CN115044637A - Biocatalytic preparation method of beta-D-arabinosylguanosine analog - Google Patents
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Abstract
The invention discloses a biocatalytic preparation method of 2 '-deoxy-2' -fluoro-beta-D-arabinoguanosine analogs, which comprises the following steps: a step (a): in a liquid reaction system, taking a compound shown in a formula II and a compound shown in a formula III as substrates, and carrying out reaction under the catalysis of deoxyribotransferase to form a compound shown in a formula I; step (b): separating the compound of formula I from the reacted reaction system of step (a); wherein the deoxyribotransferase has a Genbank accession number of Q74LQ9.1. The invention also provides a reaction system. The method has the advantages of short steps, mild conditions and high substrate conversion rate.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a biocatalytic preparation method of a 2 '-deoxy-2' -fluoro-beta-D-arabino guanosine analog.
Background
The 2 '-deoxy-2' -fluoro-beta-D-arabinoguanosine analog is a nucleoside analog which has good antiviral and antitumor activities and can be effectively embedded into a DNA chain and an RNA chain of a virus, thereby inhibiting division and replication of cells.
Currently, 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analogs are generally synthesized by chemical synthesis methods and enzymatic methods. The chemical synthesis method is a multi-step process, needs a process of protecting and deprotecting groups in order to realize regioselectivity and stereoselectivity, has complex operation steps and high cost, is easy to generate toxic and harmful substances, and causes pollution to the environment. The enzyme synthesis is more and more emphasized by people due to factors such as high regioselectivity, stereoselectivity, mild reaction conditions, environmental protection and no pollution, but the enzyme synthesis of the 2 '-deoxy-2' -fluoro-beta-D-arabino guanosine analogue has the defect of low substrate conversion rate, which seriously limits the application of the enzyme synthesis in preparation and production.
Therefore, there is a need in the art to develop a novel method for the preparation of 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analogs.
Disclosure of Invention
The invention aims to provide a preparation method of a 2 '-deoxy-2' -fluoro-beta-D-arabinosylguanosine analogue, which has the advantages of high substrate conversion rate, mild reaction conditions, simple and convenient operation and easy industrial production.
In a first aspect of the invention, there is provided a biocatalytic preparation method of a 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analogue, comprising the steps of:
step (a): in a liquid reaction system, taking a compound (2 '-deoxy-2' -fluoro-beta-D-arabinofuranosyl uracil) shown in a formula II and a compound (guanine analogue) shown in a formula III as substrates, and carrying out reaction under the catalysis of deoxyribotransferase to form a compound (2 '-deoxy-2' -fluoro-beta-D-arabinosyl guanosine analogue) shown in a formula I; wherein R is selected from O, S or F;
step (b): separating the compound of formula I from the reaction system after the reaction of step (a);
wherein the Genbank number of the deoxyribotransferase is Q74LQ9.1.
In some embodiments, the concentration of the compound of formula II in the reaction system is from 0.1 to 500 g/L. Preferably, the concentration of the compound of formula II in the reaction system is 0.1g/L or more, 0.2g/L or more, 0.5g/L or more, 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula II is 1-500 g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula II is 10-400 g/L; most preferably, the concentration of the compound of formula II in the reaction system is 50-350 g/L.
In some embodiments, the molar ratio of the compound of formula II to the compound of formula III in the reaction system is 1: (1-10). Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.1-5). More preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.5 to 3).
In some embodiments, in the reaction system, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.01 to 10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.1-5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.5-2): 1.
in some embodiments, the reaction system is an aqueous system. Preferably, the aqueous system contains a buffer.
In some embodiments, the buffer of the reaction system is Phosphate Buffered Saline (PBS), Tris-methylamine salt buffer (Tris-HCl), or Tris-methylamine sulfate buffer (Tris-H) 2 SO 4 ). It is preferable thatThe buffer solution of the reaction system is trihydroxymethyl methylamine salt buffer solution (Tris-HCl).
In some embodiments, in step (a), the reaction temperature is from 15 ℃ to 70 ℃. Preferably, the reaction temperature is 25 ℃ to 65 ℃. More preferably, the reaction temperature is 45-60 ℃.
In some embodiments, in step (a), the reaction time is 1 to 72 hours. Preferably, the reaction time is 2 to 48 hours. More preferably, the reaction time is 4 to 36 hours. Most preferably, the reaction time is 8 to 24 hours.
In some embodiments, in step (a), the reaction system has a pH of 6.0 to 9.0. Preferably, the pH is 6.5 to 8.0. More preferably, the pH is 6.5 to 7.5.
In some embodiments, the deoxyribotransferase is present in the reaction system in the form of: free form, immobilized enzyme or enzyme in the form of bacterial cells.
In some embodiments, in step (b), the method of separating comprises: after the reaction is terminated, the reaction solution is centrifuged or filtered, the filtrate is extracted with an extraction solvent, and the organic layer is concentrated.
In some embodiments, in the step (b), the operation of terminating the reaction is adding methanol or a methanol-water mixed solution having a concentration of 50% by volume or more.
In some embodiments, in step (b), ≧ 40% of the compound of formula II is converted into the compound of formula I in the reaction system after the reaction. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.
In a second aspect, the present invention provides a reaction system comprising:
(1) an aqueous solvent;
(2) a substrate 1, wherein the substrate 1 is a compound of formula II (2 '-deoxy-2' -fluoro-beta-D-arabinofuranosyluracil);
(3) a substrate 2, said substrate 2 being a compound of formula III (guanine analog);
wherein R is selected from O, S or F;
(4) deoxyribotransferase, Genbank accession number Q74LQ9.1.
In some embodiments, the concentration of the compound of formula II in the reaction system is from 0.1 to 500 g/L.
Preferably, the concentration of the compound of formula II in the reaction system is 0.1g/L or more, 0.2g/L or more, 0.5g/L or more, 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula II is 1-500 g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula II is 10-400 g/L; most preferably, the concentration of the compound of formula II in the reaction system is 50-350 g/L.
In some embodiments, the molar ratio of the compound of formula II to the compound of formula III in the reaction system is 1: (1-10). Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.1-5). More preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.5-3).
In some embodiments, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II in the reaction system is (0.01-10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.01-5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.5-2): 1.
in some embodiments, the aqueous solvent is a buffer.
In some embodiments, the buffer of the reaction system is Phosphate Buffered Saline (PBS)) Tris-hydroxymethyl methylamine salt buffer (Tris-HCl) or Tris-hydroxymethyl methylamine sulfate buffer (Tris-H) 2 SO 4 ). Preferably, the buffer of the reaction system is Tris (hydroxymethyl) methylamine salt buffer (Tris-HCl).
In some embodiments, ≧ 40% of the compound of formula II is converted into the compound of formula I in the reaction system after the reaction. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.
The reaction system provided by the invention can carry out enzymatic reaction and prepare the 2 '-deoxy-2' -fluoro-beta-D-arabinosylguanosine analog with high conversion rate. In the above reaction system, the concentration of the compound of formula II, the molar ratio of the compound of formula II to the compound of formula III, and the mass ratio (w/w) of the oxyribotransferase to the compound of formula II all refer to the initial parameters and/or the feeding parameters of the reaction system.
In a third aspect, the present invention also provides a biocatalytic preparation method of a 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analog comprising: carrying out an enzymatic reaction using a reaction system according to the second aspect of the invention, thereby producing a compound of formula I:
in some embodiments, the concentration of the compound of formula II in the reaction system is from 0.1 to 500 g/L.
Preferably, the concentration of the compound of formula II in the reaction system is 0.1g/L or more, 0.2g/L or more, 0.5g/L or more, 1g/L or more, 10g/L or more, 25g/L or more, 50g/L or more, 75g/L or more, 100g/L or more, 125g/L or more, 150g/L or more, 175g/L or more, 200g/L or more, 220g/L or more, 240g/L or more, 260g/L or more, 280g/L or more, 300g/L or more, 320g/L or more, 350g/L or more, 400g/L or more, or 450g/L or more.
Preferably, in the reaction system, the concentration of the compound shown in the formula II is 1-500 g/L; more preferably, in the reaction system, the concentration of the compound shown in the formula II is 10-400 g/L; most preferably, the concentration of the compound of formula II in the reaction system is 50-350 g/L.
In some embodiments, the molar ratio of the compound of formula II to the compound of formula III in the reaction system is 1: (1-10). Preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.1-5). More preferably, the molar ratio of the compound of formula II to the compound of formula III is 1: (1.5 to 3).
In some embodiments, in the method, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II in the reaction system is (0.01-10): 1. preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.01-5): 1. more preferably, the mass ratio (w/w) of the deoxyribotransferase to the compound of formula II is (0.5-2): 1.
in some embodiments, the method comprises converting ≥ 40% of the compound of formula II into the compound of formula I in the reaction system after the reaction. Preferably, not less than 50% of the compound of formula II is converted into the compound of formula I.
Technical terms
Deoxyribotransferase
In the present invention, the "deoxyribotransferase" is an enzyme capable of transferring a sugar group in a deoxynucleoside to a free purine or pyrimidine to synthesize a different deoxynucleoside compound. A typical deoxyribotransferase is identified in Genbank accession number Q74LQ9.1. In the reaction system of the present invention, the above-mentioned deoxyribotransferase may be used in the form of wet cells, crude enzyme solution, crude enzyme powder or pure enzyme, and may be commercially available products, or may be produced and prepared by constructing recombinant plasmids and recombinant engineering bacteria with reference to "molecular cloning laboratory Manual" and the like.
Chemical bond(s)
In this application, chemical bond
Represents a single bond or a double bond. With C in the structure of a compound of formula I
R is for example, when the substituent R is a monovalent radical, e.g. F, Cl, Br, OH, NH 2 When the C and the R are connected through a single bond; when the substituent R is a divalent group, such as O or S, C and R are connected by a double bond, i.e. C = O, C = S is formed.
Biocatalytic preparation method
The invention provides a method for preparing a compound shown in a formula I by catalyzing a compound shown in a formula II and a compound shown in a formula III through deoxyribotransferase. The reaction formula is shown as follows:
a specific implementation process of the biocatalytic preparation method is as follows: adding a compound shown in a formula II, a compound shown in a formula III and deoxyribosyltransferase into a Tris-HCl buffer solution, uniformly mixing, maintaining the temperature at 15-70 ℃, and reacting under stirring for 1-72 hours. After the reaction is finished, adding a methanol-water solution into the reaction solution, centrifuging and/or filtering by using kieselguhr, taking supernatant or filtrate, and concentrating the collected solution to obtain a product.
The present inventors have conducted extensive and intensive studies and, as a result of extensive screening experiments, have unexpectedly developed for the first time a method for the biological production of a 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analogue having a high conversion rate, in which deoxyribotransferase exhibits specificity and selectivity for a substrate of the reaction.
The main advantages of the invention include:
1) compared with the prior art, the deoxyribotransferase screened by the application and the preparation method thereof are used for preparing the compound shown in the formula I, the conversion rate of the substrate compound shown in the formula II is improved, and the reaction cost is reduced;
2) the deoxyribotransferase screened by the application has selectivity to different reaction substrates;
3) the deoxyribotransferase screened by the method can be used for preparing the compound shown in the formula I from the compound shown in the formula II and the compound shown in the formula III through one-step reaction, the reaction steps are short, the reaction conditions are mild, the post-treatment only needs centrifugal filtration, the operation is simple and convenient, and the industrial production is easy.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise specified, the test materials of the present invention are commercially available reagents and are commercially available.
Example 1: preparation of deoxyribotransferase
The method for producing deoxyribotransferase provided in this example is suitable for producing various deoxyribotransferases described in the present application (Genbank numbers of deoxyribotransferase are Q74LQ9.1, WP _016896513.1, WP _ 010022667.1). And (3) customizing and synthesizing recombinant plasmids with the gene sequences of the deoxyribotransferase, respectively inserting the recombinant plasmids into host bacteria for expression in a gene recombination mode, and preparing freeze-dried enzyme powder.
1.1 construction and transformation of recombinant strains
Coli BL21(DE3) pET-28a-LjNDT was constructed as an example
The Kinzymenia corporation of Suzhou was entrusted to synthesize a recombinant plasmid pET-28a-LjNDT having a deoxyribotransferase gene sequence (Genbank accession number Q74LQ9.1).
Activating E.coli DH5 a/pET-28 a (+) strain [ E.coli DH5 a from Novenza Biotech Co., Ltd ], adding 3-5. mu.L of pET-28a-LjnDT (50 ng/. mu.L) to 50-100. mu.L of E.coli BL21(DE3) competent cells (from Novenza Biotech Co., Ltd.), placing on ice for 20min, performing heat shock at 42 ℃ for 90sec, rapidly returning to ice for 5min, adding 800. mu.L of non-resistant LB culture solution, 37 ℃, 200rpm, culturing for 1h, spreading on LB agar plate medium containing kanamycin resistance, and culturing overnight at 37 ℃ for 12h to obtain a recombinant strain E.coli BL21(DE3) containing pET-28 a-LjnDT.
The formula of the non-resistant LB culture solution is as follows: 5g/L of yeast powder, 10g/L of sodium chloride and 10g/L of peptone;
the formulations of the LB agar plate medium containing the kanamycin resistance were as follows: 5g/L of yeast powder, 10g/L of sodium chloride, 10g/L of peptone, 20g/L of agar powder and 25 mu g/mL of sodium bicarbonate.
1.2 culture and inducible expression of recombinant bacteria
Taking the culture and induced expression of E.coli BL21(DE3) recombinant bacteria containing pET28a-LjNDT as an example
The recombinant bacterium E.coli BL21(DE3) containing pET-28a-LjnDT obtained in 1.1 above was directly spread on a solid LB plate containing 25. mu.g/mL kanamycin resistance, and cultured at 37 ℃ for 12 to 14 hours to obtain a monoclonal colony. A single colony of E.coli BL21(DE3) containing pET28a-LjNDT was picked from a Carna plate, inoculated in 1mL of liquid LB medium containing 25. mu.g/mL of Carna resistance, and cultured with shaking at 37 ℃ for 12 hours. Then inoculating the strain into 1L of liquid LB culture medium containing 25 mug/mL kanamycin resistance according to the inoculation amount of 2 percent (v/v), culturing at 37 ℃ until the OD600 is about 0.6-0.8, adding IPTG (purchased from Shanghai Aladdin Biotechnology Co., Ltd.) until the final concentration is 1.0mmol/L, performing induced expression at 200rpm at 25 ℃ for 20h, centrifuging (4 ℃, 4000rpm, 30 min), removing supernatant, and washing the obtained bacterial sludge with 30mL of 0.9 percent NaCl solution for re-suspension for later use.
1.3 preparation of lyophilized enzyme powder
Preparation of lyophilized enzyme powder of deoxyribotransferase (Genbank accession number Q74LQ9.1)
Washing the bacterial liquid obtained in the step 1.2 twice by using 50mmol/L Tris-HCl, then suspending the thallus in 50mmol/L Tris-HCl (pH8.0) buffer solution, ultrasonically crushing cells in ice bath (amplitude transformer 6 with the power of 500W, switching on for 2s, switching off for 5s, and 30 minutes, centrifuging the ultrasonically crushed sample at 12000rpm for 30min at 4 ℃, taking the supernatant, putting the supernatant into a freeze dryer at-80 ℃ for freeze drying for 24h, and grinding the obtained freeze-dried sample to obtain freeze-dried enzyme powder.
Example 2: effect of deoxyribotransferase on reaction with various substrates
The reaction formula is as follows:
reaction operation: to a 10mL reaction flask was added 4mL Tris-HCl buffer (pH 7.0, 50 mM), and then 2mg of the compound of formula II (8.12 mmol, 1 eq), the compound of formula III (20.3 mmol, 2.5 eq) and 2mg of deoxyribosyltransferase (lyophilized enzyme powder) were added, mixed well, the temperature was adjusted to 60 ℃ and the reaction was stirred for 24 hours. After the reaction is finished, 50 mu L of reaction liquid is taken and added into a 1.5mL centrifuge tube, then 1mL of 50% methanol-water post-treatment liquid is added to stop the reaction, the centrifuge tube is centrifuged at 10000rpm, and the result of the centrifuged supernatant is detected by HPLC. The detection conditions are as follows: the chromatographic column is a Waters Xbridge C18 chromatographic column; the mobile phase is: mobile phase A: containing 10mM NH 4 5% acetonitrile solution of OAc, and 95% acetonitrile solution of mobile phase B; the temperature is 40 ℃; the flow rate was 1.3 mL/min. The results of the reaction of the substrates for the different R substituents are shown in Table 1.
Definition of enzyme activity unit: catalyzing the formation of 1ug 2 '-deoxy-2' -fluoro- β -D-arabino-guanosine analogs (R = O, S, F, Cl, NH) at 60 ℃ and pH 7.0 per minute 2 ) The amount of enzyme required is defined as one deoxyribotransferase activity unit U.
Specific activity definition: the enzyme activity unit of the deoxyribotransferase per mg is U/mg under the conditions of 60 ℃ and pH value of 7.0.
TABLE 1
As can be seen from the comparison of experiments 1-5, the effect of deoxyribotransferase (Genbank accession number Q74LQ9.1) on the reaction of substrates with different R substituents is significantly different.
Example 3: effect of different deoxyribotransferases on the reaction
The reaction formula is as follows:
reaction operation: the same substrate was used, and the same reaction procedure as in example 2 was carried out using a different deoxyribotransferase. The reaction results are shown in table 2.
Definition of enzyme activity unit: the enzyme amount required for the catalytic production of 1ug 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine at 60 ℃ and pH 7.0 per minute was defined as a deoxyribotransferase activity unit.
Specific activity definition: the enzyme activity unit of the deoxyribotransferase per mg is U/mg under the conditions of 60 ℃ and pH value of 7.0.
TABLE 2
As can be seen from the comparison of experiments 1, 6 and 7, the reaction effects of different deoxyribotransferases are significantly different.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A biocatalytic preparation method of 2 '-deoxy-2' -fluoro-beta-D-arabinosylguanosine analogs is characterized by comprising the following steps:
a step (a): in a liquid reaction system, taking a compound shown in a formula II and a compound shown in a formula III as substrates, and carrying out reaction under the catalysis of deoxyribotransferase to form a compound shown in a formula I; wherein R is selected from O, S or F;
step (b): separating the compound of formula I from the reaction system after the reaction of step (a);
wherein the deoxyribotransferase has a Genbank accession number of Q74LQ9.1.
2. The method of claim 1, wherein the compound of formula II is present at a concentration of 0.1 to 500 g/L.
3. The method of claim 1, wherein the molar ratio of the compound of formula II to the compound of formula III is 1: (1-10).
4. The method of claim 1, wherein the mass ratio of the deoxyribotransferase to the compound of formula II is (0.01-10): 1.
5. the method of claim 1, wherein the buffer of the reaction system is a phosphate buffer, a tris buffer, or a tris buffer.
6. The process of claim 1, wherein in step (a), the reaction temperature is from 15 ℃ to 70 ℃.
7. The method of claim 1, wherein in step (a), the reaction time is 1 to 72 hours.
8. The method according to claim 1, wherein in the step (a), the pH of the reaction system is 6.0 to 9.0.
9. A reaction system, characterized in that the reaction system comprises:
(1) an aqueous solvent;
(2) a substrate 1, said substrate 1 being a compound of formula II;
(3) a substrate 2, wherein the substrate 2 is a compound of formula III;
wherein R is selected from O, S or F;
(4) deoxyribotransferase, Genbank accession number Q74LQ9.1.
10. A biocatalytic preparation method of a 2 '-deoxy-2' -fluoro- β -D-arabinoguanosine analog, the preparation method comprising: performing an enzymatic reaction using the reaction system of claim 9, thereby producing a compound of formula I:
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