A polyclonal antibody for efficiently recognizing protein arginine rhamnosylation and its preparation method
A polyclonal antibody, arginine mouse technology, applied in the field of medicinal chemistry, can solve problems such as inability to detect proteins
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Embodiment 1
[0052] The synthesis of embodiment 1 key intermediate 6
[0053] First, L-rhamnose 2 was used as raw material to obtain triacetyl-protected α-chloroglycoside 3 under the condition of acetyl chloride, and 3 was refluxed in acetonitrile under the action of potassium thiocyanate and tetrabutylammonium iodide to obtain rat The isothiocyanate 4 of lose, and then 4 is ammoniated under the action of ammonia to obtain rhamnose thiourea 5, and finally 5 is divided into two parts under the action of iodoethane, di-tert-butyl dicarbonate and triethylamine. The key intermediate 6 was obtained through one-step reaction. For specific synthetic routes, see image 3 .
Embodiment 2
[0054] Example 2 Synthesis of Arginine Rhamnosylated Polypeptide 1
[0055] The solid-phase glycosylation strategy is adopted, and the specific synthetic route is shown in Figure 4 . First, linear peptides were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS). 2-chlorotrityl resin as a carrier. Fmoc-Orn(Alloc)-OH as the initiator amino acid for glycosylation. After obtaining the linear peptide, we cut off the protecting group of the side chain amino group under the action of (triphenylphosphine) palladium to obtain compound 7. Glycopeptide 10 was then obtained by reacting amino groups with thioesters under silver catalysis on the resin. Next, the acetyl protecting group on rhamnose was removed under the action of 5% hydrazine hydrate. Finally, the resin was cleaved with 5% triisopropylsilane / trifluoroacetic acid, and the pure glycopeptide 1 was obtained by reverse-phase high performance liquid chromatography. The overall yie...
Embodiment 3
[0056] Example 3 Glycopeptide and carrier protein cross-linking
[0057] Use Sulfo-GMBS as a cross-linking agent to cross-link the carrier protein BSA with the sulfhydryl group at the N-terminal of the glycopeptide. The specific method is as follows: dissolve 20 mg of BSA in 2 ml of 10 mM K 2 HPO 4 -KH 2 PO 4 Add 420ul MBS (3mg / ml) to the buffer solution, and react for half an hour; dissolve 20mg 1 in 1ml water, slowly drop into the BSA solution, adjust the pH to 7.4, react at room temperature for 2 hours, dialyze with PBS, and set aside.
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