WO2019158007A1 - Procédé et système de synthèse d'oligonucléotides - Google Patents

Procédé et système de synthèse d'oligonucléotides Download PDF

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WO2019158007A1
WO2019158007A1 PCT/CN2019/074588 CN2019074588W WO2019158007A1 WO 2019158007 A1 WO2019158007 A1 WO 2019158007A1 CN 2019074588 W CN2019074588 W CN 2019074588W WO 2019158007 A1 WO2019158007 A1 WO 2019158007A1
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group
reaction vessel
substituted
formula
solid phase
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PCT/CN2019/074588
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English (en)
Chinese (zh)
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王勇
陈世宏
周超
孙宝策
冯利鹤
黄小罗
沈玥
李汉东
章文蔚
徐讯
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深圳华大生命科学研究院
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Priority to CN201980006472.9A priority Critical patent/CN111479818A/zh
Publication of WO2019158007A1 publication Critical patent/WO2019158007A1/fr

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    • 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
    • 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
    • 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

Definitions

  • the invention relates to the field of nucleic acid synthesis.
  • Nucleic acid is the basic genetic material in life. In vitro artificial synthesis of nucleic acids can replicate any naturally occurring nucleic acid function or create new nucleic acid functions, depending on the needs of the research and application. With the development of genomics, molecular biology, systems biology, bioinformatics and synthetic biology, synthetic nucleic acids have a wide range of applications in cell engineering, gene editing, disease diagnosis and treatment, and new material development. value.
  • a column synthesizer such as Dr. oligo 192, is a solid phase synthesis reaction on a porous reaction column of the order of centimeters by the addition of a solenoid valve control reagent.
  • the method has a low error rate, but the synthesis flux is not high. There is also more material required.
  • Microarray synthesizers such as the CustomArray synthesizer, reduce the synthesis reaction to micron-sized reaction wells, with tens of thousands of reaction wells on a single chip, which reduces feedstock consumption to a certain extent while increasing synthesis throughput. However, the yield is low, the electrochemical reaction is not easy to control, and the error rate is high.
  • the column and microarray nucleic acid synthesizers are all added to the synthesis column or the synthetic chip through the pre-laid pipeline, and the added reagent is greatly excessive, which causes the reagent to be extremely Great waste and low material usage.
  • the error rate of the single-stranded nucleic acid synthesized by the common single-base nucleic acid synthesis method rapidly increases with the increase of the length, correspondingly The yield is also drastically reduced, which results in a significant limitation on the length and yield of the nucleic acid synthesis product.
  • the commercial column synthesizer has low synthesis flux and low material utilization rate, and cannot meet the demand for large-scale low-cost nucleic acid synthesis in the future.
  • microarray chips enable high-throughput nucleic acid synthesis
  • the yield of this synthesis method is small, the error rate is high, and the product is difficult to separate from the mixture, increasing the cost of subsequent operations, such as polymerase chain reaction and Gene assembly operations.
  • both column and microarray nucleic acid synthesizers require reagent input and output lines, and the amount of reagents used in the synthesis process is large, which greatly increases the cost of synthesis.
  • nucleic acid synthesis technology needs further improvement and optimization.
  • the present invention proposes a novel method for coping with these problems, namely a "synthesis pool" nucleic acid synthesis method. More particularly, the present invention relates to synthetic pool multi-base synthesis methods (e.g., two-base nucleic acid synthesis, three-base nucleic acid synthesis, four-base nucleic acid synthesis, etc.).
  • synthetic pool multi-base synthesis methods e.g., two-base nucleic acid synthesis, three-base nucleic acid synthesis, four-base nucleic acid synthesis, etc.
  • the "synthesis pool" nucleic acid synthesis method of the present invention is used.
  • the nucleic acid single chain with longer chain length can be obtained quickly, and the synthesis efficiency can be greatly improved by the synthesis strategy, the synthetic chain length can be greatly extended correspondingly, and the synthesis error rate can be correspondingly greatly reduced.
  • the invention provides a method of synthesizing an oligonucleotide, the method comprising:
  • Nu is a single bond
  • X 1 is independently -O- or -S-;
  • X 2 is independently -O-, -S- or -NR-;
  • X 3 is independently -O-, -S-, -CH 2 - or -(CH 2 ) 2 -;
  • R 1 is a protecting group
  • R 2 is independently -H, -F, -NHR 6 , -CH 2 R 6 or -OR 6 ;
  • R 3 is independently -OCH 2 CH 2 CN, -SCH 2 CH 2 CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 ;
  • R is -H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or an amine protecting group;
  • R 6 is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a protecting group;
  • R 7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group;
  • Each B is independently a base that is modified or unmodified
  • R 13 is a solid phase carrier or -Y 1 -LY 1 -R 14 ;
  • Y 1 is a single bond, a double bond, -C(O)-, -C(O)NR 17 , -C(O)O-, -NR 17 - or -O-;
  • L is a single bond, a double bond, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;
  • R 17 is -H, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;
  • R 14 is a solid phase carrier
  • q is 0 or a positive integer
  • X 5 is -OH or -SH; the other groups are as defined above;
  • R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group;
  • R 4 and R 5 together with the nitrogen to which they are bonded form a heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl or heteroaryl group is preferably a five or six membered ring;
  • n 0 or a positive integer
  • step d) optionally, contacting the solid support with a blocking agent in a third reaction vessel comprising a blocking agent, wherein formula (II) is not reacted with the phosphoramidite monomer or multimer in step c) The deprotected solid support or the X 5 group of the stereoisomer is blocked;
  • first reaction vessel, the second reaction vessel, the third reaction vessel and the fourth reaction vessel are independent of each other;
  • step f) optionally repeating steps b), c), e) or b) to e) one or more times, wherein the final step is step b), d) or e), thereby synthesizing the desired oligonucleotide .
  • the capping step may be omitted if the sequence of the sequence of the oligonucleotide to be synthesized is If the length is long (for example, the number of cycles > 25), the capping step is usually not omitted.
  • a washing step is also included between steps b and c and between steps d and e.
  • the solid phase carrier is washed by contacting the solid phase carrier with a washing reagent in a fifth reaction vessel containing a detergent, wherein the fifth reaction vessel is combined with the first reaction vessel, the second reaction vessel, the third reaction vessel,
  • the fourth reaction vessels are independent of each other.
  • the detergent can be acetonitrile.
  • step f) each time steps b), c), e) or b) to e) are repeated, the deprotecting agent in the first reaction vessel, in the second reaction vessel
  • the activator is reused with the phosphoramidite monomer or multimer or stereoisomer thereof, the capping agent in the third reaction vessel, and/or the oxidizing agent or vulcanizing agent in the fourth reaction vessel.
  • the phrase "reuse” means that the reagents required for nucleic acid synthesis, such as deprotecting agents, activators and phosphoramidite monomers or polymers or stereoisomers thereof, blocking agents, oxidizing agents or vulcanizing agents, etc.
  • the reagents are not discarded after being contacted with the solid phase carrier and reacted once, but are again contacted and reacted with the solid phase carrier in one or more subsequent steps.
  • the reaction reagent contained in the reaction vessel is used multiple times.
  • step b) when the step b) is carried out for the first time, the solid phase carrier is contacted with the deprotecting agent in the first reaction vessel containing the deprotecting agent, and the deprotecting agent is not after the reaction is completed. Will be discarded and still remain in the first reaction vessel.
  • step b) is repeated one or more times.
  • step b) When step b) is repeatedly carried out for the first time, the deprotecting agent remaining in the first reaction vessel is used for the second time in contact with the solid phase carrier, and likewise, the deprotecting agent is not discarded after the reaction is completed. Retained in the first reaction vessel.
  • step b) is repeated a second time, the deprotecting agent remaining in the first reaction vessel is used for the third time in contact with the solid support.
  • the number of repetitions required is ultimately determined based on the length of the oligonucleotide to be synthesized. This achieves repeated use of the deprotecting agent contained in the first reaction vessel.
  • n may be selected from a positive integer of 0, 1, 2, 3, 4, 5, 6, 7, or more.
  • n 0, the method as described above is equivalent to the prior art single base nucleic acid synthesis method.
  • n is a positive integer greater than or equal to one.
  • n is 1, 2 or 3.
  • n is 1.
  • the phosphoramidite activator is selected from the group consisting of tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salt.
  • the oxidizing agent is selected from the group consisting of iodine solutions.
  • the vulcanizing agent is selected from the group consisting of 3-amino-[1,2,4]-dithiazole-5-thione or 3H-benzodithiazol-3-one 1,1-dioxide.
  • the deprotecting agent is selected from the group consisting of a solution of trichloroacetic acid in dichloromethane or a solution of trifluoroacetic acid in acetonitrile.
  • the phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof may be a phosphoramidite monomer or polymer as shown in formula (VI) or Its stereoisomers:
  • R 8 is a substituted or unsubstituted trityl group such as 4,4'-dimethoxytrityl.
  • R 10 and R 11 are each independently a substituted or unsubstituted aliphatic group.
  • R 10 and R 11 are preferably an isopropyl group.
  • m is 0, 1, or 2.
  • R 1 is an acid labile protecting group or a trialkylsilyl group, e.g. tert-butyldimethylsilyl or triisopropylsilyl silyl group.
  • R 1 is substituted or unsubstituted trityl, 9-(phenyl)xanthene (also known as “pixyl") or tetrahydropyranyl (also known as "THP"").
  • R 1 is unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, trialkoxytrityl, THP or 9 -Phenylindole.
  • R 1 is 4,4'-dimethoxytrityl (also known as "DMT").
  • R 2 represents C-allyl. In a preferred embodiment, R 2 is -H, -O or -OCH 2 CH 2 OMe.
  • R 3 is independently -OCH 2 CH 2 CN, -SCH 2 CH 2 CN, 4-cyanobut-2-enylthio, 4-cyanobut-2-enyloxy, Allylthio, allyloxy, 2-butenylthio or 2-butenyloxy. In a preferred embodiment, R 3 is -OCH 2 CH 2 CN or -SCH 2 CH 2 CN.
  • the method further comprises a treatment with a base synthetic oligonucleotides to remove from -OCH 2 CH 2 CN or -SCH 2 CH 2 CN in -CH 2 CH 2 CN.
  • each of R 4 and R 5 is isopropyl.
  • R 7 is o-chlorophenyl or p-chlorophenyl.
  • B may also be H, for example, when one or more abasic moieties are present.
  • the phosphoramidite monomer or multimer or a stereoisomer thereof of formula (III) is selected from one of the following 20 compounds or a stereoisomer thereof:
  • Bz is a benzoyl group and ib is an isobutyryl group.
  • the solid support can be any solid support suitable for solid phase oligonucleotide synthesis, such as, but not limited to, pore size controllable glass spheres (also known as "CPG"), polystyrene, microporous polyamides.
  • CPG pore size controllable glass spheres
  • polystyrene polystyrene
  • microporous polyamides polydimethylacrylamide, polyethylene glycol coated polystyrene, and polyethylene glycol supported on polystyrene, such as those sold under the tradename Tentagel.
  • the solid support is a CPG bearing an amino-modified reactive functional group.
  • the particle diameter of the CPG may be less than or equal to 5 ⁇ m, less than or equal to 25 ⁇ m, less than or equal to 50 ⁇ m, less than or equal to 100 ⁇ m, less than or equal to 200 ⁇ m, less than or equal to 500 ⁇ m or more; and the pore diameter may be less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to Or bigger.
  • the linking molecule of the modified solid phase carrier may have an ester group, a lipid group, a thioester group, an o-nitrobenzyl group, a coumarin group, a hydroxyl group, a thiol group, an anthracene ether group, a carboxyl group, an aldehyde group, an amino group, an amine group, A compound of any one or more of an amide group, an alkenyl group, or an alkynyl group.
  • the aliphatic group used in the present invention includes a linear or branched C 1 -C 18 hydrocarbon group which is fully saturated or contains one or more non-aromatic double bonds, or is fully saturated or contains one or more non-conjugated double A C 3 -C 18 cyclic hydrocarbon group of the bond.
  • the lower alkyl group is a fully saturated linear or branched C 1 -C 8 hydrocarbon group or a C 3 -C 8 cyclic hydrocarbon group.
  • the aliphatic group is preferably a lower alkyl group.
  • the aromatic groups used in the present invention include carbocyclic aromatic ring systems (e.g., phenyl) and carbocyclic aromatic ring systems fused to one or more carbocyclic aromatic or non-aromatic rings (e.g., naphthyl, anthracenyl, and 1,2,3,4-tetrahydronaphthyl).
  • carbocyclic aromatic ring systems e.g., phenyl
  • carbocyclic aromatic ring systems fused to one or more carbocyclic aromatic or non-aromatic rings e.g., naphthyl, anthracenyl, and 1,2,3,4-tetrahydronaphthyl.
  • heteroaryl groups include heteroaryl ring systems (eg, thienyl, pyridyl, pyrazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, oxazolyl, furyl, pyrrolyl, Imidazolyl, pyrazolyl, triazolyl, pyrimidinyl, pyrazinyl, thiazolyl, isoxazolyl, isothiazolyl, tetrazolyl or oxadiazolyl) and one of the carbocyclic aromatic rings, carbocyclic non- A heteroaromatic ring system in which an aromatic ring, heteroaryl ring or heterocycloalkyl ring is fused to one or more other heteroaryl rings (eg, benzothienyl, benzimidazole, indole, tetrahydroanthracene, Azaindole, carbazole, quinoline, imidazo
  • an aralkyl group is an aromatic substituent attached to a moiety through an aliphatic group preferably having from 1 to about 6 carbon atoms.
  • heterocycloalkyl group as used herein is a non-aromatic ring system which preferably has 5 to 6 atoms and includes at least one hetero atom such as nitrogen, oxygen or sulfur.
  • heterocycloalkyl groups include morpholine, piperidine, piperazine and the like.
  • Suitable substituents for aliphatic groups, aromatic groups, aralkyl groups, heteroaryl groups and heterocycloalkyl groups include aryl groups, halogenated aryl groups, lower alkyl groups, halogenated lower alkyl groups (e.g., trifluoromethyl and Trichloromethyl), -O-(aliphatic or substituted aliphatic), -O-(aryl or substituted aryl), benzyl, substituted benzyl, halogen, cyano, nitro, - S-(aliphatic or substituted aliphatic) and -S-(aryl or substituted aryl).
  • Amine, alcohol and thiol protecting groups are known to those skilled in the art.
  • an amine protecting group see Greene et al., Protective Groups in Organic Synthesis (1991), John Wiley & Sons, Inc. (hereinafter referred to as "the book") 309-405
  • the amine is protected as an amide or carbamate. See pages 10-142 of this book for examples of alcohol protecting groups, the contents of which are hereby incorporated by reference in its entirety.
  • a preferred alcohol protecting group is tert-butyldimethylsilyl.
  • thiol protecting groups see pages 277-308 of the book, the contents of which are incorporated herein by reference in its entirety.
  • the acid labile protecting group is a protecting group which can be removed by contact with a Bronsted acid or a Lewis acid.
  • Acid labile protecting groups are known to those skilled in the art. Examples of common acid labile protecting groups include substituted or unsubstituted trityl groups (pages 60-62 of the book), substituted or unsubstituted tetrahydropyranyl groups (pages 31-34 of the book). , substituted or unsubstituted tetrahydrofuranyl (pages 36-37 of the book) or 9-phenylxanthene (p. 65 of the book).
  • a preferred acid labile protecting group is a substituted or unsubstituted trityl group such as 4,4'-dimethoxytrityl (also referred to as "DMT").
  • the trityl group is preferably substituted by an electron donating group such as an alkoxy group.
  • Nucleoside bases include naturally occurring bases such as adenine, guanine, cytosine, thymine, and uracil, as well as modified bases such as 7-deazaguanine, 7-deaza-8-aza Guanine, 5-propynylcytosine, 5-propynyluracil, 7-deaza adenine, 7-deaza-8-azadenine, 7-deaza-6-oxopurine, 6 -oxopurine, 3-deaza adenosine, 2-nitro-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo- 5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine, 4-triazole-5-methylthymidine And 4-triazole-5-methyluracil.
  • bases such as adenine,
  • a protected nucleobase is a nucleobase in which the active functional group of the base is protected.
  • nucleobases have an amine group that can be protected with an amine protecting group, such as by formation of an amide or carbamate.
  • an amine protecting group such as by formation of an amide or carbamate.
  • the amine groups of adenine and cytosine are typically protected with a benzoyl protecting group, while the amine groups of guanine are typically protected with isobutyryl, acetyl or t-butylphenoxyacetyl groups.
  • other protection schemes can also be used.
  • the adenine and guanine amine groups are protected with a phenoxyacetyl group, while the cytosine amine group is protected with an isobutyryl group.
  • the conditions for removal of the protecting group will depend on the protecting group employed.
  • the oligonucleotide may be treated with an alkali solution such as a concentrated aqueous ammonia solution, a normal methylamine solution or a t-butylamine/ammonium hydroxide solution to remove it.
  • Structural formulae as referred to herein are understood to include the corresponding stereoisomers where appropriate.
  • the deprotecting agent used in step b) depends on the R 1 group used. If R 1 is an acid labile protecting group, the deprotecting agent is selected from the group consisting of acids. If R 1 is a trialkylsilyl group such as tert-butyldimethylsilyl or triisopropylsilyl, the second intermediate can be treated with fluoride ions to remove R 1 . Typically, tert-butyldimethylsilyl and triisopropylsilyl groups are removed by treatment with a solution of tetrabutylammonium fluoride in THF.
  • the final step of the reaction cycle may be the capping step; if no capping is performed In the step, the final step of the reaction may be an oxidation or sulfurization step.
  • the final step of the reaction cycle can be removal of R 1.
  • the synthetic oligonucleotide can be an oligoribonucleotide. In a specific embodiment, the synthetic oligonucleotide can be an oligodeoxyribonucleotide.
  • the synthetic oligonucleotide can be a phosphate ester and thus has only a phosphate linkage (ie, the internucleotide phosphorus is only bonded to oxygen).
  • the synthetic oligonucleotide may be a phosphorothioate, thus having only a phosphorothioate linkage (phosphor between each nucleotide is bonded to at least one S, preferably only one S) .
  • the synthetic oligonucleotide can be a chimeric oligonucleotide that contains both a phosphate and a phosphorothioate internucleotide linkage.
  • each of the reaction vessels in steps b to e above is collectively referred to as a "synthesis cell”
  • a solid phase carrier is referred to as a “synthetic needle”
  • a plurality of solid phase carriers are referred to as “synthetic needle sets”.
  • the nucleic acid synthesis method of the present invention is also referred to as a “synthetic pool” nucleic acid synthesis method.
  • the innovative "synthetic pool” nucleic acid synthesis method of the present invention can realize large-scale control by precisely controlling the reaction time of "synthesis needle” immersed in the "synthesis tank” and the number of cycles of "synthesis pool” under the premise of satisfying the yield. Direct synthesis of long-chain nucleic acid fragments at low cost, high efficiency, and low error rate.
  • the synthesis method can greatly reduce the synthesis cost by recycling the materials in the "synthesis cell”, and can also ensure the low error rate of the synthesized product by solid phase synthesis.
  • the method does not require reagent input and output pipelines, which saves pipeline cost and reduces design complexity.
  • the "synthetic pool” method can avoid cross-contamination and further ensure the accuracy of nucleic acid synthesis; solid phase synthesis "synthesis
  • the Needle” group is scalable and easy to integrate, allowing flexible adjustment of throughput and throughput as needed, and recycling to reduce costs.
  • the nucleic acid synthesis method based on "synthesis pool” proposed by the invention improves the synthesis efficiency, and also avoids the shortcomings of the current commercial synthesizer, and provides a higher development for the future commercial synthesizer. A new way of feasibility.
  • the "synthetic pool" nucleic acid synthesis method of the present invention is well suited for use in high throughput nucleic acid synthesis, i.e., simultaneous synthesis of a variety of desired nucleic acids.
  • the invention provides a method of synthesizing an oligonucleotide, the method comprising:
  • Nu is a single bond
  • X 1 is independently -O- or -S-;
  • X 2 is independently -O-, -S- or -NR-;
  • X 3 is independently -O-, -S-, -CH 2 - or -(CH 2 ) 2 -;
  • R 1 is a protecting group
  • R 2 is independently -H, -F, -NHR 6 , -CH 2 R 6 or -OR 6 ;
  • R 3 is independently -OCH 2 CH 2 CN, -SCH 2 CH 2 CN, a substituted or unsubstituted aliphatic group, -OR 7 or -SR 7 ;
  • R is -H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or an amine protecting group;
  • R 6 is -H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, or a protecting group;
  • R 7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group;
  • Each B is independently a base that is modified or unmodified
  • R 13 is a solid phase carrier or -Y 1 -LY 1 -R 14 ;
  • Y 1 is a single bond, a double bond, -C(O)-, -C(O)NR 17 , -C(O)O-, -NR 17 - or -O-;
  • L is a single bond, a double bond, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;
  • R 17 is -H, a substituted or unsubstituted aliphatic group, or a substituted or unsubstituted aryl group;
  • R 14 is a solid phase carrier
  • q is 0 or a positive integer
  • X 5 is -OH or -SH; the other groups are as defined above;
  • R 4 and R 5 are each independently a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl group;
  • R 4 and R 5 together with the nitrogen to which they are bonded form a heterocycloalkyl or heteroaryl group, wherein the heterocycloalkyl or heteroaryl group is preferably a five or six membered ring;
  • n 0 or a positive integer
  • step c) optionally, contacting said plurality of solid phase supports with a blocking agent in a third reaction vessel comprising a blocking agent, wherein said step c) is not reacted with a phosphoramidite monomer or multimer
  • a blocking agent in a third reaction vessel comprising a blocking agent
  • first reaction vessel, the second reaction vessel, the third reaction vessel and the fourth reaction vessel are independent of each other;
  • step f) optionally repeating steps b), c), e) or b) to e) one or more times, wherein the final step is step b), d) or e), thereby synthesizing the desired oligonucleotide .
  • the invention also provides a system for nucleic acid synthesis comprising:
  • One or more reaction vessels containing a deprotecting agent One or more reaction vessels containing a deprotecting agent
  • One or more reaction vessels comprising a phosphoramidite activator and a phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof,
  • reaction vessels comprising a capping agent
  • One or more reaction vessels containing an oxidizing agent or a vulcanizing agent are provided.
  • a control system for controlling movement of the mobile device is
  • system further comprises one or more reaction vessels comprising a detergent.
  • the mobile device controls the one or more solid phase carriers to move simultaneously between the respective reaction vessels.
  • the one or more solid phase carriers are detachably secured to the mobile device.
  • the mobile device is a robotic arm.
  • control system is a computer operated programmatic control system.
  • the invention provides a system for nucleic acid synthesis comprising:
  • reaction vessel for receiving a deprotection reagent having one or more separate tanks, and the number of said tanks being at least equal to the number of said solid phase supports,
  • One or more reaction vessels comprising a phosphoramidite activator and a phosphoramidite monomer or multimer represented by formula (III) or a stereoisomer thereof,
  • reaction vessels comprising a capping agent
  • One or more reaction vessels containing an oxidizing agent or a vulcanizing agent are provided.
  • a control system for controlling movement of the mobile device is
  • the one or more separate tanks each correspond to a solid support.
  • system further comprises an injection device for adding a deprotection reagent to the tank.
  • the infusion device as described herein can be any device suitable for adding a deprotecting agent to the tank.
  • the infusion device can be an injector containing a deprotecting reagent, such as a syringe.
  • the injection device can be a reaction vessel containing a deprotection reagent in fluid communication with the tank.
  • such an injection device further comprises means for controlling the flow of deprotection reagent into the tank.
  • the means for controlling the flow of deprotection reagent into the tank may include, for example, a valve.
  • the valve's switch can be controlled by the control system.
  • the means for controlling the flow of the deprotection reagent into the tank may also include, for example, a pressure control system or the like. For example, a positive pressure can be created in the injection device such that the deprotection reagent flows from the injection device into the tank.
  • system further comprises a drain for discharging the deprotection reagent from the tank.
  • the venting device as described herein can be any device suitable for expelling the deprotecting agent from the tank.
  • the discharge device can be a pipette, such as a vacuum aspirator.
  • the discharge means can be a fluid passage in fluid communication with the tank.
  • a discharge device further comprises means for controlling the flow of deprotection reagent out of the tank.
  • the means for controlling the deprotection reagent to flow out of the tank may comprise, for example, a valve.
  • the valve's switch can be controlled by the control system.
  • the means for controlling the deprotection reagent to flow out of the tank may also include, for example, a pressure control system or the like. For example, a negative pressure can be created in the discharge device such that the deprotection reagent flows out of the tank.
  • system further comprises one or more reaction vessels comprising a detergent.
  • a deprotecting reagent is added to the sample by the injection device according to the desired oligonucleotide sequence to be synthesized on the one or more solid phase carriers.
  • the mobile device controls the one or more solid phase carriers to move simultaneously between the respective reaction vessels.
  • the one or more solid phase carriers are detachably secured to the mobile device.
  • the mobile device is a robotic arm.
  • control system is a computer operated programmatic control system.
  • reaction vessel or “synthetic pool” means any device capable of containing a reagent, including but not limited to tanks, channels, wells, test tubes, cups, dishes, and the like.
  • the reaction vessel or synthesis tank is open at one end.
  • the reaction vessel may have any suitable shape, for example, the reaction vessel may be square, spherical, conical, cylindrical, irregular, or the like.
  • the reaction vessel may have any suitable size, for example, its size may be arbitrarily adjusted according to the volume of the reaction solution to be contained, for example, it may be sized to accommodate at least 1 ⁇ L, at least 5 ⁇ L, at least 10 ⁇ L, at least 20 ⁇ L, at least 50 ⁇ L, at least 100 ⁇ L, at least 1 mL, at least 5 mL, at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, at least 200 mL, at least 500 mL, at least 1 L or more, or may be sized to accommodate up to 1 ⁇ L, up to 5 ⁇ L, up to 10 ⁇ L, Up to 20 ⁇ L, up to 50 ⁇ L, up to 100 ⁇ L, up to 1 mL, up to 5 mL, up to 10 mL, up to 20 mL, up to 50 mL, up to 100 mL, up to 200 mL, up to 500 mL, up to
  • the mobile device for example, the robot arm
  • the control manner may be an electric control, a magnetic control or an electromagnetic hybrid mode
  • the moving direction of the mobile device for example, the mechanical arm
  • the moving direction of the mobile device may be horizontal, vertical, Free movement in the direction of the circumference.
  • the first step in the preparation of oligonucleotides comprising a solid support represented by the formula (I) deprotection, i.e. removal of a 5'-protecting group represented by R 1.
  • R 1 is an acid labile protecting group
  • R 1 is removed by treatment of the oligonucleotide with an acid.
  • the 5'-protecting group is a trityl group such as 4,4'-dimethoxytrityl.
  • the oligonucleotide can be removed by treating the oligonucleotide with a solution of dichloroacetic acid, trichloroacetic acid or trifluoroacetic acid in an organic solvent such as dichloromethane, acetonitrile.
  • the second step of preparing the oligonucleotide comprises coupling the phosphoramidite multimer to the deprotected solid support represented by structural formula II.
  • the -OH or -SH group on the solid support or the 5'-deprotected group and the polymer of the nucleoside or oligonucleotide attached to the solid support are replaced by a -NR 4 R
  • the 5 group reacts.
  • the concentration of the 5'-deprotected nucleotide is typically about 0.02-2 M, and the concentration of the multimer is preferably 5'-deprotected nucleoside or about 5 of the oligonucleotide. -15 equivalents.
  • a phosphoramidite activator such as tetrazole, S-ethylthiotetrazole, dicyanoimidazole or pyridinium salt (such as pyridine chloride), which is equivalent to a 5'-deprotected nucleoside, is often added. ⁇ ) to promote the coupling reaction.
  • the reaction time is usually from about 10 seconds to about 120 seconds.
  • the third step of preparing the oligonucleotide typically involves capping the unreacted deprotected solid support to render it unreactive in the subsequent coupling step.
  • the sequences that failed in the synthesis were capped such that they were more easily separated from the full length oligonucleotide.
  • An acid anhydride such as acetic anhydride or isobutyric anhydride or an acid chloride such as acetyl chloride or isobutyryl chloride is usually used as a blocking agent in the presence of a base.
  • the fourth step in the preparation of the oligonucleotide comprises oxidizing or vulcanizing the trivalent phosphorus group of the oligonucleotide.
  • a newly formed nucleus or a 5'-deprotected group of the nucleoside or oligonucleotide attached to the -OH or -SH group or the solid support on the solid support is formed.
  • the trivalent phosphorus bond is oxidized or sulfided.
  • the oxidation reaction is usually carried out by treating the oligonucleotide with an oxidizing agent, for example, I 2 in the presence of water, or a peroxide such as t-butyl hydroperoxide in an organic solvent.
  • an oxidizing agent for example, I 2 in the presence of water, or a peroxide such as t-butyl hydroperoxide in an organic solvent.
  • the oxidizing solution typically contains from about 20 to about 100 equivalents of I 2 in the presence of a base and traces of water.
  • the reaction is carried out in an aprotic polar solvent such as THF, to which is added a base such as a tertiary alkylamine or pyridine and about 1% water.
  • the ratio of aprotic solvent to base is from about 4:1 to about 1:4 (by volume).
  • the oxidation reaction is usually carried out for about 5 seconds to about 2 minutes.
  • the trivalent phosphorus group can be sulfided with any sulfur transfer reagent known to those skilled in the art for oligonucleotide synthesis.
  • sulfur transfer reagent examples include 3H-benzobisthiazol-3-one 1,1-dioxide (also referred to as "Beaucage reagent"), dibenzoyltetrasulfide, phenylacetyl disulfide, and disulfide. N,N,N',N'-tetraethylthiuram, 3-amino-[1,2,4]-dithiazole-5-thione (see U.S. Patent No. 6,096,881, the disclosure of which is incorporated herein by reference ), or elemental sulfur.
  • 3-Amino-[1,2,4]-dithiazole-5-thione is a preferred sulfur transfer reagent.
  • the oligonucleotide is made with 3-amino-[1,2,4]-dithiazole-5-thione at a concentration of about 0.04-0.2 M in an organic solvent (eg pyridine/acetonitrile, 1:9 w/w) The solution in contact.
  • the sulfurization reaction is usually carried out for about 30 seconds to about 2 minutes.
  • Phosphoric acid oligonucleotides can be prepared by oxidizing a trivalent phosphorus group and selecting an internucleotide phosphorus protecting group (for example, a group represented by R 3 ) such that a phosphate group is formed after deprotection of the internucleotide group.
  • an internucleotide phosphorus protecting group for example, a group represented by R 3
  • R 3 an internucleotide phosphorus protecting group
  • the oxidation of trivalent phosphorus protected by ⁇ -cyanoethoxyl followed by alkali deprotection results in the formation of a phosphate group.
  • the phosphorothioate oligonucleotide can be formed by vulcanizing or oxidizing a trivalent phosphorus group and selecting an internucleotide phosphorus protecting group (for example, a group represented by R 3 ) such that the internucleotide group is deprotected.
  • an internucleotide phosphorus protecting group for example, a group represented by R 3
  • R 3 internucleotide phosphorus protecting group
  • phosphorothioate groups for example, beta-cyanoethylthio-protected trivalent phosphorus is oxidized and then deprotected with anhydrous basics to form a phosphorothioate group.
  • the chimeric oligonucleotide may be prepared by oxidizing a trivalent phosphorus group in one or more reaction cycles and vulcanizing the trivalent phosphorus group in one or more additional reaction cycles while appropriately selecting the core.
  • a phospho-phosphorus protecting group (such as the group represented by R 3 ) to form the desired phosphate or phosphorothioate group.
  • chimeric oligonucleotides can be prepared by selecting a polymer in which some internucleotide protecting groups form a phosphorothioate group upon deprotection, such as beta-cyanoethylsulfide.
  • the base protecting group, and other internucleotide phosphorus protecting groups form a phosphate bond upon deprotection, such as a beta-cyanoethoxy protecting group.
  • the oligonucleotide is oxidized after each coupling step of the reaction cycle.
  • the last step of the reaction cycle may be a capping step; if no capping is performed, the last step of the reaction may be Oxidation or vulcanization step.
  • the reaction cycle can be terminated with a deprotection step.
  • the oligonucleotide is to be purified by reverse phase high performance liquid chromatography (HPLC)
  • HPLC high performance liquid chromatography
  • the 5'-protected oligonucleotide is the desired product.
  • the 5'-deprotected oligonucleotide is typically the desired product.
  • the 5'-protected nucleoside or oligonucleotide can be carried on a solid support, usually in an amount of from about 50 to 100 ⁇ mol per gram of the carrier.
  • the carrier to which the 5'-deprotected nucleoside or oligonucleotide is bound is then contacted with the mixture for about 10 seconds to 2 minutes, preferably about 90 seconds.
  • the solid support and the oxidant for example with an I 2 /water mixture or with a peroxide (such as t-butyl hydroperoxide) in an organic solvent (eg THF, Contact in acetonitrile or toluene).
  • a peroxide such as t-butyl hydroperoxide
  • an organic solvent eg THF, Contact in acetonitrile or toluene.
  • a mixture of I 2 and water is the preferred oxidizing agent.
  • other water-miscible organic solvents may also be present.
  • the solid support bound oligonucleotide with the nucleotide conjugate trivalent phosphorus can be contacted with I 2 in a solvent mixture of water, an aprotic solvent (reagent may be mixed with water) and a base solution.
  • a typical oxidation solution is about 0.01-1.5M, preferably 0.1M of I water / pyridine / tetrahydrofuran solution 2 (2:20:78) a.
  • the solid support is typically treated with an I 2 solution for about 5 seconds to 120 seconds, preferably 30 seconds.
  • the solid support may be contacted with a solution of a sulfur transfer reagent in an organic solvent to vulcanize the trivalent phosphorus group.
  • a sulfur transfer reagent in an organic solvent to vulcanize the trivalent phosphorus group.
  • the solid support can be contacted with a solution of 3H-benzodithiazol-3-one-1,1-dioxide in an organic solvent such as acetonitrile (about 0.05-0.2 M) for about 30 seconds to 2 minutes. .
  • the deprotection step is accomplished by contacting the solid support with an acid solution for about 10 seconds to 180 seconds (e.g., 60 seconds).
  • the deprotecting agent is selected from the group consisting of a solution of trichloroacetic acid in dichloromethane or a solution of trifluoroacetic acid in acetonitrile.
  • the solid support can optionally be contacted with the phosphoramidite activator solution for about 10 seconds to 120 seconds. This reaction cycle can be repeated one or more times until the desired length of oligonucleotide is synthesized.
  • reaction cycle ends with a capping step or an oxidation or sulfurization step, a 5'-protected oligonucleotide is obtained.
  • reaction cycle is terminated with a deprotection step, a 5'-deprotected oligonucleotide is obtained.
  • the method further comprises the step of separating the synthetic oligonucleotide from the solid support.
  • the oligonucleotide can be removed from the solid support using an aminolysis process.
  • the reagent for the aminolysis method may be selected from any one of ammonia water, ammonia gas, and methylamine; the aminolysis temperature may be 25, 60, 90 ° C or any temperature therebetween; the aminolysis time is usually from about 0.5 hour to about 18 Hours or longer, such as 2h, 5h, 10h, 18h or 24h.
  • the method can further comprise purifying the synthetic oligonucleotide using a purification method selected from the group consisting of desalting, MOP, PAGE, PAGE Plus or HPLC.
  • the nucleic acid synthesis method proposed by the invention realizes low-cost, high-efficiency and accurate double-base nucleic acid synthesis by using the "synthesis pool” method for the first time.
  • the nucleic acid synthesis method of the invention can recycle the reaction reagent in the "synthesis cell” by precisely controlling the "synthesis needle” group moving, immersing and lifting, without greatly designing complicated reagent input and output pipelines, thereby greatly reducing Material cost.
  • the method can ensure the flux and yield of nucleic acid synthesis by designing different "synthetic needle” groups. By precisely controlling the "synthetic needle” group, the synthesis time can be precisely controlled and shortened, and the chemical synthesis efficiency can be fully utilized.
  • the design of the pool ensures no cross-contamination and thus controls low error rates.
  • the original four-step cycle method is used for 100 cycles to obtain 100 bp, based on the "synthesis pool" double-base nucleic acid synthesis strategy, Under the premise of ensuring low cost and low error rate, it can now produce a 200 bp nucleic acid single chain, breaking the technical bottleneck faced by the single base nucleic acid synthesis technology widely used in commercial services.
  • the novel double-base nucleic acid synthesis design scheme based on "synthesis pool” proposed by the invention firstly realizes the method of synthesizing double base nucleic acid by controlling the "synthetic needle” by a pre-design program, and verifies the practicability of the method;
  • porous glass is used as a solid phase carrier, which ensures low error rate and high reaction efficiency; more importantly, the synthesis reagent can be reused through the "synthesis cell", which greatly reduces the amount of reagent used, and at the same time There is no need to lay complex reagent input and output lines separately, which greatly reduces the synthesis cost.
  • the “synthetic needle” can be expanded on a large scale and easily integrated with other devices due to its small structure and simple fabrication, such as polymerase chain.
  • the reaction device makes the nucleic acid synthesis more convenient and efficient; finally, the "synthesis pool” double base nucleic acid synthesis method can also be extended to the "synthesis pool” of the three-base nucleic acid synthesis, the "synthesis pool” of the four-base nucleic acid Synthesis, etc.
  • a schematic of the "synthetic pool” nucleic acid synthesis system is shown in Figure 2.
  • the nucleic acid synthesis method of the present invention improves the reaction efficiency and ensures a low error rate on the basis of greatly reducing the cost of nucleic acid synthesis.
  • the "synthetic needle" group in the invention has the characteristics of being expandable and easy to integrate, and can improve the synthesis flux through optimization design, and can also be combined with module automation for function expansion, such as assembling downstream polymerase chain reaction and gene assembly technology flow.
  • the nucleic acid method of the invention integrates the advantages of the existing synthetic methods, skillfully avoids the problems exposed by other methods, and lays a foundation for the future development of the third generation low-medium-high-flux synthesizer.
  • Figure 1 shows an example of four single base monomers and 20 double base monomers.
  • Bz is a benzoyl group and ib is an isobutyryl group.
  • Figure 2 shows a schematic of a "synthetic pool” nucleic acid synthesis system.
  • the blocks shown in the left three columns are synthetic pools, and the area shown in the fourth column is the robotic arm control station, which controls the movement, immersion and lifting of the "synthetic needle” group by controlling the robot arm.
  • Figures 3a-d show an exemplary embodiment of a "synthetic pool" synthetic oligonucleotide.
  • Figure 4 shows the glass flakes after completion of the synthesis of the T5 primer in Example 1.
  • Figure 5 shows a schematic diagram of the aminolysis in Example 1.
  • Figure 6a shows the HPLC profile of the T5 reference in Example 1
  • Figure 6b shows the HPLC profile of the T5 primer in Example 1.
  • Fig. 7 shows a glass piece after completion of the synthesis of the T10 primer in Example 2.
  • Figure 8 shows a schematic diagram of the aminolysis in Example 2.
  • Figure 9a shows the HPLC profile of the T10 reference in Example 2
  • Figure 9b shows the HPLC profile of the T10 primer in Example 2.
  • the glass sheet modification process is as follows:
  • the above-mentioned glass piece to be dried was placed in a solution of 750 mg of Linker, 400 mg of HATU, 800 ⁇ L of DIPEA dissolved in 50 mL of acetonitrile, and stirred overnight in a vertical stirrer. The glass piece was taken out, washed three times with acetonitrile, and washed three times with acetone. Dry, the glass piece is finished.
  • the Linker graft density of the modified glass sheet was measured by an ultraviolet spectrophotometer to be 0.003636 nmol/mm 2 .
  • the oligonucleotide synthesis process is as follows:
  • oligonucleotide sequence to be synthesized is shown below, and the oligonucleotide was synthesized on one modified glass slide.
  • TTTTT designated as T5 primer.
  • Each piece of modified glass piece is a solid phase carrier according to the present invention, and only the first deprotection can be carried out to carry out the subsequent coupling step, and if the chain length of the nucleic acid sequence to be synthesized is short (cycle number ⁇ 25), the cap in the experimental operation The steps can be omitted. If a longer chain length nucleic acid sequence is to be synthesized (cycle number > 25), a capping step is required to reduce the error rate to obtain a sufficient number of target nucleic acids.
  • the glass sheet modification process is as follows:
  • the above-mentioned glass piece to be dried was placed in a solution of 750 mg of Linker, 400 mg of HATU, 800 ⁇ L of DIPEA dissolved in 50 mL of acetonitrile, and stirred overnight in a vertical stirrer. The glass piece was taken out, washed three times with acetonitrile, and washed three times with acetone. Dry, the glass piece is finished.
  • the Linker graft density of the modified glass sheet was measured by an ultraviolet spectrophotometer to be 0.003636 nmol/mm 2 .
  • the oligonucleotide synthesis process is as follows:
  • oligonucleotide sequence to be synthesized is shown below, and the oligonucleotide was synthesized on one modified glass slide.
  • TTTTTTTTTT T10 primer.
  • the double base synthetic monomer used is: DMT-dT-dT.
  • Each piece of modified glass piece is a solid phase carrier according to the present invention, and only the first deprotection can be carried out to carry out the subsequent coupling step, and if the chain length of the nucleic acid sequence to be synthesized is short (cycle number ⁇ 25), the cap in the experimental operation The steps can be omitted. If a longer chain length nucleic acid sequence is to be synthesized (cycle number > 25), a capping step is required to reduce the error rate to obtain a sufficient number of target nucleic acids.

Abstract

L'invention concerne un procédé et un système pour synthétiser un acide nucléique à l'aide d'une "synthèse en pool" Le procédé est basé sur la synthèse en phase solide de phosphoramidite d'un acide nucléique dans un cycle à quatre étapes, déprotection, couplage, coiffage et oxydation étant effectués dans une première cuve de réaction, une deuxième cuve de réaction, une troisième cuve de réaction et une quatrième cuve de réaction qui sont indépendantes l'une de l'autre. Le procédé concerne l'utilisation d'un nucléotide multi-base en tant que monomère de synthèse d'acide nucléique, de telle sorte qu'un acide nucléique ayant une chaîne plus longue peut être rapidement synthétisé, le taux d'erreur de synthèse est faible, et un réactif peut être utilisé de façon répétée de telle sorte que l'efficacité de synthèse est améliorée de manière considérable.
PCT/CN2019/074588 2018-02-13 2019-02-02 Procédé et système de synthèse d'oligonucléotides WO2019158007A1 (fr)

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