NHS esters are non-innocent protein acylating reagents

N-Hydroxysuccinimide (NHS)-ester derivatives represent one of the most widely used reagents in biological chemistry and chemical biology. Their perceived utility as acylating agents –intermediates that are sufficiently stable to be isolated (even in commercial form) yet active enough to allow direct and acylation simply upon addition – has seen their widespread application. Their efficacy relies critically on the exclusive chemoselectivity of activated acyl over that of the imidic acyl moieties in the succinimide, such that NHS acts simply as an innocent nucleofuge ‘leaving group’. Here, through systematic structural variation that modulates acyl reactivity, coupled with a statistically controlled ultra-rapid screen for unknown modifications in tandem mass spectra and lysine profiling within the native E. coli proteome, which presents complex lysine environments, we reveal that ring-opening reaction of the succinimide to afford several N-succinamide derivatives is a present, sometimes dominant, side-reaction of certain NHS esters. Moreover, the extent of side-reaction is shown to be lysine nucleophile and therefore site-dependent, with both side-reaction and desired reaction occurring within the same protein substrate. The resulting formation of bioconjugates with unintended, unstable linkages and modifications – that may have previously gone undetected – not only suggests re-evaluation of i) use of potentially several hundred of the many-thousand, known commercial reagents; and ii) the functional conclusions previously drawn using NHS esters where these hinge crucially on the precise identity of conjugates in areas as diverse as antibody-drug biotherapy, vaccination to cross-link-enabled structural analyses.