Synthesis of a photocaged tamoxifen for light-dependent activation of Cre-ER recombinase-driven gene modification

Abstract

We report the design of a water-soluble, quaternized tamoxifen photoprobe and demonstrate its application for light-controlled induction of green fluorescent protein expression via a Cre-ER recombinase system.

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging

“…UVA is less toxic to most mammalian cells than shorter UVB and UVC, and thus has served as a tool for active control in chemical biology such as protein activation, [3a, 5a, 15] optogenetics, [4a, 4c, 16] and photochemical internalization of biomacromolecules. [17] More recently, applications of light-controlled mechanisms for drug release [13] have been developed by numerous laboratories including ours in the design of prodrugs and drug delivery systems for various therapeutic agents including olaparib (AZD-2281), [11e] melphalan, [11f] methotrexate, [18] doxorubicin (Dox), [10b, 10c, 19] paclitaxel (taxol), [20] 5-fluorouracil, [21] tamoxifen, [4a, 8, 16] and ciprofloxacin. [22] However, efficient implementation of photocage molecules for such applications [13] is often challenging due in part to the synthetic complexity involved in drug and/or reporter conjugation which requires a multistep process.…”

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression

“…S9, ESI †). Lastly, we tested whether serum concentrations affected ONB 1 -4OHT activity, as it did for ONB-TAM, 22 and observed no dramatic difference in recombination efficiency (Fig. S10, ESI †).…”

“…While we previously used the UT MEF reporter system to validate the photorelease of photocaged TAM, 22 it required near toxic concentrations (Z8 mM). Consequently, our initial test of ONB-4OHT compounds used similarly high concentrations (5 mM), and we often observed reduced cell viability after treatment (Fig.…”

“…[17][18][19][20][21] We previously published a study validating this concept using a photocaged tamoxifen (TAM). 22 In that study, an o-nitrobenzyl (ONB)-alkylated N-quaternary salt 15,23,24 of tamoxifen (ONB-TAM) was synthesized and found to be water soluble, and its activity was neutralized until exposed to long wave ultraviolet light (UVA, 365 nm). We also generated a reporter, called UT MEFs, from mouse embryonic fibroblasts and used it to validate the photoreleased TAM by induction of GFP expression.…”

“…Mouse embryonic fibroblasts (MEFs) containing a UBC-CreER T2 transgene and the mTmG reporter (UT MEFs) were previously generated. 22 UT MEFs were maintained in media containing Dulbecco's modified Eagle's medium (DMEM) high glucose (GIBCO) and supplemented with 10% FBS (Invitrogen), 100 U mL À1 Penicillin 100 mg mL À1 Streptomycin (Hyclone), and 1Â L-glutamine (Hyclone). Cells were passaged 1 : 3 with 0.05% Trypsin (Gibco) every 3-5 days and were used below 8 passages.…”