CN117384983B - Preparation method of GSK-3 alpha inhibitor - Google Patents

Preparation method of GSK-3 alpha inhibitor Download PDF

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CN117384983B
CN117384983B CN202311672584.2A CN202311672584A CN117384983B CN 117384983 B CN117384983 B CN 117384983B CN 202311672584 A CN202311672584 A CN 202311672584A CN 117384983 B CN117384983 B CN 117384983B
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孟雨晴
张珺哲
李岩
王继刚
朱永平
刘艳青
谷丽维
邱崇
夏菲
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Abstract

The invention discloses a preparation method of a GSK-3 alpha inhibitor. A preparation method of GSK-3 alpha inhibitor comprises the following steps: step one, synthesizing key synthetase and constructing an expression vector; step two, expressing key synthetase; step three, building an enzymatic reaction system; and fourthly, separating the product to obtain the GSK-3 alpha inhibitor. The invention has the following beneficial effects: 1. the method has the advantages that the cheap and good biological affinity L-tryptophan and 2, 3-dichloro-L-phenylalanine are used as substrates, so that the participation of toxic chemicals as substrates in the existing method is avoided, the atomic utilization rate is improved, and the chemical pollution and the possibly-introduced toxic and side effects are reduced; 2. the yield of the GSK-3 alpha inhibitor SB-216763 is improved.

Description

Preparation method of GSK-3 alpha inhibitor
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a preparation method of a GSK-3 alpha inhibitor.
Background
Glycogen synthase kinase-3 alpha (GSK-3 alpha) is a kinase with strict evolutionary conservation widely existing in mammals, and has a molecular weight of 51 kDa.
GSK-3α is a major physiological substrate for Protein Kinase B (PKB) whose activity is inhibited by PKB/Akt mediated phosphorylation in response to certain growth factor stimuli, such as Nerve Growth Factor (NGF) and glial cell line-derived neurotrophic factors.
GSK-3 alpha plays an important role in many different cellular processes, including glycogen synthesis in skeletal muscle, neuronal cell survival, and alleviation of hyperglycemia by increasing glycogen synthesis and apoptosis. GSK-3 a inhibitors have become promising drugs for the treatment of diabetes, neurodegenerative diseases (e.g., alzheimer's disease), neurological disorders (e.g., bipolar disorder, stroke, inflammation, and cancer) in association with various disease processes.
SB-216763 is a novel highly potent GSK-3 alpha selective inhibitor, IC thereof 50 The molecular is maleimide with a unique structure, the isotope labeled SB-216763 can also be used as a novel probe of biomedical imaging technology Positron Emission Tomography (PET), can carry out noninvasive monitoring on enzyme GSK-3 alpha in diseases, and has extremely high application value in the field of medical research. However, SB-216763 is generally expensive due to the limitations of current methods of preparation.
At present, SB-216763 is mainly prepared by chemical synthesis, and the reaction route is as follows:
however, the above reaction scheme has the following disadvantages: 1. poor stability of the reaction intermediates, resulting in low overall yields (typically less than 30%); 2. the reaction raw materials and reaction byproducts easily cause chemical pollution, and chemical residues influence the accuracy of the results of SB-216763 in the application process of the subsequent biological scientific research field.
Disclosure of Invention
The invention aims to: aiming at the problems of low synthesis yield, chemical pollution, residue and the like in the traditional SB-216763 chemical synthesis method, the invention discloses a preparation method of a GSK-3 alpha inhibitor.
The invention reforms an actinomyceteLentzea albidaThe heme-dependent oxidase of (2) yielded the key synthase LA1263 and used the PmLAAD protein and MarC protein to co-operate with the key synthase LA1263 to produce SB-216763.
The technical scheme is as follows: a preparation method of GSK-3 alpha inhibitor comprises the following steps:
step one, synthesis of Key synthetase and construction of expression vector
(11) For actinomycetesLentzea albidaThe heme dependent oxidase is structurally modified to obtain key synthetase LA1263, and the amino acid sequence of the key synthetase LA1263 is shown as SEQ ID NO. 1;
(12) Cloning a gene fragment of key synthetase LA1263 into a vector pMCSG10 by using a Gibson splice cloning technology to obtain an expression vector pMCSG10-LA1263, cloning a gene fragment PmLAAD into a vector pET28a (+) to obtain an expression vector pET28a (+) -PmLAAD, and cloning a gene fragment MarC into a vector pET26b to obtain an expression vector pET26b-MarC, wherein:
the amino acid sequence of the gene fragment PmLAAD is shown in SEQ ID NO. 2;
the amino acid sequence of the gene fragment MarC is shown as SEQ ID NO. 3;
(13) Respectively converting the constructed expression vectors pET28a (+) -PmLAAD, the expression vectors pMCSG10-LA1263 and the expression vectors pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two, expression of key synthetase
(21) Preparing crude PmLAAD protein lysate;
(22) Preparing LA1263 protein crude lysate;
(23) Preparing MarC protein crude lysate;
step three, construction of an enzymatic reaction system
(31) The following substances were added to the reaction vessel in order:
(32) After uniform mixing, carrying out oscillation reaction for 6-8h, and keeping the reaction vessel in an open state in the whole process of the oscillation reaction;
(33) 600mL of MarC protein crude lysate, 2.1g of alpha-ketoglutaric acid, 1.32g of ascorbic acid and a proper amount of ferrous sulfate are respectively added into a reaction vessel, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1mM, and after uniform mixing, the reaction vessel is continuously subjected to shaking reaction for 2 hours to obtain a reaction liquid;
step four, separation of products
And (3) carrying out post-treatment on the reaction liquid obtained in the step (III) to obtain the GSK-3 alpha inhibitor after completion.
Further, the specific steps of step (21) are as follows:
(211) Adding 10 mu L of pET28a (+) -PmLAAD strain solution into 5mL of LB liquid culture medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET28a (+) -PmLAAD seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(212) Adding the pET28a (+) -PmLAAD seed solution obtained in the step (211) into a test container containing 1L of LB liquid medium, and then shake culturing at 37 ℃ and 200rpm in a shaking table until the bacterial solution contains the bacterial solution concentration OD of the PmLAAD expression vector 600 Until 1.1-1.3, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(213) Adding a proper amount of IPTG into the bacterial liquid treated in the step (212) to ensure that the concentration of the IPTG in the bacterial liquid is 0.1mM, mixing uniformly, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 hours;
(214) Pouring the bacterial liquid induced in the step (213) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O-washing followed by addition of 30The deposited bacteria are resuspended in mL loading buffer until no obvious bacteria mass exists to obtain bacteria weight suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 5mM imidazole, 10% v/v glycerol, the balance being water;
(215) Crushing the bacterial body weight suspension obtained in the step (214) by using an ultrasonic crusher to obtain crushed bacterial liquid, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude PmLAAD protein lysate.
Further, the specific steps of step (22) are as follows:
(221) Adding 10 mu L of pMCSG10-LA1263 strain solution into 5mL of LB liquid medium, and performing shake overnight culture at 37 ℃ and 200rpm to obtain pET26b-LA1263 seed solution, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(222) Adding the pMCSG10-LA1263 seed solution obtained in the step (221) into a test container containing 1L of LB liquid medium, and shake culturing at 37 ℃ and 200rpm in a shaker until the bacterial solution concentration OD containing LA1263 expression vector 600 Until =0.7-0.9, the test vessel was then placed in an ice bath for 30min, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(223) Adding proper amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM, and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 hours;
(224) After the induction of the step (223)Pouring the bacterial liquid into a centrifugal cup, centrifuging at 4000g for 15min, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(225) Crushing the bacterial body weight suspension obtained in the step (224) by using a ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LA1263 protein crude lysate.
Further, the specific steps of step (23) are as follows:
(231) Adding 10 mu L of pET26b-MarC strain solution into 5mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-MarC seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(232) Adding the pET26b-MarC seed solution obtained in the step (231) into a test container containing 1L of LB liquid medium, and shake culturing in a shaking table at 37 ℃ and 200rpm until the concentration of the bacterial solution OD containing the MarC expression vector is reached 600 Until =0.9-1.1, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(233) Respectively adding ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (232) to ensure that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at the temperature of 18 ℃ and the speed of 200rpm for 16 hours;
(234) Pouring the bacterial liquid induced in the step (233) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(235) Crushing the bacterial body heavy suspension by using an ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate.
Further, the NaH in step (31) 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0;
in step (31), the (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M.
Further, the specific steps of the post-treatment in the fourth step are as follows:
and (3) regulating the pH value of the reaction liquid obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding 1L of ethyl acetate into the reaction liquid to extract for at least three times, filtering an emulsion layer by using diatomite, separating the solution, combining organic phases, drying the organic phases by using anhydrous sodium sulfate, concentrating the organic phases under reduced pressure to obtain a crude product, purifying the crude product by using silica gel column chromatography, and eluting under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the GSK-3 alpha inhibitor.
The beneficial effects are that: the invention has the following beneficial effects:
1. the method has the advantages that the cheap and good biological affinity L-tryptophan and 2, 3-dichloro-L-phenylalanine are used as substrates, so that the participation of toxic chemicals as substrates in the existing method is avoided, the atomic utilization rate is improved, and the chemical pollution and the possibly-introduced toxic and side effects are reduced;
2. the yield of the GSK-3 alpha inhibitor SB-216763 is improved.
Drawings
FIG. 1 is a plasmid map of the expression vector pMCSG10-LA 1263.
FIG. 2 is a plasmid map of the expression vector pET28a (+) -PmLAAD.
FIG. 3 is a plasmid map of expression vector pET26 b-MarC.
FIG. 4 is an HPLC plot of the product prepared in example 1.
FIG. 5 is a diagram of the product of example 1 1 H NMR spectrum.
FIG. 6 is a diagram of the product of example 1 13 C NMR spectrum.
Detailed Description
The following detailed description of specific embodiments of the invention.
The "range" disclosed herein is defined in terms of lower and upper limits, with the given range being defined by the selection of a lower and an upper limit, the selected lower and upper limits defining the boundaries of the particular range. Ranges that are defined in this way can be inclusive or exclusive of the endpoints, and any combination can be made, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 10-50 are listed for a particular parameter, it is understood that ranges of 10-40 and 20-50 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3,4 and 5 are listed, the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise indicated, the range of values "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed throughout, and "0-5" is simply a shorthand representation of a combination of these values.
All embodiments and alternative embodiments of the present application may be combined with each other to form new solutions, unless specifically stated otherwise.
All technical features and optional technical features of the present application may be combined with each other to form new technical solutions, unless specified otherwise.
All steps of the present application may be performed sequentially or randomly, preferably sequentially, unless otherwise indicated. For example, the method comprises steps (a) and (b), meaning that the method may comprise steps (a) and (b) performed sequentially, or may comprise steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), may include steps (a), (c) and (b), may include steps (c), (a) and (b), and the like.
Reference herein to "comprising" and "including" means open ended, as well as closed ended, unless otherwise noted. For example, the terms "comprising" and "comprises" may mean that other components not listed may be included or included, or that only listed components may be included or included.
Unless otherwise specified, the reaction is carried out under normal temperature and normal pressure conditions.
Unless otherwise indicated, all parts or percentages are parts or percentages by weight.
In the present invention, the materials used are all known materials, and are commercially available or synthesized by known methods.
In the present invention, the devices or apparatuses used are conventional devices or apparatuses known in the art, and are commercially available.
The synthetic route of the invention is as follows:
。
a preparation method of GSK-3 alpha inhibitor comprises the following steps:
step one, synthesis of Key synthetase and construction of expression vector
(11) For actinomycetesLentzea albidaIs structured by heme-dependent oxidaseModifying to obtain key synthetase LA1263, wherein the amino acid sequence of the key synthetase LA1263 is shown as SEQ ID NO. 1;
SEQ ID NO.1（LA1263）：
NRIATRGRLVEVLREAALIELAVMLQYLYAAYSVPTYGAGAEYVRLGLWTPEQLQLACGDGGETLDEGVRSLLLGVAREEMIHFLLVNNIIMALGEPFHVPRVDFATINNELPVPLDFALERLCLGSVERFTQIEKPEHQVGDLRPRDGEHQAQRQEHGYASLSELYADIREALRQIPDLFLVEKGRGGGEHHLFLRESINHEHPDYQLEVDDLPSALHAIDVITEQGEGGVLDEDAVEDSHYTSFLLIGELLRKAPVTGPHGELWNPAYPVARNPSLNPASPAAEAVTDPDARTVMQIFNESYFTALQLMAQHFGERPDSSLRRSDLMNAAIDIMAGLMRPLAELLVTLPSGRRGTTAGPSFELPEVPAAISRPDVARRSIAMRLDDIAAQCAKCALVPARVGELSTFWADHFRAQAGGL
(12) Cloning a gene fragment of key synthetase LA1263 into a vector pMCSG10 by using a Gibson splice cloning technology to obtain an expression vector pMCSG10-LA1263, cloning a gene fragment PmLAAD into a vector pET28a (+) to obtain an expression vector pET28a (+) -PmLAAD, and cloning a gene fragment MarC into a vector pET26b to obtain an expression vector pET26b-MarC, wherein:
the amino acid sequence of the gene fragment PmLAAD is shown in SEQ ID NO. 2;
the amino acid sequence of the gene fragment MarC is shown as SEQ ID NO. 3;
SEQ ID NO.2（PmLAAD）：
MNISRRKLLLGVGAAGVLAGGAALVPMVRRDGKFVEAKSRASFVEGTQGALPKEADVVIIGAGIQGIMTAINLAERGMSVTILEKGQIAGEQSGRAYSQIISYQTSPEIFPLHHYGKILWRGMNEKIGADTSYRTQGRVEALADEKALDKAQAWIKTAKEAAGFDTPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPALARYAKQIGVKIYTNCAVRGIETAGGKISDVVSEKGAIKTSQVVLAGGIWSRLFMGNMGIDIPTLNVYLSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPRIFTSSIVKDSFLLGPKFMHLLGGGELPLEFSIGEDLFNSFKMPTSWNLDEKTPFEQFRVATATQNTQHLDAVFQRMKTEFPVFEKSEVVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMTEGPAAGEVTADIVMGKKPVIDPTPFSLDRFKK
SEQ ID NO.3（MarC）：
MLSAEDNKLLTEVAADTRMGQLLRRYWHPIAASSQLDDKHPTRLVHLLGEKLVLYKDKQGRLGLIDERCPHRRASMLYGIPEQEGLRCSYHGWLFNNAGRCLAQPYEQMEDPCSNFKDHVRIKSYPVRELGGLVFAYLGPAPAPELPAWDLLVTENLHRDIGFAVVPCNWLQIMENAADPVHAEWLHGHFANYVWERLGKPERIKPFPTHKKIGFDLSEYGIIKRRVLEGETEEHENWKFGHSLVFPNLQKGGGLQWRVPMDETRTLHVWYYTYTPAEGTVVPKDAPIPVFDVPVPALDEHGHPRWDVLDFTAGQDMVMWYTQGAVAERWKETLGRSDRGVIMYRNLLKANLEKLARGEEPMNVFRDPAKAAFIQLDTEESSGRRLYSDRARQYGPSSSNGPGGGATKYSPVLNLHKGAETVSAKEVMPETALPAAPPAARKETA
(13) Respectively converting the constructed expression vectors pET28a (+) -PmLAAD, the expression vectors pMCSG10-LA1263 and the expression vectors pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two, expression of key synthetase
(21) Preparing crude PmLAAD protein lysate;
(22) Preparing LA1263 protein crude lysate;
(23) Preparing MarC protein crude lysate;
step three, construction of an enzymatic reaction system
(31) The following substances were added to the reaction vessel in order:
(32) After uniform mixing, carrying out oscillation reaction for 6-8h, and keeping the reaction vessel in an open state in the whole process of the oscillation reaction;
(33) 600mL of MarC protein crude lysate, 2.1g of alpha-ketoglutaric acid, 1.32g of ascorbic acid and a proper amount of ferrous sulfate are respectively added into a reaction vessel, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1mM, and after uniform mixing, the reaction vessel is continuously subjected to shaking reaction for 2 hours to obtain a reaction liquid;
step four, separation of products
And (3) carrying out post-treatment on the reaction liquid obtained in the step (III) to obtain the GSK-3 alpha inhibitor after completion.
Further, the specific steps of step (21) are as follows:
(211) Adding 10 mu L of pET28a (+) -PmLAAD strain solution into 5mL of LB liquid culture medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET28a (+) -PmLAAD seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(212) Adding the pET28a (+) -PmLAAD seed solution obtained in the step (211) into a test container containing 1L of LB liquid medium, and then shake culturing at 37 ℃ and 200rpm in a shaking table until the bacterial solution contains the bacterial solution concentration OD of the PmLAAD expression vector 600 Until 1.1-1.3, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(213) Adding a proper amount of IPTG into the bacterial liquid treated in the step (212) to ensure that the concentration of the IPTG in the bacterial liquid is 0.1mM, mixing uniformly, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 hours;
(214) Pouring the bacterial liquid induced in the step (213) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 5mM imidazole, 10% v/v glycerol, the balance being water;
(215) Crushing the bacterial body weight suspension obtained in the step (214) by using an ultrasonic crusher to obtain crushed bacterial liquid, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude PmLAAD protein lysate.
Further, the specific steps of step (22) are as follows:
(221) Adding 10 mu L of pMCSG10-LA1263 strain solution into 5mL of LB liquid medium, and performing shake overnight culture at 37 ℃ and 200rpm to obtain pET26b-LA1263 seed solution, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(222) Adding the pMCSG10-LA1263 seed solution obtained in the step (221) into a test container containing 1L of LB liquid medium, and shake culturing at 37 ℃ and 200rpm in a shaker until the bacterial solution concentration OD containing LA1263 expression vector 600 Until =0.7-0.9, the test vessel was then placed in an ice bath for 30min, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(223) Adding proper amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM, and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 hours;
(224) Pouring the bacterial liquid induced in the step (223) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(225) Crushing the bacterial body weight suspension obtained in the step (224) by using a ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LA1263 protein crude lysate.
Further, the specific steps of step (23) are as follows:
(231) Adding 10 mu L of pET26b-MarC strain solution into 5mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-MarC seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(232) Adding the pET26b-MarC seed solution obtained in the step (231) into a test container containing 1L of LB liquid medium, and shake culturing in a shaking table at 37 ℃ and 200rpm until the concentration of the bacterial solution OD containing the MarC expression vector is reached 600 Until =0.9-1.1, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(233) Respectively adding ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (232) to ensure that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at the temperature of 18 ℃ and the speed of 200rpm for 16 hours;
(234) Pouring the bacterial liquid induced in the step (233) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(235) Crushing the bacterial body heavy suspension by using an ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate.
Further, the NaH in step (31) 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0;
in step (31), the (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M.
Further, the specific steps of the post-treatment in the fourth step are as follows:
and (3) regulating the pH value of the reaction liquid obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding 1L of ethyl acetate into the reaction liquid to extract for at least three times, filtering an emulsion layer by using diatomite, separating the solution, combining organic phases, drying the organic phases by using anhydrous sodium sulfate, concentrating the organic phases under reduced pressure to obtain a crude product, purifying the crude product by using silica gel column chromatography, and eluting under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the GSK-3 alpha inhibitor.
Example 1
A preparation method of GSK-3 alpha inhibitor comprises the following steps:
step one: synthesis of key synthetases and construction of expression vectors
(11) For actinomycetesLentzea albidaThe heme dependent oxidase is structurally modified to obtain key synthetase LA1263, and the amino acid sequence of the key synthetase LA1263 is shown as SEQ ID NO. 1;
(12) Cloning a gene fragment of key synthetase LA1263 into a vector pMCSG10 by using a Gibson splice cloning technology to obtain an expression vector pMCSG10-LA1263, cloning a gene fragment PmLAAD into a vector pET28a (+) to obtain an expression vector pET28a (+) -PmLAAD, and cloning a gene fragment MarC into a vector pET26b to obtain an expression vector pET26b-MarC, wherein:
the amino acid sequence of the gene fragment PmLAAD is shown in SEQ ID NO. 2;
the amino acid sequence of the gene fragment MarC is shown as SEQ ID NO. 3;
(13) Respectively converting the constructed expression vectors pET28a (+) -PmLAAD, the expression vectors pMCSG10-LA1263 and the expression vectors pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two, expression of key synthetase
(21) Preparing crude PmLAAD protein lysate;
(22) Preparing LA1263 protein crude lysate;
(23) Preparing MarC protein crude lysate;
step three, construction of an enzymatic reaction system
(31) The following substances were added to the reaction vessel in order:
(32) After uniform mixing, carrying out oscillation reaction for 7 hours, and keeping the reaction vessel in an open state in the whole process of the oscillation reaction;
(33) 600mL of MarC protein crude lysate, 2.1g of alpha-ketoglutaric acid, 1.32g of ascorbic acid and a proper amount of ferrous sulfate are respectively added into a reaction vessel, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1mM, and after uniform mixing, the reaction vessel is continuously subjected to shaking reaction for 2 hours to obtain a reaction liquid;
step four, separation of products
And (3) carrying out post-treatment on the reaction liquid obtained in the step (III) to obtain the GSK-3 alpha inhibitor after completion. The product mass was 2.84g and the yield was 38.4%.
Further, the specific steps of step (21) are as follows:
(211) Adding 10 mu L of pET28a (+) -PmLAAD strain solution into 5mL of LB liquid culture medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET28a (+) -PmLAAD seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(212) Adding the pET28a (+) -PmLAAD seed solution obtained in the step (211) into a test container containing 1L of LB liquid medium, and then shake culturing at 37 ℃ and 200rpm in a shaking table until the bacterial solution contains the bacterial solution concentration OD of the PmLAAD expression vector 600 Until =1.2, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(213) Adding a proper amount of IPTG into the bacterial liquid treated in the step (212) to ensure that the concentration of the IPTG in the bacterial liquid is 0.1mM, mixing uniformly, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 hours;
(214) Pouring the bacterial liquid induced in the step (213) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 5mM imidazole, 10% v/v glycerol, the balance being water;
(215) Crushing the bacterial body weight suspension obtained in the step (214) by using an ultrasonic crusher to obtain crushed bacterial liquid, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude PmLAAD protein lysate.
Further, the specific steps of step (22) are as follows:
(221) Adding 10 mu L of pMCSG10-LA1263 strain solution into 5mL of LB liquid medium, and performing shake overnight culture at 37 ℃ and 200rpm to obtain pET26b-LA1263 seed solution, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(222) Adding the pMCSG10-LA1263 seed solution obtained in the step (221) into a test container containing 1L of LB liquid medium, and shake culturing at 37 ℃ and 200rpm in a shaker until the bacterial solution concentration OD containing LA1263 expression vector 600 Until 0.8, the test vessel was then placed in an ice bath for 30min, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(223) Adding proper amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM, and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 hours;
(224) Pouring the bacterial liquid induced in the step (223) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(225) Crushing the bacterial body weight suspension obtained in the step (224) by using a ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LA1263 protein crude lysate.
Further, the specific steps of step (23) are as follows:
(231) Adding 10 mu L of pET26b-MarC strain solution into 5mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-MarC seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(232) Adding the pET26b-MarC seed solution obtained in the step (231) into a test container containing 1L of LB liquid medium, and shake culturing at 37 ℃ and 200rpm in a shaking table until the concentration of the bacterial solution OD containing the MarC expression vector is reached 600 Until =1, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(233) Respectively adding ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (232) to ensure that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at the temperature of 18 ℃ and the speed of 200rpm for 16 hours;
(234) Pouring the bacterial liquid induced in the step (233) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(235) Crushing the bacterial body heavy suspension by using an ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate.
Further, the NaH in step (31) 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0;
in step (31), the (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M.
Further, the specific steps of the post-treatment in the fourth step are as follows:
and (3) regulating the pH value of the reaction liquid obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding 1L of ethyl acetate into the reaction liquid to extract for at least three times, filtering an emulsion layer by using diatomite, separating the solution, combining organic phases, drying the organic phases by using anhydrous sodium sulfate, concentrating the organic phases under reduced pressure to obtain a crude product, purifying the crude product by using silica gel column chromatography, and eluting under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the GSK-3 alpha inhibitor.
Example 2
Substantially the same as in example 1, the difference is only that:
1. after uniformly mixing in the step (32), the oscillation reaction time is 6 hours;
2. in the step (212), the concentration OD of the bacterial liquid containing the PmLAAD expression vector in the bacterial liquid 600 Until =1.1;
3. in the step (222), the bacterial liquid concentration OD containing the LA1263 expression vector is reached 600 =0.7;
4. in the step (232), the concentration OD of the bacterial liquid containing the MarC expression vector is reached 600 =0.9;
the final product obtained in example 2 had a mass of 2.80g and a yield of 37.86%.
Example 3
Substantially the same as in example 1, the difference is only that:
1. after uniformly mixing in the step (32), the oscillation reaction time is 6-8h;
2. in the step (212), the concentration OD of the bacterial liquid containing the PmLAAD expression vector in the bacterial liquid 600 =1.1-1.3;
3. in the step (222), the bacterial liquid concentration OD containing the LA1263 expression vector is reached 600 =0.7-0.9;
4. in the step (232), the concentration OD of the bacterial liquid containing the MarC expression vector is reached 600 =0.9-1.1;
the final product obtained in example 3 had a mass of 2.86g and a yield of 38.67%.
Characterization of the properties:
1. the product prepared in example 1 was subjected to HPLC detection, and the results are shown in FIG. 4. As can be seen from FIG. 4, the product prepared in example 1 has a higher purity and almost no other impurities remain.
2. The results of subjecting the product prepared in example 1 to nuclear magnetic resonance spectroscopy are shown in FIGS. 5 and 6. From FIGS. 5 and 6, it can be seen that the product prepared in example 1 has consistent signal chemical shift, coupling constant and other parameters compared with the standard nuclear magnetic resonance spectrogram, which indicates that the molecular structure of the product is the same as SB-216763 and has higher purity.
2. Detection Kit (Mouse GSK-3 alpha ELISA Kit) and Mouse GSK-3 beta Kit for detecting activity by using Mouse GSK-3 alpha KitThe Activity detection Kit (Mouse GSK-3. Beta. ELISA Kit) determines the IC of the SB-216763 product prepared in example 1 on Mouse GSK-3. Alpha. And GSK-3. Beta 50 Values. The final concentrations of SB-216763 added to the reaction system were 2nM, 5nM, 10nM, 20nM, 40nM, 80nM and 100nM, respectively, and the IC of SB-216763 for small GSK-3. Alpha. And GSK-3. Beta. Was determined and calculated by ELISA method 50 The values were 29.8nM and 87.2nM, respectively.
The embodiments of the present invention have been described in detail. However, the present invention is not limited to the above-described embodiments, and various modifications may be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.

Claims (6)

1. A method for preparing a GSK-3 alpha inhibitor, comprising the steps of:
step one, synthesis of Key synthetase and construction of expression vector
(11) For actinomycetesLentzea albidaThe heme dependent oxidase is structurally modified to obtain key synthetase LA1263, and the amino acid sequence of the key synthetase LA1263 is shown as SEQ ID NO. 1;
(12) Cloning a gene fragment of key synthetase LA1263 into a vector pMCSG10 by using a Gibson splice cloning technology to obtain an expression vector pMCSG10-LA1263, cloning a gene fragment PmLAAD into a vector pET28a (+) to obtain an expression vector pET28a (+) -PmLAAD, and cloning a gene fragment MarC into a vector pET26b to obtain an expression vector pET26b-MarC, wherein:
the amino acid sequence of the gene fragment PmLAAD is shown in SEQ ID NO. 2;
the amino acid sequence of the gene fragment MarC is shown as SEQ ID NO. 3;
(13) Respectively converting the constructed expression vectors pET28a (+) -PmLAAD, the expression vectors pMCSG10-LA1263 and the expression vectors pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two, expression of key synthetase
(21) Preparing crude PmLAAD protein lysate;
(22) Preparing LA1263 protein crude lysate;
(23) Preparing MarC protein crude lysate;
step three, construction of an enzymatic reaction system
(31) The following substances were added to the reaction vessel in order:
；
(32) After uniform mixing, carrying out oscillation reaction for 6-8h, and keeping the reaction vessel in an open state in the whole process of the oscillation reaction;
(33) 600mL of MarC protein crude lysate, 2.1g of alpha-ketoglutaric acid, 1.32g of ascorbic acid and a proper amount of ferrous sulfate are respectively added into a reaction vessel, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1mM, and after uniform mixing, the reaction vessel is continuously subjected to shaking reaction for 2 hours to obtain a reaction liquid;
step four, separation of products
And (3) carrying out post-treatment on the reaction liquid obtained in the step (III), and obtaining the GSK-3 alpha inhibitor after finishing, wherein:
the GSK-3 alpha inhibitor is SB-216763.
2. The method of preparing a GSK-3α inhibitor according to claim 1 wherein step (21) is performed as follows:
(211) Adding 10 mu L of pET28a (+) -PmLAAD strain solution into 5mL of LB liquid culture medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET28a (+) -PmLAAD seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(212) Adding the pET28a (+) -PmLAAD seed solution obtained in the step (211) into a test container containing 1L of LB liquid medium, and then shake culturing at 37 ℃ and 200rpm in a shaking table until the bacterial solution contains the bacterial solution concentration OD of the PmLAAD expression vector 600 Until 1.1-1.3, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(213) Adding a proper amount of IPTG into the bacterial liquid treated in the step (212) to ensure that the concentration of the IPTG in the bacterial liquid is 0.1mM, mixing uniformly, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 hours;
(214) Pouring the bacterial liquid induced in the step (213) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 5mM imidazole, 10% v/v glycerol, the balance being water;
(215) Crushing the bacterial body weight suspension obtained in the step (214) by using an ultrasonic crusher to obtain crushed bacterial liquid, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude PmLAAD protein lysate.
3. The method of preparing a GSK-3α inhibitor according to claim 1 wherein step (22) is performed as follows:
(221) Adding 10 mu L of pMCSG10-LA1263 strain solution into 5mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pMCSG10-LA1263 seed solution, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(222) Adding the pMCSG10-LA1263 seed solution obtained in the step (221) into a test container containing 1L of LB liquid medium, and shake culturing at 37 ℃ and 200rpm in a shaker until the bacterial solution concentration OD containing LA1263 expression vector 600 Until =0.7-0.9, the test vessel was then placed in an ice bath for 30min, wherein:
ampicillin-containing liquid medium with concentration of 50 mug/mL;
(223) Adding proper amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM, and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 hours;
(224) Pouring the bacterial liquid induced in the step (223) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(225) Crushing the bacterial body weight suspension obtained in the step (224) by using a ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LA1263 protein crude lysate.
4. The method of preparing a GSK-3α inhibitor according to claim 1 wherein step (23) is performed as follows:
(231) Adding 10 mu L of pET26b-MarC strain solution into 5mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-MarC seed solution, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(232) Adding the pET26b-MarC seed solution obtained in the step (231) into a test container containing 1L of LB liquid mediumShake culturing at 37deg.C and 200rpm in shaking table to reach bacterial liquid concentration OD containing MarC expression vector 600 Until =0.9-1.1, the test vessel was then removed and placed in an ice bath for 30min, wherein:
LB liquid medium contains kanamycin, and the concentration of kanamycin is 50 mug/mL;
(233) Respectively adding ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (232) to ensure that the concentration of the ferrous ammonium sulfate in the bacterial liquid is 40 mu M, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25mM and the concentration of the IPTG in the bacterial liquid is 0.1mM, uniformly mixing, and then placing into a shaking table for induced culture at the temperature of 18 ℃ and the speed of 200rpm for 16 hours;
(234) Pouring the bacterial liquid induced in the step (233) into a centrifugal cup, centrifuging for 15min at 4000g, discarding the supernatant, and precipitating the surface with ddH 2 O cleaning, and adding 30mL of loading buffer solution to suspend the precipitated bacteria until no obvious bacteria mass exists, so as to obtain bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100mM Tris-HCl, 300mM NaCl, 15mM imidazole, 10% v/v glycerol, the balance being water;
(235) Crushing the bacterial body heavy suspension by using an ultrasonic crusher, wherein:
the diameter of the amplitude transformer is 6mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2s, the suspension time is 4s, and the total working time is 30min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate.
5. The method of preparing a GSK-3α inhibitor according to claim 1 wherein the NaH in step (31) 2 PO 4 -Na 2 HPO 4 The concentration of the buffer is 1M, the pH value is 8.0, and/or
In step (31), the (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M.
6. The method of preparing a GSK-3α inhibitor according to claim 1 wherein the post-treatment in step four is performed as follows:
and (3) regulating the pH value of the reaction liquid obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding 1L of ethyl acetate into the reaction liquid to extract for at least three times, filtering an emulsion layer by using diatomite, separating the solution, combining organic phases, drying the organic phases by using anhydrous sodium sulfate, concentrating the organic phases under reduced pressure to obtain a crude product, purifying the crude product by using silica gel column chromatography, and eluting under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the GSK-3 alpha inhibitor.
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