Fluorous Scavenging for Radioimaging Agent Production

Prof. John Valliant’s group at McMaster University has been one of the most active researchers in the application of fluorous techniques to the production of radioimaging agents.  Most of the work to date has used fluorous aryl tins as precursor to radioactive aryl iodides.  They used fluorous solid phase extraction (FSPE) to purify both the precursors and the products to provide “carrier-free” solutions of the radioimaging agents.  This ensures that the radioimaging agent does not have to compete with non-radioactive ligands at the target tissue.  In a research setting removal of excess reagents and ligands is easily accomplished with HPLC.  In a clinical setting, however, HPLC is not practical for a number of reasons.  SPE and simple, readily automatable processes are highly preferred which is why Prof. Valliant has used fluorous methods.  A new Chemical Communications paper from the Valliant group describes an alternative strategy to fluorous tagged substrates which they dub fluorous ligand capture (FLC).  It’s basically a fluorous scavenging strategy and in this instance was applied to the production of 99mTc-labelled compounds.

99mTc is the most widely used radionuclide in diagnostic medicine and is used in single photon emission computed tomography (SPECT) for tumor imaging, bone scans, myocardial perfusion imaging, etc.  Normally, the 99mTc is added to excess of a ligand which is specific to the tissue of interest.  The resultant mixture contains the 99mTc labelled ligand and excess ligand which can be problematic since the non-radioactive ligand can compete with the 99mTc labelled ligand for affinity with the targeted tissue.  One way around this is to use a scavenger after the addition of the metal to remove any excess ligand that is present and solid phase is an obvious choice.  However, solid phase methods generally do not perform well with radioactive compounds and have resulted in high non-specific binding of the radionuclide.  The Valliant group’s solution was to use fluorous scavenging in conjunction with FSPE.

To test this strategy they fist prepared a copper complex pictured below which was to be the fluorous scavenger A.  They then reacted bipyridyl ligand B with [99mTc(CO)3(H2O)3]+ in a microwave for 2 min to form a mixture of 99mTc complex C and unreacted B.  Fluorous scavenger A was added to this mixture to form copper complex D which was readily separated from C using FSPE to complete the fluorous ligand capture process.  The authors found that >99% of B was removed from the mixture using FLC and that 8% of the radioactivity remained on the FSPE column.  In contrast solid-pase analog of A resulted in 86-90% of B being removed and upwards of around 25% of the radioactivity binding to the solid phase.  In other words less removal of unwanted material and lower yield of desired material using solid phase vs. fluorous phase.

Having demonstrated the FLC concept then then moved onto a peptide conjugate, specifically peptide E with a bipyridyl modified lysine.  The LTVSPWY peptide is known to target erbB2 receptors.  ErbB2 is known to be overexpressed in 30% of breast cancers and can be interpreted as a sign that the cancer is metastasizing.  For tumor imaging then 99mTc-LTVSPWY conjugate could be used to visualize erbB2 overexpression in a tumor.  In this instance you can easily see why excess uncomplexed ligand would be a problem since free peptide could complex competitively with the radioimaging agent to the erbB2 receptors resulting in lower signal.  Following the FLC protocol developed previously the researchers found that 95% of the excess ligand was removed with about 12% of the radioactivity bound to the fluorous column.  Not quite as good as with the model system, but still pretty useful.  In this instance there was no comparison to the solid-phase scavenging.

The researchers also tried an on-cartridge scavenging approach which pre-loaded the fluorous scavenger A onto the FSPE cartridge and passed the mixture of E and 99mTc-E through the cartridge.  This was not as successful, however, as 33% of the radioactivity was retained on the cartridge.  The authors suggest that this may be due to non-speicific binding with free silanols that may be present on the fluorous silica gel and that they are investigating other fluorous media which may perform better.  Even with that result, however, the off-cartridge process is still pretty easy.

The bottom line is that fluorous scavenging, or FLC, is a potentially attractive method for the purification of SPECT imaging agents.  The method is selective, fast, and potentially  automatable.  All things you need in a clinical setting.  There are still some issues that would need to be sorted out in terms of getting the isolated product after FSPE into an injectable formulation obviously, but it certainly has potential.