PTFE Immobilization of Fluorous Tagged Oligosaccharides

A very interesting paper with Prof. Mamoru Mizuno as the corresponding author is now available as an accepted manuscript in Bioorganic & Medicinal Chemistry Letters.  The work presented is the recognition of influenza virus by oligosaccharides immobilized on a polytetrafluoroethylene (PTFE) or Teflon® filter.  The whole story is actually a collaboration between Prof. Hatanaka’s group at the University of Tokyo and Prof. Mizuno’s at the Noguchi Institute.  Both groups have been working on the fluorous synthesis and purification of oligosaccharides for quite some time now with Prof. Hatanaka using in vivo glycosylation of fluorous tagged sugars and Prof. Mizuno using more traditional fluorous synthetic methods.  Both have used fluorous solid phase extraction (FSPE) effectively as a purification method.

In the present report the groups first synthesized a series of oligosachharides with varying fluorous tags and evaluated their ability to be immobilized on PTFE filters and to capture proteins.  They first used standard chemistry to produce the fluorous tagged lactose derivitives shown in the figure below.  These compounds were spotted on a PTFE filter and incubated with biotin-labeled ECA, a lectin which specifically binds to lactose, in PBS buffer.  Visualization was conducted with avidin-HRP.  As you can see the PEG spacer between the lactose and fluorous chain was critical in this instance in order to observed decent binding.  In the absence of the PEG spacer, no binding with ECA was observed.  The fluorous diethyleneglycol showed no binding at all.  The authors also added free lactose and mannose as competitive ligands and as expected observed less ECA binding to the immobilized sugars in the presence of lactose.

The authors claim that the spacer was required for the immobilization onto the PTFE filter.  I’m not sure that this interpretation is entirely correct. (I’m also not sure that this entirely what they meant either, which I’ll explain.)  The inability to observe lectin binding in the absence of the PEG spacer could be due to either a) inefficient immobilization of the carbohydrates on the PTFE filter or b) inefficient binding of the ECA to the immobolized sugars.  Based on the data shown, it’s not clear whether the absence of observed carbohydrate-lectin complex is due to a or b.

The researchers then went on to compare the PEG spacer linked lactose with a corresponding alkyl spacer linked lactose and found that no binding was observed with the alkyl spacer lactose.  In this case they state that the hydrophobicity of the alkyl chain prevented binding with ECA.  So in this instance they believe that the oligosaccharide is there, but that it’s not binding which runs somewhat counter to what was previously stated.  In my opinion, this second explanation (basically hypothesis b from above) makes more sense.  I would actually expect that the alkyl chain or the shorter PEG chain would accentuate fluorous partitioning, not attenuate.

They next prepared a sialylated trisachharide using the in vivo glycosylation previously reported by Hatanaka.  They attempted to isolate the fluorous trisaccharide by FSPE but found that it was ineffective in separating the fluorous tagged compounds from the non-fluorous.  They eventually used regular silica gel chromatography to purify the trisaccharide.  What makes the FSPE failure even more surprising is that they were able to successully immobilize the fluorous trisachharide to the PTFE filter.  In any event, they finished the paper by demonstrating that the immobilized trisaccharide could capture influenza A virus through recognition of the hemmagglutinin protein.

There are some interesting aspects of this paper which I think are fairly obvious, particularly in terms of pathogen recognition.  One of the more important items to me, however, is the use of Teflon® as an immobilization surface.  Gladysz has previously shown that fluorous catalysts can be captured from reaction mixtures using Teflon®, but this is the first instance to my knowledge of a large complex being immobilized on it.  we have a sugar complexed to a lectin then visualized by a HRP labeled protein.  That’s a lot of stuff hanging on by a single fluorous chain onto Teflon®.  Since PTFE has been made into fibers, beads, membranes, coatings, etc. that opens up a vast number of ways to immobilize fluorous ligands, reagents, antigens, you name it.  Very interesting indeed.

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