H-phosphonate impurity (phospho synthesis)
During on-resin phosphorylation, the phosphoramidite intermediate can be reduced before oxidation, giving an H-phosphonate (P-H bond) instead of a phosphate (P=O). -16 Da vs. expected phosphopeptide (PO₃H₂ vs. PHO₂H₂).
Why it happens (mechanism)
P(III) phosphoramidite or its ring-opened intermediate doesn't get fully oxidized in the I₂/H₂O step → remains as H-phosphonate (P(O)(OH)H instead of P(O)(OH)₂). The -OH group is replaced by -H, so 80 - 16 = 64 effective Δm vs. unphosphorylated. Vs. expected phosphate, that's -16.
When it strikes (triggers)
Inadequate iodine oxidation. Old/wet phosphoramidite reagent. Cold oxidation step.
How to spot it (MS signature)
-16 Da from the expected phosphopeptide (or +64 instead of +80 vs. unphosphorylated). MS/MS of H-phosphonate gives -64 loss instead of -80.
How to prevent it
- Fresh I₂/H₂O/THF oxidation cocktail; ≥10 min at RT.
- Replace I₂ with tert-butyl hydroperoxide (TBHP) — milder, more selective, less side reaction.
- Pre-dry reagents; H-phosphonate is favored by traces of water in the phosphoramidite step.
If it already happened (salvage)
- Re-oxidation of the isolated H-phosphonate peptide with I₂/H₂O can sometimes convert it; usually low yield.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 12, §12.1.