Diffusion Enhanced Amide Bond Formation on Solid Support

Abstract

Performing amino acids coupling reactions on solid support using fast overhead stirring is far more efficient than the common shaking mixing methods. Stirring does not harm the polystyrene beads and allows to decrease dramatically the amount of reagents used and wasted in these transformations.

Stirring Peptide Synthesis to a New Level of Efficiency

“…14,15,25,26 While peptide synthesis can be performed with 1.5 or 1.1 equiv, using such concentrations requires extending the reaction time, which is not suitable for accelerated synthesis. 9,38 Interestingly, although the sequence contains two aspartic acids and the process was performed at high temperatures, 26 no significant aspartimide formation was observed in liquid chromatography-mass spectrometry (LC-MS).…”

“…The HTFSPS of the pentapeptide H 2 N-His-Phe-Gly-Trp-Ile (Figure 1, HFGWI), which includes a notoriously hard coupling of Fmoc-L-His(Trt)-OH amino acid to the tetrapeptide, 9 was performed under Route 3 conditions. HPLC analysis showed an overall 73% crude purity (SI Section 4.4).…”

“…Therefore, the conditions that influence the SPPS reaction efficiency are (1) the initial concentration of reagents; 19−21 (2) reaction time; 21−23 (3) temperature; 24,25 and (4) the mixing method. 2,9,10 High molar excess (3−60 equiv) and elevation of reaction temperature (75−90 °C) are commonly used to accelerate reactions on the solid support. 21,26 However, the effect of mixing efficiency on SPPS processes is rarely discussed.…”

“…21,26 However, the effect of mixing efficiency on SPPS processes is rarely discussed. 5,9,10,25 To perform an efficient SPPS process, one needs to consider all conditions that can affect the diffusion according to Fick's second law (eq 1), including mixing, which is setup-dependent. Different setups are employed for SPPS processes (Figure 1A).…”

“…We recently showed that fast overhead stirring does not break polystyrene beads, confirming that such setups are viable for laboratory-scale SPPS. 9,10 High shear stirring SPPS (Figure 1A, HSS-SPPS) enabled decreasing the molar excess of reagents used for both Fmoc deprotection and coupling to almost equimolar ratio, albeit at an extended reaction time, suggesting that improved mixing by itself is not enough for accelerating SPPS. 9,10 While heated and stirred reactors are used in the industrial-scale production of peptides to improve synthesis efficiency, low to medium mixing rates and extended reaction durations are used to enable sufficient mass and heat transfer.…”