Process for the synthesis of phosphorodiamidate morpholino oligonucleotides
By optimizing the synthesis method of phosphorylated diamine morpholino oligonucleotides, using 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin support, EtOH deprotection reagent and acetic anhydride capping reagent, the problems of low synthesis efficiency and insufficient purity of PMO in the prior art were solved, and high-yield and high-purity PMO synthesis was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINESE PEPTIDE CO
- Filing Date
- 2026-05-09
- Publication Date
- 2026-06-05
AI Technical Summary
Existing methods for synthesizing phosphoryl diamine morpholino oligonucleotides (PMOs) suffer from problems such as low condensation yield, long reaction time, instability of 5'-chloro-aminophosphate monomers, and difficulty in coupling, which limit their application.
The synthesis process of phosphorylated diamine morpholino oligonucleotide was optimized by using 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin as a support, improving the removal efficiency of the protecting group by using the deprotecting agent EtOH, reducing the impurity level by using acetic anhydride as a capping agent, increasing the concentration of phosphoramidite monomer in the coupling reaction, and combining appropriate neutralizing agents and lysis buffer treatment.
This improved coupling efficiency, reduced impurity levels, and significantly increased the yield and purity of phosphorylated diamine morpholino oligonucleotides, thus achieving a highly efficient synthesis method.
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Figure CN122145519A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of solid-phase synthesis technology of phosphoryldiamine morpholine oligonucleotides, specifically relating to a method for synthesizing phosphoryldiamine morpholine oligonucleotides. Background Technology
[0002] Due to their high affinity for mRNA, resistance to various nucleases, in vivo stability, and low toxicity, phosphoryldiamine morpholino oligonucleotides (PMOs) have shown promise as antisense oligonucleotide therapeutic agents. Unfortunately, these promising applications of PMOs are severely limited by the lack of efficient synthetic methodologies. In contrast to standard methods for the chemical preparation of DNA and RNA on automated synthesizers, PMOs are currently synthesized on polystyrene resins in a 5' to 3' orientation. As a first step, 5'-hydroxy-N-triphenylmethyl-morpholinonucleotide is condensed with N,N-dimethylaminodichloroaminophosphate to generate the N,N-dimethylaminochloroaminophosphate synthon. Coupling in the presence of a base produces a dimer attached to the resin. Further detriphenylmethylation with acid generates a dimer that can be prolonged by repeating the cycle. This method has several recognized problems. For example, the condensation yield is low (the recovery yield of the dithyroxine morpholino group is 45%) and the reaction time is long. Furthermore, 5'-chloro-aminophosphate monomers are unstable, and coupling two morpholino monomers is difficult in the presence of a large amount of base. Summary of the Invention
[0003] The purpose of this invention is to provide a method for synthesizing phosphorylated diamine morpholino oligonucleotides with high coupling efficiency, low impurity level, high yield, and high purity.
[0004] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for synthesizing phosphoryldiamine morpholino oligonucleotides includes: adding 9-fluorenylmethoxycarbonyl-piperazine-succinic acid to a resin and reacting to synthesize 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-resin; then, after deprotection treatment, adding phosphorus amide monomers for coupling reaction; after coupling, performing capping treatment; and then repeating the deprotection treatment, coupling reaction, and capping treatment until all phosphorus amide monomers are coupled; then removing the protecting group of the last phosphorus amide monomer; and then performing cleavage treatment and debasing treatment to obtain phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution; the amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid used is 1.0-2.0 times the amount of resin. The structural formula of phosphorylated diamine morpholino oligonucleotide is shown below: In this invention, EtOH is added to the deprotecting reagent. EtOH acts as a trapping agent, which can more effectively remove protecting groups, especially Tlt protecting groups. Increasing the concentration of phosphorous amide monomer in the coupling reaction can improve the coupling efficiency. In the capping reagent, acetic anhydride is used instead of benzoic anhydride, which can reduce the level of impurities. The method of this invention significantly improves the purity and yield of products prepared from sequences rich in guanosine.
[0005] Preferably, a capping agent is used for capping treatment in the preparation of 9-fluorenylmethyloxycarbonyl-piperazine-succinic acid-robin resin.
[0006] Preferably, the deprotection treatment uses a deprotection reagent, which includes 4-cyanopyridine, TFA, EtOH, TFE, and DCM. The volume ratio of TFA, EtOH, TFE, and DCM in the deprotection reagent is 0.5-1:0.5-2:10-30:70-90, and the mass-volume ratio of the mixture of 4-cyanopyridine and TFA, EtOH, TFE, and DCM is 0.5-2 g:50-200 mL.
[0007] Preferably, in the deprotection process, a neutralizing agent and DCM are used for washing. The neutralizing agent includes IPA, DIEA and DCM, and the volume ratio of IPA, DIEA and DCM in the neutralizing agent is 20-30:1-10:60-80.
[0008] Preferably, in the coupling reaction, the phosphoramidite monomers include morphoyl G monomer, morphoyl A monomer, morphoyl C monomer, and morphoyl T monomer.
[0009] More preferably, in the coupling reaction, the structure of the morpholine G monomer is as follows: .
[0010] More preferably, in the coupling reaction, the structure of the morpholine A monomer is as follows: .
[0011] More preferably, in the coupling reaction, the structure of the morpholine C monomer is as follows: .
[0012] More preferably, in the coupling reaction, the structure of the morpholine T monomer is as follows: .
[0013] Preferably, in the coupling reaction, the phosphorus amide monomer is prepared as a phosphorus amide monomer reagent, which includes phosphorus amide monomer and reaction aids, including DMI and NEM. The concentration of phosphorus amide monomer in the phosphorus amide monomer reagent is 0.15-0.25M, and the concentration of NEM is 0.3-0.9M.
[0014] Preferably, the pyrolysis process uses a pyrolysis solution comprising DTT, NMP, and DBU, wherein the volume ratio of DTT, NMP, and DBU in the pyrolysis solution is 10-20:60-80:20-40.
[0015] Preferably, the de-alkali protecting group treatment uses frozen ammonia water, which is pretreated at -30°C to -20°C, and then treated at 45-65°C for 8-24 hours to remove the alkali protecting group.
[0016] This invention discloses the phosphoryldiamine morpholino oligonucleotide prepared by the above method.
[0017] Preferably, in the synthesis of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin, 9-fluorenylmethoxycarbonyl-piperazine-succinic acid is added to the robin resin, followed by the addition of DCM, pyridine, and DCB. The reaction is carried out at 20-40°C for 2-8 hours. After the reaction is completed, a capping reagent is added and the reaction is carried out for 15-30 minutes. The mixture is then washed with DCM to obtain 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin.
[0018] More preferably, in the synthesis of 9-fluorenylmethyloxycarbonyl-piperazine-succinic acid-robin resin, the amount of 9-fluorenylmethyloxycarbonyl-piperazine-succinic acid used is 1.0-2.0 times the amount of robin resin.
[0019] More preferably, in the synthesis of 9-fluorenylmethyloxycarbonyl-piperazine-succinic acid-robin resin, the volume-to-mass ratio of DCM torobin resin is 10-20 mL: 0.5-2 g.
[0020] More preferably, in the synthesis of 9-fluorenyloxycarbonyl-piperazine-succinic acid-robin resin, the molar amount of Py used is 80-800% of that of 9-fluorenyloxycarbonyl-piperazine-succinic acid.
[0021] More preferably, in the synthesis of 9-fluorenyloxycarbonyl-piperazine-succinic acid-robin resin, the molar amount of DCB used is 40-400% of that of 9-fluorenyloxycarbonyl-piperazine-succinic acid.
[0022] More preferably, in the synthesis of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin, the capping reagent includes acetic anhydride, NMM and DMF, the volume ratio of acetic anhydride, NMM and DMF in the capping reagent is 5-20:1-10:70-90, and the volume of the capping reagent used is 200-500% of the volume of therobin resin.
[0023] More preferably, in the synthesis of 9-fluorenylmethyloxycarbonyl-piperazine-succinic acid-robin resin, an appropriate amount of DCM is used in the washing process.
[0024] Preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-roximate resin is swollen, then deprotected with a deprotecting agent, followed by coupling with phosphoramide monomers. After coupling, a capping agent is added for capping. The deprotecting, coupling, and capping processes are repeated until all phosphoramide monomers are coupled. Then, a deprotecting agent is added to remove the protecting group of the last phosphoramide monomer, followed by lysis with a lysis buffer, and finally, a refrigerated ammonia solution is added to remove the base protecting group, thus obtaining the phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution.
[0025] More preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, during the swelling treatment, the resin is first swollen by stirring with NMP for 2-4 hours, and then the swollen resin is washed sequentially with DCM and washing reagent. The volume of NMP used is 300-500% of the volume of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-roximate resin. The washing reagent includes TFE and DCM, and the volume ratio of TFE to DCM in the washing reagent is 20-40:60-80. Appropriate amounts of DCM and washing reagent are used during washing.
[0026] More preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, the deprotection treatment includes 4-cyanopyridine, TFA, EtOH, TFE, and DCM. The volume ratio of TFA, EtOH, TFE, and DCM in the deprotection reagent is 0.5-1:0.5-2:10-30:70-90. The mass-volume ratio of the mixture of 4-cyanopyridine with TFA, EtOH, TFE, and DCM is 0.5-2 g:50-200 mL. The volume of the deprotection reagent used is 300-500% of the volume of 9-fluorenemethoxycarbonyl-piperazine-succinic acid-roximate resin. After removing the deprotection reagent, the mixture is washed sequentially with a neutralizing agent and DCM. The neutralizing agent includes IPA, DIEA, and DCM. The volume ratio of IPA, DIEA, and DCM in the neutralizing agent is 20-30:1-10:60-80. Appropriate amounts of both the neutralizing agent and DCM are used during the washing process.
[0027] More preferably, in the synthesis of phosphoridamide dimorpholine oligonucleotide (PMO) reaction solution, in the coupling of phosphoridamide monomer, the amount of phosphoridamide monomer used is 2.0-5.0 times the initial amount of resin used. The phosphoridamide monomer is prepared as a phosphoridamide monomer reagent, which includes phosphoridamide monomer and reaction aids, including DMI and NEM. The concentration of phosphoridamide monomer in the phosphoridamide monomer reagent is 0.15-0.25M, and the concentration of NEM is 0.3-0.9M. In the coupling of phosphoridamide monomer, after the phosphoridamide monomer is coupled, it is washed sequentially with a neutralizing agent and DCM. The neutralizing agent and DCM are used in appropriate amounts during washing.
[0028] More preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, the capping treatment includes acetic anhydride, NMM and DMF, and the volume ratio of acetic anhydride, NMM and DMF in the capping reagent is 5-20:1-10:70-90. The volume of the capping reagent used is 200-500% of the initial volume of the resin used. After removing the capping reagent, the solution is washed with DMF and DCM in sequence, and the amount of DMF and DCM used in the washing is appropriate.
[0029] More preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, the lysis treatment includes DTT, NMP and DBU, and the volume ratio of DTT, NMP and DBU in the lysis solution is 10-20:60-80:20-40. The lysis solution is collected after the lysis treatment.
[0030] More preferably, in the synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution, in the debasing and protecting group treatment, frozen ammonia water is pretreated at -30°C to -20°C, and then treated at 45-65°C for 8-24 hours to remove the base protecting group. Then, deionized water is added to the ammonolysis solution and the pH is adjusted to neutral to obtain phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution.
[0031] This invention employs a solid-phase synthesis method to synthesize phosphorylated diamine morpholine oligonucleotides. By adding EtOH as a scavenging agent to the deprotecting reagent, the removing of protecting groups, especially Tlt protecting groups, is more effective. Increasing the concentration of phosphoramidite monomer improves coupling efficiency. Furthermore, using acetic anhydride instead of benzoic anhydride in the capping reagent reduces impurity levels, thereby increasing the yield and purity of phosphorylated diamine morpholine oligonucleotides. Therefore, this invention offers the following advantages: high coupling efficiency, low impurity levels, high yield, and high purity of phosphorylated diamine morpholine oligonucleotides. Thus, this invention provides a method for synthesizing phosphorylated diamine morpholine oligonucleotides with high coupling efficiency, low impurity levels, high yield, and high purity. Attached Figure Description
[0032] Figure 1 This is a flowchart of the liquid-solid phase synthesis process for PMO.
[0033] Figure 2 This is a yield chart.
[0034] Figure 3 This is a purity graph. Detailed Implementation
[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0036] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0037] The reagents used in this invention and their abbreviations are shown in Table 1.
[0038] Table 1. Reagents and their abbreviations
[0039] Example 1: Synthesis of phosphorylated diamine morpholino oligonucleotide Synthesis of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin: 9-fluorenylmethoxycarbonyl-piperazine-succinic acid was added to robin resin, followed by the addition of DCM, Py, and DCB. The reaction was carried out at 30 °C for 5 h. After the reaction was complete, a capping reagent was added and the reaction was carried out for 20 min. The mixture was washed with DCM to obtain 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin. The amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid used was 2.0 times the amount of robin resin, and the volume-to-mass ratio of DCM to robin resin was 10 mL: 1 g. The molar amount of Py used was 300% of that of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid, and the molar amount of DCB used was 200% of that of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid. The capping reagent includes acetic anhydride, NMM, and DMF. The volume ratio of acetic anhydride, NMM, and DMF in the capping reagent is 10:5:85. The volume of the capping reagent used is 300% of the volume of the resin. It is used in appropriate amounts during DCM washing.
[0040] Synthesis of phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution: 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-roximate resin was swollen, then deprotected with a deprotecting agent, followed by coupling with phosphorus amide monomers. After coupling, a capping agent was added for capping. The deprotection, coupling, and capping processes were repeated until all phosphorus amide monomers were coupled. Then, a deprotecting agent was added to remove the protecting group of the last phosphorus amide monomer. The mixture was then lysed with lysis buffer, followed by debasing with chilled ammonia to obtain the phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution.In the swelling treatment, the resin was first swollen by stirring with NMP for 3 hours. Then, the swollen resin was washed sequentially with DCM and a washing reagent. The volume of NMP used was 400% of the volume of the 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin. The washing reagent included TFE and DCM, with a volume ratio of TFE to DCM of 30:70. Appropriate amounts of DCM and washing reagent were used during washing. In the deprotection treatment, the deprotection reagents included 4-cyanopyridine, TFA, EtOH, TFE, and DCM, with a volume ratio of TFA, EtOH, TFE, and DCM of 0.5:0.9:20:7. The mass-to-volume ratio of the mixture of 9,4-cyanopyridine with TFA, EtOH, TFE, and DCM was 1 g: 100 mL. The volume of the deprotecting agent used was 400% of the volume of the 9-fluorenemethoxycarbonyl-piperazine-succinic acid-robin resin. After removing the deprotecting agent, the mixture was washed sequentially with a neutralizing agent and DCM. The neutralizing agent included IPA, DIEA, and DCM, with a volume ratio of IPA, DIEA, and DCM of 25:5:70. Appropriate amounts of both the neutralizing agent and DCM were used during washing. In the coupling of the phosphorus amide monomer, the molar amount of phosphorus amide monomer used was equal to the initial amount used at the reaction sites on the rod resin. 200% of the phosphorus amide monomer is used to prepare a phosphorus amide monomer reagent, which includes phosphorus amide monomer and reaction aids, including DMI and NEM. The concentration of phosphorus amide monomer in the phosphorus amide monomer reagent is 0.2M, and the concentration of NEM is 0.6M. In the coupling of phosphorus amide monomer, after coupling, it is washed sequentially with a neutralizing agent and DCM. The neutralizing agent and DCM are used in appropriate amounts during washing. In the capping treatment, the capping reagent includes acetic anhydride, NMM, and DMF. The volume ratio of acetic anhydride, NMM, and DMF in the capping reagent is 10:5:85. The volume of the reagent used was 300% of the initial volume of the resin used. After removing the capping reagent, the solution was washed sequentially with DMF and DCM, with appropriate amounts of DMF and DCM used during washing. In the lysis treatment, the lysis buffer included DTT, NMP, and DBU, with a volume ratio of 15.4:70:30. The lysis buffer was collected after the lysis treatment. In the debasing treatment, frozen ammonia water was pretreated at -20°C, then treated at 55°C for 12 hours to remove the base protecting groups. The ammonia solution was then added to deionized water, and the pH was adjusted to neutral to obtain the phosphoryldiamine morpholine oligonucleotide (PMO) reaction solution. The solid-phase synthesis process of the phosphoryldiamine morpholine oligonucleotide (PMO) reaction solution in this embodiment is as follows. Figure 1 As shown, the structure of the phosphorylated diamine morpholino oligonucleotide coupled in this embodiment is as follows: .
[0041] Example 2: Synthesis method of phosphorylated diamine morpholino oligonucleotide The difference between this embodiment and Example 1 lies in the synthesis of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin. In the synthesis of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin, the amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid used was adjusted from 2.0 times the initial amount of robin resin to 1.5 times. The remaining steps and conditions were the same as in Example 1.
[0042] Example 3: Synthesis method of phosphorylated diamine morpholino oligonucleotide The difference between this embodiment and Example 1 is that the deprotection treatment was performed during the synthesis of the phosphoryldiamine morpholino oligonucleotide reaction solution.
[0043] In the deprotection treatment, the deprotection reagents included 4-cyanopyridine, TFA, EtOH, TFE, and DCM. The volume ratio of TFA, EtOH, TFE, and DCM in the deprotection reagent was 1:0.9:20:79. The mass-to-volume ratio of the mixture of 4-cyanopyridine and TFA, EtOH, TFE, and DCM was 1 g:100 mL. The volume of the deprotection reagent used was 400% of the volume of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-roximate resin. The remaining steps and conditions were the same as in Example 1.
[0044] Example 4: Synthesis of phosphorylated diamine morpholino oligonucleotide The difference between this embodiment and Example 3 is that the deprotection treatment was performed during the synthesis of the phosphoryldiamine morpholine oligonucleotide reaction solution.
[0045] In the synthesis of the phosphoryldiamine morpholino oligonucleotide reaction solution, the composition of the deprotecting reagent was further modified. The mass-to-volume ratio of 4-cyanopyridine to the mixture of TFA, EtOH, TFE, and DCM was adjusted from 1 g:100 mL to 1 g:75 mL, while the volume ratio of TFA, EtOH, TFE, and DCM remained unchanged at 1:0.9:20:79. The remaining steps and conditions were the same as in Example 3.
[0046] Comparative Example 1: Synthesis of phosphorylated diamine morpholino oligonucleotide The difference between this comparative example and Example 1 lies in the synthesis of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin.
[0047] In the synthesis of 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin, DCB was not used as a condensing agent; instead, an equimolar amount of DCC (dicyclohexylcarbodiimide) was used. All other steps and conditions were the same as in Example 1.
[0048] Comparative Example 2: Synthesis of phosphorylated diamine morpholino oligonucleotide The difference between this comparative example and Example 1 lies in the removal of protection treatment.
[0049] In the synthesis of the phosphoryldiamine morpholino oligonucleotide reaction solution, the deprotecting reagents included 4-cyanopyridine, TFA, EtOH, TFE, and DCM. The volume ratio of TFA, EtOH, TFE, and DCM in the deprotecting reagents was 0.5:0.9:20:79. The mass-to-volume ratio of the mixture of 4-cyanopyridine and TFA, EtOH, TFE, and DCM was 0.5 g:100 mL. The remaining components and amounts were the same as in Example 1. The remaining steps and conditions were also the same as in Example 1.
[0050] Comparative Example 3: Synthesis of phosphorylated diamine morpholino oligonucleotide The difference between this comparative example and Example 1 lies in the coupling step of the phosphoramidite monomer.
[0051] In the synthesis of the phosphoryldiamine morpholino oligonucleotide reaction solution, in the coupling step of the phosphoramide monomer, NEM was not used as the reaction aid; instead, an equal concentration of NMI (N-methylimidazole) was used. The remaining steps and conditions were the same as in Example 1.
[0052] Experimental example: In this invention, the phosphoryldiamine morpholino oligonucleotide (PMO) reaction solutions prepared in Examples 1-4 and Comparative Examples 1-3 were purified, and their yields and purity were calculated and determined. The results are as follows: Figure 2 (yield) and Figure 3 (Purity) is shown in the figure, where S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, D1 is Comparative Example 1, D2 is Comparative Example 2, and D3 is Comparative Example 3. From Figure 2 and Figure 3 The results show that the PMO prepared in Examples 1-4 of this invention all have high yield and purity. Among them, Example 4 has the best effect, with the highest yield and purity. Example 3 is second, and Example 1 ranks third. The yield and purity of Example 2 are relatively low but still maintain a good level. The yield and purity of Comparative Examples 1-3 are significantly lower than those of the examples.
[0053] In the synthesis of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-robin resin, the molar amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid used has a significant impact on the linker's connection efficiency. In Example 2, after adjusting the amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid from 2.0 times to 1.5 times the amount of robin resin, the yield decreased compared to Example 1, indicating that reducing the amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid affects the connection efficiency.
[0054] In deprotection treatment, the composition of the deprotection reagent is crucial to the removal efficiency of the Trt protecting group and the stability of the base protecting group. This invention reveals that TFA and 4-cyanopyridine need to work synergistically within a specific ratio range to achieve the best deprotection effect. In Example 3, the volume ratio of TFA, EtOH, TFE, and DCM was adjusted to 1:0.9:20:79, while the concentration of 4-cyanopyridine was maintained at 1 g:100 mL. The yield and purity were superior to those of Example 1, indicating that appropriately increasing the TFA ratio helps improve deprotection efficiency. In Example 4, based on Example 3, the concentration of 4-cyanopyridine was further adjusted to 1 g:75 mL, achieving the highest yield and purity. This shows that appropriately increasing the 4-cyanopyridine concentration while increasing the TFA ratio can ensure efficient removal of the Trt protecting group. Therefore, TFA and 4-cyanopyridine in the deprotection reagent need to be within a specific ratio range to obtain the optimal effect.
[0055] In the coupling step of phosphorous amide monomers, the choice of reaction promoter has a decisive impact on the coupling efficiency. Example 1 used NEM as the reaction promoter, achieving high yield and purity; however, in Comparative Example 3, replacing NEM with NMI significantly reduced both yield and purity. NEM, as a mild non-nucleophilic base, primarily functions through basic catalysis and does not trigger side reactions; while NMI, as a strong nucleophilic catalyst, although accelerating coupling, may induce side reactions in the specific system of PMO synthesis, leading to decreased coupling efficiency and byproduct accumulation. Therefore, NEM is more suitable as a reaction promoter for the PMO synthesis process of this invention.
[0056] In the linker connection step, the choice of condensing agent is equally crucial. Example 1 used DCB as the condensing agent, achieving high yield and purity; however, in Comparative Example 1, replacing DCB with DCC significantly reduced both yield and purity. DCC readily generates insoluble byproducts in solid-phase synthesis, affecting the efficiency of subsequent reactions, thus making it unsuitable for the solid-phase synthesis of PMO. DCB, as an optimized condensing agent, has easily removable byproducts, making it more suitable for the synthesis system of this invention.
[0057] In the deprotection process, the addition of 4-cyanopyridine is crucial to the deprotection efficiency. In Comparative Example 2, the amount of 4-cyanopyridine added was halved, resulting in the lowest yield and purity. This is because 4-cyanopyridine, as a nucleophilic catalyst, accelerates the deprotection reaction by attacking the carbonyl or phosphate ester bonds activated by TFA. Insufficient 4-cyanopyridine will severely affect the product quality. Therefore, a strict quantitative ratio of 4-cyanopyridine to TFA is required to maintain the optimal nucleophilic catalytic environment and ensure efficient deprotection.
[0058] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0059] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method for synthesizing phosphorylated diamine morpholino oligonucleotides, characterized in that, include: 9-fluorenylmethoxycarbonyl-piperazine-succinic acid was added to a king resin and reacted to synthesize 9-fluorenylmethoxycarbonyl-piperazine-succinic acid-king resin. Then, after deprotection treatment, phosphoramide monomers were added for coupling reaction. After coupling, capping treatment was performed. Then, deprotection treatment, coupling reaction and capping treatment were repeated until all phosphoramide monomers were coupled. Then, the protecting group of the last phosphoramide monomer was removed. Then, cleavage treatment and debase protecting group treatment were performed to obtain phosphoryldiamine morpholino oligonucleotide (PMO) reaction solution. The amount of 9-fluorenylmethoxycarbonyl-piperazine-succinic acid used is 1.0-2.0 times the amount of Wang resin; The structural formula of phosphorylated diamine morpholino oligonucleotide is shown below: 。 2. The synthesis method according to claim 1, characterized in that, In the preparation of the 9-fluorenemethyloxycarbonyl-piperazine-succinic acid-robin resin, a capping agent is used for capping treatment.
3. The synthesis method according to claim 1, characterized in that, The deprotection treatment uses deprotection reagents, including 4-cyanopyridine, TFA, EtOH, TFE, and DCM. The volume ratio of TFA, EtOH, TFE, and DCM in the deprotection reagent is 0.5-1:0.5-2:10-30:70-90. The mass-volume ratio of the mixture of 4-cyanopyridine and TFA, EtOH, TFE, and DCM is 0.5-2 g:50-200 mL.
4. The synthesis method according to claim 1, characterized in that, In the deprotection process, a neutralizing agent and DCM are used for washing. The neutralizing agent includes IPA, DIEA and DCM, and the volume ratio of IPA, DIEA and DCM in the neutralizing agent is 20-30:1-10:60-80.
5. The synthesis method according to claim 1, characterized in that, In the coupling reaction, the phosphoramidite monomers include morphoyl G monomer, morphoyl A monomer, morphoyl C monomer, and morphoyl T monomer.
6. The synthesis method according to claim 1, characterized in that, In the coupling reaction, the phosphorus amide monomer is prepared into a phosphorus amide monomer reagent, which includes phosphorus amide monomer and reaction aids, including DMI and NEM. The concentration of phosphorus amide monomer in the phosphorus amide monomer reagent is 0.15-0.25M, and the concentration of NEM is 0.3-0.9M.
7. The synthesis method according to claim 1, characterized in that, The pyrolysis process uses a pyrolysis solution comprising DTT, NMP, and DBU, with a volume ratio of 10-20:60-80:20-40.
8. The synthesis method according to claim 1, characterized in that, The de-alkali protection treatment uses frozen ammonia water, which is pretreated at -30°C to -20°C, and then treated at 45-65°C for 8-24 hours to remove the alkali protection groups.
9. The phosphoryldiamine morpholino oligonucleotide prepared by any of the synthetic methods described in claims 1-8.