Influence of freeze-concentration effect on proteinase-catalysed peptide synthesis in frozen aqueous systems

Freezing of the reaction mixture is a powerful tool in proteinase-catalysed peptide synthesis. In this study, the considerable yield-increasing effect of freezing has been analysed by physical and analytical methods. 1H-NMR relaxation time measurements have been used to determine the amount of unfrozen water in partially frozen systems thus quantifying the extent of the 'freeze concentration effect' for the first time. Comparative studies in ice and at room temperature verify the importance of freeze-concentration which, however, is not sufficient for a complete understanding of the observed effects. Furthermore, the phase behaviour of frozen systems is discussed.