Azabicyclic vinyl sulfones for residue-specific dual protein labelling

Abstract

We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation.

Stable Pyrrole‐Linked Bioconjugates through Tetrazine‐Triggered Azanorbornadiene Fragmentation

“…A second‐order rate constant ( k ) of 0.026 (±0.002) m −1 s −1 at 37 °C was determined for the triple‐cascade reaction with the initial cycloaddition step as rate limiting (see Figure S10). This value is similar to the one determined for iEDDA ligation between DPTz and azanorbornene of type C (Scheme ) . Similar reaction rates were also reported for iEDDA reactions with aza/oxabenzonorbornadienes .…”

“…The sulfone 2 showed high stability and no r DA reaction was observed after 1 week at 37 °C in CDCl 3 , in agreement with the high activation barrier of Δ G ≠ ≈28 kcal mol −1 calculated for an N ‐Ac‐azabicycle analogue in chloroform (see Figure S13). This result contrasts with the 7‐azanorbornene analogue of type C , which we found to be more prone to undergoing slow r DA breakdown under the same reaction conditions (calculated activation barrier of Δ G ≠ ≈25 kcal mol −1 in chloroform) . The exceptional reactivity of the bromovinyl sulfone moiety embedded in a [2.2.1]bicyclic skeleton over a less‐strained bicyclic system was confirmed through reaction of N ‐Boc‐Cys‐OMe with differently bridged bicyclic derivatives ( 3 — 5 ; Scheme ).…”

“…We have recently developed a method for residue‐specific dual protein labelling that consists of cysteine‐selective thio‐Michael addition to the 7‐azanorbornadiene system B followed by bioorthogonal iEDDA labelling of the resulting 7‐azanorbornene conjugate with fluorogenic tetrazines (Scheme ) . The ability to perform the iEDDA reaction enabled the incorporation of a second label and also avoided collateral r DA of the 7‐azanorbornene C .…”

Stable Pyrrole‐Linked Bioconjugates through Tetrazine‐Triggered Azanorbornadiene Fragmentation

“…This value is similar to the one determined for iEDDA ligation between DPTz and azanorbornene of type C (Scheme 2). [19] Similar reaction rates were also reported for iEDDA reactions with aza/oxabenzonorbornadienes. [17] Finally, [2.2.1]bicyclic analogues (7-9) reacted with 10 in a similar manner to 2 (see Scheme S3).…”

“…This result contrasts with the 7-azanorbornene analogue of type C, which we found to be more prone to undergoing slow rDA breakdown under the same reaction conditions (calculated activation barrier of DG°% 25 kcal mol À1 in chloroform). [19] The exceptional reactivity of the bromovinyl sulfone moiety embedded in a [2.2.1]bicyclic skeleton over a less-strained bicyclic system was confirmed through reaction of N-Boc-Cys-OMe with differently bridged bicyclic derivatives (3-5; Scheme 3). The compounds 3 and 4, which feature a [2.2.1]bicyclic skeleton, reacted rapidly with N-Boc-Cys-OMe (calculated activation barrier with MeS À of DG°= 11 and 14 kcal mol À1 , respectively; see Figure S12), whereas the less-strained [2.2.2]bicyclic derivative 5 (calculated activation barrier with MeS À of DG°% 16 kcal mol À1 ; Figure S12) did not react under the same reaction conditions.…”