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Synthesizing hydrophobic peptides is often difficult.  Inter-chain hydrogen bonding in the hydrophobic regions causes poor solvation of the peptide chains and slowed reaction rates.  Measures to disrupt this hydrogen bonding often significantly improve the purity and yield of the crude peptide.

Heating disrupts the hydrogen bonding between peptide chains leading to increased reaction rates in addition to rate acceleration normally observed due to increased temperature.  Microwave heating directly increases the vibration of molecules within the solution resulting in rapid temperature increase.  The rapid temperature increase can lead to overshooting the desired temperature.  Microwave heating (Figure 1A) accelerates peptide synthesis and improves the purity of crude peptides.  Rapid conventional heating (Figure 1B), although slower within a minute of microwave heating, produces nearly identical results as microwave heating in improving the purity and yield of crude “difficult” peptides.6  Heating, either conventionally or by microwaves, also accelerates the racemization of the easily-racemized cysteine and histidine residues.

 

Temperature Profiles of Microwave, Conventional, Gradient and Delayed Gradient Heating for the Coupling Reaction

Mcrowave heating         gradient heating      delayed heating gradient

Fig. 1A                                     Fig. 1B                                 Fig. 1C                                  Fig. 1D

Slower heating on a temperature gradient (Figure 1C and 1D) also improved the yield and purity of crude peptides but with less racemization of cysteine.  Peptide chains on the surface of the resin beads are exposed to the reaction mixture and react rapidly at room temperature or with mild heating while racemization is slow.  Usually 80-90% of coupling to reactive sites occurs within the first 2 to 4 minutes of reaction time.  Heating is only required to accelerate the reactions at the remaining sites.  If heating causes 4% racemization, then rapid heating, as with microwaves, will result in approximately 4% racemization in nearly all of the peptide.  With gradient heating, racemization affects only the 10-20% of peptide chains that had not already reacted at lower temperature.  This results in a reduction in diastereomeric impurities by 80-90%.

With microwave and conventional heating, the accelerated racemization of cysteine is competitive with the coupling rate leading to increased formation of diastereomeric impurities.  In gradient heating, the majority of the peptide chains react at lower temperatures where the racemization reactions are slow and less diasteriomeric impurities are formed.  Advancing on this finding, we allowed the coupling to proceed at room temperature for 2 minutes before starting the temperature gradient (Figure 1D).  With this delayed gradient procedure, the yield and purity of the crude peptides was maximized and the racemization of cysteine residues was reduced.