Strain-promoted sydnone bicyclo-[ 6 . 1 . 0 ]-nonyne cycloaddition

We report the strain-promoted sydnone bicyclo-[6.1.0]-nonyne cycloaddition and demonstrate that this bioorthogonal reaction enables site-specific protein labelling.

Western blots were performed with an antibody against the hexahistidine tag (Cell Signaling Technology, His Tag 27E8 mouse mAb (HRP conjugate) #9991).

Protein Mass Spectrometry
Protein LC-MS (ESI) was carried out using a Phenomenex Jupiter C4 column (150 x 2 mm, 5 µm) and samples were analyzed in the positive mode, following protein UV absorbance at 214 and 280 nm.Total protein masses were calculated by deconvolution within the MS ChemStation software (Agilent Technologies).

E.coli Lysate Labeling with Sydnone-BODIPY-FL
E.coli DH10B cells containing either psfGFP 150TAG PylT-His 6 and pBKBCNRS or psfGFP 150TAG PylT-His 6 and pBKPylRS were inoculated into LB containing ampicillin (for pBKBCNRS, 100 µg/mL) or kanamycin (for pBKPylRS, 50 µg/mL) and tetracycline (25 µg/mL).The cells were incubated for 16 h at 37 ˚C (220 rpm).5 mL of overnight culture was use to inoculate into 100 mL of LB supplemented with ampicillin (50 µg/mL) and tetracycline (12 µg/mL) and incubated at 37 ˚C (220 rpm).At OD 600 = 0.4-0.5, 3 mL culture ) to a final concentration of 2 mM.After 30 min of incubation at 37 ˚C (220 rpm), protein expression was induced by the addition of arabinose to a final concentration of 0.2%.After 2 h of expression, 1 mL cell suspensions were harvested by centrifugation (16000 g, 10 min) and frozen at -80 ˚C until required.Cell pellets were thawed on ice, suspended in in 500 µL of PBS buffer, centrifuged (16000 g, 10 min) and the supernatant discarded.This process was repeated two times.
Finally, the washed cell pellet was suspended in 100 µL of PBS buffer and incubated with 1 µL of PheSyd-BODIPY-FL (6, 5 mM in DMSO) at 37 ˚C for 6 h.The cell suspension was centrifuged (16000 g, 10 min) and the supernatant discarded.The cell pellet was suspended in 100 µL of 1X NuPAGE LDS sample buffer supplemented with 5% β-mercaptoethanol and BCN (2, final concentration of 1 mM), incubated at 37 ˚C for 2 h, heated at 93 ˚C for 10 min and centrifuged at 16000 g for 10 min.The crude cell lysates were analyzed by 4-12% SDS-PAGE to assess protein levels.Gels were either Coomassie stained or scanned with a Typhoon imager in order to visualize fluorescent bands.

Electronic
Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S1.The proposed mechanism for the 1,3-dipolar cycloaddition of Nphenyl sydnone with BCN (2).We propose that the reaction proceeds via initial suprafacial [3+2] cycloaddition of a sydnone tautomer to afford an initial diaza-[2.2.1]bicyclic lactone, which undergoes cycloreversion with the extrusion of carbon dioxide to afford a cyclooctane-fused N-phenyl pyrazole 3. Supplementary Figure S2.LC/MS (254 nm) showing the traceless formation of N-phenyl pyrazole 3 from the reaction of N-phenyl sydnone 1 with 1 eq. of BCN 2 in MeOH (c = 80 mM, rt, 30 min).Molecular mass is quoted in Daltons (Da). .UV-vis spectra of N-phenyl sydnone (1), BCN (2) and the product of the cycloaddition N-phenyl pyrazole (3) after reaction 1 with 50 eq of 2 in 55:45 MeOH:H 2 O after 24 h at room temperature.The rate constant for the reaction was determined under pseudo-first order conditions by following the exponential decay in phenyl sydnone absorbance at 310 nm over time upon reaction with a 10-80 fold excess of BCN in 55:45 MeOH:H 2 O.The mean of the observed reaction rate constants k' were plotted against the concentration of BCN to afford a linear plot with gradient k.The calculated rate constant for the reaction was 0.054 M -1 s -1 (±0.00067M -1 s -1 ) at 21 ˚C.(ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S4.Protein MS characterization of sfGFP-5 150 .All masses are given in Daltons (Da).The minor green peak represents proteolysis of the N-terminal methionine (-131 Da).
Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014 room temperature, stirred for 16 h and concentrated under reduced pressure.Residual DMF was removed by azeotropic evaporation with toluene.The crude reaction mixture was dissolved in 25 mL of 70:30:0.7 water:acetonitrile:formic acid and purified by reversed-phase HPLC.Appropriate fractions were concentrated under reduced pressure and freeze-dried to Small Molecule KineticsRate constant k for the reaction of phenyl sydnone with BCN was measured under pseudo- Electronic Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S6.Protein MS characterization of sfGFP-5 150 quantitatively labeled with PheSyd-BODIPY-FL (6).All masses are given in Daltons (Da).The minor green peak represents proteolysis of the N-terminal methionine (-131 Da).Electronic Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014 Supplementary Figure S6.Protein MS characterization of sfGFP-4 150 .All masses are given in Daltons (Da).The minor green peak represents proteolysis of the N-terminal methionine (-131 Da).Electronic Supplementary Material (ESI) for Chemical Science This journal is © The Royal Society of Chemistry 2014