Method for synthesizing cyclic peptide nucleic acid by one-pot method and cyclic peptide nucleic acid

Abstract

The invention discloses a method for synthesizing cyclic peptide nucleic acid by a one-pot method, which comprises the following steps: introducing aryl diamine into a linker to fix on resin based on a solid-phase flow peptide nucleic acid synthesis mode, then coupling with a peptide nucleic acid monomer to form a peptide nucleic acid chain, and then forming an aryl triazole structure under the activation of isoamyl nitrite to obtain the cyclic peptide nucleic acid. And then carrying out intramolecular cyclization in an alkaline environment, and removing a base protecting group from a product obtained by cyclization to obtain the cyclic peptide nucleic acid. The invention also discloses a cyclic peptide nucleic acid with an excellent transmembrane effect, the base sequence of the cyclic peptide nucleic acid is Cyclo-TCCACGCACAAA, the bases A, T, C and G are connected with a cyclic skeleton through methylene carbonyl, and the cyclic skeleton is formed by connecting N-(2-aminoethyl) glycine units through amido bonds.

Description

Technical Field

[0001] The invention relates to the field of cyclic peptide nucleic acids, and in particular to a one-pot method for synthesizing cyclic peptide nucleic acids and the cyclic peptide nucleic acids. Background Art

[0002] Circular nucleic acids (CNAs) are ubiquitous in nature. They are closed-loop conformations formed by intramolecular covalent bonds at both ends of linear nucleic acids. Compared with linear nucleic acids, CNAs exhibit stronger biological and thermodynamic stability, unique nucleic acid and protein recognition capabilities, and can serve as effective templates for DNA and RNA polymerases. These advantages have prompted researchers to develop synthetic cyclic nucleic acid analogs, including phosphorothioate analogs, triazole cycloalkane analogs, 2'-F analogs, and cyclic peptide nucleic acids (PNAs).

[0003] PNAs are a type of DNA / RNA analogues in which the polypeptide backbone replaces the sugar-phosphate backbone and is electrically neutral, making PNAs more stable and having stronger binding affinity to DNA / RNA. They are widely used in therapeutic agents, anticancer drugs, molecular tools, biosensors, and nanotechnology.

[0004] For example, Zhao et al. reported a sequence-specific linear PNA (TCCACGCACAAA, C' to N') 1 , combined with the light scattering (LS) technology of carbon nanotubes, it can be used as a nanosensor to quickly detect single nucleotide polymorphisms (SNPs) (Xu, W.; Xing, S.; Xu, X.; Xu, M.; Fu, P.; Gao, T.; Zhao, C., Peptide Nucleic Acid-Assisted Label-free Detection of Single-Nucleotide Polymorphisms Based on LightScattering of Carbon Nanotubes. ACS Omega 2018, 3(12), 17835-17841).

[0005] Recently, researchers have theoretically demonstrated that cyclic PNAs are significantly better than other small molecule agonists in binding to STING protein, indicating that cyclic PNAs may become a research hotspot in the field of small molecule agonists in the future.

[0006] Circular PNAs with longer sequences have potential application value in antiviral, metal ion detection and self-assembly.

[0007] Although cyclic PNAs have shown great potential in many fields, the inefficient synthesis methods have greatly restricted their development.

[0008] At present, there are four main methods for preparing cyclic PNAs, namely, the fully protected skeleton strategy, the "hybrid" strategy, the natural chemical synthesis method, and the solid phase synthesis method. Among them: the first two methods involve complex orthogonal protection and deprotection steps, which are cumbersome and inefficient; the synthesis length of the natural chemical synthesis method is limited to less than 6 monomers, and the highest cyclization rate is only 66%; the solid phase synthesis strategy requires pre-derivatization of the solid phase carrier, the synthesis length range is 3-6 residues, and the cyclization rate is only 11% to 14%.

[0009] In summary, the existing synthesis strategies all have the disadvantages of short synthesis chain, low efficiency and many steps. Summary of the invention

[0010] In view of the above technical problems and the shortcomings in the art, the present invention provides a one-pot method for synthesizing cyclic peptide nucleic acids, which is a method for preparing cyclic PNAs by using aromatic diamine immobilized linkers, and can synthesize cyclic PNAs and their derivatives of 2-12 or even more monomers. It is a synthesis method with easy-to-obtain raw materials, simple steps, and no intermediate purification steps. The method does not require the use of dangerous reagents such as strong acids and strong bases, and the conditions are mild, which is easy to carry out industrial production. At the same time, the present invention realizes automated high-temperature flow synthesis, effectively shortens the reaction time, and can meet the needs of high-throughput screening.

[0011] A one-pot method for synthesizing cyclic peptide nucleic acid, based on the solid phase flow synthesis of peptide nucleic acid, an aromatic diamine is introduced into a linker and fixed on a resin, and then a peptide nucleic acid monomer is coupled to form a peptide nucleic acid chain, and then an aromatic triazole structure is formed under the activation of isopentyl nitrite, and then intramolecular cyclization occurs in an alkaline environment, and the base protecting group of the cyclized product is removed to obtain a cyclic peptide nucleic acid;

[0012] The aromatic diamine introduced linker has a structure shown in the following formula (I):

[0013]

[0014] The aryl triazole structure is shown below:

[0015]

[0016] Wherein, L represents a peptide nucleic acid chain.

[0017] In the present invention, the monomers constituting the peptide nucleic acid chain may include any one or more of monomer A, monomer T, monomer C, and monomer G having the following structure:

[0018]

[0019]

[0020] Furthermore, the base sequence of the cyclic peptide nucleic acid may be Cyclo-TCCACGCACAAA (SEQ ID NO: 1).

[0021] The present invention is also applicable to the automated high temperature flow synthesis of circular PNA. In the process of connecting to form peptide nucleic acid chains, the reaction temperature can be 40-70°C.

[0022] In some embodiments, in the method of the present invention, during the process of connecting to form a peptide nucleic acid chain, the delivery and coupling temperature of the monomers constituting the peptide nucleic acid chain can be 70°C.

[0023] In some embodiments, in the method described in the present invention, during the process of connecting to form a peptide nucleic acid chain, the delivery and coupling time of the monomers constituting the peptide nucleic acid chain can be 55 to 120 s, wherein, further, the delivery time can be 1 to 2 s, and the coupling time can be 58 to 118 s.

[0024] In the method of the present invention, the condensing agent used in the process of connecting to form a peptide nucleic acid chain can be PyAOP or HBTU.

[0025] The method of the present invention can be used to treat the cyclization product with a mixed solution of trifluoroacetic acid (TFA), water and triisopropylsilane (TIPS) to remove the base protecting group. Further, the volume ratio of trifluoroacetic acid, water and triisopropylsilane in the mixed solution of trifluoroacetic acid, water and triisopropylsilane can be 94:3:3.

[0026] As a general inventive concept, the present invention also provides a cyclic peptide nucleic acid, the base sequence of which is Cyclo-TCCACGCACAAA, wherein the bases A, T, C, and G are connected to a cyclic backbone via a methylene carbonyl group, and the cyclic backbone is formed by connecting N-(2-aminoethyl)glycine units via amide bonds.

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

[0028] The invention provides a general method for synthesizing cyclic PNAs of various compositions and lengths, enriches the sequences of cyclic PNAs, and lays a foundation for developing cyclic peptide nucleic acids with biological activity.

[0029] The cyclic peptide nucleic acid Cyclo-TCCACGCACAAA provided by the present invention can further improve the passive membrane penetration effect while retaining the original nanosensor function of the linear peptide nucleic acid linear-TCCACGCACAAA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the liquid chromatography (LC) chart (260nm) of the crude product of cyclic peptide nucleic acid cyclo-TCCACGCACAAA.

[0031] Figure 2 This is the mass spectrum (MS) of cyclic peptide nucleic acid cyclo-TCCACGCACAAA. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0033] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0034] A method for synthesizing cyclic peptide nucleic acid of the present invention, the synthesis route is shown as follows:

[0035]

[0036] The specific steps include:

[0037] (1) The carboxyl group of compound (a) is condensed with the amino group on the Rink Amide resin to obtain product (b).

[0038] (2) The nitro group of product (b) is reduced with boronic acid to give product (c).

[0039] (3) Product (c) is coupled with PNA monomers (monomer A, monomer T, monomer C, monomer G), the condensing agent is (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAOP) or O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), and the activating agent is N,N-diisopropylethylamine (DIEA); after the reaction, 20% piperidine in N,N-dimethylformamide (DMF) solution is added for deprotection. Repeat the above steps to complete the extension of the PNA chain to obtain product (d).

[0040] (4) Product (d) is activated by isoamyl nitrite to obtain product (e);

[0041] (5) The product (e) was cyclized in a 2% DIEA solution in DMF;

[0042] (6) The protecting group Bhoc on the PNA monomer is removed from the cyclized product to obtain cyclo-PNA (f).

[0043] The following specifically introduces two methods for synthesizing cyclic PNA: Cyclo-TCCACGCACAAA.

[0044] The manual synthesis route is as follows:

[0045] 1) 0.36mmol / g Rink Amide resin (200mg, 0.072mmol) was placed in a 10mL syringe with a sieve plate, and then 5mL DMF and 5mL dichloromethane (DCM) were added to swell for 30min respectively. After being drained, 5-nitro-6-aminonicotinic acid or 3-nitro-4-aminobenzoic acid (0.29mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (109mg, 0.29mmol), and DIEA (75μL, 0.43mmol) in DMF solution were added and stirred for 2h. After the reaction was completed, it was washed three times with DMF and DCM respectively.

[0046] 2) Boric acid (19 mg, 0.22 mmol) and 4,4'-bipyridine (1 mg, 0.007 mmol) were dissolved in 2 mL DMF, added to the resin obtained in 1), and reacted on a shaker for 30 min. After the reaction, the resin was washed three times with DCM and DMF respectively. This step was repeated twice to achieve complete reduction of the nitro group.

[0047] 3) Take the aromatic diamine resin (c) (20 mg) and place it in a 3 mL syringe with a sieve plate, add 1.5 mL DMF and 1.5 mL DCM respectively and swell for 30 minutes.

[0048] 4) The reaction steps and conditions for PNA chain extension are as follows:

[0049] Add PNA monomer A (4.0 eq.), condensing agent PyAOP (3.8 eq.) and activating agent DIEA (8.0 eq.) to the resin and react for 1 hour. After completion, rinse with DMF and DCM three times respectively. Subsequently, add 20% piperidine to remove the protecting group Fmoc twice, each time for 5 minutes. Then add DMF and DCM to rinse three times respectively. Repeat the above steps to continue coupling monomers, thereby obtaining a PNA chain containing NH2-TCCACGCACAAA.

[0050] 5) Cyclization: Add 50% isoamyl nitrite in DMF to the resin and stir for 1.5 hours. After the reaction is completed, drain the solvent and wash with DMF and DCM for 5 times respectively. Then add 2% DIEA in DMF to the resin and react for 2 hours. After the reaction is completed, remove the solvent by rotary evaporation.

[0051] 6) Deprotection: Add 1 mL of TFA:H2O:triisopropylsilane (TIPS) = 94:3:3 (volume ratio) solution to the cyclized product and stir for 50 min. The obtained product was washed with ether three times to precipitate a crude product. Purification was performed by HPLC. The LCMS analysis results of the crude product of Cyclo-TCCACGCACAAA can be found in Figure 1 , Figure 2 , as follows: MS (ESI) exact mass calculation. for C 127 H 157 N 71 O 32 [M+3H] 3+ :1063.76;Found:1064.27.

[0052] The high temperature flow automation synthesis route is as follows:

[0053] 1) Aryl diamine resin (c) (20 mg) was placed in a reactor.

[0054] 2) The reaction conditions are shown in the following table.

[0055]

[0056] Description in the table:

[0057] a) PNA long chain preparation steps:

[0058] Pump 1: 0.05 M PNA monomer solution in DMF;

[0059] Pump 2: 0.0475 M HBTU in DMF (due to the side reactions when PyAOP is used as a condensation agent under high temperature conditions, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) was used instead);

[0060] Pump 3: DIEA, 99.9% purity.

[0061] b) Deprotection step:

[0062] Pump 2: 20% (volume ratio) piperidine and 0.5% (volume ratio) HCOOH in DMF;

[0063] c) Activation step:

[0064] Pump 3: 50% (volume ratio) isoamyl nitrite in DMF solution;

[0065] d) Cyclization step:

[0066] Pump 3: 2% (volume ratio) DIEA in DMF solution;

[0067] The volumes shown in the table refer to the volume of a single pump. If three pumps are used at the same time, the total volume will be three times that in the table.

[0068] In this embodiment, the linear PNA sequence linear-TCCACGCACAAA mentioned above with nanosensor function is cyclized to effectively improve its passive membrane penetration effect.

[0069] The cyclic PNA of this embodiment has good penetration ability in the parallel artificial membrane permeability assay (PAMPA). Specifically, the passive membrane penetration experiment of the cyclic PNA includes the following steps:

[0070] 1) PAMPA permeability test was performed using a 96-well donor plate (MilliporeMAIPNTR10) with a 0.45 μm hydrophobic Immobilon-P membrane support and a 96-well Teflon acceptor plate (Millipore MSSACCEPTOR).

[0071] 2) Prepare 5% dimethyl sulfoxide (DMSO) in PBS (pH = 7.4) and add 300 μL to each well of the receptor plate. app =3.9×10 -6 cm / s) was dissolved in a 5% DMSO PBS solution to a concentration of 10 μM, and ultrasonically mixed. Prepare a 1% (w / v) soybean lecithin solution in n-dodecane (90%), ultrasonicate for 5 minutes, and apply 5 μL to the bottom of each donor plate well membrane. After about 15 minutes, add 150 μL of sample solution to the wells of the donor plate. Set up three parallel tests for each sample. Place the donor plate on top of the receptor plate so that the artificial membrane is in contact with the buffer solution below, and ensure that no bubbles are formed under the membrane. Cover the donor plate with a cover and leave the system at room temperature overnight. Place a wet paper towel next to it to prevent evaporation.

[0072] 3) After about 16 hours, separate the donor plate and the acceptor plate, and transfer 100 μL of the solution in each well of the donor plate and the acceptor plate to a 96-well plate. Determine the sample content in the solution by UPLC. Effective permeability P app The calculation formula is as follows:

[0073]

[0074]

[0075]

[0076] P app (cm / s) = -C*ln(1-%T)[4]

[0077] in:

[0078] I a is the UV absorption intensity of the receptor plate solution;

[0079] I d is the UV absorption intensity of the donor plate solution;

[0080] The effective surface area of ​​the membrane: M sa =240mm 2 ;

[0081] Acceptor plate pore volume: V a =300 μL;

[0082] Donor plate well volume: V d =150μL;

[0083] Experiment duration: T S =16hr.

[0084] The average value of the three parallel groups was obtained to obtain the P value of Cyclo-TCCACGCACAAA. app =1.25×10 -6 cm / s, while its linear precursor linear-TCCACGCACAAA has a P app =1.74×10 -7 cm / s. It can be seen that the membrane permeability of this cyclic PNA is improved by an order of magnitude compared to that of linear PNA. When it is used as a nanosensor or for other intracellular applications, it does not need to be combined with additional membrane-penetrating peptides to assist in membrane permeation.

[0085] In addition, it should be understood that after reading the above description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A one-pot method for synthesizing cyclic peptide nucleic acid, characterized in that: Based on the solid phase flow synthesis of peptide nucleic acid, aromatic diamine is introduced into the linker and fixed on the resin, and then the peptide nucleic acid monomer is coupled to form a peptide nucleic acid chain, and then an aromatic triazole structure is formed under the activation of isopentyl nitrite, and then intramolecular cyclization occurs in an alkaline environment, and the base protecting group of the cyclized product is removed to obtain a cyclic peptide nucleic acid; The aromatic diamine introduced linker has a structure shown in the following formula (I): The aryl triazole structure is shown below: Wherein, L represents a peptide nucleic acid chain.

2. The method according to claim 1, characterized in that The monomers constituting the peptide nucleic acid chain include any one or more of monomer A, monomer T, monomer C, and monomer G having the following structure:

3. The method according to claim 2, characterized in that The base sequence of the cyclic peptide nucleic acid is Cyclo-TCCACGCACAAA.

4. The method according to claim 1, characterized in that: During the process of connecting to form a peptide nucleic acid chain, the reaction temperature is 40 to 70°C.

5. The method according to claim 1, characterized in that During the process of connecting to form the peptide nucleic acid chain, the delivery and coupling temperature of the monomers constituting the peptide nucleic acid chain is 70° C. and the time is 55 to 120 seconds.

6. The method according to claim 1, characterized in that In the process of connecting to form peptide nucleic acid chains, the condensation agent used is PyAOP or HBTU.

7. The method according to claim 1, characterized in that The product obtained by cyclization is treated with a mixed solution of trifluoroacetic acid, water and triisopropylsilane to remove the base protecting group.

8. The method according to claim 7, characterized in that The volume ratio of trifluoroacetic acid, water and triisopropylsilane in the mixed solution of trifluoroacetic acid, water and triisopropylsilane is 94:3:

3.

9. A cyclic peptide nucleic acid, characterized in that The base sequence of the cyclic peptide nucleic acid is Cyclo-TCCACGCACAAA, wherein the bases A, T, C, and G are connected to the cyclic skeleton through a methylene carbonyl group, and the cyclic skeleton is formed by connecting N-(2-aminoethyl)glycine units through amide bonds.

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