Phosphorylated arginine analogue, synthesis method and application thereof
A technology for phosphorylated arginine and analogues, applied in the field of phosphorylated arginine analogues and their synthesis
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Embodiment 1
[0068] Example 1: Synthesis of phosphorylated arginine analogue N-(2-ethylamine)-2-phosphonic acid acetamidine (pAIE)
[0069] The overall synthetic route is as Figure 4 shown.
[0070] Synthesis of compound 1 (2-diethylphosphono-thioacetamide):
[0071]Diethyl cyanomethylphosphonate (5.30g, 30mmol), triethylamine (6.05g, 60mmol), tetrabutylammonium bromide (125mg), and toluene (25mL) were added to the round bottom flask. Under the protection of argon, dry hydrogen sulfide (H 2 S) gas, stirred and reacted at 10°C for 5h, then reacted at room temperature for 12h. After the reaction was completed, excess nitrogen gas was introduced into the reaction solution, cooled at 4° C. for 3 h, and filtered to obtain a yellow solid. The resulting yellow solid was filtered and washed with toluene under nitrogen protection to obtain the product compound 1 (2.34 g, yield 37%).
[0072] 1 H NMR (500MHz, CDCl 3 ):δ8.45(s,1H,NH protons)and 7.73(s,1H,NH protons),4.19(dq,J=14.2,7.1Hz,4H,-O...
Embodiment 2
[0092] Example 2: Using the phosphorylated arginine analogue pAIE as a hapten to prepare an antibody against phosphorylated arginine
[0093] Chemical coupling and immunization of pAIE with carrier protein:
[0094] Such as Figure 5 As indicated, pAIE and keyhole limpet hemocyanin (KLH) were dissolved in coupling buffer (0.1M Na 2 CO 3 , 0.15M NaCl, pH 8.5) and mixed in proportion, where the final concentration of pAIE is 15mM, the final concentration of KLH is 2mg / mL, slowly add 25% glutaraldehyde to a final concentration of 0.5%, after incubation at room temperature for 2h, add NaBH 4 The solid was reacted at room temperature to a final concentration of 10 mg / mL for 2 hours, and then dialyzed overnight in PBS 7.4, and the protein concentration was determined by the Bradford method.
[0095] The prepared pAIE-KLH was used as an immune antigen, mixed and emulsified with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant, and injected at 200 μg / mo...
Embodiment 3
[0101] Example 3: Purification of Mouse Anti-Phosphorylated Arginine Polyclonal Antibody
[0102] Binding / washing buffer: 0.15M NaCl, 20mM Na 2 HPO 4 , pH 8.0.
[0103] Elution buffer: 0.1M glycine, pH 2.5.
[0104] Neutralization buffer: 1M Tris-HCl, pH 8.5.
[0105] Take 20 μL of protein A agarose microbeads (50% suspension) in a micro-purification column, drain naturally, and use 5 times the column volume (50 μL) of ddH 2 Wash with 0 and 5 column volumes (50 μL) of binding buffer. Mouse anti-phosphorylated arginine serum (10 μL) was diluted with 9-fold (90 μL) binding buffer and carefully added to the top of the column gel to collect the flow-through, and then wash the beads with 10 times the column volume (100 μL) of washing buffer, Receive the washing components in 20 μL per tube; then wash the resin with 10 column volumes (100 μL) of elution buffer, receive the elution components in 20 μL per tube, and immediately neutralize the collected elution components with 2 μ...
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