Antifreeze glycoproteins [AFGPs: {Ala-Thr(Galβ1-3GalNAcα)-Ala}_n, n=4-50] in the serum of polar fishes have been known to depress the freezing point of their blood and help them to survive at the temperature below-1.9℃[1]. Though it is widely accepted that AFGPs bind to the ice surface and prevent its growth, the definitive molecular mechanism of the action has not been solved yet. In this study, structure-activity relationships in AFGPs were studied toward understanding of the mechanism, thorough chemical syntheses, activity evaluations, and conformational analyses of the AFGP and its analogues, in which sugar or peptide moieties were different from those of the wild-type AFGP. The AFGP (1) and its analogues (2-8) were successfully synthesized via simple polymerization of their repeating unit using a DPPA method without any protection of sugar -OH groups [2]. Additionally, sialyl T-bearing glycopolypeptide (9) was successfully prepared through enzymatic elongation of a sialic acid residue to C-3 position of the galactose residue in the AFGP (1), indicating the broad applicability of this methodology for the syntheses of various glycopeptide polymers. In the evaluation assay of the antifreeze activity, artificial AFGP (1) showed similar activity as that of the wild-type AFGP. On the contrary, neither an AFGP repeating unit glycopeptide (19) nor a sugar-lacked polypeptide (poly-ATA, 8) shows the activity. Interestingly, AFGP analogs having GalNAc (2) or LacNAc (3) as sugar moiety showed similar activities as AFGP, while that of Galactose (4), Lactose (5), β-O-linked (6), and ASA (7) did not. These results clearly indicate the importance of i) repeating structure of glycopeptide, ii) NHAc group at C-2 position of the sugar moiety directly attached to the peptide, iii) α-glycosidic linkage between the sugar and the threonyl residue, for the specific interaction with ice. Furthermore, secondary structural analyses of the synthetic compounds were carried out by means of a CD spectroscopic method, in order to clarify the relationships between their conformations and their activities. The CD spectra of active compounds (1-3) were obviously different from those of inactive analogues (4-8), suggesting the presence of an ordered structure, but not α-helix.
