Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester
A cytarabine monoester and whole-cell catalyst technology, which is applied in the field of cell catalysts, can solve the problem of low selectivity by-products, etc., and achieve the effects of easy control of product structure, low preparation cost, and no need for group protection and deprotection
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Embodiment 1
[0018] will be cultivated Pseudomonas fluorescens GIM1.209 seed solution was transferred to the liquid medium containing Tween 80 enzyme-producing inducer for culture. The composition of the liquid medium is: 20 g / L Tween 80, 0.5 g / L yeast extract, 0.2 g / L MgSO 4 ·7H 2 O , 2 g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after 24 hours of shake flask culture at 20°C, washed twice with citrate buffer at pH 5.5, and the whole cell catalyst was obtained after vacuum freeze-drying for 12 hours. Add the prepared cell catalyst (amount of 5 g / L) into hexane pyridine with a cytarabine content of 25 mmol / L and a molar ratio of carboxylate to cytarabine of 15:1, and the synthesis reaction temperature When the temperature is 60 ℃, the shaking speed of the reaction process is 60 rpm, and the reaction time is 24 h, cytarabine monoester can be obtained. The regioselectivity of the catalyzed cytarabine acylation reaction of the ...
Embodiment 2
[0020] will be cultivated Pseudomonas fluorescens GIM1.209 seed liquid was transferred to liquid medium containing Tween 85 enzyme-producing inducer for culture. The composition of the liquid medium is: 30 g / L Tween 85, 40 g / L yeast extract, 4 g / L MgSO 4 ·7H 2 O , 15 g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after 72 hours of shake flask culture at 40°C, washed twice with citrate buffer at pH 5.5, and vacuum freeze-dried for 24 hours to obtain the whole cell catalyst. The prepared cell catalyst (amount of 50 g / L) was added to hexane-pyridine with a cytarabine content of 50 mmol / L and a molar ratio of carboxylate to cytarabine of 18:1, and the synthesis reaction The temperature is 60 ℃, the shaking speed of the reaction process is 60 rpm, and the reaction time is 24 h, and cytarabine monoester can be obtained. The regioselectivity of the catalyst for catalyzing the cytarabine acylation reaction is 97%, and the...
Embodiment 3
[0022] will be cultivated Pseudomonas fluorescens GIM1.209 seed liquid was transferred to liquid medium containing Span 60 enzyme-producing inducer for culture. The composition of the liquid medium is: 25 g / L Span 60, 30 g / L yeast extract, 2 g / L MgSO 4 ·7H 2 O , 10g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after cultured in shake flasks at 30°C for 48 hours, washed twice with pH 5.5 phosphate buffer, and vacuum freeze-dried for 24 hours to obtain the whole-cell catalyst. The prepared cell catalyst (amount of 100 g / L) was added to hexane-isopropyl ether with a cytarabine content of 2 mmol / L and a molar ratio of carboxylate to cytarabine of 50:1. The synthesis reaction temperature is 20°C, the oscillation speed of the reaction process is 100 rpm, and the reaction time is 12 hours, and cytarabine monoester can be obtained. The regioselectivity of the catalyst for catalyzing the cytarabine acylation reaction is 97...
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