1. Preparation of Fmoc-Ser(tBu)-WangResin
Put 1.71g Wang Resin into the reaction column, wash it once with DMF, and drain it; use an appropriate amount of dry dichloromethane to swell at room temperature, blow and stir with N2 for 30 minutes until the resin is fully swollen, and drain the liquid; wash it once with DMF, and drain it; re-evaporate DMF for washing Once, drain. Add 5ml of DMF/DCM (1:1) solution of 3.07gFmoc-Gly-OH, 1.30gHOBt, and 1.49ml DIC. After reacting for 5 minutes, 0.10 g of DMAP was added and reacted for 4 hours. After the reaction, the reaction solution was drained, washed with DMF three times and drained, and a little resin was taken out for substitution degree detection. Then add 200ml of blocking reagent (AC2O/pyridine (7:6, V:V)), react for 8-16h, wash twice with DMF; wash twice with anhydrous methanol (10min/time), and dry under vacuum to constant weight. The degree of substitution of Fmoc-Ser(tBu)-WangResin was measured to be 0.71mmol/g.
2. Preparation of Matrixyl-WangResin
1) In the Fmoc-Ser(tBu)-WangResin reaction column, add DCM to swell for 20-30min, then pump it off. Add DBLK solution (piperidine/DMF (V/V)=1:4) for deprotection twice (5+10min), add DMF to wash for 1min between two deprotections, and remove. After taking off the protection twice, wash it with DMF, methanol, DCM, and DMF once each, pick a little resin with a glass rod, check it with indene, and it turns dark purple, indicating that Fmoc has been removed and is ready to be fed. 2) In the process of deprotection, weigh 0.93g Fmoc-Lys(Boc)-OH and 0.81gHOBt into a beaker, add 2ml of refined DMF to dissolve, after the complete dissolution, put the beaker in an ice bath to cool for 2min, and then Add 0.93ml DIC to activate the solution for 8 minutes. If white flocculent foam appears, the activation is complete. Use a Buchner funnel to filter out the small white solid particles in the activation solution, pour the obtained clear filtrate into the reaction column, and then add DMF In the reaction column, adjust the N2 so that the resin is evenly blown up, put on a drying tube, and keep no wall hanging phenomenon during the reaction process, if there is wall hanging, use a little refined DMF to flush the resin on the wall to in the reaction solution. React at room temperature for 2 hours. After passing the indene test, the reaction solution can be removed and the next amino acid can be connected.
3. Cleavage of Matrixyl-WangResin
Put 0.10 g of Matrixyl Wang Resin dried to constant weight in a glass flask, add 4 ml of TFA-sulfide anisole-anisole-EDT (volume ratio 95:5:3:2) under stirring in an ice bath, react for 30 minutes, and gradually rise to to room temperature, reacted at room temperature for 2h. Filter, wash the resin with a little TFA, pour the filtrate into 40ml of anhydrous glacial ether, precipitate a white solid, centrifuge at 4000rpm for 4min, discard the supernatant, repeat twice, and dry in vacuum to obtain 46.6mg of crude peptide.
4. Analysis and identification of Matrixyl crude peptide
The crude Matrixyl was analyzed by RPHPLC. The HPLC-ESI-MS analysis of the purified Matrixyl showed that its m/z was 402.3{[M+H]2+}, and the molecular weight of Matrixyl was 802.05. The theoretical value was consistent with the actual value.
