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Nouvelles sondes glycolipidiques pour l'imagerie membranaire – CARBOHYDRPROBES

Organic synthesis: At first, the regioselective modification of natural di- and trisacharides was achieved. Direct azidation following a method developed in our group afforded the carbohydrate azides ready to be further functionalized by “click” reactions. To this end, a mono and dialkyne version of a push-pull chromophore was synthesized. The two entities were then connected by a careful tuning of the Huisgen Cu(I) catalyzed reaction conditions.
Optical properties: The new series different structures gave a red fluorescent signal with a maximum emission wavelength close to 690 nm. This is interesting since, due to physical reasons, biology favors dyes emitting in the red region. They also showed a very large Stokes shift as well as a strong environmental dependence.
Membrane insertion properties: Using a model system, the Langmuir monolayer balance technique, the insertion abilities of the compounds were evaluated. Both kinetic an efficiency data were collected and showed diverse behavior for the different probes due to the varying overall structure of the molecules. Three compounds consisting of one fluorophore attached to either disaccharidic or trisaccharidic polar heads were found to insert faster and more efficiently than the others.
Biological experiments: When used on muscle cells, cells which undergo contraction when damaged, these three probes proved to be very efficient membrane markers, for which no cytotoxicity was noticed during the biological experiments. When compared to commonly used dye for membrane imaging, they showed similar fluorescence intensities but with a sharper pattern. More recently, these compounds were used in TPEF and SHG experiments in which they demonstrated a very good membrane staining.

This work, financed by the ANR, has allowed the synthesis and study of two families of carbohydrate based probes. A first family (not discussed yet) for which the optical properties were unfortunately not adapted for the desired experiments. These compounds were used in a new approach for their photo-induced fragmentation properties in collaboration with the LASIM lab (Lyon 1). Based on the preliminary results an ANR funding with YB was obtained. Collaboration between LASIM and SC has also started. For the second family of probes (discussed in the previous chapter) which possess the appropriate optical behavior, this work validated the carbohydrate/chromophore design. It also allowed a better rational of the structure/property relationship and showed the potency of such probes both in linear and non-linar imaging experiments (in collaboration with Prof. Clays, Leuven and S Pouvreau, Lyon 1, respectively.


The first family of probes was described in two papers referring to their photo-induced fragmentation. One particular compound was selected as a model and was used in a patent in which YB is one of the authors. The second series was described in a paper submitted to Bioconjuguate Chem. and a second one referring to the non linear imaging experiments will be submitted soon. An article which concerns the use of another series of probes is also in preparation. This work was presented in several meetings concerning either carbohydrates or non-linear imaging.

Submission summary

In this interdisciplinary project, we wish to mimic the overall structure of natural glycolipids to beneficiate of their well known membrane specificity in order to produce new tools for membrane imaging by second harmonic generation microscopy. Being very sensitive to variation in membrane potential and with a signal originating only from molecules asymmetrically distributed in the cell membrane, SHG microscopy proved to be an unparallel technique for cell membrane imaging and for the direct time resolved measurement of membrane potential in living cells. Unfortunately lack of probes specifically devoted to the new emerging nonlinear optical imaging methods strongly limits its use. Carbohydrates are especially present at the surface of cells as glycoproteins or glycolipids. We wish to introduce carbohydrate on efficient second order nonlinear chromophores and show the advantages of the use of carbohydrate based probes in the prospect of cell membrane imaging. Due to their intrinsic properties, carbohydrates will bring to the probes the required water solubility and at the same time the amphilicity necessary for a good membrane staining and improved residency time in the outside lipid leaflet. The membrane penetration properties of the new objects that will be synthesized in this work will be characterized using the Langmuir monolayer technique. The surface morphology of the film inserting the probe will be observed by Brewster angle microscopy allowing a direct visualization of the lipid domain morphology formed in the monolayer and allow a better understanding of the effect of each structural modification on the behavior of the probes. Finally, nonlinear microscopy will be performed on live cell to identify the best probes. This work will be done in close collaboration with a team specialized in nonlinear imaging. This interdisciplinary project involved three partners: two organic chemists specialized in carbohydrate chemistry (project coordinator) and in the design of nonlinear chromophores, and one biochemist that will be in charge of the studies on Langmuir films. We wish in this proposal to use the concept of 'click' chemistry to graft carbohydrates moieties to adequately functionalized nonlinear chromophores. Two reactions are envisaged: Cu(I) catalysed 1,3-dipolar Huisgen cycloaddition of azide compounds with alkynes and thiol-ene coupling between a sulfhydryl group and an alkene. To that end we will study the scope of the Mitsunobu reaction employing HN3 for the production of different regioselectively functionalized carbohydrate azides. Using the "click" reaction, the obtained carbohydrate azides will then be attached to the alkyne chromophores for the production of the first generation of nonlinear glycoprobes. This reaction will then be extended to the conception and the synthesis of thio- and azido-thio carbohydrates for obtaining bisfuctionalized scaffold. To get more insights into the structural requirements and try to find better probes one should first find an access to bis-functionalized carbohydrate scaffolds bearing two functions which could orthogonally react. In this purpose, we propose to extend the work on Mitsunobu functionalization of carbohydrates to another type of function, thioesters, which can be used to generate thiols functions. To mimic natural compounds such as glycosphingolipids, a second lipophilic residue could be attached to the thiol function while the azide would be kept for grafting the chromophore. Such modifications would bring a higher membrane affinity to the probe and this will probably give better results for the GSH imaging experiments. The requirement of obtaining larger carbohydrate entities on the probes can be to use a multiple presentation of the same carbohydrate structure on one tether. To this end it would be of great interest to try reacting multi-allylated carbohydrate structures with carbohydrate thiols, using here the thiol-ene coupling method. In a second time, work will focus on the conception and the synthesis of new thioglycoside donors and their use for the synthesis of Tn antigen decorated probes. In this proposal, the synthesis of new thiolactonic or thiolactamic glycoside will be considered as well as the study of their use in glycosylation reactions. The second part of this task will be the synthesis of the Tn Antigen, this later will then be attached to the best scaffold. The complex object obtained will be used for the possible detection of a change in SHG signal when a recognition event occurs at the surface of a cell. This ambitious project will be run in close collaboration with biophysicist. We will bring our expertise in the field of carbohydrate chemistry, nonlinear chromophore design and in surface biochemistry. With this proposal, we wish to participate to the development of a functional nonlinear voltage-sensitive imaging of the cell membrane.

Project coordinator

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


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Beginning and duration of the scientific project: - 0 Months

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