The Liu group at the University of North Carolina has been using fluorous tags in the chemoenzymatic synthesis of heparan sulfate, an important oligosaccharide which has been implicated in a variety of biological processes. The function and effect of heparan sulfate is dependent on the location and degree of sulfation on the sugars. The exact effect of sulfation on function is not entirely clear, so the preparation of distinctly sulfated and characterized heparans would be very helpful in the study and understanding of the role of various sulfation states.
In the latest report from Liu, these same researchers use heparan sulfate sulfotransferases as a method to selectively sulfate heparan derivatives at specific hydroxyls. Once again they used fluorous tagging in order to facilitate purification post-reaction using fluorous solid phase extraction (FSPE). Given the number of enzyme mediated reactions using fluorous tagged substrates that we have covered in F-Blog recently, we won’t go into all the details here. Suffice it to say that, in general, fluorous tags do not substantially interfere with substrate specificity or activity and that this has been demonstrated many times for many types of enzymes.
Instead let’s address the characterization of these sulfated hexasaccharides by MS. Not surprisingly, MS/MS is the method choice in determining which monosaccharide units are sulfated in these reactions and which were not. Other methods such as NMR would be very difficult to interpret. When using MS/MS the last thing you want is to make the interpretation more difficult due to fragmentation at other parts of the molecule, such as the tag. So one great thing about fluorous tags is that the tag itself is non-fragmentary. On top of that the tags actually can make things easier. Unlike peptide fragmentation in MS/MS, the individual monomer units in oligosaccharides are quite similar in mass. The fluorous tag, however, adds additional mass to one end of the oligosaccharide thereby providing additional confidence regarding the fragmentation site. The same thing is observed in peptide fragmentation, of course, and can be seen by a distinct shift in either the b or y-ions depending on the site of the fluorous tag.
Modern biology and analytical chemistry rely increasingly on mass spectrometry as the primary method for identification and characterization of compounds. That holds true for proteomics, metabolomics, glycomics, etc. and in all these disciplines fluorous tags fit in quite well with modern MS techniques. First, the tag provides a method to enrich a sample in the particular analyte or class of analytes of interest. Second, the tag is non-fragmentary and does not unnecessarily complicate the MS. Third, fluorine is not found endogenously in biological systems while also containing a mass defect, that is unlike carbon, hydrogen, nitrogen, sulfur, etc., fluorine is slightly lighter than it atomic mass number. The defect is small, but adds up when you use a C6F13 tag so that one can tell if you the compound being analyzed has a fluorous tag or not. Finally, the fluorous tag’s mass is easy to incorporate into protein and peptide identification software, such as MASCOT or SEQUEST.