Reagents
| Reagent | Role | Amount |
|---|---|---|
| Fmoc-amino acid | Acyl donor | 3.0 eq |
| DMTMM·BF₄ | Coupling reagent | 3.0 eq |
| NMM (N-methylmorpholine) | Base | 6.0 eq |
Conditions
- Solvent
- NMP or DMF/NMP mixtures (DMTMM·BF₄ has limited solubility in pure DMF)
- Temperature
- 20–25 °C (room temperature)
- Reaction time
- 30–60 min per coupling
- Atmosphere
- Ambient (no inert gas required)
Procedure
- Swell resin in DMF (5 mL/g resin) for 15 min. Drain.
- Deprotect Fmoc group: treat with 20% piperidine/DMF (2 × 5 min). Wash resin with DMF (5×).
- Dissolve Fmoc-amino acid (3.0 eq) and DMTMM·BF₄ (3.0 eq) in minimum DMF.
- Add NMM (6.0 eq) to the solution. Mix briefly to ensure dissolution.
- Add activated solution to drained resin. Agitate (shaking or N₂ bubbling) for 30–60 min.
- Drain coupling solution. Wash resin with DMF (5×).
- Monitor coupling completion by Kaiser test (ninhydrin) or chloranil test. Recouple if necessary.
- Repeat deprotection–coupling cycle for each residue.
Expected outcome
Coupling efficiency comparable to PyBOP/HATU in published comparisons (Kamiński et al., 2005). Lower epimerization than HATU/HBTU + DIPEA when using NMM as base. Crude purity typically improved vs TBTU or PyBOP in automated synthesis.
Analytical monitoring
Kaiser/chloranil test after each coupling. HPLC and MS analysis of cleaved crude peptide.
Troubleshooting
- Incomplete coupling (positive Kaiser test)
- Recouple with fresh reagent solution. Extend reaction time to 90 min. For very hindered residues, increase to 5.0 eq reagent.
- Elevated epimerization
- If using DIPEA as base, switch to NMM or collidine. Reduce coupling temperature to 0–10 °C for sensitive residues.
- Poor resin swelling
- Pre-swell in DCM before switching to DMF. Ensure DMF is fresh and dry.
Notes & applicability
- Store DMTMM·BF₄ in a desiccator. Weigh quickly to minimize moisture exposure.
- DMTMM·BF₄ has limited solubility in pure DMF. Use NMP or DMF/NMP co-solvent mixtures. Verify dissolution before adding to resin.
- For fragment condensation, 1.5 eq reagent with extended reaction time (2–4 h) is often sufficient.
- Base selection is critical for epimerization control: NMM or collidine are preferred. Epimerization advantages over uronium reagents were demonstrated in specific model systems (Kamiński et al., 2005).
- Compatible with standard Fmoc/tBu protection strategy.