Synthesis and activity of glycopeptides of interest for drug design of inhibitors of Trypanosoma cruzi trans-sialidase.

Author(s)

Vanessa Leiria Campo

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2007-12-10

Examining board members

Ivone Carvalho; Joao Luis Callegari Lopes; Sergio Schenkman; Carlos Henrique Tomich de Paula da Silva; Gil Valdo Jose da Silva

• Advisor: Ivone Carvalho

Abstract

Trypanosoma cruzi trans-sialidase (TcTS) belongs to the family of glycoproteins expressed on the surface of the parasite and constitutes one of the few examples of natural surface glycosyltransferases found in eucariotes. T. cruzi can not synthesize sialic acid itself and uses a trans-sialidase enzyme to scavenge this monosaccharide from host glycoconjugates to sialylate acceptors molecules, such as GPI (glycosylphosphatidylinositol)-anchored mucins, that are present in parasite plasma membrane. This enzyme is specific to catalise, preferentially, the transference of sialic acid to mucin glycoproteins, originating -2,3- linkages with acceptor galactose molecules in the parasite surface. The sialylated mucin molecules are involved in the attachment and subsequent penetration of the parasite into host cells. Given the heterogeneity of T. cruzi mucin molecules, there are no suitable synthetic occurring sources of TcTS glycopeptide substrates. Thus, the objective of this work was the development of chemical and chemoenzymatic methods of synthesis of T. cruzi mucin glycopeptides in order to investigate the nature of the molecular interactions involved in recognition and processing of containing sialic acid glycosides, in the presence of trans-sialidase. A better understanding of these interactions led to drug design of selective potential inhibitors for T. cruzi trans-sialidase. Some of the main glycopeptides obtained by methods of solid phase and chemoenzymatic synthesis with 1,4-galactosyltransferase (1,4-GalT) enzyme were: NH2(Thr)2-(LacNAc)- (Thr)3-GlyOH 2, NH2(Thr)2-(LacNAc)-(Thr)3-GlyOH 4 and NH2(Thr)2-(LacNAc)-(Thr)3- (LacNAc)-(Thr)3-GlyOH 5. As precursors of these glycopeptides, the building blocks GlcNAc-FmocThrOH 24, GlcNAc-FmocThrOH 25, Gal-FmocThrOH 27 and LacNAc- FmocThrOH 75 were synthesized by glycosylation reactions of the threonine amino acid 18, containing the protecting groups N-Fmoc and O-Bn, with the correspondent sugars GlcNAcCl 12, GalBr 13 and LacN3Cl 33, followed by hydrogenolysis reactions (10% Pd- C/ H2) for deprotection of O-Bn group and later coupling into the peptide chain rich in threonine sequence. The glycosylated amino acids GlcNAc-ThrOH 22 and GlcNAc-ThrOH 23, totally deprotected, were employed in enzymatic reactions with 1,4-GalT, being obtained the disaccharide glycosides LacNAc-ThrOH 53 and LacNAc-ThrOH 41 (also chemically synthesized). The disaccharide glycosides 41 and 53, the glycopeptides 2 and 4 and the glycosylated amino acid Gal-ThrOH 28 were submitted to enzymatic assays with TcTS enzyme, being verified the sialylation of all tested acceptors in high yields, which confirmed that these compounds can act as potential acceptors substrates for TcTS enzyme. (AU)