General steps:
4.2 General Procedure A: Resin Loading
Solid phase peptide synthesis was performed manually in a sintered polypropylene syringe. The 2-chlorotrityl chloride (CTC) resin was pre-immersed in dichloromethane (DCM) for 15 minutes and drained. The first amino acid in a DCM solution of 0.4 M N,N-diisopropylethylamine (DIPEA) was added and the mixture was stirred for 3 hours. After draining the solvent, the resin was treated with a 17:2:1 DCM/methanol (MeOH)/DIPEA (3 × 3 mL × 5 min) solution, and then any free 2-CTC resin joints were blocked with an 8:1:1 N,N-dimethylformamide (DMF)/DIPEA/acetic anhydride (2 × 3 mL × 10 min) solution. The resin was finally washed with DCM (2 × 3 mL × 1 min), DMF (2 × 3 mL × 1 min), DCM (2 × 3 mL × 1 min), and DMF (2 × 3 mL × 1 min).
4.3 General method B: Fmoc deprotection
The resin was stirred with a 10% piperidine solution of DMF (2 x 3mL x 3 min) and subsequently washed with DMF (3 x 3mL x 1 min), DCM (3 x 3mL x 1 min), and DMF (5 x 3mL x 1 min). The deprotected solutions were combined and diluted appropriately (100-fold, 0.05 mmol resin loading). Resin loading was estimated by measuring the absorbance of piperidine-fulvene adduct with 10% piperidine in DMF as reference (λ = 301 nm; ε = 7800 M-1cm-1).
4.4 Generalized method C: peptide coupling with HBTUs
Solutions of amino acids protected by appropriate Fmoc (3 eq. relative to resin loading) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (2.9 eq. relative to resin loading) were prepared in minimal amounts of DMF. DIPEA (6 equivalents relative to resin loading) was added and the resin was stirred for 1.5 hours. The resin was then drained and washed with DMF (3 x 3 mL x 1 min), DCM (3 x 3 mL x 1 min) and DMF (5 x 3 mL x 1 min).
4.5 Generalized Method D: Peptide Coupling with HATUs
Solutions of amino acids protected by appropriate Fmoc (3 equivalents relative to resin loading) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (2.9 equivalents relative to resin loading) were prepared in minimal DMF. DIPEA (6 equiv. vs. resin loading) was added to the solution and the mixture was immediately added to the resin and stirred. The reaction time was varied based on the coupled residues: Phe (NMe) and Ala (2 × 2 h); Thr and Sta (1 × 2 h); Asn, Leu, D-Val, and L-Val (2 × 2 h); and DMVal (3 × 3 h). Once the reaction was complete, the resin was drained and washed with DMF (3 × 3 mL × 1 min), DCM (3 × 3 mL × 1 min), and DMF (3 × 3 mL × 1 min).
4.6 Generalized Method E: Peptide Coupling to DICs
A solution of amino acids protected by appropriate Fmoc (1.5 equivalents relative to resin loading), 1-hydroxybenzotriazole (HOBt) (1.5 equivalents relative to resin loading), and N,N'-diisopropylcarbodiimide (DIC) (1.5 equivalents relative to resin loading) in minimal DMF was prepared. The solution was stirred for 20 minutes, then added to the resin and stirred overnight. The resin was drained and washed with DMF (3 × 3 mL × 1 min), DCM (3 × 3 mL × 1 min), and DMF (5 × 3 mL × 1 min). Application of coupled fluorinated amino acids followed by double coupling of the next amino acid.
4.7 Generalized Procedure F: Resin Cleavage
After final Fmoc deprotection, the resin was washed with DMF (3 x 3mL x 1 min) and DCM (3 x 3mL x 1 min) and then vacuum dried. The resin was stirred with 95:2.5:2.5 trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/H2O (3 mL) solution for 2 hours. The resin was drained and washed with the same TFA mixture described above (2 × 3 mL × 1 min). The combined lysis solutions were concentrated under a stream of nitrogen. Ether was added and the supernatant was decanted off (3×). The residue was then dried under vacuum to give a crude linear peptide.
