ORIGINAL ARTICLE One-pot preparation of labelled mannan–peptide conjugate, model for immune cell processing Pavol Farkaš 1 & Alžbeta Čížová 1 & Peter Bystrický 1 & Lucia Paulovičová 1 & Ema Paulovičová 1 & Slavomír Bystrický 1 Received: 2 October 2015 /Revised: 30 November 2015 /Accepted: 2 December 2015 /Published online: 14 December 2015 # Springer Science+Business Media New York 2015 Abstract An efficient method for preparation of fluorescently labelled mannan–peptide glycoconjugates has been devel- oped. After selective Dess–Martin periodinane oxidation of mannan, it was conjugated to the fluorescent label alone and a peptide with the label via reductive amination. Prepared glycoconjugates were characterised by HPSEC, FTIR-ATR and UV-VIS spectroscopy. Finally, the fluorescently labelled mannan and mannan–peptide conjugate were used for micro- scopic visualization of their accumulation in intracellular or- ganelles of RAW 264.7 cells. Keywords Mannan . Glycoconjugate . Fluorescent label . Candida albicans . 2-aminobenzamide Introduction Nowadays glycoconjugates represent an important class of efficient and safe glycoconjugate vaccines. The conjugation of a polysaccharide antigen with a protein or peptide via co- valent linkage is one of the main recent trends to overcome shortcomings of old polysaccharide vaccines, such as a T-cell independence. However, while the traditional mechanism of action of glycoconjugates considered peptides generated from the carrier protein to be responsible for T-cell help recruitment, only recently the evidence of the active involvement of the carbohydrate part in determining the T-cell help has been shown [1]. Moreover, it was identified using radiolabelled glycoconjugates the means by how glycoconjugates are proc- essed by B-cells [2]. For example, fluorescently labelled pneu- mococcal polysaccharides were used to determine the location of the polysaccharide component of the glycoconjugate in the antigen presenting cells (APCs) [3]. In another example radiolabelled ( 3 H) group B streptococcal type III polysaccha- ride coupled to a carrier protein was used for processing by APCs, resulting in presentation of carbohydrate epitopes that were able to stimulate CD4 + T cells [2]. Thus, labelled glycoconjugates represent effective tools for tracking antigen exposure in immunobiological studies. However the involve- ment of intracellular vesicles in the trafficking of Candida mannan antigen is not known. The fluorescently labelled man- nan is a useful tool for the investigation of uptake of the Candida albicans mannan and two-component conjugate sys- tems (mannan as Candida saccharide antigen and Candida specific peptide antigen) and could effectively serve for intra- cellular trafficking by the APCs. Our goal was to prepare a series of fluorescently labelled mannan glycoconjugates, namely mannan–peptide–label and mannan–label with well-defined composition as well as pro- portionally identical composition of saccharide, peptide and the fluorescent label. In the presented work, fluorescently labelled mannan and mannan–peptide conjugates were prepared and characterized by FTIR-ATR spectroscopy, HPSEC and UV-VIS colorime- try. The cell penetration and transport of conjugates was suc- cessfully monitored using fluorescence microscopy imaging. We suppose that proposed synthesis of glycoconjugates is versatile, simple and can be used in various phases of artificial vaccine development and related fields. This is especially Electronic supplementary material The online version of this article (doi:10.1007/s10719-015-9644-0) contains supplementary material, which is available to authorized users. * Pavol Farkaš chempalo@savba.sk 1 Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, 845 38 Bratislava, Slovak Republic Glycoconj J (2016) 33:113–120 DOI 10.1007/s10719-015-9644-0