CN113004363A - Production process for library building joint of DNA coding compound library - Google Patents
Production process for library building joint of DNA coding compound library Download PDFInfo
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- CN113004363A CN113004363A CN202110231891.1A CN202110231891A CN113004363A CN 113004363 A CN113004363 A CN 113004363A CN 202110231891 A CN202110231891 A CN 202110231891A CN 113004363 A CN113004363 A CN 113004363A
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 34
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 30
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 28
- 238000011049 filling Methods 0.000 claims abstract description 16
- -1 phosphoramidite triester Chemical class 0.000 claims abstract description 12
- 230000008878 coupling Effects 0.000 claims abstract description 9
- 238000010168 coupling process Methods 0.000 claims abstract description 9
- 238000005859 coupling reaction Methods 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 53
- 239000000047 product Substances 0.000 claims description 50
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 42
- 239000007790 solid phase Substances 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 28
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 21
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 21
- 230000008569 process Effects 0.000 claims description 18
- BMTZEAOGFDXDAD-UHFFFAOYSA-M 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium;chloride Chemical compound [Cl-].COC1=NC(OC)=NC([N+]2(C)CCOCC2)=N1 BMTZEAOGFDXDAD-UHFFFAOYSA-M 0.000 claims description 14
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 14
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 14
- 238000006482 condensation reaction Methods 0.000 claims description 14
- 239000012043 crude product Substances 0.000 claims description 14
- 238000010511 deprotection reaction Methods 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 14
- 238000001819 mass spectrum Methods 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 239000000178 monomer Substances 0.000 claims description 14
- 230000003647 oxidation Effects 0.000 claims description 14
- 238000007254 oxidation reaction Methods 0.000 claims description 14
- 150000008300 phosphoramidites Chemical class 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 claims description 14
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 7
- 230000004913 activation Effects 0.000 claims description 7
- 230000000903 blocking effect Effects 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 claims description 7
- 125000006239 protecting group Chemical group 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000004743 Polypropylene Substances 0.000 claims description 2
- 229920001155 polypropylene Polymers 0.000 claims description 2
- 239000002994 raw material Substances 0.000 abstract description 4
- 239000000376 reactant Substances 0.000 abstract description 2
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- 238000010276 construction Methods 0.000 description 6
- 238000007877 drug screening Methods 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 229940042040 innovative drug Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a production process for a DNA coding compound library-building joint, which comprises the steps of preparing a synthetic column, filling a carrier and CPG into the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate. According to the production process for the DNA coding compound library-establishing joint, the core primer raw material constructed by the DNA coding compound library is used for connecting the small molecules and the AOP-Headpiece-primer of the DNA coding compound library, and the Headpiece-primer is synthesized by using a phosphoramidite triester method, so that the advantages of high efficiency and rapid coupling and stable initial reactant are achieved, compared with the AOP-Headpiece-primer produced by the traditional production method, the yield is high, the cost is low, the period is short, stable batch products are obtained, and compared with small-specification multi-batch synthesis, the cost is low, and the period is short.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a production process for a DNA coding compound library building joint.
Background
Drug screening comprises high-throughput drug screening and DNA coding molecular library drug screening, wherein the DNA coding molecular library drug screening is to screen target proteins and hundreds of millions of compound libraries, the screened targets of the molecular libraries are various and cover basically all disease types, but the traditional single compound is not screened in sequence, so the screening in the mode has obvious speed and cost advantages. The DNA coding molecule library drug screening technology is gradually developed and matured in recent years and becomes one of the leading technologies in the field of innovative drug discovery, scientists have successfully screened a series of high-activity drug candidate compounds for important malignant tumor drug targets, the starting point and support of the technology are DNA coding compound libraries, and AOP-Headpiece-primer is a core raw material for constructing DNA coding compound libraries.
The invention provides a production process for a DNA coding compound library construction joint, which can directly provide an AOP-Headpiece-primer for the downstream DNA coding compound library construction through a solid phase phosphoramidite triester method, saves the operation, time and cost for obtaining the AOP-Headpiece-primer by enzyme connection for the downstream library construction, and can use raw materials with more stable batches.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a production process for a DNA coding compound library-establishing joint, and solves the problems of high cost and long period of the traditional preparation of a Headpiece-primer for the DNA coding compound library-establishing joint.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a production process for a DNA coding compound library building joint specifically comprises the following steps:
preparing a synthetic column, filling a carrier and CPG (chlorinated Polypropylene) into the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds, wherein the eluted color of the normal DMT is orange red in the synthetic process;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 70-90 ℃, soaking for 30-50 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer;
step five, dissolving the Headpiece-primer in a borate buffer solution, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, and standing for 10-12 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
and step seven, adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, placing the mixed solution at minus 20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into a piperidine aqueous solution for deprotection, performing centrifugal washing on the deprotected product with ethanol to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain the target product of the AOP-Headpiece-primer.
Preferably, in the first step, the prepared synthesis column is subjected to quality inspection to ensure that the carrier in the synthesis column is complete and the CPG does not leak.
Preferably, in the second step, each cycle is divided into four steps, specifically as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
Preferably, in step two, the synthesis efficiency before the cycle is judged by the shade of the color of the eluted normal DMT in the synthesis.
Preferably, in step four, the obtained Headpiece-primer is subjected to standard detection, and the mass spectrum of the Headpiece-primer shows that the purity is more than 90%.
Preferably, in the fifth step, the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, and the pH value of the borate solution is 9.4.
Preferably, in step six, the consumption mass ratio of AOP to DMT-MM is 1: 1.
Preferably, in the seventh step, the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, and the consumption of the piperidine aqueous solution is 2.5 times of the total mass of the freeze-dried powder.
Preferably, in the seventh step, the standard detection is performed on the obtained AOP-Headpiece-primer target product, and the mass spectrum of the AOP-Headpiece-primer target product shows that the purity is more than 90%, and the HPLC purity and the CGE purity are more than 90%.
(III) advantageous effects
The invention provides a production process for a DNA coding compound library-building joint. Compared with the prior art, the method has the following beneficial effects:
the production process for the DNA coding compound library construction joint is characterized in that the core primer raw material constructed by the DNA coding compound library is used for connecting small molecules and the AOP-Headpiece-primer of the DNA coding compound library, the traditional production mode is that Headpiece is firstly produced and then connected with AOP to obtain AOP-Headpiece, then the AOP-Headpiece and the primer annealed into double chains are connected under the action of T4-DNA ligase, the Headpiece-primer is synthesized by a phosphoramidite triester method, the production process has the advantages of high efficiency and rapid coupling and stable initial reactants, compared with the AOP-Headpiece-primer produced by the traditional production method, the yield is high, the cost is low, and the period is short, the mode of producing the Headpiece with the primer by the chemical synthesis method simplifies the downstream DNA coding compound library construction operation, further shortens the library construction period of the DNA coding compound library, and even can promote the development of downstream small molecule drugs, the Headpiece-primer is produced by a chemical synthesis method of a large-specification synthesizer, stable batch products are obtained, and meanwhile, compared with small-specification multi-batch synthesis, the synthesis cost is low and the period is short.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A production process for a DNA coding compound library building joint specifically comprises the following steps:
step one, preparing a synthetic column, checking the quality of the prepared synthetic column to ensure that a carrier in the synthetic column is complete and CPG does not leak outside, filling the carrier and the CPG in the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds in each cycle, wherein the eluted color of the normal DMT is orange red in the synthetic process, and judging the synthetic efficiency before the cycle by the depth of the eluted color of the normal DMT in the synthesis;
step three, soaking the synthesized synthetic column with the Headpiece-primer in ammonia water, heating the ammonia water to 70 ℃, soaking for 30 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer, and performing standard detection on the obtained Headpiece-primer, wherein the purity of the Headpiece-primer shown by a mass spectrum of the Headpiece-primer is 92.56%;
step five, dissolving the Headpiece-primer in a borate buffer solution, wherein the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, the pH value of the borate solution is 9.4, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, wherein the consumption mass ratio of AOP to DMT-MM is 1:1, and standing for 10 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, wherein the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, the consumption amount of the piperidine solution is 2.5 times of the total mass of the freeze-dried powder, placing the mixed solution at-20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into the piperidine solution for deprotection, performing ethanol centrifugal washing on the deprotected product to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain a target product of the AOP-Headpiece-primer, performing standard detection on the obtained target product of the AOP-Headpiece-primer, wherein the mass spectrum of the target product of the AOP-Headpiece-primer shows that the purity is 93.15%, and the HPLC purity and the CGE purity are both greater than 90%.
In the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
Example 2
A production process for a DNA coding compound library building joint specifically comprises the following steps:
step one, preparing a synthetic column, checking the quality of the prepared synthetic column to ensure that a carrier in the synthetic column is complete and CPG does not leak outside, filling the carrier and the CPG in the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds in each cycle, wherein the eluted color of the normal DMT is orange red in the synthetic process, and judging the synthetic efficiency before the cycle by the depth of the eluted color of the normal DMT in the synthesis;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 75 ℃, soaking for 35 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer, and performing standard detection on the obtained Headpiece-primer, wherein the purity of the Headpiece-primer shown by a mass spectrum of the Headpiece-primer is 91.26%;
step five, dissolving the Headpiece-primer in a borate buffer solution, wherein the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, the pH value of the borate solution is 9.4, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, wherein the consumption mass ratio of AOP to DMT-MM is 1:1, and standing for 10.5 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, wherein the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, the consumption amount of the piperidine solution is 2.5 times of the total mass of the freeze-dried powder, placing the mixed solution at-20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into the piperidine solution for deprotection, performing ethanol centrifugal washing on the deprotected product to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain a target product of the AOP-Headpiece-primer, performing standard detection on the obtained target product of the AOP-Headpiece-primer, wherein the mass spectrum of the target product of the AOP-Headpiece-primer shows that the purity is 93.51%, and the HPLC purity and the CGE purity are both greater than 90%.
In the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
Example 3
A production process for a DNA coding compound library building joint specifically comprises the following steps:
step one, preparing a synthetic column, checking the quality of the prepared synthetic column to ensure that a carrier in the synthetic column is complete and CPG does not leak outside, filling the carrier and the CPG in the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds in each cycle, wherein the eluted color of the normal DMT is orange red in the synthetic process, and judging the synthetic efficiency before the cycle by the depth of the eluted color of the normal DMT in the synthesis;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 80 ℃, soaking for 40 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer, and performing standard detection on the obtained Headpiece-primer, wherein the purity of the Headpiece-primer shown by a mass spectrum of the Headpiece-primer is 93.56%;
step five, dissolving the Headpiece-primer in a borate buffer solution, wherein the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, the pH value of the borate solution is 9.4, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, wherein the consumption mass ratio of AOP to DMT-MM is 1:1, and standing for 11 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, wherein the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, the consumption amount of the piperidine solution is 2.5 times of the total mass of the freeze-dried powder, placing the mixed solution at-20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into the piperidine solution for deprotection, performing ethanol centrifugal washing on the deprotected product to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain a target product of the AOP-Headpiece-primer, performing standard detection on the obtained target product of the AOP-Headpiece-primer, wherein the mass spectrum of the target product of the AOP-Headpiece-primer shows that the purity is 95.34%, and the HPLC purity and the CGE purity are both greater than 90%.
In the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
Example 4
A production process for a DNA coding compound library building joint specifically comprises the following steps:
step one, preparing a synthetic column, checking the quality of the prepared synthetic column to ensure that a carrier in the synthetic column is complete and CPG does not leak outside, filling the carrier and the CPG in the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds in each cycle, wherein the eluted color of the normal DMT is orange red in the synthetic process, and judging the synthetic efficiency before the cycle by the depth of the eluted color of the normal DMT in the synthesis;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 85 ℃, soaking for 45 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer, and performing standard detection on the obtained Headpiece-primer, wherein the purity of the Headpiece-primer shown by a mass spectrum of the Headpiece-primer is 96.57%;
step five, dissolving the Headpiece-primer in a borate buffer solution, wherein the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, the pH value of the borate solution is 9.4, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, wherein the consumption mass ratio of AOP to DMT-MM is 1:1, and standing for 11.5 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, wherein the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, the consumption amount of the piperidine solution is 2.5 times of the total mass of the freeze-dried powder, placing the mixed solution at-20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into the piperidine solution for deprotection, performing ethanol centrifugal washing on the deprotected product to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain a target product of the AOP-Headpiece-primer, performing standard detection on the obtained target product of the AOP-Headpiece-primer, wherein the mass spectrum of the target product of the AOP-Headpiece-primer shows that the purity is 98.35%, and the HPLC purity and the CGE purity are both greater than 90%.
In the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
Example 5
A production process for a DNA coding compound library building joint specifically comprises the following steps:
step one, preparing a synthetic column, checking the quality of the prepared synthetic column to ensure that a carrier in the synthetic column is complete and CPG does not leak outside, filling the carrier and the CPG in the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds in each cycle, wherein the eluted color of the normal DMT is orange red in the synthetic process, and judging the synthetic efficiency before the cycle by the depth of the eluted color of the normal DMT in the synthesis;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 90 ℃, soaking for 50 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer, and performing standard detection on the obtained Headpiece-primer, wherein the purity of the Headpiece-primer shown by a mass spectrum of the Headpiece-primer is 91.35%;
step five, dissolving the Headpiece-primer in a borate buffer solution, wherein the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, the pH value of the borate solution is 9.4, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, wherein the consumption mass ratio of AOP to DMT-MM is 1:1, and standing for 12 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, wherein the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, the consumption amount of the piperidine solution is 2.5 times of the total mass of the freeze-dried powder, placing the mixed solution at-20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into the piperidine solution for deprotection, performing ethanol centrifugal washing on the deprotected product to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain a target product of the AOP-Headpiece-primer, performing standard detection on the obtained target product of the AOP-Headpiece-primer, wherein the purity shown by a mass spectrum of the target product of the AOP-Headpiece-primer is 92.15%, and the purity of HPLC and the CGE are both greater than 90%.
In the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A process for producing a library adaptor for a DNA encoding compound, comprising: the method specifically comprises the following steps:
preparing a synthetic column, filling a carrier and CPG (chlorinated Polypropylene) into the synthetic column, filling the prepared synthetic column into a synthetic plate, and marking a synthetic batch number on the synthetic plate;
step two, checking the amount and the position of a synthetic reagent and the state of a synthesizer, opening a synthetic program, introducing a Headpiece-primer sequence, starting the synthetic program to start synthesis, adopting a solid-phase phosphoramidite triester method during synthesis, sequentially synthesizing from the 3 'end to the 5' end of the Headpiece-primer, connecting a phosphoramidite monomer and a product connected to a solid-phase carrier in the previous cycle through 3 'to 5' phosphodiester bonds, wherein the eluted color of the normal DMT is orange red in the synthetic process;
step three, soaking the synthesized column with the Headpiece-primer in ammonia water, heating the ammonia water to 70-90 ℃, soaking for 30-50 minutes, and cutting the Headpiece-primer connected to the CPG to obtain a product a;
step four, purifying the product a to obtain a Headpiece-primer;
step five, dissolving the Headpiece-primer in a borate buffer solution, and uniformly stirring and mixing to obtain a base solution b;
step six, firstly adding AOP into the base liquid b, then adding DMT-MM, and standing for 10-12 hours at room temperature to obtain a crude product of AOP-Headpiece-primer;
and step seven, adding a NaCl solution and a glacial ethanol solution into the crude product of the AOP-Headpiece-primer, placing the mixed solution at minus 20 ℃ for 1 hour, performing centrifugal treatment to collect precipitates, performing freeze-drying treatment on the precipitates to obtain freeze-dried powder, putting the freeze-dried powder into a piperidine aqueous solution for deprotection, performing centrifugal washing on the deprotected product with ethanol to remove a solvent and piperidine, and finally removing impurities through high performance liquid chromatography to obtain the target product of the AOP-Headpiece-primer.
2. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the first step, the quality of the prepared synthetic column is checked to ensure that the carrier in the synthetic column is complete and CPG does not leak.
3. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the second step, each cycle is divided into four steps, which are as follows:
step a1, deprotection: the protecting group DMT connected on the solid phase carrier reacts with trichloroacetic acid to be removed, and free 5' hydroxyl is obtained;
step a2, activation and coupling: mixing a phosphoramidite monomer with an activating agent to obtain a phosphorous acid intermediate with an activated 3 'end, and carrying out a condensation reaction on the phosphorous acid intermediate and free 5' hydroxyl on a solid phase carrier;
step A3, capping: blocking 5 'hydroxyl which does not participate in condensation reaction by using acetic anhydride and 1-methylimidazole, so that the 5' hydroxyl can not participate in reaction in the subsequent step;
step A4, oxidation: under the oxidation of iodine, the labile phosphite is oxidized to a stable phosphotriester.
4. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the second step, the synthesis efficiency before circulation is judged by the shade of the eluted color of the normal DMT in the synthesis.
5. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the fourth step, the obtained Headpiece-primer is subjected to standard detection, and the mass spectrum of the Headpiece-primer shows that the purity is more than 90%.
6. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the fifth step, the consumption mass ratio of the Headpiece-primer to the borate buffer solution is 1:3.2, and the pH value of the borate solution is 9.4.
7. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the sixth step, the consumption mass ratio of AOP to DMT-MM is 1: 1.
8. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: in the seventh step, the consumption mass ratio of the NaCl solution to the glacial ethanol solution is 2:1, and the consumption of the piperidine aqueous solution is 2.5 times of the total mass of the freeze-dried powder.
9. The process of claim 1 for the production of a library adaptor for DNA encoding compounds, wherein: and seventhly, performing standard detection on the obtained AOP-Headpiece-primer target product, wherein the purity of the AOP-Headpiece-primer target product shown by the mass spectrum is more than 90%, and the HPLC purity and the CGE purity are more than 90%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964794A (en) * | 2018-09-29 | 2020-04-07 | 成都先导药物开发股份有限公司 | Primers suitable for sequencing DNA coding compound sequencing library |
| CN112575389A (en) * | 2019-09-30 | 2021-03-30 | 成都先导药物开发股份有限公司 | Liquid chromatography purification method of DNA coding compound library |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130197117A1 (en) * | 2012-01-30 | 2013-08-01 | National University Corporation Nagoya University | Linker and support for solid phase synthesis of nucleic acid |
| US20130281324A1 (en) * | 2010-04-16 | 2013-10-24 | Nuevolution A/S | Bi-functinal complexes and methods for making and using such complexes |
| CN110325491A (en) * | 2017-03-17 | 2019-10-11 | 成都先导药物开发股份有限公司 | The synthetic method and composition of code database |
-
2021
- 2021-03-02 CN CN202110231891.1A patent/CN113004363A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130281324A1 (en) * | 2010-04-16 | 2013-10-24 | Nuevolution A/S | Bi-functinal complexes and methods for making and using such complexes |
| US20130197117A1 (en) * | 2012-01-30 | 2013-08-01 | National University Corporation Nagoya University | Linker and support for solid phase synthesis of nucleic acid |
| CN110325491A (en) * | 2017-03-17 | 2019-10-11 | 成都先导药物开发股份有限公司 | The synthetic method and composition of code database |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964794A (en) * | 2018-09-29 | 2020-04-07 | 成都先导药物开发股份有限公司 | Primers suitable for sequencing DNA coding compound sequencing library |
| CN112575389A (en) * | 2019-09-30 | 2021-03-30 | 成都先导药物开发股份有限公司 | Liquid chromatography purification method of DNA coding compound library |
| CN112575389B (en) * | 2019-09-30 | 2023-09-19 | 成都先导药物开发股份有限公司 | Liquid chromatography purification method of DNA coding compound library |
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