Non-natural amino acid, application thereof, recombinant protein containing non-natural amino acid, and recombinant protein conjugate
A non-natural amino acid, recombinant protein technology, applied in the field of biopharmaceuticals, can solve the problems of harsh process conditions, increased production costs, complex process, etc., and achieves the effects of good safety, not easy inactivation, and high expression efficiency.
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Embodiment 1
[0073] The preparation of embodiment 1 unnatural amino acid NOBK
[0074] The structural formula of NOBK is as follows:
[0075]
[0076] The reaction process is as follows:
[0077]
[0078] The preparation process includes the following steps:
[0079] a) In the reaction flask, add 3-buten-1-ol (2.00g, 27.74mmol), add solvent DCM (15.0mL), add CDI (4.50g, 27.74mmol) at 0C °, the mixture is at room temperature After reacting for 18 hours, 10 mL of water was added, extracted three times with DCM, the organic phases were combined, dried by adding anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 1-1 (4.50 g, yield 98%).
[0080] b) In a reaction flask, add product 1-1 (0.55g, 3.31mmol) and Fmoc-lysine hydrochloride (1.12g, 2.76 mmol), add solvent dioxane 9mL and water 3mL (v / v =3:1), then added triethylamine (0.70g, 6.9mmol), and reacted the mixture at room temperature for 24 hours, then added 20mL of 1M HCl solution, adjuste...
Embodiment 2
[0084] The preparation of embodiment 2 unnatural amino acids NOHK
[0085] The structural formula of NOHK is as follows:
[0086]
[0087] The reaction process is as follows:
[0088]
[0089] The preparation process includes the following steps:
[0090] a) In a reaction flask, add 5-oxohexanoic acid (2.60g, 20.0mmol), N-hydroxysuccinimide (2.30g, 20.0mmol), EDCI (3.83g, 20.0mmol), add solvent DCM (100 mL), the mixture was reacted at room temperature for 18 hours, washed with water three times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain product 2-1 (4.62 g, yield 102%), which was directly used in the next step without purification.
[0091] b) In a reaction flask, add product 2-1 (4.62g, 20.35mmol) and Boc-lysine (4.42g, 16.96mmol), then add DIPEA (4.07mL, 42.4mmol), add solvent DMF (50mL) , the mixture was reacted at 40°C for 24 hours, washed with water 5 times, dried by adding anhydrous sodium sulfate, filtered, concent...
Embodiment 3
[0094] The preparation of embodiment 3 unnatural amino acids NOPK
[0095] The structural formula of NOPK is as follows:
[0096]
[0097] The reaction process is as follows:
[0098]
[0099] The preparation process includes the following steps:
[0100] a) In the reaction flask, add propylene bromide (18.15g, 0.15mol), add DMSO (60mL), cool down to 0°C, add ethylene glycol (37.26g, 0.6mol), DMSO (120mL), KOH (10.11g , 0.18mol) and water (30mL), reacted at this temperature for 1 hour, raised to room temperature and stirred for 18 hours, added water (30mL) to the reaction solution, extracted 3 times with DCM, combined the organic phases, and washed 2 times with water , dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure at about 0°C, and finally purified by column chromatography (eluent: PE:EA=10:1) to obtain product 3-1 (6.30g, yield 25%) .
[0101] b) In the reaction flask, add p-nitrophenyl chloroformate (1.0g, 4.90mmol), add solvent...
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