Polymerase enzyme from 9°n
a polymerase and enzyme technology, applied in the field of molecular biology, can solve the problems of inferior sequencing ladder compared to wild type enzymes, insufficient incorporation rate of modified nucleotides, and difficulty in incorporating modified nucleotides, so as to improve the efficiency of certain dna sequencing methods and improve the incorporation rate of such 3′ substituted nucleotide analogues
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example 1
Synthesis of 3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine (2)
[0121]5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine (1) (2.0 g, 5.6 mmol) was dissolved in a mixture consisting of DMSO (10.5 mL), acetic acid (4.8 mL), and acetic anhydride (15.4 mL) in a 250 mL round bottom flask, and stirred for 48 hours at room temperature. The mixture was then quenched by adding saturated K2CO3 solution until evolution of gaseous CO2 was stopped. The mixture was then extracted with EtOAc (3×100 mL) using a separating funnel. The combined organic extract was then washed with a saturated solution of NaHCO3 (2×150 mL) in a partitioning funnel, and the organic layer was dried over Na2SO4. The organic part was concentrated by rotary evaporation. The reaction mixture was finally purified by silica gel column chromatography.
example 2
Synthesis of 3′-O-(ethyldithiomethyl)-2′-deoxythymidine (4)
[0122]Compound 2 (1.75 g, 4.08 mmol), dried overnight under high vacuum, dissolved in 20 mL dry CH2CI2 was added with EtsN (0.54 mL, 3.87 mmol) and 5.0 g molecular sieve-3A, and stirred for 30 min under Ar atmosphere. The reaction flask was then placed on an ice-bath to bring the temperature to sub-zero, and slowly added with 1.8 eq 1M SO2CI2 in CH2CI2 (1.8 mL) and stirred at the same temperature for 1.0 hour. Then the ice-bath was removed to bring the flask to room temperature, and added with a solution of potassium thiotosylate (1.5 g) in 4 mL dry DMF and stirred for 0.5 hour at room temperature.
[0123]Then 2 eq EtSH (0.6 mL) was added and stirred additional 40 min. The mixture was then diluted with 50 mL CH2CI2 and filtered through celite-S in a funnel. The sample was washed with adequate amount of CH2CI2 to make sure that the product was filtered out. The CH2CI2 extract was then concentrated and purified by chromatography...
example 3
Synthesis of the triphosphate of 3′-O-(ethyldithiomethyl)-2′-deoxythymidine (5)
[0124]In a 25 mL flask, compound 4 (0.268 g, 0.769 mmol) was added with proton sponge (210 mg), equipped with rubber septum. The sample was dried under high vacuum for overnight. The material was then dissolved in 2.6 mL (MeO)3P0 under argon atmosphere. The flask, equipped with Ar-gas supply, was then placed on an ice-bath, stirred to bring the temperature to sub-zero. Then 1.5 equivalents of POCI3 was added at once by a syringe and stirred at the same temperature for 2 hours under Argon atmosphere. Then the ice-bath was removed and a mixture consisting of tributylammonium-pyrophosphate (1.6 g) and Bu3N (1.45 mL) in dry DMF (6 mL) was prepared. The entire mixture was added at once and stirred for 10 min. The reaction mixture was then diluted with TEAB buffer (30 mL, 100 mM) and stirred for additional 3 hours at room temperature. The crude product was concentrated by rotary evaporation, and purified by CI ...
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