CN116463362B - Preparation method of cell division inhibitor - Google Patents

Preparation method of cell division inhibitor Download PDF

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CN116463362B
CN116463362B CN202310708460.9A CN202310708460A CN116463362B CN 116463362 B CN116463362 B CN 116463362B CN 202310708460 A CN202310708460 A CN 202310708460A CN 116463362 B CN116463362 B CN 116463362B
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concentration
bacterial
marc
viob
laad
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CN116463362A (en
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孟雨晴
张珺哲
王继刚
刘艳青
朱永平
谷丽维
邱崇
刘丹丹
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Institute of Materia Medica of CAMS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms

Abstract

The invention discloses a preparation method of a cell division inhibitor, which comprises the following steps: step one, synthesizing key synthetase and constructing a carrier; step two, expressing and preprocessing key synthetase; step three, building an enzymatic reaction system; and step four, regulating the pH value of the reaction liquid obtained in the step three to be 1, adding ethyl acetate 1L 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, concentrating under reduced pressure to obtain a crude product, purifying the crude product by using silica gel column chromatography, and eluting to obtain an orange-red product to obtain the cell division inhibitor. The invention has the following beneficial effects: 1. the reaction steps are few; 2. l-tryptophan and L-histidine with low cost and good biological affinity are used as substrates, so that the atom utilization rate is improved, and chemical pollution and possibly introduced toxic and side effects are reduced; 3. realizes the synthesis of gram-grade products, saves the cost of the subsequent preparation of the medicines and reduces the price of the medicines.

Description

Preparation method of cell division inhibitor
Technical Field
The invention belongs to the field of drug synthesis, and particularly relates to a preparation method of a cell division inhibitor.
Background
In recent years, dimeric amino acid active natural products have received widespread attention for pharmaceutical use due to their generally good biological activity. The tryptophan-histidine heterodimer represented by DDM-A (Didemnidine A) from natural sources shows outstanding activity in inhibiting cell division G2 phase check points and the like, and meanwhile, the tryptophan-histidine heterodimer also has certain topoisomerase I and protein kinase C inhibition effects, and the biological activity makes the tryptophan-histidine heterodimer an ideal molecular model and a patent medicine precursor of medicines such as anticancer medicines, antimalarial medicines and the like. thestructuralformulaofDDM-Aisasfollows:
。
currently,DDM-aofnaturaloriginismainlyderivedfromthechordaeintheocean,ascidians,themainfunctionofDDM-ainnaturebeingtoalertanddefendpotentialpredators. inrecentyears,theyieldofnaturalsourcesofDDM-Ahasbeenextremelylimitedduetoexcessivedevelopmentofmarineresourcesandenvironmentalpollution.
DDM-Aanditsderivativesarepreparedbythecurrentcommonchemicalsynthesismethod,andthesyntheticrouteisasfollows:
however, the technical scheme has the following defects: 1. the preparation route is longer, the method is a multi-step reaction, the method is complex and tedious, and the separation and purification are required to be carried out independently after each step of reaction is finished; 2. the reaction raw materials and the reaction byproducts are often toxic, so that chemical pollution is easy to cause, and the biological safety is doubtful; 3. theefficiencyofsynthesizingDDM-Aisnothigh,theoverallrecoveryyieldisgenerallylowerthan50%,andthereactionscaleisusuallyintheorderofhundredmilligrams,sothatmassproductioncannotberealized.
Disclosure of Invention
The invention aims to: thepresentinventionhasbeenmadeinviewoftheabove-mentionedproblemsoccurringinthepriorart,anditisanobjectofthepresentinventiontodiscloseamethodforpreparingacelldivisioninhibitor,whichiscapableofdirectlypreparingDDM-aasacelldivisioninhibitorbyaone-pottwo-stepmethod.
The technical scheme is as follows: a preparation method of a cell division inhibitor comprises the following specific steps:
step one: synthesis and vector construction of key synthetases
(11) Synthesizing LAAD gene fragments, vioB gene fragments and MarC gene fragments for expressing key synthetase, wherein:
the amino acid sequence of the LAAD gene fragment is shown as SEQ ID NO. 1;
the amino acid sequence of the VioB gene fragment is shown as SEQ ID NO. 2;
the amino acid sequence of the MarC gene fragment is shown as SEQ ID NO. 3;
(12) Cloning the LAAD gene fragment into a pET28a (+) vector by using a Gibson splicing cloning technology to obtain an expression vector pET28a (+) -LAAD, and cloning the VioB gene fragment and the MarC gene fragment into a pET26b vector to obtain an expression vector pET26b-VioB and an expression vector pET26b-MarC, wherein:
the LAAD gene fragment, the VioB gene fragment and the MarC gene fragment are positioned between NdeI and HindIII restriction enzyme sites;
(13) Respectively converting the constructed expression vectors pET28a (+) -LAAD, pET26b-VioB and pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two: expression and pretreatment of key synthetases
(21) Preparation of LAAD protein crude lysate
(211) Adding 10 uL of pET28a (+) -LAAD strain solution into 5 mL of LB liquid culture medium, and shaking overnight at 37 ℃ and 200rpm for culture to obtain pET28a (+) -LAAD seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(212) Adding the pET28a (+) -LAAD 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 concentration of bacterial solution OD containing LAAD expression vector 600 Until 1.1-1.3, the test vessel was then removed and placed in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 0.1 and mM, uniformly mixing, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 h;
(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 adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 5 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LAAD protein crude lysate;
(22) Preparation of VioB protein crude lysate
(221) Adding 10 uL of pET26b-VioB strain solution into 5 mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-VioB seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(222) Adding the pET26b-VioB seed solution obtained in the step (221) 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 bacterial solution OD containing the VioB expression vector 600 Until =0.7-0.9, the test vessel was then placed in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(223) Adding proper amounts of ammonium ferrous sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ammonium ferrous sulfate in the bacterial liquid is 40 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude VioB protein lysate.
(23) Preparation of MarC protein crude lysate
(231) Adding 10 uL of pET26b-MarC strain solution into 5 mL 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 with the concentration of 50 ug/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 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate;
step three: construction of enzymatic reaction System
(31) The following substances were added to the reaction vessel separately:
，
wherein:
the NaH is 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0;
said (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M;
(32) After uniform mixing, carrying out oscillation reaction for 6-8h, wherein the reaction vessel is kept in an open state (namely, the oxygen concentration at the upper part of the reaction vessel is the same as the oxygen concentration in the air) in the whole process of the oscillation reaction;
(33) Adding 600 mL of MarC protein crude lysate prepared in the step (23), 2.1g of alpha-ketoglutaric acid, 1.32 g of ascorbic acid and a proper amount of ferrous sulfate into a reaction vessel respectively, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1 mM, mixing uniformly, and continuing to shake and react for 2 hours to obtain a reaction liquid;
step four: separation and purification of the product
And (3) regulating the pH value of the reaction solution obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding ethyl acetate 1L to the reaction solution for extraction 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 the crude product under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the cell division inhibitor.
The beneficial effects are that: the invention has the following beneficial effects:
1. the reaction steps are few, namely, on the basis of the research results of the synthesis biology, a 2-step synthesis method taking key enzymes LAAD, vioB and MarC as catalytic conditions is developed;
2. the cheap and good biological affinity L-tryptophan and L-histidine 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, the chemical pollution is reduced, and the possibly introduced toxic and side effects are reduced;
3. in a reaction system as small as possible, the synthesis of gram-grade products is realized, the recovery yield is higher than that of the existing chemical synthesis means, the cost of the subsequent preparation of the medicines is saved, and the medicine price is reduced.
Drawings
FIG. 1 is a vector map of the expression vector pET28a (+) -LAAD.
FIG. 2 is a vector map of the expression vector pET26 b-VioB.
FIG. 3 is a vector map of the 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.
FIG. 7 is a single crystal growth chart under a microscope of the product prepared in example 1.
Detailed Description
The following detailed description of specific embodiments of the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
1. Aiming at the problem of longer route of the current chemical synthesis method, a 2-step synthesis method taking key enzymes LAAD, vioB and MarC as catalytic conditions is developed on the basis of the research results of the synthesis biology, and the specific reaction steps are as follows:
2. the method takes the L-tryptophan and the L-histidine with low cost and good biological affinity as the substrates, avoids the participation of toxic chemicals as the substrates in the existing method, improves the atomic utilization rate, reduces the chemical pollution and the possibly introduced toxic and side effects.
3. The invention also needs to solve the problem of low yield caused by the increase of synthesis steps in the prior art route, realize the synthesis of gram-grade products in a reaction system as small as possible, and have recovery yield higher than that of the prior chemical synthesis means. So as to save the cost of the subsequent preparation of the medicines and reduce the price of the medicines.
Example 1
A preparation method of a cell division inhibitor comprises the following specific steps:
step one: synthesis and vector construction of key synthetases
(11) Synthesizing LAAD gene fragments, vioB gene fragments and MarC gene fragments for expressing key synthetase, wherein:
the amino acid sequence of the LAAD gene fragment is shown as SEQ ID NO. 1;
the amino acid sequence of the VioB gene fragment is shown as SEQ ID NO. 2;
the amino acid sequence of the MarC gene fragment is shown as SEQ ID NO. 3;
(12) Cloning the LAAD gene fragment into a pET28a (+) vector by using a Gibson splicing cloning technology to obtain an expression vector pET28a (+) -LAAD, and cloning the VioB gene fragment and the MarC gene fragment into a pET26b vector to obtain an expression vector pET26b-VioB and an expression vector pET26b-MarC respectively, wherein the specific steps are shown in figures 1-3, and the specific steps are as follows:
the LAAD gene fragment, the VioB gene fragment and the MarC gene fragment are positioned between NdeI and HindIII restriction enzyme sites;
(13) Respectively converting the constructed expression vectors pET28a (+) -LAAD, pET26b-VioB and pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two: expression and pretreatment of key synthetases
(21) Preparation of LAAD protein crude lysate
(211) Adding 10 uL of pET28a (+) -LAAD strain solution into 5 mL of LB liquid culture medium, and shaking overnight at 37 ℃ and 200rpm for culture to obtain pET28a (+) -LAAD seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(212) Adding the pET28a (+) -LAAD 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 concentration of bacterial solution OD containing LAAD expression vector 600 Stop incubation at=1.2, then take out the test vessel and place in ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 0.1 and mM, uniformly mixing, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 h;
(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 adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 5 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LAAD protein crude lysate;
(22) Preparation of VioB protein crude lysate
(221) Adding 10 uL of pET26b-VioB strain solution into 5 mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-VioB seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(222) Adding the pET26b-VioB seed solution obtained in the step (221) 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 bacterial solution OD containing the VioB expression vector 600 Stop incubation at=0.8, then place the test vessel in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(223) Adding proper amounts of ammonium ferrous sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ammonium ferrous sulfate in the bacterial liquid is 40 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude VioB protein lysate.
(23) Preparation of MarC protein crude lysate
(231) Adding 10 uL of pET26b-MarC strain solution into 5 mL 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 with the concentration of 50 ug/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 Stop incubation at=1.0, then take out the test vessel and place in ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate;
step three: construction of enzymatic reaction System
(31) The following substances were added to the reaction vessel separately:
，
wherein:
the NaH is 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0;
said (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M;
(32) After uniform mixing, vibrating and reacting for 7 hours, wherein the whole vibrating and reacting process keeps the reaction container in an open state (namely, the oxygen concentration at the upper part of the reaction container is the same as the oxygen concentration in the air);
(33) Adding 600 mL of MarC protein crude lysate prepared in the step (23), 2.1g of alpha-ketoglutaric acid, 1.32 g of ascorbic acid and a proper amount of ferrous sulfate into a reaction vessel respectively, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1 mM, mixing uniformly, and continuing to shake and react for 2 hours to obtain a reaction liquid;
step four: separation and purification of the product
And (3) regulating the pH value of the reaction solution obtained in the step (III) to be 1 by using 1N dilute hydrochloric acid, adding ethyl acetate 1L to the reaction solution for extraction 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 the crude product under the condition of DCM/MeOH=20:1 to obtain an orange-red product, thus obtaining the cell division inhibitor. After weighing, the weight of the cell division inhibitor was 2.65 and g, and the yield was 63.3%.
In another embodiment, after the mixing in the step (32), the shaking reaction is performed for 6 hours, and the reaction vessel is kept open during the shaking reaction.
In yet another embodiment, after the mixing in step (32), the reaction vessel is kept open during the shaking reaction for 8 hours.
Verification test and analysis
1. HPLCandnuclearmagneticresonancespectroscopyanalysiswereperformedontheorange-redproductpreparedinexample1,andtheresultsareshowninFIGS.4to6,confirmingthatthefinalorange-redproductwasDDM-A.
2. theorange-redproductwasobservedunderamicroscope,andtheresultisshowninFIG.7,whereinthebar-shapedobjectinthesquareframeinFIG.7isDDM-Acrystal.
3. Determination of the IC of the orange-red product prepared in example 1 on PKC and PKA, respectively, using a Protein Kinase C (PKC) activity detection kit (PKC Kinase Activity Assay Kit) and a Protein Kinase A (PKA) activity detection kit (PKA Kinase Activity Assay Kit) 50 Values. Meanwhile, the orange-red product prepared in example 1 was examined against plasmodium falciparumPlasmodium falciparumAnd inhibitory activity of murine melanoma cell B16. The detection results obtained are as follows:
(31) Inhibition activity assay for Protein Kinase C (PKC): coating a micropore plate with a purified human PKC antibody by using a double-antibody sandwich method to prepare a solid-phase antibody, sequentially adding PKC into micropores coated with monoclonal antibodies, combining with the PKC antibody marked by HRP to form an antibody-antigen-enzyme-labeled antibody complex, and adding a substrate TMB for color development after thorough washing. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. Absorbance was measured with a microplate reader at a wavelength of 450nm, which correlated positively with the PKC content in the sample. The PKC concentration in the sample was calculated by a standard curve. theDDM-Asubstrateconcentrationsusedwere5uM,10uM,20uM,50uM,100uMand200uM,respectively,withareactiontimeof15minandareactiontemperatureof37℃.
(32) Inhibition activity assay for Protein Kinase A (PKA): and (3) simultaneously incubating the to-be-detected object and the TXA2 antibody marked by biotin by using an ELISA method, washing, adding the HRP marked by avidin, and adding a substrate TMB for color development after washing. Absorbance was measured with a microplate reader at a wavelength of 450nm, which correlated positively with the PKC content in the sample. The PKA concentration in the samples was calculated by standard curve. theDDM-Asubstrateconcentrationsusedwere5uM,10uM,20uM,50uM,100uMand200uM,respectively,withareactiontimeof10minandareactiontemperatureof37℃.
(33) For plasmodium falciparumPlasmodium falciparumIs determined by the inhibition activity of (a): DDM-awasdissolvedindmso,dilutedgradientwithassaymedium(rpmi1640mediumwith5%albumaxii,0.2%w/vglucose,0.03%l-glutamine)andaddedtoeachwellofthemicrotiterplate. Subsequently cultured plasmodium falciparumPlasmodium falciparumEach well was added to a final volume of 100 uL (final DMSO concentration. Ltoreq.0.25%). Plates were incubated at 37℃for 48 hours and 0.1. Mu. Ci was added to each well 3 H-hypoxanthine. The plates were mixed and incubated for an additional 24 hours. The reaction was stopped by placing the plate in a-80℃refrigerator. The cells were thawed using 96-well plates and transferred to glass fiber filter pads. Bound radioactivity was counted using a liquid Betalux scintillation counter and IC was calculated 50 Values. theDDM-Asubstrateconcentrationsusedwere1uM,2uM,5uM,10uM,20uMand50uM,respectively.
(34) Determination of inhibitory Activity on murine melanoma cell B16: 200 mousemelanomacellsB16andagradientdilutedDDM-ADMSOsolutionareaddedintotheuLreactionsystem,andthefinalconcentrationoftheDDM-Ais0.1uM,0.2uM,0.5uM,1uM,2uMand5uMrespectively. After incubation for 1h with shaking at 37deg.C, the survival of B16 cells was determined by MTT method and IC was calculated 50 Values.
Example 2
Substantially the same as in example 1, the difference is only that:
1. in the step (212), the bacterial liquid concentration OD containing the LAAD expression vector is reached 600 Stop culturing at=1.1;
2. in the step (222), the bacterial liquid concentration OD containing the VioB expression vector is reached 600 Stop culturing at=0.7;
3. in the step (232), the concentration OD of the bacterial liquid containing the MarC expression vector is reached 600 Culture was stopped at=0.9.
Example 2 the final orange-red product gave a cell division inhibitor. After weighing, the weight of the cell division inhibitor was 2.60. 2.60 g, and the yield was 62.1%.
Example 3
Substantially the same as in example 1, the difference is only that:
1. step by stepIn the step (212), the bacterial liquid concentration OD containing the LAAD expression vector is reached 600 Stop culturing at=1.3;
2. in the step (222), the bacterial liquid concentration OD containing the VioB expression vector is reached 600 Stop culturing at=0.9;
3. in the step (232), the concentration OD of the bacterial liquid containing the MarC expression vector is reached 600 Culture was stopped when=1.1.
Example 3 the final orange-red product gave a cell division inhibitor. After weighing, the weight of the cell division inhibitor was 2.62. 2.62 g, and the yield was 62.6%.
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 (5)

1. A method for preparing a cell division inhibitor, comprising the steps of:
step one, synthesizing key synthetase and constructing a carrier:
(11) Synthesizing LAAD gene fragments, vioB gene fragments and MarC gene fragments for expressing key synthetase, wherein:
the amino acid sequence of the LAAD gene fragment is shown as SEQ ID NO. 1;
the amino acid sequence of the VioB gene fragment is shown as SEQ ID NO. 2;
the amino acid sequence of the MarC gene fragment is shown as SEQ ID NO. 3;
(12) Cloning the LAAD gene fragment into a pET28a (+) vector by using a Gibson splicing cloning technology to obtain an expression vector pET28a (+) -LAAD, and cloning the VioB gene fragment and the MarC gene fragment into a pET26b vector to obtain an expression vector pET26b-VioB and an expression vector pET26b-MarC respectively;
(13) Respectively converting the constructed expression vectors pET28a (+) -LAAD, pET26b-VioB and pET26b-MarC into competent cells BL21 (DE 3) to obtain strain solutions, and respectively storing at-80 ℃;
step two, expressing and preprocessing key synthetase:
(21) Preparing LAAD protein crude lysate;
(22) Preparing a crude VioB protein lysate;
(23) Preparing MarC protein crude lysate;
step three, building an enzymatic reaction system:
(31) The following substances were added to the reaction vessel separately:
；
(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) Adding 600 mL of MarC protein crude lysate prepared in the step (23), 2.1g of alpha-ketoglutaric acid, 1.32 g of ascorbic acid and a proper amount of ferrous sulfate into a reaction vessel respectively, so that the concentration of the ferrous sulfate in bacterial liquid is 0.1 mM, mixing uniformly, and continuing to shake and react for 2 hours to obtain a reaction liquid;
step four, separating and purifying a product:
the reaction solution obtained in the third step is adjusted to pH=1 by using 1N diluted hydrochloric acid, then ethyl acetate 1L is added to the reaction solution for extraction for at least three times, the emulsion layer is separated after being filtered by using diatomite, the organic phases are combined and dried, the crude product is obtained by decompression concentration, then the crude product is purified by using silica gel column chromatography, and the orange-red product is obtained by eluting under the conditions of DCM/MeOH=20:1, wherein the cell division inhibitor is obtained, wherein:
the step (23) is as follows:
(231) Adding 10 uL of pET26b-MarC strain solution into 5 mL 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 with the concentration of 50 ug/mL;
(232) Adding the pET26b-MarC seed solution obtained in the step (231) into a test container containing 1L of LB liquid medium, and placing the test container in a shaking table at 37℃,Shake culturing at 200rpm until the concentration of bacterial liquid OD containing MarC expression vector 600 Until =0.9-1.1, the test vessel was then removed and placed in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(236) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain MarC protein crude lysate.
2. The method of claim 1, wherein the LAAD gene fragment, the VioB gene fragment, and the MarC gene fragment of step (12) are located between NdeI and HindIII restriction sites.
3. The method of preparing a cell division inhibitor according to claim 1, wherein the step of step (21) is as follows:
(211) Adding 10 uL of pET28a (+) -LAAD strain solution into 5 mL of LB liquid culture medium, and shaking overnight at 37 ℃ and 200rpm for culture to obtain pET28a (+) -LAAD seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(212) Adding the pET28a (+) -LAAD 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 concentration of bacterial solution OD containing LAAD expression vector 600 Until 1.1-1.3, the test vessel was then removed and placed in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/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 0.1 and mM, uniformly mixing, and then placing into a shaking table to perform induction culture at 18 ℃ and 200rpm for 16 h;
(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 adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 5 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(216) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain LAAD protein crude lysate.
4. The method of claim 1, wherein step (22) comprises the steps of:
(221) Adding 10 uL of pET26b-VioB strain solution into 5 mL of LB liquid medium, and performing shake culture at 37 ℃ and 200rpm overnight to obtain pET26b-VioB seed solution, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(222) Adding the pET26b-VioB seed solution obtained in the step (221) 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 bacterial solution OD containing the VioB expression vector 600 Until =0.7-0.9, the test vessel was then placed in an ice bath for 30 min, wherein:
LB liquid medium contains kanamycin with the concentration of 50 ug/mL;
(223) Adding proper amounts of ammonium ferrous sulfate, 5-aminolevulinic acid and IPTG into the bacterial liquid treated in the step (222) respectively, so that the concentration of the ammonium ferrous sulfate in the bacterial liquid is 40 uM, the concentration of the 5-aminolevulinic acid in the bacterial liquid is 0.25 mM, and the concentration of the IPTG in the bacterial liquid is 0.1 and mM, uniformly mixing, and then placing into a shaking table for induced culture at 18 ℃ and 200rpm for 16 h;
(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 washing followed by adding 30 mL loading buffer to suspend the precipitated bacteria to no distinct clumps to obtain a bacterial heavy suspension, wherein:
the pH value of the loading buffer solution is 8.0;
the loading buffer solution comprises: 100 mM Tris-HCl, 300 mM NaCl, 15 mM 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 6 mm;
the crushing procedure is as follows: the energy is 40%, the ultrasonic circulation working time is 2 s, the suspension time is 4 s, and the total working time is 30 min;
(226) Centrifuging the crushed bacterial liquid at 10000g for 15min at low temperature, and taking supernatant to obtain crude VioB protein lysate.
5. The method of claim 1, wherein the NaH in step (31) is 2 PO 4 -Na 2 HPO 4 The concentration of the buffer solution is 1M, and the pH value is 8.0; said (NH) 4 ) 2 SO 4 The concentration of the stock solution was 2M.
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