Antifreeze glycopeptide diastereomers

Abstract

Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4-55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments.

Keywords: bioorganic chemistry; circular dichroism; glycopeptides; ice recrystallization; microwave chemistry.

Scheme 1

Synthesis of the monosaccharide-substituted threonine building block (A = allo-L-threonine; B = D-threonine). (a) Ag₂CO₃, AgClO₄, CH₂Cl₂/toluene (1:1), rt, 40 h, 68% (A) and 67% (B); (b) AcSH/pyridine (2:1), toluene, 50 °C, 30 min, 56% (A) and 42% (B); (c) TFA/H₂O (95:5), 90 min, 96% (A) and 90% (B).

Scheme 2

Structures of monosaccharide-substituted AFGP analogues containing allo-L-threonine and L-alanine 5, the corresponding aglycon 6, the L-glycopeptide comprising D-threonine and D-alanine 7, and the aglycon 8.

Figure 1

CD spectra of the monosaccharide-substituted AFGP analogues (5 and 7) and their corresponding aglycons (6 and 8) measured at a concentration of 0.2 mg mL⁻¹ in water at 20 °C. For comparison, the spectra of monosaccharide-substituted glycopeptide with naturally occurring sequence 9 and its correlate aglycon 10 are shown [16]. Capital letters symbolize L-amino acids (A = L-Ala, T = L-Thr), lowercase letters D-amino acids (a = D-Ala, t = D-Thr).

Figure 2

(a) Temperature dependent CD spectra of the glycosylated allo-L-Thr containing peptide 5 from −10 to 80 °C in water, revealing an isodichroic point at 207 nm; and (b) the difference spectra between +80 and −10 °C, indicating the contribution of a β-like structure.

Figure 3

(a) Temperature-dependent CD spectra of the aglycon comprising allo-L-Thr 6 from −10 to 80 °C in water, revealing an isodichroic point at 209 nm; and (b) the difference spectra between +80 and −10 °C indicating the contribution of a β-like structure.

Figure 4

(a) Temperature-dependent CD spectra of the glycosylated D-Thr and D-Ala containing peptide 7 from −10 to 80 °C in water, revealing an isodichroic point at 203 nm; and (b) the difference spectra between +80 and −10 °C.

Figure 5

Optical microphotographs taken after 0 and 120 min during the recrystallization process of polycrystalline ice samples at −8 °C formed in aqueous 45 wt % sucrose solutions. (a) Negative control solution without peptides. (b) Peptide 5 (c 1000 µg mL⁻¹). (c) Peptide 7 (c 1000 µg mL⁻¹). (d) Positive control solution containing 200 µg mL⁻¹ of the ice recrystallization inhibiting peptide 9 ([AAT(GalNAc)]₄AA). The pictures were contrast-enhanced for better visibility.