Fmoc-Lys(Mtt)-Wang resin (200-400 mesh, 0.5-0.8 mmol/g)

Synthesis of GGGKDex from Fmoc-Lys(Mtt)-Wang Resin^[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Resin initiation, swelling, and loading: Place the Fmoc-Lys(Mtt)-Wang resin into a solid-phase synthesis reaction vessel/reactor. Allow the resin to fully swell under infiltration conditions in N-methylpyrrolidone (NMP; containing 25% piperidine) or dichloromethane (containing 2% trifluoroacetic acid; TFA). Drain the solvent and set aside for subsequent steps.
2. Peptide chain elongation: Using 5 equiv of Fmoc-Gly-OH, 5 equiv of PyClock, and 10 equiv of DIEA in NMP, perform microwave-accelerated coupling on a microwave peptide synthesizer to sequentially graft Fmoc-Gly residues, yielding Fmoc-GGGK(Mtt)-resin (at this stage, the C-terminus is a Wang-ester-anchored lysine, with its ε-NH still protected by Mtt). Treat the resin with 25% piperidine in NMP, drain, and wash thoroughly with NMP followed by DCM before proceeding to the next coupling or operation. Repeat this procedure until the triglycine sequence GGG is fully assembled at the N-terminus of the peptide chain, obtaining Fmoc-GGGK(Mtt)-resin (N-terminus of the main chain: Fmoc-Gly-Gly-Gly-; side chain still: –NH-Mtt).
3. Selective Mtt deprotection and side-chain conjugation with SDex: Treat the Fmoc-GGGK(Mtt)-resin with 2% TFA in DCM and incubate for 20min to selectively remove the Mtt (4-methyltrityl) group from the lysine ε-position. Subsequently, continue washing the resin with 2% TFA in DCM until the Mtt is essentially completely removed. Mix the above resin with 2.5 equiv of SDex-COOH + 2.5 equiv of HATU + 2.5 equiv of HOAt + 5 equiv of DIEA + 5 equiv of 2,6-lutidine in 2.5mL of NMP. Place on an orbital shaker at room temperature overnight to complete the amidation labeling of SDex onto the side-chain ε-NH₂, yielding Fmoc-GGGKDex-resin. After the reaction, wash the resin thoroughly with NMP and DCM.
4. N-terminal final processing, resin cleavage, and product purification: Treat the Fmoc-GGGKDex-resin again with 25% piperidine in NMP to remove the N-terminal Fmoc group. Immerse the resin in cleavage cocktail (81.5% TFA / 5% thioanisole / 5% phenol / 5% H₂O / 2.5% ethanedithiol / 1% TIS) and incubate at 38°C for 30min, simultaneously cleaving the Wang-ester bond and removing all acid-labile side-chain protecting groups. Discharge/add the cleavage solution into 40mL of ice-cold diethyl ether to precipitate the crude peptide. After centrifugation/separation, resuspend the precipitate in water and lyophilize to obtain the crude peptide. Purify using a VYDAC C18 column (250mm × 10mm ID) with a linear gradient of acetonitrile/water (containing 0.1% TFA). Collect the target fraction and lyophilize to obtain the purified product GGGKDex.
Precautions:
(1) Mtt deprotection must be strictly controlled at a low TFA concentration.
(2) The 81.5% TFA final cleavage system contains highly corrosive TFA along with sulfur-containing/phenolic scavengers; operations must be performed in a fume hood with a face shield, acid-resistant gloves, and a lab coat. Keep the diethyl ether precipitation step away from open flames and heat sources. Organic waste containing TFA/aromatic fragments should be collected and disposed of separately as strong-acid halogenated organic waste.
(3) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Li M, Tao Y, Shu Y, et al. Discovery and characterization of a peptide that enhances endosomal escape of delivered proteins in vitro and in vivo. J Am Chem Soc. 2015 Nov 11;137(44):14084-93.