General protocol for post-synthetic phosphorylation

The use of DMF is preferred to acetonitrile in the phosphinylation step, since tetrazole is far more soluble in this solvent, enabling the reaction to be conducted at a much higher concentration. Anhydrous DMF is commercially available stored under nitrogen in bottles fitted with a septum. 

Only resublimed DNA-grade tetrazole should be used this is available from Sigma in septum-sealed, argon-filled vials containing 300 mg of material, sufficient for carrying out a 0.1-mmol-scale reaction. Dibenzyl-Af,N-diisopropylphosphoramidite is available in argon-filled one-shot vials from Novabiochem. 

Place the peptidyl resin 0.1 mmol) in the peptide synthesis vessel and dry overnight at 40°C in vacuo. 

Seal the reaction vessel with a rubber septum. 

Open the tap and flush the vessel with a stream of argon delivered via a needle inserted through the septum. Close tap. 

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