Figure 1.

Synthetic schemes of HFPmn, HFPdm, HFPtr, and HFPte. FP ≡ AVGIGALFLGFLGAAGSTMGARS. A black circle represents a resin bead, lines are drawn to clarify chemical functionalities, an arrow signifies a chemical reaction, and two arrows signify multiple sequential chemical reactions. All reactions were carried out at ambient temperature. The specific reactions are described as follows. Reaction a: Fmoc deprotection in 3 mL of 20% piperidine/DMF (v/v), 15 minutes/cycle, 2 cycles. Reaction b: Peptide synthesis with Fmoc chemistry. For HFPmn syntheses, 2-hour single couplings were used for each amino acid with the following exceptions: 4-hour single couplings for Trp, Ser and Arg residues; 6-hour single couplings for the Leu-12 to Leu-7 residues; double coupling with 3-hours per coupling for the ¹³CO labeled residue. For the HFPdm(Cys) synthesis, 4-hour single couplings were used for each amino acid with the following exceptions: 8-hour single couplings for Trp, Ser, and Arg residues; double couplings with 4-hours per coupling for the ¹³CO labeled residue and for the Leu-12 to Leu-7 residues. Reaction c: Cleavage from the resin using a 4 mL solution containing TFA/thioanisole/ethanedithiol/anisole in 90:5:3:2 volume ratio. After 2.5 hours reaction time, TFA was removed with nitrogen gas and peptide was precipitated with cold methyl t-butyl ether. Reaction d: Coupling using PyAOP and DIPEA (1:2 molar ratio) in 4 mL DMF with 6 hour reaction times for Cys and 2 hour reaction time for Lys. Reaction e: Cross-linking in 5 mM DMAP, pH = 8.4, open to the air. HFPdm or HFPte conditions: 1 µmol HFPmn(Cys) or HFPdm(Cys) in 400 µL solution overnight. HFPtr conditions: 1 µmol HFPmn(Cys) and 1.5 µmol HFPdm(Cys) in 400 µL solution for 2.5 hours. Reaction f: Selective deprotection of Mtt in 3 mL of 1% TFA/DCM (v/v), 6 minutes/cycle, 6 cycles.