Optimized Synthesis of Isotopically Labeled M2TM

Due to the requirements for a large quantity of isotopically labeled peptides and the hydrophobic nature of M2TM, we developed an optimized procedure that delivers crude peptide with >80% purity. Problems encountered in obtaining high-yields and purity included aspartamide formation at residue 44 and slow coupling near the center of the chain. M2TM(22–46) with uniformly ¹³C, ¹⁵N-labeled V27, A30 and G34 (VAG-M2TM) was synthesized using Fmoc chemistry at elevated temperature (75°C for both coupling and deprotection) in a semiautomated Quest synthesizer using Rink Amide Chemmatrix resin (Matrix Innovation Inc, Canada). Coupling reagent were 5 eq amino acid, 5 eq HCTU, 10 eq DIEA in NMP for 5 mins coupling. 5% piperazine and 0.1 M HOBt in DMF were used as the deprotection solution in order to minimize aspartamide formation. The peptide was cleaved from the resin using 95% TFA, 2.5% Tris, 2.5% H₂O and precipitated from ether after removal of TFA. Ether was decanted after centrifugation and the peptide was washed with cold ether again. The final peptide was dissolved in 50% B′ (59.9% isopropanol, 30% acetonitrile, 10% H₂O, and 0.1% TFA) and 50% A (99.9% H₂O, 0.1% TFA) and purified by preparative C4 reverse phase HPLC with a linear gradient of 70% B′ to 85% B′. The peptide was eluted at 78% B′. The purity and identify of the peptide was confirmed by analytical HPLC (>98% purity) and MALDI-MS. Calculated MS: 2782.38, Observed MS: 2782.90.