Novel C-linked antifreeze glycoprotein (AFGP) analogues as potent recrystallization inhibitors: Preparation, assessment and in vitro studies

Abstract

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found primarily in the plasma of Teleost fish inhabiting in sub-zero temperature environments. These compounds have the ability to inhibit the growth and recrystallization of ice, thus ensuring the survival of these organisms in subzero environments. This characteristic property is highly desirable for medical and commercial applications. However, the limited bioavailability and the inherent instability of native AFGPs have precluded their commercialization. Consequently, rationally designed C-linked AFGP analogues are very attractive. This work describes the preparation of stable C-linked AFGP analogues and the evaluation of their antifreeze-specific activities. The in vitro behaviors of AFGP8 and C-linked AFGP analogues are also described. A new series of C-linked AFGP analogues was prepared to evaluate the functions of the length and rigidity of the side chain between the carbohydrate moiety and the polypeptide backbone, the acetamide group in the galactosamine residue, and the polypeptide backbone. C-linked AFGP analogues containing three and four methylene groups in the side chain were prepared using an olefin cross-metathesis (OCM) method. C-linked AFGP analogues containing a C-glycosylated L-serine residue were prepared using a catalytic asymmetric hydrogenation (CAH) strategy. A neoglycopolymer with the polymeric chain composed of alternating ethylene groups and cyclopentane rings was prepared using the ring-opening metathesis polymerization (ROMP). The new series of C-linked AFGP analogues was evaluated for their antifreeze-specific activities, including thermal hysteresis (TH) and recrystallization inhibition (RI). Although no TH activity was displayed in any analogues, some analogues demonstrated potent RI activity. These examples with tailored RI and TH activities suggest that it is possible to design AFGP analogues for different applications. The behaviors of AFGP8 and two C-linked AFGP analogues with potent RI activity were studied in vitro. High concentration AFGP8 demonstrated significant toxic effects to human liver and kidney cells at both physiological and cryogenic temperatures. In contrast, C-linked AFGP analogues did not show any cytotoxicity in human cell lines under the same conditions. A caspase-3/7 assay and an independent fluorescence apoptosis assay verified that the cytotoxicity caused by AFGP8 was due to the induction of apoptosis, while C-linked AFGP analogues could inhibit the activation of caspase 3 and 7. In addition, an internalization study demonstrated that both AFGP8 and the C-linked AFGP analogues shared the same cellular uptake mode and intracellular pathway. The cytotoxicity of AFGP8 may limit its applications in the biological systems. In contrast, our C-linked AFGP analogues are not cytotoxic and can inhibit the activation of caspase 3 and 7. In addition, since they have no TH activity, no detrimental ice spicules will be formed. Therefore, these C-linked AFGP analogues have an advantage for being used as cryoprotectants for cell preservation.