Diffusion-Enhanced Amide Bond Formation on a Solid Support

Abstract

Mixing of polystyrene resins in solid-phase synthesis is performed by shaking or gentle agitation of the reaction vessel to avoid breaking the brittle beads. These mixing strategies result in poor diffusion to and into the beads. Using a large excess of reagents is the common way to compensate for these deficiencies. We use fast overhead stirring for performing coupling reactions on a solid support. We show that fast overhead stirring enhances the efficiency of amide bond formation on the solid support compared to the state-of-the-art mixing method, while preserving the integrity of the beads. We find that fast overhead stirring minimizes the effect of decomposition of the activated species by increasing the diffusion-dependent coupling reaction. This allows decreasing the excess of reagents used for the multistep synthesis of peptides, thus providing a greener and more sustainable alternative for peptide synthesis on solid supports.

Stirring Peptide Synthesis to a New Level of Efficiency

“…This is in line with our previous observation that high shear stirring and long reaction times lead to a high purity. 4 Furthermore, the result proves that given enough time, an almost complete conversion is achieved in each step when using HSS-SPPS even while using low concentrations of reagents. The crude purity of peptide-a synthesized via Route-2 (30 sec reactions, 30 °C) was 91% (Fig.…”

“…1 SPPS, like other processes on insoluble support, is diffusion-dependent. [4][5][6] The solid phase process is heterogeneous, hence it obeys Ficks' second law of diffusion (Equation 1). 7,8 Eq.…”

“…Therefore, the conditions that influence the SPPS reaction efficiency are 1) the initial concentration of reagents; [10][11][12] 2) reaction time; [12][13][14] 3) temperature; 15,16 and 4) the mixing method. 2,4,5 Increased reaction durations, high reagent concentrations, and elevation of reaction temperature are commonly applied to accelerate reactions on a solid support. 12,17 However, the effect of mixing efficiency on SPPS processes is not commonly discussed.…”

“…12,17 However, the effect of mixing efficiency on SPPS processes is not commonly discussed. 4,5,16 To perform an efficient SPPS process, one needs to consider all conditions that can affect the parameters related to Fick's second law of diffusion (Equation 1), including mixing, which is setup-dependent.…”

“…We recently showed that fast overhead stirring does not break the polystyrene beads, confirming that such setups are viable for SPPS. 4,5 High sheer stirring SPPS (Fig 1A , HSS-SPPS) enabled decreasing the concentration of reagents used for both Fmoc deprotection and amino acid coupling steps, albeit with extended reaction times suggesting that improved mixing by itself is not enough for accelerating SPPS. 4,5 After realizing the effect of both mixing (HSS-SPPS) and temperature (Mw-SPPS and AFPS), we hypothesized that their combination should enable accelerated SPPS even at low concentrations by maximizing the effect of diffusion.…”