Novel AFGP8 analogues: Synthesis and biological evaluation of potent recrystallization inhibitors

Abstract

This dissertation describes the study of native and synthetic antifreeze glycoproteins (AFGPs). Antifreeze glycoproteins (AFGPs) are biological antifreezes found in several species of Atlantic and Antarctic teleost fishes. These compounds have the ability to inhibit the growth of ice and protect organisms from cryoinjury and death. They exhibit two main physical properties associated with antifreeze activity. First, they possess the ability to create a non-colligative depression of the freezing point below that of the melting point; this phenomenon is referred to as thermal hysteresis (TH). Second, they possess the ability to inhibit the growth of ice, referred to as recrystallization inhibition (RI). Consequently the rational design and de novo synthesis of chemically and biologically stable AFGP analogues have become an attractive challenge. These properties have made AFGPs of great interest for medical, industrial and commercial applications. AFGPs molecules have been thoroughly studied in order to understand their molecular mechanism of action. It is believed that conformation of these compounds plays an essential function during the ice adsorption process which is required for antifreeze activity. While several conformation studies of AFGP8 molecules have been published using different spectroscopic methods, an accepted conformation has failed to emerge and its role on antifreeze activity needs to be addressed. Chapter 3 examines the solution aggregation of AFGP8 using dynamic light scattering (DLS) and investigates if this phenomenon have any influence on the conformations of AFGP8 using circular dichroism (CD). Although structure activity relationship (SAR) studies seem to be one of the most appropriate tools to understand the molecular mechanism of action of AFGPs, only few syntheses of AFGPs analogues, have been reported in the literature. Amongst these publications, even fewer have tested their analogues for thermal hysteresis activity and none have tested them for recrystallization inhibition. Recrystallization inhibition might be considered as the most relevant activity for cell storage at sub-zero temperatures, when dynamic ice shaping (DIS) and thermal hysteresis (TH) potentially damage cell membranes. Recently, it has been demonstrated that there is no correlation between these two activities. However, the required structural features of AFGPs for RI, TH and DIS activities are still to be elucidated. Using O-linked glycopeptide analogues, the required structural feature for TH and RI activity are investigated in Chapter 4. Although AFPG8 displays attractive RI activity, it also exhibits undesired properties for cryopreservation. For instance, it favours the formation of needle shape ice crystals below the TH gap. Furthermore, AFGPs molecules are not truly stable under basic, acid or enzymatic conditions due to their exocyclic oxygen. Consequently, chapter 5 describes the synthesis and study of stable C-linked analogues in order to obtain better cryoprotectants with potent RI activity and no DIS activity.