Synthesis and conformational preferences of short analogues of antifreeze glycopeptides (AFGP)

Antifreeze glycoproteins are a class of biological agents which enable living at temperatures below the freezing point of the body fluids. Antifreeze glycopeptides usually consist of repeating tripeptide unit (-Ala-Ala-Thr*-), glycosylated at the threonine side chain. However, on the microscopic level, the mechanism of action of these compounds remains unclear. As previous research has shown, antifreeze activity of antifreeze glycopeptides strongly relies on the overall conformation of the molecule as well an on the stereochemistry of amino acid residues. The desired monoglycosylated analogues with acetylated amino termini and the carboxy termini in form of N-methylamide have been synthesized. Conformational nuclear magnetic resonance (NMR) studies of the designed analogues have shown a strong influence of the stereochemistry of amino acid residues on the peptide chain stability, which could be connected to the antifreeze activity of these compounds. A better understanding of the mechanism of action of antifreeze glycopeptides would allow applying these materials, e.g., in food industry and biomedicine.


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Matrix Assisted Laser Desorption/Ionization -Time of Flight
Mass spectra were recorded on Voyager DE Instrument (PE Biosystems), using 2,5-dihydroxybenzoic acid as a matrix.

Reactions:
Preparation of 3,4,6-tri-O-acetyl-D-galactal 3,4,6-tri-O-acetyl-D-galactal was obtained from D-(+)-galactose (1eq.), acetic anhydride (5.07eq.), Et3N (5.07eq.) and DMAP (0.01eq.) in methylene chloride as a solvent in an overnight reaction at room temperature. The crude product was washed with water, 1M HCl, sat. NaHCO3 and NaCl solution. The organic layer was dried over Na2SO4 and DCM was removed in vacuo resulting β-D-galactose pentaacetate. The product was dissolved in DCM and HBr 33% in AcOH was added dropwise to the solution at 0°C, maintaining this temperature conditions for an hour longer and left overnight at room temperature until the starting material was completely consumed according to TLC. The reaction solution was diluted with DCM, washed with ice-cold water, 1% aq. NaHCO3, sat. NaHCO3 3 times and NaCl solutions. Organic layer was dried over Na2SO4 and solvent was evaporated to give 2,3,4,6-tetra-O-acetyl-α-bromo-D-galactose. The crude product (1eq) was dissolved in glacial acetic acid, after cooling the mixture to 0°C, CuSO4 (0.43eq) in water and activated (I) zinc dust (15.5eq) were added. The reaction conditions were maintained for 30 min, afterwards the suspension was stirred overnight at room temperature.

Glycosylation using Glycosyl Halides
The mixture of Fmoc-Thr-OAllyl (1.0 eq), Ag2CO3 (1.5 eq) and freshly activated 4Å molecular sieve in toluene and DCM (1:2) was stirred at -10 °C for 30 min. Then AgClO4 (0.25 eq), dissolved in toluene, was slowly added. Afterwards 3,4,6-Tri-O-acetyl-2-azido-2-desoxy-α-D-galactopyranosyl chloride (1.5 eq) in a mixture of toluene and DCM (1:1) was gradually added to the solution. The reaction mixture was stirred in the dark under argon at room temperature overnight. After diluting with DCM the suspension was filtered through Celite, washed with water and sat. NaHCO3 solution. The organic layer was dried over Na2SO4, the solvent was removed in the vacuo and the crude product purified by column chromatography. Scheme S3. Glycolysation of threonine residue where e) AgClO4, Ag2CO3, CH2Cl2, toluene, -10°C (30min) -rt, in the dark with argon, overnight Reduction of the azido group on carbon C2 of the sugar was carried out with thioacetic acid and pyridine (2:1) stirred at 50°C for 30min. The reaction mixture was directly purified on a chromatographic column (PE:EtOAc, 1:1  1:2) Scheme S4. Reduction of the azido group on carbon C2, where f) pyridine, AcSH , 50°C; 30min.
Ready building block, used in SPPS, was obtained by removal of the allyl group with the palladium catalyst (0.03eq.) and phenylsilane (1.1eq.) as a scavenger in CH2Cl2. Subsequently the mixture was washed with NH4Cl solution and water. Organic layer was dried over Na2SO4 and solvent was removed in vacuum. Crude product was purified on column (DCM:MeOH:AcOH; 95:4.5:0.5).