Continuous flow preparation method and continuous flow preparation device of morpholino oligonucleotide

Abstract

The invention relates to a continuous flow preparation method and a continuous flow preparation device of morpholino oligonucleotide, and belongs to the technical field of morpholino oligonucleotide preparation. The invention relates to a continuous flow preparation method of morpholino oligonucleotide. A continuous flow preparation device comprises an automatic synthesizer with a synthesis column; the continuous flow preparation method comprises the following steps: (1) loading morpholino phosphoramidite protected by a protecting group on a solid-phase carrier, filling the solid-phase carrier into a synthetic column, pumping 3-7cv of a deprotection agent into the synthetic column at a first flow rate of 150-240cm / h, and pumping 7-10cv of the deprotection agent into the synthetic column at a second flow rate of 120-150cm / h; the difference between the first flow velocity and the second flow velocity is not less than 30cm / h; (2) pumping a morpholino phosphoramidite monomer reagent and an activating reagent into the synthetic column, heating the synthetic column and the post-column of the synthetic column, keeping the temperature at 30-60 DEG C, and circularly carrying out a coupling reaction; (3) pumping a capping reagent for capping reaction to form a basic group; and (4) circulating the steps (1)-(3) for each basic group to generate the morpholino oligonucleotide. The coupling efficiency is effectively improved, and the purity of the product after ammonolysis is improved.

Description

Technical Field

[0001] This application relates to the technical field of the preparation of morpholino oligonucleotides, and particularly relates to a continuous flow preparation method and a continuous flow preparation device for morpholino oligonucleotides. Background Art

[0002] The synthesis of morpholino oligonucleotides is greatly affected by environmental humidity, and high humidity in different regions and seasons will significantly affect product quality and batch - to - batch stability. Especially in the scale - up stage, the consequences are serious, and commercial companies are difficult to solve, resulting in high product prices.

[0003] The synthesis of morpholino oligonucleotides often uses the traditional batch process. The traditional batch process is cumbersome in operation, low in efficiency, long in synthesis cycle. When scaled up, the time consumption doubles, lengthening the entire synthesis cycle; difficult to clean, high in reagent consumption; and it is a solid - phase synthesis, which may have a scale - up effect. For the early R & D stage, even small - scale products require continuous and intensive manual operations, with extremely high R & D labor costs and high failure risks, resulting in extremely expensive small - scale products.

[0004] Currently, there are already automatic synthesis methods to replace the traditional batch process. Automatic synthesis is simple in operation, does not require intensive manual operations, has low costs, and short synthesis cycles. However, the purity of the product after ammonolysis prepared by the existing automatic synthesis methods is not high, the raw material consumption is relatively high, the reaction conditions are harsh, and the synthesis scale is limited, which does not meet the actual R & D needs. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the embodiments of this application is to provide a continuous flow preparation method and a continuous flow preparation device for morpholino oligonucleotides to improve the problems that although the existing automatic synthesis methods have short synthesis cycles, the purity of the product after ammonolysis prepared is not high, the raw material consumption is relatively high, the reaction conditions are harsh, and the synthesis scale is limited.

[0006] In a first aspect, the embodiments of this application provide a continuous flow preparation method for morpholino oligonucleotides. The continuous flow preparation device includes an automatic synthesizer, and the automatic synthesizer has a synthesis column. The continuous flow preparation method includes:

[0007] (1) Deprotection: Load the morpholino phosphoramidite protected by a protecting group onto a solid support, pack it into the synthesis column, and then pump a deprotecting agent into the synthesis column to remove the protecting group on the amino group of the morpholine ring. The deprotecting agent is first pumped at a first flow rate of 150 - 240 cm / h for 3 - 7 cv, and then at a second flow rate of 120 - 150 cm / h for 7 - 10 cv; and the difference between the first flow rate and the second flow rate is not less than 30 cm / h;

[0008] (2) Activation and coupling: The morpholino phosphoramidite monomer reagent and the activation reagent are simultaneously pumped into the synthesis column for mixing. The synthesis column and the post-column of the synthesis column are heated and kept warm at 30 - 60 °C, and the coupling reaction is carried out cyclically;

[0009] (3) Capping: The capping reagent is pumped in for the capping reaction to form a base;

[0010] (4) Each base cycles through steps (1)-(3), and finally a morpholino oligonucleotide is produced.

[0011] In this application, by first pumping in an appropriate amount of deprotecting agent at a high flow rate and then pumping in an appropriate amount of deprotecting agent at a low flow rate, the protecting group on the amino group of the morpholine ring can be efficiently removed, and the coupling reaction is carried out with an appropriate temperature, effectively improving the coupling efficiency, and at the same time controlling the content of impurities, so as to improve the purity of the product after ammonolysis, with less raw material consumption, mild reaction conditions, and a large synthesis scale.

[0012] In some embodiments of this application, the morpholino oligonucleotide produced in step (4) is subjected to ammonolysis treatment, and the ammonolysis treatment includes: S1, first taking out the solid-phase carrier in the synthesis column after the reaction in step (4) and placing it in an organic reagent containing 0.8 - 1.2 M of organic base and 0.3 - 0.8 M of scavenger, reacting at 20 - 30 °C for 2 - 3 h and then filtering; S2, adding ammonia water and reacting at 40 - 65 °C for 12 - 36 h and then filtering, and the ammonolysis-treated morpholino oligonucleotide is obtained in the filtrate.

[0013] In this application, by ammonolysis-treating the produced morpholino oligonucleotide through the above method, the ammonolysis treatment can be efficiently carried out to obtain a morpholino oligonucleotide with a relatively high purity after ammonolysis treatment.

[0014] In some embodiments of this application, the solid-phase carrier includes synthetic resin or controlled-pore glass.

[0015] In this application, by using the above solid-phase carrier, it is beneficial to improve the reaction efficiency and product purity.

[0016] In some embodiments of this application, the loading amount of the morpholino phosphoramidite protected by the protecting group on the solid-phase carrier is 200 - 600 μmol / g.

[0017] In this application, by loading an appropriate amount of morpholino phosphoramidite protected by the protecting group on the solid-phase carrier, it is beneficial to improve the subsequent reaction efficiency and the purity of the product produced by the reaction.

[0018] In some embodiments of the present application, the specific conditions for step (2) are as follows: 2.5 - 7.5 eq of morpholino phosphoramidite monomer reagent and 5 - 16 eq of activating reagent are simultaneously pumped into a synthesis column at a flow rate of 150 - 300 cm / h for mixing. The synthesis column and the post-column of the synthesis column are heated and kept warm at 30 - 60 °C, and circulated for 60 - 120 min to carry out a coupling reaction.

[0019] By simultaneously pumping an appropriate amount of morpholino phosphoramidite monomer reagent and activator into the synthesis column at an appropriate flow rate and carrying out a coupling reaction at an appropriate temperature for an appropriate time, the present application can achieve an efficient coupling reaction using a lower amount of morpholino phosphoramidite monomer reagent, and at the same time, the content of impurities can be controlled.

[0020] In some embodiments of the present application, the specific conditions for step (3) are as follows: a capping reagent of 0.5 - 3 cv is pumped in at a flow rate of 60 - 240 cm / h for a capping reaction.

[0021] By pumping an appropriate capping reagent at an appropriate flow rate, the present application is conducive to an efficient capping reaction.

[0022] In some embodiments of the present application, the deprotecting agent is a dichloromethane solution containing 0.1 - 0.5 mol / L of tetracyanopyridine trifluoroacetate dissolved in 1% ethanol and 20% trifluoroethanol.

[0023] The present application uses the above deprotecting agent to remove the protecting group on the morpholine ring amino group of morpholino phosphoramidite, which is conducive to efficiently removing the protecting group on the morpholine ring amino group.

[0024] In some embodiments of the present application, the monomer reagent is a solvent containing 0.11 - 0.5 mol / L of morpholino phosphoramidite monomer.

[0025] The present application uses the above monomer reagent to facilitate the coupling reaction to form a base.

[0026] In some embodiments of the present application, the activating reagent is a 1,3 - dimethyl - 2 - imidazolidinone solution containing 0.4 - 1.3 mol / L of N - ethylmorpholine and 0.1 - 0.8 mol / L of lithium bromide.

[0027] The present application uses the above activating reagent to facilitate the efficient activation of morpholino phosphoramidite monomer for an efficient coupling reaction.

[0028] In some embodiments of the present application, the capping reagent comprises capping reagent A and capping reagent B with a mass ratio of 1:1 - 2. Capping reagent A is an N,N-dimethylformamide solution containing 0.8 - 1.2 mol / L of benzoic anhydride, and capping reagent B is a solution containing 0.1 - 0.5 mol / L of 4-dimethylaminopyridine. The solvent of capping reagent B includes N-methylimidazole, 2,6-dimethylpyridine, and N,N-dimethylformamide.

[0029] By using the above capping reagent in the present application, especially by using 4-dimethylaminopyridine and 2,6-dimethylpyridine in capping reagent B, the capping efficiency can be effectively improved, thereby improving the overall efficiency of preparing morpholino oligonucleotides and reducing the preparation cycle.

[0030] In some embodiments of the present application, the volume percentage ratio of N-methylimidazole, 2,6-dimethylpyridine, and N,N-dimethylformamide is 10% - 15%:10% - 15%:75% - 80%.

[0031] By using a suitable mixture of appropriate components in a suitable ratio as the solvent of capping reagent B in the present application, an efficient capping reaction can be ensured.

[0032] In some embodiments of the present application, a cleaning step is included after each of steps (1) - (3); the cleaning step includes pumping 2 - 5 cv of a cleaning reagent at a flow rate of 100 - 400 cm / h.

[0033] By pumping an appropriate amount of a cleaning reagent at a suitable flow rate after each step in the present application, the pipeline and the synthesis column can be cleaned more thoroughly, avoiding the influence of the reagent residue in the previous step on the reaction in the subsequent step.

[0034] In some embodiments of the present application, the cleaning reagent is selected from dichloromethane or N,N-dimethylformamide.

[0035] By using the above suitable cleaning reagent in the present application, a more thorough cleaning of the pipeline and the synthesis column can be ensured.

[0036] In a second aspect, the embodiments of the present application provide a continuous flow preparation device for morpholino oligonucleotides, which is applicable to the continuous flow preparation method of morpholino oligonucleotides provided in the first aspect, and includes an automatic synthesizer, a heating device, and a constant temperature device. There is a synthesis column in the automatic synthesizer. An inlet pipe is connected between the inlet of the synthesis column and the automatic synthesizer, and an outlet pipe is connected between the outlet of the synthesis column and the automatic synthesizer; the heating device is coated on the surface of the synthesis column; the constant temperature device is coated on the outer wall of the outlet pipe.

[0037] In this application, by covering and arranging a heating device on the surface of the synthesis column, the synthesis column can be heated to an appropriate temperature to improve the coupling reaction efficiency and control the impurity content. At the same time, by covering and arranging a constant temperature device on the outer wall of the liquid outlet pipe, the reagent circulating during the coupling reaction can always be at an appropriate temperature, thereby effectively improving the coupling reaction efficiency and controlling the impurity content.

[0038] In some embodiments of this application, both the liquid inlet pipe and the liquid outlet pipe are metal pipelines.

[0039] In this application, since the liquid inlet pipe and the liquid outlet pipe adopt metal pipelines, they have better thermal conductivity, enabling the constant temperature device covered on the outer wall of the liquid outlet pipe to efficiently heat the reagent in the pipeline, so as to ensure that the reagent circulating in the liquid inlet pipe and the liquid outlet pipe is always at an appropriate temperature.

[0040] In some embodiments of this application, the length of the constant temperature device covering the liquid outlet pipe is not less than 1 / 2 of the length of the liquid outlet pipe.

[0041] In this application, by setting the appropriate length of the constant temperature device covering the liquid outlet pipe, it is conducive to efficiently heating the reagent in the liquid outlet pipe and ensuring that the reagent is always at an appropriate temperature.

[0042] In some embodiments of this application, the volume of the liquid outlet pipe does not exceed 80% of the volume of the synthesis column.

[0043] In this application, by setting the liquid outlet pipe to an appropriate volume, it is conducive to the efficient circulation and heating of the reagent, which is beneficial to the efficient coupling reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of this application, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of this application, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0045] Figure 1 It is a schematic diagram of the continuous flow preparation device of morpholino oligonucleotide provided for the embodiments of this application.

[0046] Reference numerals: 1 - automatic synthesizer; 11 - synthesis column; 12 - liquid inlet pipe; 13 - liquid outlet pipe; 2 - heating device; 3 - constant temperature device; 4 - reagent bottle.

[0047] Figure 2 It is the spectrum of the morpholino oligonucleotide after ammonolysis treatment provided for Example 1 in Test Example 1 of this application.

[0048] Figure 3It is the spectrum of the morpholino oligonucleotide provided in Example 2 of Test Example 1 of this application after ammonolysis treatment.

[0049] Figure 4 It is the spectrum of the morpholino oligonucleotide provided in Example 3 of Test Example 1 of this application after ammonolysis treatment.

[0050] Figure 5 It is the spectrum of the morpholino oligonucleotide provided in Example 4 of Test Example 1 of this application after ammonolysis treatment. Detailed implementation manners

[0051] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0052] In order to improve the problems of the current automatic synthesis method, although the synthesis cycle is short, the purity of the ammonolysis product prepared is not high, the raw material consumption is relatively high, the reaction conditions are harsh, and the synthesis scale is limited. The embodiments of this application provide a continuous flow preparation method for morpholino oligonucleotides. The continuous flow preparation device includes an automatic synthesizer, and a synthesis column is provided in the automatic synthesizer. The continuous flow preparation method includes:

[0053] (1) Deprotection: The protected morpholino phosphoramidite is loaded on a solid support and packed into the synthesis column, and then a deprotecting agent is pumped into the synthesis column to remove the protecting group on the amino group of the morpholine ring; the deprotecting agent is first pumped at a first flow rate of 150 - 240 cm / h for 3 - 7 cv, and then at a second flow rate of 120 - 150 cm / h for 7 - 10 cv; and the difference between the first flow rate and the second flow rate is not less than 30 cm / h.

[0054] (2) Activation and coupling: The morpholino phosphoramidite monomer (PMO) reagent and the activation reagent are simultaneously pumped into the synthesis column for mixing, and the synthesis column and the post-column of the synthesis column are heated and kept warm at 30 - 60 °C, and the coupling reaction is carried out cyclically.

[0055] (3) Capping: A capping reagent is pumped in for capping reaction to form one base.

[0056] (4) Each base cycle repeats steps (1) - (3), and finally a morpholino oligonucleotide is produced.

[0057] First, pump in a deprotecting agent at a high flow rate of 150 - 240 cm / h for 3 - 7 cv, and then pump in the deprotecting agent at a low flow rate of 120 - 150 cm / h for 7 - 10 cv. Can the protecting group on the morpholine ring amino group be efficiently removed? And cooperate to keep the temperature in the synthesis column and after the synthesis column at 30 - 60 °C to ensure that the coupling reaction is carried out at 30 - 60 °C, effectively improving the coupling efficiency and simultaneously controlling the content of impurities to achieve the improvement of the purity of the product after ammonolysis. Exemplarily, the protecting group can be a trityl (Tr) protecting group.

[0058] In some embodiments of the present application, the equivalent amount of the deprotecting agent pumped in is 100 - 200 eq. Exemplarily, the equivalent amount of the deprotecting agent pumped in can be but is not limited to 100 eq, 110 eq, 120 eq, 130 eq, 140 eq, 150 eq, 160 eq, 170 eq, 180 eq, 190 eq, 200 eq. It should be noted that "equivalent amount" refers to the molar amount per unit volume. "cv" refers to the volume pumped into the synthesis column.

[0059] Exemplarily, the first flow rate of pumping in the deprotecting agent can be but is not limited to 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h. The second flow rate of pumping in the deprotecting agent can be but is not limited to 120 cm / h, 130 cm / h, 140 cm / h, 150 cm / h. The volume of the deprotecting agent pumped in at the first flow rate can be but is not limited to 3 cv, 4 cv, 5 cv, 6 cv, 7 cv. The volume of the deprotecting agent pumped in at the second flow rate can be but is not limited to 7 cv, 8 cv, 9 cv, 10 cv.

[0060] In some embodiments of the present application, the morpholino oligonucleotide generated in step (4) is subjected to ammonolysis treatment. The ammonolysis treatment includes: S1, first take out the solid phase carrier in the synthesis column after the reaction in step (4), place it in an organic solvent containing 0.8 - 1.2 M of an organic base and 0.3 - 0.8 M of a capturing agent, react at 20 - 30 °C for 2 - 3 h and then filter; S2, then add ammonia water, react at 40 - 65 °C for 12 - 36 h and then filter, and the ammonolysis-treated morpholino oligonucleotide is obtained in the filtrate. The above method can efficiently carry out ammonolysis treatment on the prepared morpholino oligonucleotide to obtain a morpholino oligonucleotide with a relatively high purity after ammonolysis treatment.

[0061] Exemplarily, the organic base can be but is not limited to 1,8-diazabicyclo(5,4,0)-7-undecene (DBU), triethylamine (TEA), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), and tetramethylguanidine. The scavenger can be but is not limited to dithiothreitol, thymine, and pyridine-2-carboxaldehyde oxime. The organic reagent can be but is not limited to N-methylpyrrolidone, pyridine, and N,N-dimethylformamide.

[0062] In some embodiments of the present application, the solid support includes synthetic resin or controlled pore glass (CPG). The synthetic resin can be polystyrene (PS). Using the above solid support is beneficial to improving the reaction efficiency and product purity. Exemplarily, polystyrene can be but is not limited to being sourced from Lanxiao or Cytiva.

[0063] In some embodiments of the present application, the loading amount of the morpholino phosphoramidite protected by the protecting group on the solid support is 200 - 600 μmol / g. Exemplarily, the loading amount of the morpholino phosphoramidite protected by the protecting group on the solid support can be but is not limited to 200 μmol / g, 220 μmol / g, 250 μmol / g, 260 μmol / g, 280 μmol / g, 300 μmol / g, 320 μmol / g, 346 μmol / g, 350 μmol / g, 365 μmol / g, 380 μmol / g, 400 μmol / g, 420 μmol / g, 450 μmol / g, 480 μmol / g, 500 μmol / g, 520 μmol / g, 550 μmol / g, 580 μmol / g, 600 μmol / g. Loading an appropriate amount of the morpholino phosphoramidite protected by the protecting group on the solid support is beneficial to improving the subsequent reaction efficiency and the purity of the product generated by the reaction. And in combination with the type of the above solid support, a large-scale synthesis can be more precisely controlled and scaled up, which is beneficial to ensuring batch-to-batch consistency and stability.

[0064] In some embodiments of the present application, the specific conditions of step (2) are as follows: 2.5 - 7.5 eq of the morpholino phosphoramidite monomer reagent and 5 - 16 eq of the activating reagent are simultaneously pumped into the synthesis column at a flow rate of 150 - 300 cm / h for mixing. The synthesis column and the post-column of the synthesis column are heated and kept warm at 30 - 60 °C for 60 - 120 min for the coupling reaction. Pumping an appropriate amount of the morpholino phosphoramidite monomer reagent and the activator into the synthesis column at an appropriate flow rate and performing the coupling reaction at an appropriate temperature for an appropriate time can achieve an efficient coupling reaction using a lower amount of the morpholino phosphoramidite monomer reagent, and at the same time, the content of impurities can be controlled.

[0065] Exemplarily, the equivalent amount of the morpholino phosphoramidite monomer reagent pumped in can be, but is not limited to, 2.5 eq, 3 eq, 3.5 eq, 4 eq, 4.5 eq, 5 eq, 5.5 eq, 6 eq, 6.5 eq, 7 eq, 7.5 eq. The equivalent amount of the activating reagent pumped in can be, but is not limited to, 5 eq, 6 eq, 7 eq, 8 eq, 9 eq, 10 eq, 11 eq, 12 eq, 13 eq, 14 eq, 15 eq, 16 eq. The flow rate of the morpholino phosphoramidite monomer reagent and the activating reagent pumped in can be, but is not limited to, 150 cm / h, 160 cm / h, 170 cm / h, 180 cm / h, 190 cm / h, 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 270 cm / h, 280 cm / h, 290 cm / h, 300 cm / h. The coupling reaction can be, but is not limited to, cycling for 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min.

[0066] In some embodiments of the present application, the specific conditions of step (3) are: a capping reagent is pumped in at a flow rate of 60 - 240 cm / h for 0.5 - 3 cv for capping reaction. Exemplarily, the flow rate of the capping reagent pumped in can be, but is not limited to, 60 cm / h, 80 cm / h, 100 cm / h, 120 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 180 cm / h, 200 cm / h, 220 cm / h, 240 cm / h. The volume of the capping reagent pumped in can be, but is not limited to, 0.5 cv, 1 cv, 1.5 cv, 2 cv, 2.5 cv, 3 cv. Pumping in an appropriate capping reagent at an appropriate flow rate is conducive to an efficient capping reaction.

[0067] In some embodiments of the present application, the deprotecting agent is a dichloromethane solution containing 0.1 - 0.5 mol / L of tetracyanopyridine trifluoroacetate dissolved in 1% ethanol and 20% trifluoroethanol. Exemplarily, the concentration of tetracyanopyridine trifluoroacetate in the deprotecting agent can be, but is not limited to, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L. Using the above deprotecting agent to remove the protecting group on the morpholino ring amino group of the morpholino phosphoramidite is conducive to efficiently removing the protecting group on the morpholino ring amino group.

[0068] In some embodiments of the present application, the monomer reagent is a solvent containing 0.11 - 0.5 mol / L of morpholino phosphoramidite monomer. Exemplarily, the concentration of morpholino phosphoramidite monomer in the monomer reagent can be, but is not limited to, 0.11 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, 0.5 mol / L. Using the above monomer reagent is beneficial for carrying out a coupling reaction to form a base. Exemplarily, the solvent can be, but is not limited to, 1,3 - dimethyl - 2 - imidazolidinone, acetonitrile, N,N - dimethylformamide (DMF), tetrahydrofuran (THF).

[0069] In some embodiments of the present application, the activation reagent is a 1,3 - dimethyl - 2 - imidazolidinone solution containing 0.4 - 1.3 mol / L of N - ethylmorpholine and 0.1 - 0.8 mol / L of lithium bromide. Exemplarily, the concentration of N - ethylmorpholine in the activation reagent can be, but is not limited to, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L. The concentration of lithium bromide in the activation reagent can be, but is not limited to, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L. Using the above activation reagent is beneficial for efficiently activating the morpholino phosphoramidite monomer to carry out the coupling reaction efficiently.

[0070] In some embodiments of the present application, the capping reagent includes capping reagent A and capping reagent B with a mass ratio of 1:1 - 2. Capping reagent A is an N,N - dimethylformamide solution containing 0.8 - 1.2 mol / L of benzoic anhydride, and capping reagent B is a solution containing 0.1 - 0.5 mol / L of 4 - dimethylaminopyridine. The solvent of capping reagent B includes N - methylimidazole, 2,6 - dimethylpyridine, and N,N - dimethylformamide. Exemplarily, the concentration of benzoic anhydride in capping reagent A can be, but is not limited to, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L. The concentration of 4 - dimethylaminopyridine in capping reagent B can be, but is not limited to, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L. Using the above capping reagent, especially using 4 - dimethylaminopyridine and 2,6 - dimethylpyridine in capping reagent B, can effectively improve the capping efficiency, thereby improving the overall efficiency of preparing morpholino oligonucleotides and reducing the preparation cycle.

[0071] In some embodiments of the present application, the volume percentage ratio of N-methylimidazole, 2,6-dimethylpyridine, and N,N-dimethylformamide is 10%-15%:10%-15%:75%-80%. Using a suitable component mixture in a suitable ratio as the solvent for capping reagent B can ensure efficient capping reaction.

[0072] In some embodiments of the present application, a cleaning step is included after each of steps (1)-(3); the cleaning step includes pumping 2-5 cv of a cleaning reagent at a flow rate of 100-400 cm / h. By way of example, the flow rate of the cleaning reagent pumped can be, but is not limited to, 100 cm / h, 120 cm / h, 140 cm / h, 150 cm / h, 160 cm / h, 180 cm / h, 200 cm / h, 220 cm / h, 240 cm / h, 250 cm / h, 260 cm / h, 280 cm / h, 300 cm / h, 320 cm / h, 340 cm / h, 350 cm / h, 360 cm / h, 380 cm / h, 400 cm / h. The volume of the cleaning reagent pumped can be, but is not limited to, 2 cv, 2.5 cv, 3 cv, 3.5 cv, 4 cv, 4.5 cv, 5 cv. Pumping a suitable amount of the cleaning reagent at a suitable flow rate after each step can thoroughly clean the pipeline and the synthesis column, avoiding the influence of the reagent residue in the previous step on the reaction in the subsequent step.

[0073] In some embodiments of the present application, the cleaning reagent is selected from dichloromethane or N,N-dimethylformamide. Using the above-mentioned suitable cleaning reagent can ensure thorough cleaning of the pipeline and the synthesis column.

[0074] The embodiment of the present application provides a continuous flow preparation device for morpholino oligonucleotides, which is applicable to the above-mentioned continuous flow preparation method of morpholino oligonucleotides, as Figure 1 shown, including an automatic synthesizer 1, a heating device 2, and a constant temperature device 3. The automatic synthesizer 1 has a synthesis column 11. A liquid inlet pipe 12 is connected between the inlet of the synthesis column 11 and the automatic synthesizer 1, and a liquid outlet pipe 13 is connected between the outlet of the synthesis column 11 and the automatic synthesizer 1; the heating device 2 is wrapped on the surface of the synthesis column 11; the constant temperature device 3 is wrapped on the outer wall of the liquid outlet pipe 13.

[0075] A heating device 2 is coated on the surface of the synthesis column 11, which can heat the synthesis column 11 to an appropriate temperature to improve the coupling reaction efficiency and control the impurity content. At the same time, a constant temperature device 3 is coated on the outer wall of the liquid outlet pipe 13, which can keep the circulating reagent at an appropriate temperature during the coupling reaction, thus effectively improving the coupling reaction efficiency and controlling the impurity content. In addition, the constant temperature device 3 is arranged on the outer wall of the liquid outlet pipe 13, which is beneficial for the non-circulating steps such as removing the protecting group on the morpholine ring amino group and capping. During these steps, the reagent is only heated in the synthesis column and not heated in front of the column, so as to ensure that the reagent in the non-circulating steps such as removing the protecting group on the morpholine ring amino group and capping will not be overheated, avoiding the generation of more degradation impurities that affect the reaction effect.

[0076] Exemplarily, the automatic synthesizer 1 can be but is not limited to oligopilot plus100 of Cytiva. The synthesis column 11 can be but is not limited to FineLINE 35 Oligo synthesis column 11.

[0077] Exemplarily, the heating device 2 can be a heating bag or a heating sheet. It is beneficial to closely adhere to the surface of the synthesis column 11 to efficiently heat the synthesis column 11. The temperature of the heating device 2 is controlled at 30 - 60 °C to ensure that the temperature inside the synthesis column 11 is 30 - 60 °C.

[0078] Exemplarily, the constant temperature device 3 can be an oil bath device. The temperature of the constant temperature device 3 is controlled at 30 - 60 °C to ensure that the reagent circulating in the liquid outlet pipe 13 is maintained at 30 - 60 °C.

[0079] Exemplarily, both the heating device 2 and the constant temperature device 3 include temperature probes, which can detect the temperature to facilitate temperature control and maintenance.

[0080] In some embodiments of the present application, both the liquid inlet pipe 12 and the liquid outlet pipe 13 are metal pipelines. The use of metal pipelines for the liquid inlet pipe 12 and the liquid outlet pipe 13 has better thermal conductivity, enabling the constant temperature device 3 coated on the outer wall of the liquid outlet pipe 13 to efficiently heat the reagent in the pipeline, ensuring that the reagent circulating in the liquid inlet pipe 12 and the liquid outlet pipe 13 is always at an appropriate temperature.

[0081] In some embodiments of the present application, the length of the constant temperature device 3 coated on the liquid outlet pipe 13 is not less than 1 / 2 of the length of the liquid outlet pipe 13. Setting an appropriate length of the constant temperature device 3 coated on the liquid outlet pipe 13 is beneficial for efficiently heating the reagent in the liquid outlet pipe 13 and ensuring that the reagent is always at an appropriate temperature.

[0082] In some embodiments of the present application, the length of the liquid outlet pipe 13 can be 1 - 1.5 m, and the pipe diameter of the liquid outlet pipe 13 can be 1 - 5 cm. Exemplarily, the length of the liquid outlet pipe 13 can be, but is not limited to, 1 m, 1.1 m, 1.2 m, 1.3 m, 1.4 m, 1.5 m. The pipe diameter of the liquid outlet pipe 13 can be, but is not limited to, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4.5 cm, 5 cm. In other embodiments, the length and pipe diameter of the liquid outlet pipe 13 can be adjusted as needed.

[0083] In some embodiments of the present application, the volume of the liquid outlet pipe 13 does not exceed 80% of the volume of the synthesis column 11. Setting the liquid outlet pipe 13 to an appropriate volume is beneficial for the efficient circulation and heating of the reagent, and is conducive to an efficient coupling reaction.

[0084] In some embodiments of the present application, the automatic synthesizer 1 is further connected to a plurality of reagent bottles 4. The reagent bottles 4 are respectively used to load morpholino phosphoramidite monomer reagents, activation reagents, deprotecting agents, capping reagents, cleaning reagents, etc. Exemplarily, the automatic synthesizer 1 is connected to the reagent bottles 4 through polytetrafluoroethylene pipelines.

[0085] The features and properties of the present application are further described in detail below in conjunction with embodiments.

[0086] Example 1

[0087] This example provides a continuous flow preparation method for morpholino oligonucleotides. The continuous flow preparation device includes an oligopilot plus 100 automatic synthesizer from Cytiva, and the automatic synthesizer has a FineLINE 35 Oligo synthesis column; the continuous flow preparation method includes: oligopilot plus 100 automatic synthesizer, and the automatic synthesizer has a FineLINE 35 Oligo synthesis column; the continuous flow preparation method includes:

[0088] (1) Deprotecting the group: Loading morpholino phosphoramidite protected by the Tr protecting group at 300 μmol / g on polystyrene (crosslinking degree - 1%) synthetic resin (cytiva - 5G), packing 6.7 g of the synthetic resin into the synthesis column at a density of 0.1 g / mL, with the packed column volume being 67.3 mL, and then pumping a deprotecting agent containing 0.2 mol / L tetracyanopyridine trifluoroacetate dissolved in a dichloromethane solution of 1% ethanol and 20% trifluoroethanol into the synthesis column to remove the Tr protecting group on the amino group of the morpholine ring; the deprotecting agent is first pumped in at a first flow rate of 240 cm / h for 3 cv, and then at a second flow rate of 120 cm / h for 10 cv, and the pumped equivalent is 150 eq;

[0089] (2) Activation and coupling: 5 eq of the morpholino phosphoramidite monomer reagent in a 1,3-dimethyl-2-imidazolidinone solution containing 0.31 mol / L of morpholino phosphoramidite monomer and 16 eq of the activation reagent in a 1,3-dimethyl-2-imidazolidinone solution containing 0.8 mol / L of N-ethylmorpholine and 0.4 mol / L of lithium bromide were simultaneously pumped into the synthesis column at a flow rate of 240 cm / h for mixing. The synthesis column and the post-column of the synthesis column were heated and kept warm at 50 °C, and the coupling reaction was carried out for 90 min in a cycle;

[0090] (3) Capping: 2 cv of the capping reagent was pumped in at a flow rate of 120 cm / h for the capping reaction to form a base; the capping reagent included capping reagent A and capping reagent B with a mass ratio of 1:1. Capping reagent A was an N,N-dimethylformamide solution containing 1.0 mol / L of benzoic anhydride, and capping reagent B was a solution containing 0.3 mol / L of 4-dimethylaminopyridine. The solvent of capping reagent B included N-methylimidazole, 2,6-dimethylpyridine, and N,N-dimethylformamide with a volume percentage ratio of 10%:15%:75%;

[0091] (4) After each step of steps (1)-(3), 3 cv of dichloromethane cleaning reagent was pumped in at a flow rate of 200 cm / h for cleaning;

[0092] (5) Each base cycled through steps (1)-(4), and finally morpholino oligonucleotide was produced.

[0093] The morpholino oligonucleotide prepared in this example was 5'-GCTATTACCTTAACCCAG-3'. The synthesis scale was 2.02 mmol.

[0094] Example 2

[0095] This example provides a continuous flow preparation method of morpholino oligonucleotide. The difference from Example 1 is that the morpholino phosphoramidite protected by the Tr protecting group was loaded on a polystyrene (crosslinking degree -1%) synthesis resin at 365 μmol / g, and 3.84 g of the synthesis resin was packed into the synthesis column at a density of 0.08 g / mL, and the packed column volume was 48 mL.

[0096] The morpholino oligonucleotide prepared in this example was 5'-GTTGCCTCCGGTTCTGAA GGTGTTC-3'. The synthesis scale was 1.40 mmol.

[0097] Example 3

[0098] This example provides a continuous flow preparation method of morpholino oligonucleotides, which is different from Example 1 in that the morpholino phosphoramidite protected by the Tr protecting group is loaded on a polystyrene (crosslinking degree -1%) synthetic resin (Lanxiao) at 260 μmol / g, and 3.84 g of the synthetic resin is loaded into a synthesis column at a density of 0.08 g / mL, and the loaded column volume is 48.1 mL.

[0099] The morpholino oligonucleotide prepared in this example is 5'-AGCTATTACCTTAACCCA G-3'. The synthesis scale is 1.00 mmol.

[0100] Example 4

[0101] This example provides a continuous flow preparation method of morpholino oligonucleotides, which is different from Example 1 in that the morpholino phosphoramidite protected by the Tr protecting group is loaded on a polystyrene (crosslinking degree -1%) synthetic resin at 346 μmol / g, and 3.84 g of the synthetic resin is loaded into a synthesis column at a density of 0.08 g / mL, and the loaded column volume is 48.1 mL.

[0102] The morpholino oligonucleotide prepared in this example is 5'-TAAAGTTCGTTTAGAGAA-3'. The synthesis scale is 1.33 mmol.

[0103] Comparative Example 1

[0104] This comparative example provides a continuous flow preparation method of morpholino oligonucleotides, which is different from Example 1 in that the deprotecting agent is pumped in at a flow rate of 150 cm / h for 13 cv, and the pumped equivalent is 150 eq.

[0105] The continuous flow preparation methods of morpholino oligonucleotides provided in Examples 1-4 and Comparative Example 1 are shown in Table 1.

[0106] Table 1 Continuous flow preparation methods of morpholino oligonucleotides

[0107]

[0108]

[0109] Test Example 1

[0110] In this test example, the morpholino oligonucleotides produced in Examples 1-4 and Comparative Examples 1-2 were subjected to ammonolysis treatment. The ammonolysis treatment included: S1, first, the solid-phase carriers in the synthesis columns after the reactions in Examples 1-4 and Comparative Examples 1-2 were taken out and placed in an N-methylpyrrolidone solution containing 1.0 M of 1,8-diazabicyclo(5,4,0)-7-undecene and 0.5 M of dithiothreitol, and reacted at 250 °C for 2 h and then filtered; S2, then ammonia water was added, and the reaction was carried out at 55 °C for 24 h and then filtered, and the ammonolysis-treated morpholino oligonucleotides were obtained in the filtrate. The results are as Figures 2 - 5 shown, and the molecular weight of the product was determined according to mass, and the purity of the ammonolysis-treated morpholino oligonucleotides was calculated. The results are shown in Table 2.

[0111] Table 2 Purity of the product after ammonolysis

[0112] Purity (%) Example 1 49.0 Example 2 53.2 Example 3 48.1 Example 4 67.8

[0113] As can be seen from the results in Table 2, for the continuous flow preparation method of the morpholino oligonucleotides provided in this application, by first pumping an appropriate amount of deprotecting agent at a high flow rate and then pumping an appropriate amount of deprotecting agent at a low flow rate, the protecting groups on the amino group of the morpholine ring can be efficiently removed, and the coupling reaction is carried out in combination with an appropriate temperature, effectively improving the coupling efficiency and simultaneously controlling the content of impurities. The morpholino oligonucleotides prepared by it reach a relatively high purity after ammonolysis treatment.

[0114] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of this application claimed, but merely represents selected embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application.

Claims

1. A continuous flow preparation method for morpholino oligonucleotides, characterized in that: The continuous flow preparation device comprises an automatic synthesizer, wherein the automatic synthesizer has a synthesis column; the continuous flow preparation method comprises: (1) Removing protecting groups: loading the morpholino phosphoramidite protected by protecting groups on a solid phase carrier and loading the solid phase carrier into the synthetic column, and then pumping a deprotecting agent into the synthetic column to remove the protecting groups on the amino groups of the morpholino ring; the deprotecting agent is first pumped at a first flow rate of 150-240 cm / h for 3-7 cv, and then at a second flow rate of 120-150 cm / h for 7-10 cv; and the difference between the first flow rate and the second flow rate is not less than 30 cm / h; (2) Activation and coupling: the morpholino phosphoramidite monomer reagent and the activation reagent are simultaneously pumped into the synthesis column for mixing, the synthesis column and the column post of the synthesis column are heated and kept at 30-60° C., and the coupling reaction is cyclically performed; (3) Capping: Pump in the capping reagent to perform a capping reaction to form a base; (4) Each base is cycled through steps (1) to (3) to ultimately produce the morpholino oligonucleotide.

2. The continuous flow preparation method according to claim 1, characterized in that: The morpholino oligonucleotide produced in step (4) is subjected to an aminolysis treatment, and the aminolysis treatment comprises: S1, first taking out the solid phase carrier in the synthesis column after the reaction in step (4), placing it in an organic reagent containing 0.8-1.2M organic base and 0.3-0.8M capture agent, reacting at 20-30°C for 2-3h and then filtering; S2, adding ammonia water, reacting at 40-65°C for 12-36h and then filtering, and obtaining the morpholino oligonucleotide after the aminolysis treatment in the filtrate.

3. The continuous flow preparation method according to claim 1, characterized in that: The solid phase carrier comprises a synthetic resin or a controllable microporous glass; Optionally, the solid phase carrier has a loading capacity of 200-600 μmol / g for the morpholino phosphoramidite protected by a protecting group.

4. The continuous flow preparation method according to claim 1, characterized in that: The specific conditions of step (2) are as follows: 2.5-7.5 eq of morpholino phosphoramidite monomer reagent and 5-16 eq of activation reagent are simultaneously pumped into the synthesis column at a flow rate of 150-300 cm / h for mixing, and the synthesis column and the column post of the synthesis column are heated and kept at 30-60° C. for 60-120 min to carry out coupling reaction; And / or, the specific conditions of step (3) are: pumping 0.5-3 cv of the capping reagent at a flow rate of 60-240 cm / h to carry out the capping reaction.

5. The continuous flow preparation method according to claim 1, characterized in that: The deprotecting agent is a dichloromethane solution containing 0.1-0.5 mol / L tetracyanopyridine trifluoroacetate dissolved in 1% ethanol and 20% trifluoroethanol; and / or, the monomer reagent is a solvent containing 0.11-0.5 mol / L morpholino phosphoramidite monomer; And / or, the activation reagent is a 1,3-dimethyl-2-imidazolidinone solution containing 0.4-1.3 mol / L ethylmorpholine and 0.1-0.8 mol / L lithium bromide; And / or, the capping reagent comprises a capping reagent A and a capping reagent B in a mass ratio of 1:1-2, the capping reagent A is an N,N-dimethylformamide solution containing 0.8-1.2 mol / L benzoic anhydride, the capping reagent B is a solution containing 0.1-0.5 mol / L 4-dimethylaminopyridine, and the solvent of the capping reagent B comprises nitrogen-methylimidazole, 2,6-lutidine and N,N-dimethylformamide; optionally, the volume percentage ratio of the nitrogen-methylimidazole, the 2,6-lutidine and the N,N-dimethylformamide is 10%-15%:10%-15%:75%-80%.

6. The continuous flow preparation method according to claim 1, characterized in that: Each of the steps (1) to (3) includes a cleaning step; the cleaning step includes pumping 2-5 cv of a cleaning reagent at a flow rate of 100-400 cm / h; Optionally, the cleaning reagent is selected from dichloromethane or N,N-dimethylformamide.

7. A continuous flow preparation device for morpholino oligonucleotides, characterized in that: A continuous flow preparation method for the morpholino oligonucleotide according to any one of claims 1 to 6, comprising: An automatic synthesizer, wherein the automatic synthesizer has the synthesis column, a liquid inlet pipe is connected between the inlet of the synthesis column and the automatic synthesizer, and a liquid outlet pipe is connected between the outlet of the synthesis column and the automatic synthesizer; A heating device, the heating device is coated on the surface of the synthetic column; A constant temperature device is coated on the outer wall of the liquid outlet pipe.

8. The continuous flow preparation device according to claim 7, characterized in that: The liquid inlet pipe and the liquid outlet pipe are both metal pipes.

9. The continuous flow preparation device according to claim 7 or 8, characterized in that: The length of the thermostatic device wrapped around the liquid outlet pipe is not less than 1 / 2 of the length of the liquid outlet pipe.

10. The continuous flow preparation device according to claim 7 or 8, characterized in that: The volume of the liquid outlet pipe does not exceed 80% of the volume of the synthesis column.

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