Initial capping oligonucleotide primer containing open-loop nucleoside structure
A nucleoside structure and oligonucleotide technology, which is applied in the fields of chemical and biological engineering, can solve the problems of insufficient capping efficiency of in vitro transcription yield, and achieve high capping efficiency, high synthesis efficiency, and low immunogenicity
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Synthetic example 1
[0054] The synthetic route of A-G-P used in Synthesis Example 1 is: take 5kg of 2'OMe-rA phosphoramidite monomer in a single-neck flask, dissolve with 50L of methylene chloride, then add 2.73kg of 2',3' acetylguanosine, The temperature was lowered to 25±2°C, 880 g of tetrazolium was added under nitrogen blowing, and the temperature was raised to 25±2°C for reaction. After monitoring the reaction, add 1.2eq of iodopyridine solution to the reaction solution, spin dry after monitoring the reaction, dissolve the concentrated ointment in 4L of dichloromethane, add 1.1eq of trifluoroacetic acid, and monitor the reaction after the end. , spin dry, beat petroleum ether / dichloromethane according to a certain proportion, and filter to obtain intermediate A2; dissolve A2 in 4L acetonitrile, add 1.2eq of phosphine reagent and 1.2eq of tetrazolium to fully stir the reaction, monitor the reaction after the end , then add 1.2eq of iodopyridine solution to the reaction solution, spin dry afte...
Synthetic example 2
[0056] The synthetic method of A-UrG-P used in Synthesis Example 2 refers to the A-G-P synthetic method in Example 1, the reaction route flow of A-UrG-P, following equation (3):
[0057]
[0058] The synthesis of D refers to the synthesis steps of intermediate J; the preparation of E2 includes the following steps: weigh 20g of compound D and dissolve it in acetonitrile, add 3eq of triethylamine, cool the reaction solution to 4°C, slowly add acetic anhydride dropwise, and react After the end, 2eq of TBAF was added, and the TBS protecting group was removed by spin-dry column chromatography to obtain compound E2; compound E2 was substituted for bi-substituted guanosine to obtain A-UrG-P.
Synthetic example 3
[0059] The synthetic method of the UrA-G-P used in Synthesis Example 3 refers to the A-G-P synthetic method in Example 1, the reaction route flow of UrA-G-P, the following equation (4),
[0060]
[0061] The synthesis of D refers to the synthesis steps of intermediate J; the preparation of F4 includes the following steps: (1) Weigh 10g of compound D and dissolve it in DMF, take an ice bath, slowly add 1.2eq of NaH, stir at low temperature for 2h, slowly add dropwise Iodomethane 2eq, react at room temperature for 3 hours, add water to quench the reaction, filter to obtain the crude product of compound F1, and purify by reverse chromatography; (2) Weigh 2g of compound F and disperse it in 30mL of methanol, add 2eq of TBAF, after 2 hours After the reaction is over, spin-dried and directly react in the next step; dissolve the spin-dried solid in 30 ml of DCM, add 1.2 eq of triethylamine, stir in an ice bath for 20 min, slowly add the DCM solution of DMTr-Cl, and after the dropwi...
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