Extended Data Fig. 1: Synthesis of bis functionalized NPBF based photocleavable linker and the photo-sensitive tag (PsT).
From: Single-cell analysis of regions of interest (SCARI) using a photosensitive tag
(a) Reagents and conditions for synthesis of the NPBF cage: a) bromoacetaldehyde diethyl acetal, KOH, NMP, 70 oC, 15 hours, 90%; b) polyphosphoric acid, toluene, 111 oC, 16 hours, 33%; c) BBr3, DCM, 1 hour at - 78 oC then 1 hour at room temperature, 89%; d) TBDMSCl, imidazole, DMF, 1 hour, room temperature, 70%; e) (i) n-BuLi, THF, -78 oC, 1 hour, (ii) triisopropyl borate, 30 minutes at -78 oC then 30 minutes at room temperature; f) Pd(PPh3)4, K2CO3, THF/H2O, 75 oC, 18 hours, 75%; g) HF-pyridine, THF, room temperature, 1 hour, 77%; h) K2CO3, propargyl bromide, room temperature, 5 hours, 86%; i) NaBH4, 1,4-dioxane/methanol, room temperature, 1 hour, 0 oC to room temperature, 88%; j) N,N’-disuccinimidyl carbonate, Et3N, DMF, room temperature, 4 hours, 79%. (b) Reagents and conditions for synthesis of the PsT: a) 1% TFA, DCM, room temperature, 30 minutes (2x); b) 1, DiPEA, DMF, room temperature, 18 hours; c) 20% piperidine, DMF, room temperature, 2 minutes (3x), d) TFA, TIS, H2O 95/2.5/2.5, room temperature, 3 hours; e) CuSO4, sodium ascorbate, THPTA, AF594-N3, H2O, t-BuOH room temperature, 2 hours. (c) Uncaging efficiency of the PsT over time. 10µM PsT (in water) was uncaged with 420-nm light using a 1W LED-lamp for the indicated times and reaction products were measured using LC-MS analysis. Data points represent the mean +/- s.d. of technical triplicates (n=3) from a single experiment.
