Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies

Abstract

Antifreeze glycoproteins enable life at temperatures below the freezing point of physiological solutions. They usually consist of the repetitive tripeptide unit (-Ala-Ala-Thr-) with the disaccharide alpha-D-galactosyl-(1-3)-beta-N-acetyl-D-galactosamine attached to each hydroxyl group of threonine. Monoglycosylated analogues have been synthesized from the corresponding monoglycosylated threonine building block by microwave-assisted solid phase peptide synthesis. This method allows the preparation of analogues containing sequence variations which are not accessible by other synthetic methods. As antifreeze glycoproteins consist of numerous isoforms they are difficult to obtain in pure form from natural sources. The synthetic peptides have been structurally analyzed by CD and NMR spectroscopy in proton exchange experiments revealing a structure as flexible as reported for the native peptides. Microphysical recrystallization tests show an ice structuring influence and ice growth inhibition depending on the concentration, chain length and sequence of the peptides.

Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

“…These monosaccharide-based AFGP analogues with 3–4 repeating units and sequential variations of the primary structure using glycine, proline, and serine instead of alanine exhibit decreased antifreeze activity compare to the native AFGP sequence. These studies also suggest that the terminal galactose in native AFGP is not necessary for significant activity and underscore the importance of having periodic turns in the peptide sequence [74,75,78,79,80,81]. …”

“…Because routine production of AFGPs using conventional solid-phase peptide synthesis has been limited to small building blocks, microwave-assisted solid phase peptide synthesis was accessed by Heggemann et al and Peltier et al . [74,75,78] in order to investigate the effect of the sequential mutation of peptide backbones on either RI or TH activity using a common monosaccharide building block (Figure 5D,E). These monosaccharide-based AFGP analogues with 3–4 repeating units and sequential variations of the primary structure using glycine, proline, and serine instead of alanine exhibit decreased antifreeze activity compare to the native AFGP sequence.…”

Recent progress in the field of neoglycoconjugate chemistry

“…In an attempt to develop tumor-associated carbohydrate antigens with multivalent display and thus enhanced presentation of the underlying peptide aglycon to the immune system, Vichier-Guerre et al (51) used Koenigs-Knorr condensation, as Heggemann et al (42), but with an a-1-chloro (instead of 1-bromo) derivative of N-acetylgalactosamine and silver carbonate/perchlorate to favor a-anomeric configuration in the end product, which was then incorporated to an Fmoc-protected homoserine acceptor, prepared according to Shiori et al (52). Other attempts to mimic naturally occurring, dense glycocalyx structures have involved glycoclusters made by coupling trihydroxy amine compounds to trichloracetimidate-functionalized sugars, or trivalent carboxylic acids with aminoethyl-functionalized carbohydrates (53).…”

“…This type of glycosylation is produced by many epithelial tissues in vertebrates and serves a variety of functions including protection of the underlying tissue or antifreezing properties by means of Tantigen containing tripeptide repeats w(Ala-Ala-Thr) n ; n up to 50x. To prepare the latter, Heggemann et al (42) have converted tri-O-acetyl galactal into a 2-azido-1-bromoderivative which by conjugation to Fmoc-Thr and acetamidation of the azido group renders the glycoaminoacid as an a/ b epimer mixture in an overall ;50% yield. This building block can then be incorporated onto any SPPS-generated sequence.…”

Design and Synthesis of Antifreeze Glycoproteins and Mimics

“…[43] As reported recently, this technique was applied to the synthesis of monosaccharide AFPG analogues that include Pro residues as a polyproline II helix-inducing element. [44] The peptides were prepared in 4-33 % yield, and required limited purification; this suggests that applications to longer oligomers should be possible.…”