SYNTHESIS OF UNNATURAL ALFA-N-LINKED GLYCOPEPTIDES
Creators: name: COLOMBO, CINZIA
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: SYNTHESIS OF UNNATURAL ?-N-LINKED GLYCOPEPTIDES
Scuola di Dottorato in Scienze e Tecnologie Chimiche
 Dottorato in Scienze Chimiche XXIV ciclo
PhD Thesis
Cinzia Colombo, Matr. N. R08329
Tutor: Prof. Anna Bernardi
Coordinator: Prof. Silvia Ardizzone

The synthesis of neo-glycoconjugates has been gaining much attention in recent years due to the relevance of natural glycopeptides and glycoproteins in human health and disease [1]. Our group has been actively investigating the synthesis of ?-N-linked glycosylamides and glycopeptides [2]. These compounds are practically unknown in nature and may lead to modifications tolerated by biological systems and yet less susceptible to chemical or enzyme-mediated hydrolysis. In particular, here we concentrate on the synthesis of ?-N-linked glycopeptides. These N-linked glycopeptides are unnatural molecules, since they display an ? linkage between the peptide side chain and the sugar moiety, unlike natural glycopeptides which connect the peptide to the glycan through a ?-N-glycosidic bond. This novel type of peptides glycosylation might introduce structural diversity, leading to modifications that can mimic and/or interfere with molecular recognition events. Direct glycosylation of peptide chains is not viable for the synthesis of molecules with ?-N-linked configuration, since the corresponding ?-glycosyl amines isomerise to the ?-anomers. Hence, forced to employ ?-N-linked glycosyl amino acids to be linearly incorporated into a peptide sequence, we initially dedicated our efforts to the development of an efficient stereoselective synthesis of preformed N?-Fmoc-protected glycosyl amino acids, as suitable building blocks for solid phase synthesis applications. Using the DeShong methodology [3] and further elaborations, we succeeded in the synthesis of ?-N-linked glycosyl asparagine derivatives. In particular, N?-Fmoc-protected gluco and galacto pyranosyl-L-asparagine derivatives were obtained in good yields [4]. With these novel building blocks we initially optimized coupling conditions and further performed the solution synthesis of models of a galactosyl tripeptide and of a galactosyl pentapeptide, which were subjected to computational, conformational and molecular interaction studies. The synthesis of more complex ?-N-linked glycopeptides was performed using solid phase methodology. In particular, we developed the synthesis of mimics of antifreeze glycopeptides, which display repeating units of general structure (Ala-Asn (?-N-Gal)-Ala). The major problems towards the synthesis of such structures were observed in the coupling reactions of the ?-N-galactosyl asparagine building block, which, especially with increasing peptide length, reacted with poor yields and in long reaction time, due principally to its steric hindrance. In the end, ?-N-linked glycopeptides were obtained with modest yields, especially the longer ones. However, this work constitutes the first attempt for the synthesis of unnatural ?-N-linked glycopeptides and has been particularly useful in the understanding of behaviours, sometimes unexpected, of these molecules, in terms of reactivity and stability. Methods for the isolation and purification of glycopeptides up to 15 residues and 5 sugars have been identified, which allowed us to purify even very heterogeneous mixtures. Finally, the methods developed can now be used to include ?-N-glycosylated building blocks in other, possibly less challenging, peptide sequences. The observations accumulated so far indicate that ?-N-glycosylation may not perturb the 3D structure of the peptide, or of the sugar, and may find a number of applications, for instance to improve the water solubility of peptides without modifying their intrinsic properties, or to improve the carbohydrate affinity for interesting receptors. It remains to be established whether ?-N-linked glycopeptides are protected against enzymatic hydrolysis of the sugar moiety, a likely possibility, given the absence of this motif from natural glycoproteins. The synthetic availability of these molecules, which is the result of my PhD work, allows to explore this fascinating class of novel, unnatural glycoconjugates and to answer some of these questions. 

References 
[1] a) O. Seitz, ChemBiochem 2000, 1, 214-246; b) D. P. Gamblin, E. M. Scanlan, B. G. Davis, Chem. Rev. 2009, 109, 131-163.
[2] a) A. Bianchi, A. Bernardi, J. Org. Chem. 2006, 71, 4565-4577. b) F. Nisic, M. Andreini, A. Bernardi, Eur. J. Org. Chem. 2009, 5744-5751. c) F. Nisic, A. Bernardi, Carbohydr. Res. 2011, doi:10.1016/j.carres.2010.12.020.
[3] F. Damkaci, P. DeShong, J. Am. Chem. Soc. 2003, 125, 4408-4409.
[4] Colombo, C.; Bernardi, A. Eur. J. Org. Chem. 2011, 3911?3919.