CRITICAL The C-terminal amino acid should be a glycine (or an amino acid or linker that is not chiral on the carbon adjacent to the carboxyl) to avoid racemisation during coupling to the azido or alkyne amines.
Trifluoroacetyl protection. Synthesis of compound 2
1] Add 25 mg of peptide PKKKRKVG (1) to 10mL round-bottomed flask containing a Teflon-coated magnetic stirrer bar.
2] Transfer 5 mL of methanol to the flask.
3] Put the flask to the ice-bath and turn on stirring.
4] Add dropwise, 0.3 mL of ethyl trifluoroacetate (by use of a 0.5-1 mL syringe).
5] Add 0.1 mL of triethylamine dropwise (by use of a 0.5-1 mL syringe).
6] Close the flask with the glass stopper and keep stirring for a total of 3 days.
7] Remove the solvent under reduced pressure on a rotatory evaporator.
8] Purify and analyze products by HPLC at 50 ºC on a Vydac RP-C18 column (218TP510, 10x250 mm,) using a flow rate of 4.0 mL/min and first a linear gradient from 15% to 35% aqueous acetonitrile in 40 minutes followed by 35 to 70% acetonitrile in 10 minutes (with 0.1 % trifluoroacetic acid in all solutions).
Synthesis of TFA protected clickable peptides 3a or 3b
1] Add 0.18mg HBTU to a 1.5 mL eppendorf tube.
2] Add 0.43mg N-methylmorpholine to the tube.
3] Add 25 μL DMF to the tube.
4] Add 3 mg of peptide 2 to the tube.
5] Close the tube and seal it.
6] Keep the tube shaking on a vortex for 30 min.
7] Add 25 μL of a THF solution (made from stock) containing 2-azidoethylamine (0.18 mg) or propargylamine (0.12 mg) using an auto pipette.
8] Keep shaking for 2h.
9] Lyophilize the solution.
10] Dissolved the crude mixture in the HPLC starting buffer.
11] Purify and analyze products by HPLC at 50 ºC on a Vydac RP-C18 column (218TP510, 10x250 mm,) using a flow rate of 4.0 mL/min and first a linear gradient from 15% to 35% aqueous acetonitrile in 40 minutes followed by 35 to 70% acetonitrile in 10 minutes (with 0.1 % trifluoroacetic acid in all solutions). Fractions containing product were collected and the acetonitrile was removed under reduced pressure whereupon the remaining solution was lyophilized.
Synthesis of unprotected clickable peptides 4a or 4b
1] Add 0.18 mg of HBTU to a 1.5 mL eppendorf tube.
2] Add 0.43 mg N-methylmorpholine to the tube.
3] Add 25 mL DMF to the tube.
4] Add 3 mg of peptide 2 to the tube.
5] Close the tube and seal it.
6] Keep the tube shaking on a vortex for 30 min.
7] Add 25 mL of a THF solution (made from stock) containing 2-azidoethylamine (0.18 mg) or propargylamine (0.12 mg) using an automated pipette.
8] Keep shaking for 2h.
9] Transfer the solution to a 10mL glass vial.
10] Add 2 mL of conc. ammonia using a disposable syringe.
11] Seal the glass vial and keep at 55 ºC overnight.
12] Lyophilize the crude peptide mixture.
13] Purify and analyze products by HPLC at 50 ºC on a Vydac RP-C18 column (218TP510, 10x250 mm,) using a flow rate of 4.0 mL/min and first a linear gradient from 15% to 35% aqueous acetonitrile in 40 minutes followed by 35 to 70% acetonitrile in 10 minutes (with 0.1 % trifluoroacetic acid in all solutions). Fractions containing product were collected and the acetonitrile was removed under reduced pressure whereupon the remaining solution was lyophilized.