Labeled peptides and wang resin peptide synthesis are often used by researchers in binding studies, substrate specificity determination, and receptor cross-linking inquiries. A new method based on Rink amide 4-methylbenzhydrylamine resin combined with Fmoc-Lys(Dde)-OH is proposed. The main advantage of this method is that additional labels, such as fluorescein, methyl coumarin, dansyl groups, and possibly fluorophores and quenchers utilized in fluorescence resonance energy transfer (FRET), can be directly inserted into peptides.
Two ways can produce labeled peptides in two ways: The peptide can be synthesized using labeled amino acids. Even the label can be added after the solid phase peptide synthesis resins has been generated.
For the direct synthesis of this sequence, Fmoc-Lys(biotin)-OH is an ideal choice. Use a Rink amide MBHA resin paired with Fmoc-Lys(Dde)-OH to preserve the side chain of a lysine residue orthogonal to Fmoc/t-Bu. In this method, the peptide is first synthesized, then the label is applied for coupling to the Lys, followed by cleavage and deprotection.

Steps of Peptide Synthesis

1. HBTU/HOBt/DIEA activation is used to link rink amide MBHA resin with Fmoc-Lys(Dde)-OH.
2. Except for the N-terminal His, all other residues are integrated as Fmoc-amino acids using the same activation chemistry, with Boc for Lys and OtBu for Glu serving as side-chain protecting groups.
3. t-Boc-His incorporates the N-terminal His. The resin is rinsed twice with N, N-dimethylformamide once the synthesis is completed.
4. The Dde side-chain protection is eliminated by treating the protected peptide resin with 2 percent hydrazine in DMF (7 mL/0.1 moles; 2 times, 5 minutes each).
5. The resin is washed repeatedly with DMF before being washed twice with DMF: dimethyl sulfoxide (DMSO; 1:1, v/v).
6. In 5 mL of DMF: DMSO, a 10-fold molar concentration of biotin is dissolved (1:1). To completely dissolve the biotin, the mixture must be warmed and vortexed for several minutes.
7. After that, 2.1 mL of 0.45-M HBTU/HOBt in DMF is added to the biotin solution, preceded by 0.3 mL of diisopropylethylamine (DIEA).
8. The resin is mixed with the activated biotin solution overnight.
9. The resin is washed three times with DMF: DMSO (1:1), then with dichloromethane: methanol (1:1; 2 times).
10. After adequately drying the resin, the peptide is cleaved, then deprotected with Reagent-K.