Pyrophosphate side product
Two phosphate groups condense head-to-tail to form a P-O-P pyrophosphate linkage (instead of the desired single phosphate). +80 Da on top of an already-phosphorylated peptide (so +160 vs. unphosphorylated).
- +160 — +160 Da, double pyrophosphate
Why it happens (mechanism)
If phosphorylation is performed twice without proper monitoring, the existing -OPO₃H₂ on Ser/Thr/Tyr can attack a second phosphoramidite reagent. Result: -O-P(O)(OH)-O-P(O)(OH)₂, a pyrophosphate diester. +80 Da relative to monophosphate.
When it strikes (triggers)
Excess phosphoramidite + long coupling time + multiple phosphorylation cycles. Free phosphate (e.g., from H-phosphonate) re-attacked by reagent.
How to spot it (MS signature)
+80 Da on top of phosphopeptide mass. MS/MS shows characteristic -98 loss × 2 (each phosphate eliminates separately).
How to prevent it
- Use stoichiometric phosphoramidite (not excess).
- Single phosphorylation cycle only, with fresh reagent.
- Monitor by analytical HPLC after each phosphorylation — pyrophosphate elutes earlier than the monophosphate.
If it already happened (salvage)
- Pyrophosphate is sometimes hydrolyzable to phosphate by mild base + heat, but yields are mediocre. Re-synthesize for production-quality material.
Source
Yi Yang, Side Reactions in Peptide Synthesis (Elsevier, 2016), Chapter 12, §12.2.