RESUMEN
Amide-linked homopolymers of sialic acid offer the advantages of stable secondary structure and increased bioavailability making them useful constructs for pharmaceutical design and drug delivery. Defining the structural characteristics that give rise to secondary structure in aqueous solution is challenging in homopolymeric material due to spectral overlap in NMR spectra. Having previously developed computational tools for heteroologomers with resolved spectra, we now report that application of these methods in combination with circular dichroism, NH/ND NMR exchange rates and nOe data has enabled the structural determination of a neutral, δ-amide-linked homopolymer of a sialic acid analogue called Neu2en. The results show that the inherent planarity of the pyranose ring in Neu2en brought about by the α,δ-conjugated amide bond serves as the primary driving force of the overall conformation of the homooligomer. This peptide surrogate has an excellent bioavailability profile, with half-life of â¼12 h in human blood serum, which offers a viable peptide scaffold that is resistant to proteolytic degradation. Furthermore, a proof-of-principle study illustrates that Neu2en oligomers are functionalizable with small molecule ligands using 1,3-dipolar cycloaddition chemistry.
Asunto(s)
Secuencia de Aminoácidos , Estructura Secundaria de Proteína , Amidas/química , Dicroismo Circular , Humanos , Datos de Secuencia Molecular , Conformación Proteica , SolucionesRESUMEN
A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.
Asunto(s)
Epítopos de Linfocito T/química , Proteína gp120 de Envoltorio del VIH/síntesis química , Antígenos HLA-DR/química , Epítopos Inmunodominantes/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Epítopos de Linfocito T/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Antígenos HLA-DR/inmunología , Epítopos Inmunodominantes/inmunología , Vacunas contra la Malaria/química , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The analysis of heparan sulfate glycosaminoglycans (HSGAGs) variations in human serum at the disaccharide level has a great potential for disease diagnosis and prognosis. However, the lack of available analytical methodology for the compositional analysis of HSGAGs in human serum remains to be addressed to delineate the possible role of HSGAGs on the onset and/or progression of a disease. In this study, we have developed a method for the in-depth compositional analysis of the 12 heparin/HS-derived disaccharides from human serum using a combination of technologies--fractionation, exhaustive digestion, solid phase extraction, and LC-MS/MS. The method exhibits high recovery (72-110%) and good reproducibility (standard deviation of less than 5%) with a low limit of detection and quantification. Errors from the method validation were within 1.1%. Nondetectable non- or low-sulfated disaccharides in human serum were also detected using the optimized protocol. Further applying this method, the comprehensive analysis of HSGAGs compositions in human sera from female donors showed considerable variations in disaccharide patterns and compositions.