Analysis of lichenic Galf-containing polysaccharides using IRMPD and ion-mobility MS – ALGAIMS

The analytical methods involved in the ALGAIMS project share the ability to differentiate isomeric sugars according to their conformation. These are multi-photon infrared spectroscopy (IRMPD, Lyon) and ion mobility mass spectrometry (IM-MS, Nantes). Correlations between two techniques will be established by molecular modeling in order to better understand the impact of conformational changes on physicochemical properties. The project is divided into 5 steps:
- Synthesis of small oligosaccharides with perfectly defined structures (Rennes) as reliable analytical references (databases).
- Recording of URMPD spectra (Lyon) and IM-MS (Nantes).
- Correlations between two techniques by molecular modeling in order to better understand the impact of conformational changes on physicochemical properties (Lyon, Nantes).
- Applications to complex matrices containing these units, isolated from lichens (Rennes), specific enzymatic hydrolyses which do not affect the furanosides of interest.
Miniaturization of the detection of furanosidic patterns on lichen sections in order to collect information in chemical ecology.

More than fifty intermediates and products have been chemically synthesized to prepare disaccharides, characterized by a galactofuranose-mannopyranose junction (Galf-Manp). In order to establish the potential impact of galactose configuration modulation, the equivalent Galp-Manp disaccharides were also synthesized.
In parallel, lichen samples were produced from four organisms (L. pustulata, C. islandica, L. pygmaea and R. fuciformis). Different polysaccharide extraction protocols have been tested. Five extracts were obtained for each of the lichenic species. The fractions were analyzed by NMR and only the fractions soluble in hot water of the lichens L. pustulata, C. islandica and R. fuciformis contain Galf motifs.
The reference IRMPD signatures of the synthetic monosaccharides Galf and Galp were obtained and different charge states were compared in order to obtain clear diagnoses for both the size of the cycle and the anomeric configuration. The results suggest that an unique signature can be obtained for each isomer.
The second approach aims to explore the potential of ion mobility to selectively characterize Galf in complex natural environments. Six of the eight disaccharides were analyzed in low resolution ion mobility in MS and MS / MS modes. This highlighted that the low resolution ion mobility was insufficient to differentiate the isomers and anomalies in the structure of the synthesized compounds.
Characterizations were also carried out on biological samples. Polyhexoses have been observed (MALDI) in somefractions (degrees of polymerization from 6 to 53). Enzyme digestion tests have also been carried out using an endo-mannanase. No degradation product was however detected by MALDI mass spectrometry.

This project aims to develop fast analytical approaches to increase understanding of exchange phenomena of more or less complex carbohydrates in symbiotic organisms such as lichens. The first step is to acquire data for the IRMPD and the IMS on the basis of synthetic di- and trisaccharides, and samples from extractions.
Beyond the analytical developments, these data will also provide information on the biosynthesis even of rare sugars of interest. In addition, methodological developments will be intended to be extended to other natural organisms likely to produce (or not) galactofuranosides.

The publication strategy is currently being evaluated by the partners of the ALGAIMS project team.

Sugars in their furanose form are not usual in nature but of high interest while analytical tools are limited to recognize them easily, particularly when they are in complex mixtures. So we have created an unprecedented multidisciplinary team-project around the ALGAIMS project to develop innovative and rapid analysis of rare hexofuranosides contained in symbiotic organisms such as lichens. The analytical methods which will be involved have in common the ability to differentiate isomeric sugars according to their conformation. These are infrared multi-photon dissociation spectroscopy (IRMPD, Lyon) and ion mobility mass spectrometry (IM-MS, Nantes). Correlations between both techniques will be established by molecular modeling to better understand the impact of conformational changes on physicochemical properties. Small synthetic oligosaccharides with perfectly defined structures (Rennes) will guarantee reliable analytical results (data banks). Complex matrices containing these units, isolated from lichens (Rennes), will be subjected to specific enzymatic hydrolysis without any impact on the targeted furanosides. Once the method validated, the miniaturization of the detection should allow recognizing the patterns furanose on lichen slices, which is very informative in chemical ecology.
This fundamental project is a springboard in many areas:
- From the analysis: by focusing on rare sugars distinguished by the ring size, the results obtained will make it possible to build consistent databases to distinguish molecules of the same mass but of different shapes. The experience thus acquired will reinforce teams in the field of advanced natural product analysis, especially sugars;
- Natural products: this project will reinforce the still poorly documented knowledge about lichenic polysaccharides and, subsequently, their roles in symbiotic organisms. It will also provide valuable information on the possible presence of specific enzymes useful for subsequent biotechnological developments.