synthesized in microorganisms. However, this method is inefficient, time and labor consuming to construct strains, and only one or a few of the analogs per strain can be produced, which is difficult to use for constructing large libraries of staurosporine analogs.

Therefore, there is a need in the art to develop a synthetic route capable of producing not only the core structural unit K252c but also a large amount of K252c analogues to construct a large amount of staurosporine analogue library.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a catalytic synthesis system and a catalytic synthesis method of a staurosporine intermediate K252c and derivatives thereof. The catalytic synthesis system and the catalytic synthesis method utilize an in vitro enzymatic synthesis technology, are suitable for constructing a staurosporine analogue library, can screen out compounds with high selectivity and high activity from the library, and further develop safe and effective new drugs of kinase inhibitors, thereby meeting the great clinical treatment requirements of cancer patients.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a catalytic synthesis system for staurosporine intermediate K252c and derivatives thereof, comprising: synthesizing raw materials of L-tryptophan and/or L-tryptophan derivatives, L-tryptophan oxidase, CPA synthetase, cytochrome P450 enzyme, monooxygenase, cofactor and buffer solution;

wherein the L-tryptophan oxidase is VioA, the CPA synthetase is VioB, and the monooxygenase is SpcC.

The currently known L-tryptophan oxidase includes a natural synthetase StaO and its isozyme such as RebO, NokA, SpcO, AtmO, or VioA, etc., and the known CPA synthetase includes a natural synthetase StaD and its isozyme such as RebD, NokB, SpcD, AtmD, or VioB, etc.; however, not all reported L-tryptophan oxidase and CPA synthetase have a good catalytic effect in the synthetic pathway of CPA; for example, AtmO and AtmD, which in combination do not catalyze L-tryptophan to give CPA; and the yields of different enzymes and different combinations in the preparation process are greatly different. The VioA and VioB specifically selected in the invention are used as L-tryptophan oxidase and CPA synthetase in a CPA synthetic path, and then are combined with monooxygenase SpcC with high efficiency, so that L-tryptophan and/or L-tryptophan derivatives can be efficiently catalyzed to generate a staurosporine intermediate.

In the invention, L-tryptophan and different chemically synthesized L-tryptophan derivatives are used as synthesis raw materials, and are added into a synthetase system one by one or mixed for incubation for a certain time to generate a series of staurosporine analogs with different structures. The L-tryptophan oxidase is VioA, the CPA synthetase is VioB, the combination of the L-tryptophan oxidase and the CPA synthetase has high reaction efficiency, reaction raw materials are provided for subsequent cytochrome P450 enzyme and monooxygenase, and the synthesis of a staurosporine intermediate K252c and derivatives thereof is facilitated.

In a preferred embodiment of the present invention, the cytochrome P450 enzyme comprises StaP or RebP.

Preferably, in the catalytic synthesis system, the L-tryptophan oxidase is VioA, the CPA synthetase is VioB, the cytochrome P450 enzyme is RebP, and the monooxygenase is SpcC.

As a preferred embodiment of the present invention, the cofactor comprises reduced coenzyme and/or protein cofactor. For the present invention, both reduced coenzyme and protein cofactor are essential in the reaction system, and both are present at the same time to make the whole enzyme system active. However, in the specific experimental process, the added enzyme solution is crude enzyme solution, namely cell lysate, and is not purified; therefore, even if the reduced coenzyme and the protein cofactor are not added in the experimental process, the enzyme solution also contains the two cofactors. If a reduced coenzyme and/or a protein cofactor is additionally added, the synthesis activity of the whole enzyme system will be higher.

Preferably, the reduced coenzyme comprises NADH and/or NADPH.

Preferably, the proteinaceous cofactor comprises FldA and/or FnR.

Preferably, the catalytic synthesis system comprises the cofactors NADPH, FldA and FnR.

In the invention, the addition of the cofactor can obviously increase the yield of K252c, and meanwhile, the reaction efficiency of reduced coenzyme I (NADH) and reduced coenzyme II (NADPH) is compared in the invention, and the combination of Escherichia coli-derived FldA-FnR and spinach-derived Ferrodexin-Reductase is combined, so that the final result shows that the combination of the cofactor NADPH, FldA and FnR has the best auxiliary effect.

In the present invention, the enzyme added to the reaction system may be a purified or unpurified enzyme; when the crude enzyme solution is added, the enzyme solution inevitably contains other foreign proteins in addition to the target protein. In the invention, escherichia coli is taken as a host to produce target protein, the obtained crude enzyme solution is added into a reaction system, the adding amount of each enzyme solution is expressed by the content of the total protein in the reaction system, namely the total protein content of the enzyme solution is multiplied by the ratio of the adding volume of the enzyme solution to the total volume of the reaction system, and the following steps are performed:

as a preferable embodiment of the present invention, in the catalytic synthesis system, the total protein mass concentration of the L-tryptophan oxidase solution is 2 to 10mg/mL, and may be, for example, 2mg/mL, 3mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL or 10mg/mL, and preferably 4 to 8 mg/mL.

Preferably, in the catalytic synthesis system, the mass concentration of the total protein of the CPA synthetase solution is 1-8 mg/mL, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL or 8mg/mL, and preferably 4-6 mg/mL.

Preferably, in the catalytic synthesis system, the mass concentration of the total protein of the cytochrome P450 enzyme solution is 10 to 20mg/mL, for example, 10mg/mL, 12mg/mL, 13mg/mL, 15mg/mL, 17mg/mL, 18mg/mL, 19mg/mL or 20mg/mL, and preferably 14 to 16 mg/mL.

Preferably, in the catalytic synthesis system, the mass concentration of the total protein of the monooxygenase solution is 30-40 mg/mL, for example, 31mg/mL, 32mg/mL, 34mg/mL, 35mg/mL, 37mg/mL, 38mg/mL, 39mg/mL or 40mg/mL, preferably 33-36 mg/mL.

Preferably, in the catalytic synthesis system, the molar concentration of the cofactor NADPH is 5 to 15mM, for example, 6mM, 7mM, 9mM, 10mM, 11mM, 13mM, 14mM, or 15mM, and preferably 8 to 12 mM.

Preferably, in the catalytic synthesis system, the total protein mass concentration of the cofactor FldA protein solution is 2 to 6mg/mL, for example, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, 5mg/mL, 5.5mg/mL or 6mg/mL, and preferably 3 to 5 mg/mL.

Preferably, in the catalytic synthesis system, the total protein mass concentration of the cofactor FnR protein solution is 0.5-1.5 mg/mL, for example, 0.6mg/mL, 0.7mg/mL, 0.9mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL or 1.5mg/mL, and preferably 0.8-1 mg/mL.

As a preferred technical scheme of the invention, the buffer comprises Tris-HCl buffer.

Preferably, the Tris-HCl buffer has a molarity of 50-200 mM, and may be, for example, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200 mM.

Preferably, the pH value of the Tris-HCl buffer is 6.8-7.2, such as 6.8, 6.9, 7.0, 7.1 or 7.2.

Preferably, the Tris-HCl buffer further comprises sodium chloride.

Preferably, the molar concentration of sodium chloride in the Tris-HCl buffer is 100-200 mM, such as 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200 mM.

Preferably, the buffer solution further comprises DTT, and the DTT is added into the buffer solution to improve the stability of the enzyme.

Preferably, the molar concentration of DTT in the buffer is 0.5-1.5 mM, and may be, for example, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, or 1.5 mM.

Preferably, the buffer further comprises DMSO.

Preferably, the DMSO concentration in the buffer solution is 5 to 15% by mass, and may be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or the like.

In a second aspect, the present invention also provides a catalytic synthesis method of staurosporine intermediate K252c and its derivatives, comprising:

respectively constructing carriers for expressing L-tryptophan oxidase VioA, CPA synthetase VioB, cytochrome P450 enzyme and monooxygenase, performing induced expression and preparing enzyme solution;

mixing synthetic raw materials of L-tryptophan and/or L-tryptophan derivatives, L-tryptophan oxidase, CPA synthetase, cytochrome P450 enzyme, monooxygenase and cofactor to obtain a catalytic synthesis system as described in the first aspect, and reacting to obtain staurosporine intermediates K252c and/or K252c derivatives.

In a preferred embodiment of the present invention, the reaction time is 8 to 20 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

Preferably, the reaction temperature is 20-37 ℃, for example, can be 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ or 37 ℃, preferably 25 ℃.

As a preferred technical scheme of the invention, the cytochrome P450 enzyme in the catalytic synthesis system is RebP, the monooxygenase is SpcC, and the cofactors are NADPH, FldA and FnR.

Preferably, the vector comprises an E.coli expression vector.

Preferably, the vector comprises the expression vector pET-30a (+).

As a preferable technical scheme of the invention, the catalytic synthesis method further comprises a heating step after the reaction is finished.

Preferably, the heating temperature is 80-85 ℃, for example, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃ or 85 ℃. The heating may be performed using a metal bath to inactivate the enzymes in the reaction system, and may also be beneficial for the subsequent extraction step.

Preferably, the heating time is 10-15 min, for example, 10min, 10.5min, 11min, 11.5min, 12min, 12.5min, 13min, 14min or 15 min.

Preferably, the catalytic synthesis method further comprises an extraction step after the reaction is completed.

Preferably, the extractant used for the extraction comprises ethyl acetate.

Preferably, the extraction temperature is 30-37 ℃, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃.

Preferably, the extraction time is 20-40 min, for example, 20min, 22min, 24min, 25min, 28min, 30min, 32min, 35min, 36min, 38min or 40 min.

As a preferred technical scheme of the invention, the catalytic synthesis method comprises the following steps:

(1) respectively synthesizing nucleotides for coding L-tryptophan oxidase VioA, CPA synthetase VioB, cytochrome P450 enzyme and monooxygenase, connecting the nucleotides to an escherichia coli expression vector, constructing a vector for expressing the L-tryptophan oxidase VioA, CPA synthetase VioB, cytochrome P450 enzyme and monooxygenase, and performing induced expression to prepare enzyme solution;

(2) mixing synthetic raw materials including L-tryptophan and/or L-tryptophan derivatives, L-tryptophan oxidase, CPA synthetase, cytochrome P450 enzyme, monooxygenase and cofactors to obtain a catalytic synthesis system, reacting at 20-37 ℃ for 8-20 h, heating at 80-85 ℃ for 10-15 min, and extracting with ethyl acetate to obtain staurosporine intermediates K252c and/or K252c derivatives.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a method for synthesizing a staurosporine intermediate K252c and derivatives thereof based on an in vitro enzymatic technology, which is mainly used for synthesizing staurosporine and analogues thereof; the method is based on multi-enzyme catalysis, the selected enzyme combination has a good catalytic effect on various tryptophan derivatives, and biosynthesis is carried out in vitro, so that the problems of low yield and efficiency and lack of varieties of staurosporine analogues can be solved;

in the invention, L-tryptophan and different chemically synthesized L-tryptophan derivatives are used as synthesis raw materials, and are added into a synthetase system one by one or mixed for incubation for a certain time to generate a series of staurosporine analogs with different structures. The catalytic synthesis method is suitable for efficiently synthesizing the staurosporine analogue library, and can be widely applied to discovery and research of new drugs such as kinase inhibitors.

Drawings

FIG. 1 is a LC-MS detection spectrum obtained in example 3; wherein, the figure I is an ultraviolet absorption spectrum of an extracted sample of a reaction system at 270nm, and the figure II is an ion spectrum of a fermentation liquor extracted sample with the mass-to-charge ratio (m/z) of 386.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

In the following examples, unless otherwise specified, reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.

Example 1 construction of expression plasmid

This example was used to construct expression plasmids for staurosporine analogue natural synthetases, isoenzymes and protein cofactors.

The staurosporine analogue natural synthetase, isozyme and protein cofactor thereof are 19 in total, and are specifically shown in the following table 1:

TABLE 1

According to the codon preference of Escherichia coli (Escherichia coli), 19 protein DNA coding sequences are subjected to codon optimization and synthesis, and are respectively loaded into an expression vector pET-30a (+) through NdeI and HindIII double enzyme digestion to obtain corresponding expression plasmids.

Example 2 preparation of enzyme solution

The expression plasmids constructed in example 1 were introduced into a host strain BL21(DE3) to obtain corresponding expression strains.

The above expression strains were cultured in 5L fermentors (Shanghai Bailun) containing TB medium, respectively, and OD was obtained₆₀₀Adding an inducer IPTG at 0.4-0.6, wherein the induction expression conditions are shown in Table 2.

After completion, the cells were collected by centrifugation, washed 2 times with Buffer B (100mM Tris-HCl, 150mM NaCl, pH7), and resuspended in a Buffer (100mM Tris-HCl, 150mM NaC)l, 20% Glycerin, pH7), OD₆₀₀About 500 a. After the bacterial suspension is fully crushed by a high-pressure cell crusher (Shanghai permanent connection), the supernatant is obtained by centrifugation, namely enzyme liquid or protein liquid (cell lysate).

The protein concentration of the enzyme solution or the protein solution was measured using a Bradford kit (Shanghai Producer), and the protein concentration is shown in Table 2.

TABLE 2

Example 3 Synthesis of CPA

Using L-tryptophan (Trp) as a synthetic raw material, reaction systems were prepared as shown in table 3 to compare the activity of 6 groups of enzymes to synthesize CPA.

The reaction temperature is 25 ℃, and the reaction time is 20 h.

After the reaction is finished, heating the mixture for 15min at 85 ℃ in a metal bath, cooling the mixture to room temperature, adding 2 times of methanol in volume, fully mixing the mixture, and centrifuging the mixture to collect supernatant.

The supernatant was subjected to LCMS detection of a reaction product, Chromopyrrocolic acid (CPA), which strongly absorbs at 270nm, so that the concentration of CPA was proportional to the area of the ultraviolet absorption peak at 270 nm.

In this example, comparison was made using a combination of another L-tryptophan oxidase and CPA synthase as a comparative example; wherein, the total protein of the added StaO isozyme liquid keeps consistent with that of the added StaO isozyme liquid, and the total protein of the added StaD isozyme liquid keeps consistent with that of the added StaD isozyme liquid.

TABLE 3

Note: L-Trp: l-tryptophan; buffer: 0.1M Tris-HCl, 150mM NaCl, pH 7.0.

The LC-MS detection results are shown in FIG. 1: in the reaction system of the examples, the peak area of the target product CPA [ M/z 386, M +1] at 270nm is maximum, the peak-off time is 3.348min (figure I), and the mass/charge ratio (M/z) of the corresponding ion, namely [ M +1], of the peak is 386 (figure II);

the experimental results prove that the isoenzyme combination of the VioA-VioB is far greater than that of other reaction systems; next, comparative reaction system 3, comparative reaction system 1 and comparative reaction systems 2 and 4, and CPA was not detected in comparative reaction system 5.

In conclusion, the highest reaction efficiency is the VioA-VioB combination, and the worst is the AtmO-AtmD combination; it can also be shown that there are more distinct differences between the different enzymes.

Example 4 comparison of Activity of three groups of enzymes to synthesize K252c

In this example, CPA was used as a synthetic raw material, and a reaction system was prepared as shown in Table 4, and the reaction activities of the three groups of enzymes were compared at a reaction temperature of 25 ℃ for 20 hours.

The addition volume of the RebP enzyme solution is kept consistent with that of the StaP enzyme solution, and the addition volume of the SpcC enzyme solution is kept consistent with that of the StaC enzyme solution in the same way.

After the reaction is finished, heating the metal bath at 85 ℃ for 15min, cooling to room temperature, adding 2 times of ethyl acetate, fully mixing and extracting, shaking at 37 ℃ for 30min, centrifuging, collecting the organic phase in the supernatant, and repeating the steps once.

The organic phases obtained by two extractions are combined, evaporated and dried at 40 ℃, dissolved in 50 μ L DMSO and subjected to LCMS detection.

The reaction product K252c has strong absorption at 290nm, so the concentration of K252c is in direct proportion to the area of the ultraviolet absorption peak at 290 nm.

TABLE 4

LCMS monitoring showed that K252c formation [ M/z 312, M +1] was detected in all three reactions. Comparing the peak areas, it can be seen that the catalytic efficiency of the RebP-SpcC combination (example reaction system 4) is higher than that of the StaP-SpcC combination (example reaction system 2) and much higher than that of the StaP-StaC combination (example reaction system 3). This result indicates that SpcC catalytic activity is much greater than StaC and RebP catalytic activity is greater than StaP.

EXAMPLE 5 screening of cofactors in the reaction System

StaP catalytic activity is dependent on multiple cofactors, including: 1) reduced coenzyme NADH or NADPH; 2) redox proteins such as spinach-derived Ferredoxin (Ferredoxin) or escherichia coli-derived Flavodoxin (flavedoxin, flava), and the like; 3) an oxidoreductase such as spinach-derived ferredoxin Reductase (Reductase) or Escherichia coli-derived Flavodoxin Reductase (fnR or the like).

In this example, the reaction efficiencies of reduced coenzyme I (NADH) and reduced coenzyme II (NADPH), the reaction efficiencies of the Escherichia coli-derived FldA-FnR combination and the spinach-derived Ferrodexin-Reductase combination, and the influences of DTT and DMSO on the reaction efficiencies were compared, and specific reaction systems are shown in tables 5 and 6. Subsequently, the UV absorption peak area of K252c at 290nm was detected using LC-MS.

TABLE 5

Note: the Buffer solution was 0.1M Tris-HCl, 150mM NaCl, pH 7.0.

TABLE 6

LCMS detection shows that K252c [ M/z 312, M +1] is detected in the reaction system.

Comparing the area of the ultraviolet absorption peak at 290nm, it can be seen that the reaction efficiency of the FnR-FldA combination (example reaction system 6) is much higher than that of the Ferredoxin-reduction combination (example reaction system 5), the reaction efficiency of NADPH (example reaction system 8) is higher than that of NADH (example reaction system 7), and the reaction efficiency can be significantly improved by adding 1mM DTT and 10% DMSO (example reaction system 10).

Example 6 comparison of reaction time and temperature

In this example, a catalytic reaction was carried out using a combination of enzymes (VioA, VioB, RebP, SpcC) using L-tryptophan as a substrate, as shown in Table 7 below.

LCMS detects the area of the ultraviolet absorption peak of K252c at 290 nm. LCMS detection shows that K252c is detected in all five reaction systems.

TABLE 7

By comparing the peak areas, the optimal reaction time is 8-20 h, and the optimal reaction temperature is 25 ℃.

In conclusion, the optimal enzyme combination (VioA, VioB, RebP, SpcC), the optimal adaptive cofactor combination (FnR, FldA, NADPH), the reaction system combination of the reaction efficiency promoter 1mM DTT and 10% DMSO is the optimal reaction system, the optimal reaction temperature is 25 ℃, and the optimal reaction time is 8-20 h.

Example 7 Synthesis of Staurosporine analogs

In this example, several staurosporine analogues were synthesized using L-tryptophan derivatives as the starting material using the optimal reaction system (see Table 8) and the optimal reaction conditions (reaction temperature 25 ℃ C., reaction time 20 hours) obtained in the above examples, and the results are shown in Table 9.

TABLE 8

Note: the sample loading volume of RebP and SpcC can be adjusted according to the volume of tryptophan derivatives.

TABLE 9

Serial number	Substrate	Product of
			1	4-fluoro-L-tryptophan	4, 4' -difluoro-K252 c
2	6-methyl-L-tryptophan	6, 6' -dimethyl-K252 c
			3	5-methoxy-L-tryptophan	5, 5' -dimethoxy-K252 c

In addition, by using the reaction system and the reaction conditions and using L-tryptophan and an L-tryptophan derivative as synthetic raw materials, LCMS detection results show that the reaction product hybridized by two substrates can be detected besides K252c and the derivative thereof corresponding to the two substrates, as shown in Table 10:

watch 10

Serial number	Substrate 1	Substrate 2	Hybrid products
				1	4-fluoro-L-tryptophan	L-tryptophan	4-fluoro-K252 c
2	6-methyl-L-tryptophan	L-tryptophan	6-methyl-K252 c
				3	5-methoxy-L-tryptophan	L-tryptophan	5-methoxy-K252 c

As can be seen from the above table, the enzyme combination provided by the invention has better catalytic effect on different tryptophan derivatives, so that the problems of low yield and poor variety of staurosporine analogues can be solved.

In conclusion, the invention provides a synthesis method of a staurosporine intermediate K252c and derivatives thereof based on multi-enzyme catalysis, and can solve the problems of low yield and poor varieties of staurosporine analogues.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims

1. a catalytic synthesis system of staurosporine intermediate K252c and derivative thereof, is characterized in that, described catalytic synthesis system comprises: synthetic raw material L-tryptophan and/or L-tryptophan derivative, L-tryptophan - Tryptophan oxidase, CPA synthase, cytochrome P450 enzymes, monooxygenases, cofactors and buffers;

Wherein, the L-tryptophan oxidase is VioA, the CPA synthase is VioB, and the monooxygenase is SpcC.

2. The catalytic synthesis system according to claim 1, wherein the cytochrome P450 enzyme comprises StaP or RebP;

Preferably, in the catalytic synthesis system, the L-tryptophan oxidase is VioA, the CPA synthase is VioB, the cytochrome P450 enzyme is RebP, and the monooxygenase is SpcC.

3. The catalytic synthesis system according to claim 1 or 2, wherein the cofactors comprise reduced coenzymes and/or protein cofactors;

Preferably, the reduced coenzyme includes NADH and/or NADPH;

Preferably, the protein cofactors include FldA and/or FnR;

Preferably, the catalytic synthesis system includes cofactors NADPH, FldA and FnR.

4. The catalytic synthesis system according to any one of claims 1 to 3, wherein in the catalytic synthesis system, the total protein concentration of the L-tryptophan oxidase solution is 2 to 10 mg/mL, preferably 4～8mg/mL;

Preferably, in the catalytic synthesis system, the total protein concentration of the CPA synthase solution is 1-8 mg/mL, preferably 4-6 mg/mL;

Preferably, in the catalytic synthesis system, the total protein concentration of the cytochrome P450 enzyme solution is 10-20 mg/mL, preferably 14-16 mg/mL;

Preferably, in the catalytic synthesis system, the total protein concentration of the monooxygenase solution is 30-40 mg/mL, preferably 33-36 mg/mL;

Preferably, in the catalytic synthesis system, the molar concentration of the cofactor NADPH is 5-15 mM, preferably 8-12 mM;

Preferably, in the catalytic synthesis system, the total protein concentration of the cofactor FldA protein solution is 2-6 mg/mL, preferably 3-5 mg/mL;

Preferably, in the catalytic synthesis system, the total protein concentration of the cofactor FnR protein solution is 0.5-1.5 mg/mL, preferably 0.8-1 mg/mL.

5. The catalytic synthesis system according to any one of claims 1 to 4, wherein the buffer comprises Tris-HCl buffer;

Preferably, the molar concentration of the Tris-HCl buffer is 50-200 mM;

Preferably, the pH value of the Tris-HCl buffer is 6.8-7.2;

Preferably, the Tris-HCl buffer also includes sodium chloride;

Preferably, the molar concentration of sodium chloride in the Tris-HCl buffer is 100-200 mM;

Preferably, the buffer also includes DTT;

Preferably, the molar concentration of DTT in the buffer is 0.5-1.5 mM;

Preferably, the buffer also includes DMSO;

Preferably, the mass concentration of DMSO in the buffer is 5-15%.

6. a catalytic synthesis method of staurosporine intermediate K252c and derivative thereof, is characterized in that, described catalytic synthesis method comprises:

Constructing vectors expressing L-tryptophan oxidase VioA, CPA synthase VioB, cytochrome P450 enzyme and monooxygenase respectively, inducing expression and preparing enzyme liquid;

The synthetic raw material L-tryptophan and/or L-tryptophan derivative, L-tryptophan oxidase, CPA synthase, cytochrome P450 enzyme, monooxygenase and cofactor are mixed to obtain as claimed in claim 1 The catalytic synthesis system described in any one of ~5, through reaction, obtains staurosporine intermediate K252c and/or K252c derivative.

7. catalytic synthesis method according to claim 6, is characterized in that, the time of described reaction is 8～20h;

Preferably, the temperature of the reaction is 20-37°C, preferably 25°C.

8. catalytic synthesis method according to claim 6 or 7, is characterized in that, in described catalytic synthesis system, cytochrome P450 enzyme is RebP, described monooxygenase is SpcC, and described cofactor is NADPH, FldA and FnR;

Preferably, the vector comprises an E. coli expression vector;

Preferably, the vector comprises the expression vector pET-30a(+).

9. The catalytic synthesis method according to any one of claims 6 to 8, wherein the catalytic synthesis method further comprises a step of heating after the reaction is completed;

Preferably, the heating temperature is 80-85°C;

Preferably, the heating time is 10-15 min;

Preferably, after the reaction is completed in the catalytic synthesis method, the step of extraction is also included;

Preferably, the extractant used in the extraction comprises ethyl acetate;

Preferably, the temperature of the extraction is 30-37°C;

Preferably, the extraction time is 20-40 min.

10. The catalytic synthesis method according to any one of claims 6 to 9, wherein the catalytic synthesis method comprises the following steps:

(1) synthesizing nucleotides encoding L-tryptophan oxidase VioA, CPA synthase VioB, cytochrome P450 enzyme and monooxygenase respectively and connecting them to an E. coli expression vector to construct and express the L-tryptophan The carrier of acid oxidase VioA, CPA synthase VioB, cytochrome P450 enzyme and monooxygenase, inducing expression and preparing enzyme liquid;

(2) Mixing synthetic raw materials L-tryptophan and/or L-tryptophan derivatives, L-tryptophan oxidase, CPA synthase, cytochrome P450 enzyme, monooxygenase and cofactors to obtain catalytic In the synthesis system, the reaction is carried out at 20-37 °C for 8-20 h, heated at 80-85 °C for 10-15 min, and then extracted with ethyl acetate to obtain the staurosporine intermediate K252c and/or K252c derivative.