[Babcock_Hall]

We are attempting to modify the cysteine residue of an enzyme to change it to E-CH₂-S-CH₂-PO₃H₂.  We added this phosphonomethyl group to cysteine residues of other proteins (see below), but both the enzyme and the electrophilic agent are new ones in our hands.  The reagent is C₆F₅S(O)₂-OCH₂PO₃H₂, PMFB.  We synthesized it some time ago, and we checked the purity by NMR again very recently and did not notice anything out of order.  Very rough approximations for the two pK_a values of the phosphonate group would be 1-2 for the first dissociation constant and 6-7 for the second dissociation.  The enzyme has four cysteine residues, including the active site nucleophile.

We performed an inactivation on the enzyme plus PMFB, and we also set up a dummy inactivation experiment using methylphosphonic acid, which was chosen because it is inert but would mimic the acidic properties of PMFB.  In both cases Bis Tris buffer was present and two equivalents of triethylamine were also present.  The concentration of PMFB is much larger than the concentration of enzyme.  We did the experiment in two ways, half the time following the inactivation reaction with a reaction with iodoacetamide, and half the time not doing the iodoacetamide step.  The iodoacetamide should react with any cysteine residue that was not previously alkylated with PMFB.

In one of our initial attempts to perform mass spectrometry on the peptides produced by digestion with trypsin, we saw a shift of 228 mass units (relative to what a cysteine residue would produce) when we came to the portion of the sequence in the peptide where cysteine or modified cysteine should be.  Our modification should produce a shift of approximately 94 mass units.  We checked a mass of 228 against a list of common protein modifications, but we did not find much of anything that looked realistic.  On the other hand, if there were a nucleophilic displacement of one of the fluoride atoms on PMFB (vertical arrow in diagram), this would produce a different intermediate.  Subsequent hydrolysis would produce a modification with a shift of +228 mass units, relative to cysteine.  My question is how plausible is a nucleophilic aromatic substitution reaction on the pentafluorinated, sulfonated ring?

We have also worked with a related reagent, phosphonomethyl trifluoromethansulfonate (PMT).  When PMT reacts with free cysteine or with a cysteine residue within a protein, the trifluoromethanesulfonate group leaves, producing the desired modification of cysteine to phosphonomethylcysteine.  PMT also inactivates our enzyme, but phosphonomethyl trifloroethanesulfonate does not.