Use of polyether ether ketone as a carrier for DNA synthesis

Abstract

The application discloses application of polyether ether ketone in DNA synthesis carrier, wherein the polyether ether ketone is a chemically modified polyether ether ketone, and has a structural formula shown in the following formula I: wherein n is an integer between 200 and 500. The chemically modified polyether ether ketone provided by the application can be used as a DNA synthesis carrier for DNA synthesis, and enriches the types of DNA synthesis carrier materials. The DNA synthesis carrier provided by the application is a chemically modified polyether ether ketone, the polyether ether ketone is cheap and easy to obtain, and the preparation method is simple.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to the application of polyether ether ketone as a DNA synthesis carrier. Background Technology

[0002] Currently, DNA chemical synthesis mainly employs solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis typically utilizes the phosphoamide triester method. The specific steps of DNA chemical synthesis based on the solid-phase phosphoamide triester method are as follows: First, a nucleotide (usually contained within a synthesis column) with its 5'-hydroxyl group protected by dimethoxytriphenylmethyl (DMT) and attached to a solid support is deprotected by adding trichloroacetic acid, thus obtaining a free 5'-hydroxyl terminus for the next synthesis reaction, adding a monomer to the DNA chain. Second, the phosphoramidite monomer is mixed with a tetrazolium activator and introduced into the synthesis column to form a phosphoramidite tetrazolium active intermediate (its 5' end remains protected by DMT, while the 3' end is activated). Third, a coupling reaction occurs in the sequence with the 5'-hydroxyl group of the nucleoside previously attached to the solid support, forming a phosphoamide triester, thereby extending the synthetic sequence chain by one base. Fourth, an iodine-tetrahydrofuran solution is used to hydrolyze the unstable phosphoamide bond, which is easily hydrolyzed by acids and bases, converting it into a stable pentavalent phosphate bond. After repeating the same cyclic steps as described above, the DNA text sample required for storage can be obtained. In short, the DNA chemical synthesis based on the solid-phase phosphoamyl triester method consists of four reaction steps forming a cycle to complete the synthesis of one monomer. That is, with each additional cycle, one base is added to the oligonucleotide chain that is continuously growing on the solid support.

[0003] Literature reports on DNA synthesis vectors mainly focus on pore-controlled glass (CPG), polystyrene, as well as modified glass and modified silicon wafers, indicating a significant limitation in the selection of DNA synthesis vectors. Summary of the Invention

[0004] In order to address the problems existing in the prior art, the purpose of this invention is to provide a new DNA synthesis vector.

[0005] To achieve the above objectives, the present invention employs the following technical means:

[0006] The application of polyetheretherketone (PEEK) as a DNA synthesis carrier, wherein the PEEK is a chemically modified PEEK having the structural formula shown in Formula I:

[0007]

[0008] In the formula, n is an integer between 200 and 500. The structure of the Linker is as follows:

[0009]

[0010] R is an alkyl, phenyl, or substituted phenyl group.

[0011] Preferably, the method for preparing the chemically modified polyetheretherketone includes the following steps:

[0012] S1, Hydroxyl modification: Using dimethyl sulfoxide as solvent and sodium borohydride as reducing agent, react with polyether ether ketone at 100-140℃ for 3h. Reduce the aldehyde group in polyether ether ketone with sodium borohydride to obtain hydroxyl-modified polyether ether ketone, so as to further modify it to obtain the target product.

[0013] S2, Isocyanate group modification: Using toluene as solvent and triethylenediamine as base, the hydroxyl-modified polyether ether ketone obtained in step S1 is reacted with 4,4-dicyclohexylmethane diisocyanate in a nitrogen atmosphere at room temperature for 3 days. The isocyanate group-modified polyether ether ketone is obtained by reacting the hydroxyl group with one of the isocyanate groups, so as to further modify it to obtain the target product.

[0014] S3, Amino modification: Under room temperature conditions, the polyether ether ketone modified with isocyanate groups obtained in step S2 is reacted with sodium hydroxide in a 1,4-dioxane. Carbon dioxide is removed by the reaction of sodium hydroxide with isocyanate groups to obtain amino-modified polyether ether ketone, so as to further modify it to obtain the target product.

[0015] S4, Linker grafting: At room temperature, the amino-modified polyether ether ketone obtained in step S3 is reacted with Linker in an acetonitrile solution containing 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine overnight to obtain Linker-grafted polyether ether ketone.

[0016] Preferably, in step S1, the surface area of ​​the polyetheretherketone is 200 cm². 2 .

[0017] Preferably, in step S1, the polyetheretherketone, sodium borohydride, and dimethyl sulfoxide are in the following mass-volume ratio: polyetheretherketone: sodium borohydride: dimethyl sulfoxide = 0.5g: 0.1g: 50mL.

[0018] Preferably, in step S2, the triethylenediamine, hydroxyl-modified polyetheretherketone, 4,4-dicyclohexylmethane diisocyanate, and toluene are in the following mass-volume ratio: hydroxyl-modified polyetheretherketone: triethylenediamine: 4,4-dicyclohexylmethane diisocyanate: toluene = 0.5g: 2.15mg: 1mL: 19mL.

[0019] Preferably, in step S3, the isocyanate-modified polyether ether ketone, sodium hydroxide, and 1,4-dioxane are in the following mass ratio: isocyanate-modified polyether ether ketone: sodium hydroxide: 1,4-dioxane = 0.5:1:1.

[0020] Preferably, in step S4, the amino-modified polyetheretherketone, Linker, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, N,N-diisopropylethylamine, and acetonitrile are in the following mass-volume ratio: amino-modified polyetheretherketone: Linker: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate: N,N-diisopropylethylamine: acetonitrile = 500mg: 200mg: 160mg: 400uL.

[0021] A method for DNA synthesis includes the step of using the chemically modified polyether ether ketone as a DNA synthesis carrier to synthesize DNA.

[0022] Specifically, the following steps were taken: Using an ABI 8909 DNA synthesizer, chemically modified polyether ether ketone was placed in a Luer connector, and the TCA phosphorus amide synthesis program was set to perform automated DNA synthesis.

[0023] The synthesis procedure is as follows:

[0024]

[0025]

[0026] Beneficial effects of the present invention

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The chemically modified polyetheretherketone (PEEK) provided in this invention can be used as a DNA synthesis carrier, enriching the variety of DNA synthesis carrier materials. The DNA synthesis carrier provided by this invention is a chemically modified PEEK, which is inexpensive, readily available, and simple to prepare. Furthermore, the DNA synthesis carrier provided by this invention offers strong designability, allowing the synthesis of DNA molecule sequences of different fragment lengths according to specific practical needs. It also develops a novel chemical modification method for carriers to synthesize DNA molecules, demonstrating strong practicality. Attached Figure Description

[0029] Figure 1 The image shows a quality control diagram of the DNA synthesized in Example 2 of this invention using PAGE denaturing gel electrophoresis.

[0030] Figure 2The HPLC analysis chromatogram of the DNA synthesized in Example 2 of this invention is shown;

[0031] Figure 3 The diagram shows the DNA synthesis results of Example 2 and Comparative Examples 1-3 of the present invention using different DNA synthesis vectors. Detailed Implementation

[0032] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions disclosed in these documents concerning synthetic techniques, product and processing design, polymers, comonomers, initiators, or catalysts in the art. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0033] The numerical ranges in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For example, if a component, physical, or other property (such as molecular weight, melt index, etc.) is described as 100 to 1000, it means that all individual values, such as 100, 101, 102, etc., are explicitly listed, as well as all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the minimum and maximum values ​​listed are considered to be clearly stated in this application.

[0034] When referring to chemical compounds, unless explicitly stated otherwise, the singular includes all isomers and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). Additionally, unless explicitly stated otherwise, nouns described with "an," "a," or "the" also include their plural forms.

[0035] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0036] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0037] Example

[0038] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0040] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0041] 1,4-Dioxane, triethylenediamine, N,N-diisopropylethylamine, dimethyl sulfoxide, sodium borohydride, 4,4-dicyclohexylmethane diisocyanate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, sodium hydroxide, xylene, decapping agents, activators, oxidants, capping agents, and A, T, G, and C monomers used in DNA synthesis are generally commercially available products that are readily available on the market.

[0042] Example 1

[0043] This embodiment provides a chemically modified polyetheretherketone having the structural formula shown in Formula I:

[0044]

[0045] In the formula, R is phenyl (-Ph), and n is 400.

[0046] This embodiment also provides a method for preparing chemically modified polyether ether ketone, the method comprising the following steps:

[0047] S1, Preparation of hydroxyl-modified polyetheretherketone: Add 10 mL of dimethyl sulfoxide (DSMO) and 0.05 g of sodium borohydride (NaBH4) to a 20 mL round-bottom flask, heat at 120 °C until completely dissolved, then add 0.5 g of polyetheretherketone (PEEK) material (200 cm⁻¹). 2 The PEEK material was completely immersed in a reaction solution at 120°C for 3 hours. After the reaction was complete, the PEEK material was removed from the round-bottom flask and rinsed three times each with methanol, deionized water, 0.5 mol / L hydrochloric acid, deionized water, and ethanol, respectively. After rinsing, the PEEK material microplate was dried under vacuum at 60°C for 3 hours to obtain the corresponding hydroxyl-modified polyetheretherketone (PEEK) material. The reaction process is shown in Formula I below:

[0048]

[0049] S2, Preparation of isocyanate-modified polyetheretherketone: Under nitrogen atmosphere, 10 mL of dimethylbenzene, 0.5 mL of 4,4-dicyclohexylmethane diisocyanate (HMDI), and 1.08 g of triethylenediamine (DABCO) were added to a sealed reaction vessel and stirred thoroughly until completely dissolved. The hydroxyl-modified polyetheretherketone obtained in step S1 was then completely immersed in the stirred reaction solution and soaked at room temperature for 3 days. After the reaction, the polyetheretherketone material sample was washed three times each with xylene and 1,4-dioxane solvent under nitrogen atmosphere to obtain isocyanate-modified polyetheretherketone (PEEK-NCO). The reaction process is shown in Formula II below:

[0050]

[0051] S3, Preparation of amino-modified polyetheretherketone: Under room temperature conditions, the isocyanate-modified polyetheretherketone obtained in step S2 was immersed in a mixed solution of 0.5 mol / L sodium hydroxide (NaOH) solution and 1,4-dioxane (1,4-dioxane) in a volume ratio of 1:1 for 5 hours. After the reaction was complete, it was rinsed three times with deionized water and 1,4-dioxane solution respectively. The rinsed material was dried under vacuum for 3 hours to obtain the corresponding amino-modified polyetheretherketone (PEEK-NH2). The reaction process is shown in Equation III below:

[0052]

[0053] S4, Preparation of Linker-grafted polyetheretherketone: At room temperature, add 100 mg Linker, 80 mg 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 200 μL N,N-diisopropylethylamine, and 10 mL acetonitrile (CH3CN) to a 20 mL round-bottom flask and shake well. Place the amino-modified polyetheretherketone obtained in step S3 into a round-bottom flask and stir overnight. After the reaction is complete, remove the material and wash it three times with acetonitrile and 1,4-dioxane, respectively, and air dry to obtain the corresponding Linker-grafted polyetheretherketone (PEEK-Linker) material. The reaction process is shown in Formula IV below:

[0054]

[0055] Example 2

[0056] This embodiment aims to provide the application of the chemically modified polyetheretherketone obtained in Example 1 as a DNA synthesis carrier:

[0057] (1) DNA synthesis: Using an ABI 8909 DNA synthesizer, the chemically modified polyether ether ketone obtained in Example 1 was placed in a Luer connector, and the TCA phosphorus amide synthesis program was set to perform automatic DNA synthesis.

[0058] The synthesis procedure is shown in Table 1 below:

[0059] Table 1, Synthesis Procedure 1

[0060]

[0061]

[0062] (2) Post-processing and quality control of synthesized DNA

[0063] After synthesis, the PEEK material was removed from the ABI 8909 DNA synthesizer and placed in 500 μL of ammonia water for deprotection and cleavage treatment. After 2 hours at 70°C, it was removed. The ammonia solution was aspirated, and 1.5 mL of ice-cold ethanol solution was added, followed by 50 μL of sodium acetate (3M concentration). After the synthesized product precipitated, it was centrifuged at 6000 g, and the supernatant was discarded. 80% ethanol water was added, and the precipitate was washed by shaking. After another centrifugation, the washing process was repeated. The final precipitate was dried at 60°C for 5 minutes. The dried synthesized product was dissolved in 10 mM TrisCl solution, and the concentration was measured to be 15 ng / μL using a Nanodrop instrument, with a total product concentration of 120 pmol. The PAGE denaturing gel electrophoresis and HPLC quality control results are as follows: Figure 1 and Figure 2 As shown.

[0064] Comparative Example 1

[0065] The difference between Comparative Example 1 and Example 2 is that Comparative Example 1 used existing carrier porous glass beads (CPG) material for DNA synthesis, and the synthesized product was tested using an Agilent 2100 bioanalyzer. Figure 3 As shown.

[0066] Comparative Example 2

[0067] Comparative Example 2 differs from Example 1 in that it uses unmodified PEEK material for DNA synthesis, and the results are as follows: Figure 3 As shown (unmodified PEEK material cannot be used as a vector for DNA synthesis).

[0068] Comparative Example 3

[0069] Comparative Example 3 differs from Example 1 in that it uses polyphenylene sulfide (PPS) material for DNA synthesis, and the results are as follows: Figure 3 As shown (polyphenylene sulfide material, cannot be used as a carrier for DNA synthesis).

[0070] Depend on Figure 2 It is known that neither unmodified PEEK materials nor polyphenylene sulfide materials can be used as carriers for pseudoDNA synthesis.

[0071] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of polyether ether ketone as a carrier for DNA synthesis, characterized in that, The polyether ether ketone is a chemically modified polyether ether ketone, having a structural formula as shown in the following formula I: Formula I In the formula, n is an integer between 200 and 500, wherein the structure of Linker is as follows: R is an alkyl group, a phenyl group or a substituted phenyl group.

2. Use of polyether ether ketone according to claim 1 as a DNA synthesis support, characterized in that, The preparation method of the chemically modified polyether ether ketone comprises the following steps: S1, hydroxyl modification: reacting with polyether ether ketone at 100-140℃ for 3h using dimethyl sulfoxide as solvent and sodium borohydride as reducing agent to obtain hydroxyl modified polyether ether ketone; S2, isocyanate group modification: reacting with 1,6-hexane diisocyanate under nitrogen atmosphere for 3 days at room temperature using toluene as solvent and triethylenediamine as base to obtain isocyanate group modified polyether ether ketone; S3, amino modification: reacting with sodium hydroxide in 1,4-dioxane at room temperature to obtain amino modified polyether ether ketone; S4, Linker grafting: reacting with Linker in acetonitrile solution containing 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine at room temperature overnight to obtain Linker grafted polyether ether ketone.

3. Use of polyether ether ketone according to claim 2 as a DNA synthesis support, characterized in that, The surface area of the polyether ether ketone in step S1 was 200 cm 2 .

4. Use of the polyether ether ketone according to claim 2 as a carrier for DNA synthesis, characterized in that, In step S1, the mass-volume ratio of the polyether ether ketone, sodium borohydride and dimethyl sulfoxide is as follows: polyether ether ketone: sodium borohydride: dimethyl sulfoxide = 0.5g: 0.1g: 50mL.

5. Use according to claim 4, characterized in that, In step S2, the mass-volume ratio of the hydroxyl modified polyether ether ketone, triethylenediamine, 1,6-hexane diisocyanate and toluene is as follows: hydroxyl modified polyether ether ketone: triethylenediamine: 1,6-hexane diisocyanate: toluene = 0.5g: 2.15mg: 1mL: 19mL.

6. Use according to claim 4, characterized in that, In step S3, the mass ratio of the isocyanate group modified polyether ether ketone, sodium hydroxide and 1,4-dioxane is as follows: isocyanate group modified polyether ether ketone: sodium hydroxide: 1,4-dioxane = 0.5: 1:

1.

7. A method of synthesizing DNA, characterized by, The method comprises the following steps: placing the chemically modified polyether ether ketone in a luer connector of a DNA synthesizer, setting a TCA method phosphoramidite synthesis program, and performing automatic DNA synthesis.

8. The method of synthesis of DNA according to claim 7, characterized in that, The method comprises the following steps: placing the chemically modified polyether ether ketone in a luer connector of a DNA synthesizer, setting a TCA method phosphoramidite synthesis program, and performing automatic DNA synthesis.

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