A Minimal, Unstrained S-Allyl Handle for Pre-Targeting Diels-Alder Bioorthogonal Labeling in Live Cells

Abstract

The unstrained S-allyl cysteine amino acid was site-specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5-tetrazines by means of an inverse-electron-demand Diels-Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre-targeting approach. The small size, easy chemical installation, and selective reactivity of the S-allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.

Keywords: Diels-Alder reactions; apoptosis; bioorthogonal labeling; pre-targeting; unstrained alkenes.

Figure 1

a) General reaction showing the application of IEDDA reactions for protein labeling with a site‐specifically installed S‐allyl handle. b–d) Structures of S‐allyl Cys, tetrazine cores, and fluorophores used in this study. e) Rate constants for the reaction of 2–5 with the model amino acid S‐allyl Cys 1 were measured following the consumption of tetrazines by UV/Vis (f) or fluorescence (g).

Figure 2

Chemical approach used for protein labeling through IEDDA reaction between S‐allyl Cys‐tagged proteins and tetrazines. a) The unstrained alkene handle was site‐specifically installed into AnxV by allylation of Cys315 through a [2,3]‐sigmatropic rearrangement with allyl selenocyanate. A tetrazine probe could selectively react with the installed alkene. b) Deconvoluted ESI‐MS spectrum of AnxV S‐allyl Cys. c) Deconvoluted ESI‐MS spectrum of the product of the reaction between AnxV S‐allyl Cys and PyTz 2. d) Deconvoluted ESI‐MS spectrum of the product of the reaction between AnxV S‐allyl Cys and BnNH₂‐Tz 3. e) MS/MS spectrum of the m/z 652.12 doubly charged ion of the Cys‐modified peptide KALLLLCGEDD from AnxV. The underscore relates to the modified amino acid.

Figure 3

a) Pre‐targeting of apoptotic cells with AnxV S‐allyl Cys followed by IEDDA labeling with fluorogenic Tz‐Cy3 5. b) SDS‐PAGE of AnxV labeled with Tz‐Rhod 4 and Tz‐Cy3 5 for 12 h at 37 °C. The labeled protein was purified by dialysis. Left image shows Coomassie blue‐stained gels and the right image presents the fluorescent imaging of the same gels before staining with Coomassie blue. c) ESI‐MS spectrum of the product of the reaction of AnxV S‐allyl Cys with Tz‐Cy3 5. d) Specific labeling of apoptotic cells (ii) pre‐targeted with AnxV S‐allyl Cys and subsequently labeled with Tz‐Cy3 5. Cells were imaged 3 h after addition of the fluorogenic dye 5. Non‐apoptotic cells (i) were included as a control. The specificity of the labeling was confirmed through blocking of cells with non‐labeled AnxV (iii) prior to incubation with AnxV S‐allyl Cys or by treating cells only with the tetrazine probe Tz‐Cy3 5 (iv) without preincubation with the targeting protein AnxV S‐allyl Cys. Apoptotic cells are shown red, while the nuclei counterstained with Hoechst 33 342 are shown blue. Scale bar=100 μm.