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.


Synthesis of the rhodamine precursor Rhod-NH 2
Rhod-NH 2 was prepared according to a published protocol. [1] Rhodamine B (1.
The second-rate constant for the reaction between 5-norbornene-2-methanol (Norb) and Tz-Cy3 5 was also determined to be used as a reference ( Figure S4). Supporting Information S17

Fluorogenicity of the tetrazine probes
A 0.2 mM solution of the tetrazine-fluorophores Tz-Rhod 4 and Tz-Cy3 5 in PBS buffer pH 7.6 was reacted with 1000 equivalents of Norb at 37 º C. Emission spectra were recorded before and after 2 h reaction ( Figure S5).

Control: reaction of Ellman's reagent with AnxV
Control studies to proof quantitative modification of the free cysteines of the AnxV and C2Am were performed using the Ellman's test. As an example, the ESI-MS spectra for the reaction between AnxV and the Ellman's reagent are shown below.

Direct alkylation with allyl chloride at pH 8.0
AnxV was prepared as a 0.25 mg/mL solution in 50 mM sodium phosphate buffer at pH 8.0 and 20 µL (0.140 nmol) were transferred to a 0.5 mL eppendorf tube. Allyl chloride        Reaction between AnxV S-allyl Cys and Py-Tz 2 was performed in triplicate with similar results (Figure S14 and S15).

Reaction of tetrazine Py-Tz 2 with AnxV
During the reaction between the tetrazine cores and AnxV S-allyl Cys it was observed the formation of a bis-ligation product. To check if the double addition was result of cross-reactivity with other amino acids on the protein, the tetrazine reaction was performed with the unmodified AnxV using the same conditions (200 eq, 37 º C, 96 h, Figure S19). These control experiments revealed that there is no ligation of the tetrazine to the protein if the allyl handle is not present precluding any cross-reactivity with other amino acid residues (controls for C2Am are shown in Figure S33).

Reaction of tetrazine Py-Tz 2 with AnxV S-dimethylallyl Cys and AnxV S-propargyl Cys
Control experiments shown above revealed that there is no reaction between AnxV (no S-allyl present) and Py-Tz 2. To further corroborated that the bis-addition is not crossreactivity with other amino acids on the protein, the Cysteine 316 of AnxV was alkylated with dimethylallyl bromide and propargyl chloride (Figures S20 and S21). This control was to verify if modification of the cysteine may induce conformational changes in the structure of the protein, exposing any buried amino acid that could potentially react with the tetrazine. As observed before, upon reaction of AnxV S-dimethylallyl Cys ( Figure   S22) and AnxV S-propargyl Cys with Py-Tz 2 ( Figure S23) the starting protein was detected unaltered after 48 h at 37 º C.

Reaction of 3,3-dimethylallyl bromide with AnxV
AnxV was prepared as a 0.12 mg/mL solution in 50 mM sodium phosphate buffer at pH 11.0 and 50 µL (0.168 nmol) were transferred to a 0.5 mL eppendorf tube.

Reaction of propargyl chloride with AnxV
AnxV was prepared as a 0.4 mg/mL solution in 50 mM sodium phosphate buffer at pH 11.0 and 50 µL (0.559 nmol) were transferred to a 0.5 mL eppendorf tube. Propargyl

Reaction of tetrazine-dye Tz-Rhod 4 with AnxV S-allyl Cys
AnxV S-allyl Cys was prepared as a 0.19 mg/mL solution in PBS buffer at pH 7.

Figure S28
ESI-MS of the reaction of allyl selenocyanate with C2Am after 2 h at room temperature.

NuPAGE Bis-Tris gels
AnxV S-allyl Cys was reacted with an excess of Tz-Rhod 4 and Tz-Cy3 5 as previous described (Sections 2.4.7. and 2.4.8.). After 12 h at 37 ºC, the reaction mixture was Fluorescence microscopy was performed using an inverted epifluorescent microscope (Olympus IX-71) connected to a F-view digital camera (Soft Imaging System). Images were acquired in the Tritc and Hoechst channels and analyzed using the software Cell-F. Identical Supporting Information S51 image acquisition settings were used for the control, experimental and blocking data sets.

Computational Details
Full geometry optimizations were carried out with Gaussian 09 [4] using the M06-2X hybrid functional [5] and 6-31G(d) basis set. Bulk solvent effects in water were considered implicitly through the IEF-PCM polarizable continuum model. [6] The possibility of different conformations was taken into account for all structures. Frequency analyses were carried out at the same level used in the geometry optimizations, and the nature of the stationary points was determined in each case according to the appropriate number of negative eigenvalues of the Hessian matrix. Scaled frequencies were not considered. Scaled frequencies were not considered. Mass-weighted intrinsic reaction coordinate (IRC) calculations were carried out by using the Gonzalez and Schlegel scheme [7,8] in order to ensure that the TSs indeed