Photoswitchable Probes of Oxytocin and Vasopressin

Oxytocin (OT) and vasopressin (VP) are related neuropeptides that regulate many biological processes. In humans, OT and VP act via four G protein-coupled receptors, OTR, V1aR, V1bR, and V2R (VPRs), which are associated with several disorders. To investigate the therapeutic potential of these receptors, particularly in the receptor-dense areas of the brain, molecular probes with a high temporal and spatial resolution are required. Such a spatiotemporal resolution can be achieved by incorporating photochromic moieties into OT and VP. Here, we report the design, synthesis, and (photo)pharmacological characterization of 12 OT- and VP-derived photoprobes using different modification strategies. Despite OT’s and VP’s sensitivity toward structural changes, we identified two photoprobes with good potency and photoswitch window for investigating the OTR and V1bR. These photoprobes should be of high value for producing cutting-edge photocontrollable peptide probes for the study of dynamic and kinetic receptor activation processes in specific regions of the brain.

To assess the cycle performance, the solutions were irradiated alternating with 340 nm and 420 nm (8-17) or 528 nm (18 and 19).A UV/vis spectrum was measured after each switching step.This was repeated ten times to demonstrate the stability of the compounds.The absorption of the maximum of the E-isomer was plotted against the cycle number.To determine the PSS of the photoswitches, the samples (in 0.1 mM in HEPES buffer + 1% DMSO, pH 7.5) were irradiated first with 340 nm to get the Z-isomer.Afterwards, the sample was irradiated with 420 nm (8-17) or 528 nm (18 and 19), respectively, to get back to the E-isomer.The samples were measured at the isosbestic points.

Thermal Half-lives
Thermal half-lives were measured in a 96-well plate in a Thermo Scientific Multiskan® Spectrum at 25 °C (and at 37 °C for compounds 10 and 12).The solutions (50 µM in HEPES buffer + 0.25% DMSO) were pre-irradiated with 340 nm.The absorption at 335 nm was measured every 3 h.The data was analyzed using Origin 2021.

Figure S17 .
Figure S17.PSS of compound 11 after irradiation with nm.

Figure S18 .
Figure S18.PSS of compound 12 after irradiation with nm.

Figure S19 .
Figure S19.PSS of compound 12 after irradiation with nm.

Figure S20 .
Figure S20.PSS of compound 13 after irradiation with nm.

Figure S24 .
Figure S24.PSS of compound 15 after irradiation with 340 nm (here: Z-isomer has longer retention times than E-isomer).

Figure S40 .
Figure S40.PSS of compound 15 after irradiation with 420 nm (here: Z-isomer has longer retention times than E-isomer).

Figure S27 .
Figure S27.PSS of compound 17 after irradiation with nm.

Figure S28 .
Figure S28.PSS of compound 18 after irradiation with nm.

Figure S29 .
Figure S29.PSS of compound 18 after irradiation with nm.

Figure S30 .
Figure S30.PSS of compound 19 after irradiation with nm.

Figure S33 .
Figure S33.Two-point evaluation (100 nM and 10 µM) of compounds 13-19 at V1aR.Data represent mean values ± SEM from at least three independent experiments performed in triplicate.

Figure S34 .
Figure S34.Two-point evaluation (100 nM and 10 µM) of compounds 15-19 at V1bR.Data represent mean values ± SEM from at least three independent experiments performed in triplicate.

Figure S35 .
Figure S35.Two-point evaluation (100 nM and 10 µM) of compound 12 at OTR.Data represent mean values ± SEM from at least three independent experiments performed in triplicate.