Transmembrane signal transduction by cofactor transport

Information processing and cell signalling in biological systems relies on passing chemical signals across lipid bilayer membranes, but examples of synthetic systems that can achieve this process are rare. A synthetic transducer has been developed that triggers catalytic hydrolysis of an ester substrate inside lipid vesicles in response to addition of metal ions to the external vesicle solution. The output signal generated in the internal compartment of the vesicles is produced by binding of a metal ion cofactor to a head group on the transducer to form a catalytically competent complex. The mechanism of signal transduction is based on transport of the metal ion cofactor across the bilayer by the transducer, and the system can be reversibly switched between on and off states by adding cadmium(ii) and ethylene diamine tetracarboxylic acid input signals respectively. The transducer is also equipped with a hydrazide moiety, which allows modulation of activity through covalent conjugation with aldehydes. Conjugation with a sugar derivative abolished activity, because the resulting hydrazone is too polar to cross the bilayer, whereas conjugation with a pyridine derivative increased activity. Coupling transport with catalysis provides a straightforward mechanism for generating complex systems using simple components.

1 H and 13 C NMR NMR spectra were recorded on a 400-MHz Bruker® spectrometer. Chemical shifts are reported as δ values in ppm. Flash chromatography was carried out on an automated system (Combiflash® Rf+ LumenTM) using pre-packed cartridges of silica (25 μm PuriFlash® Column) or neutral alumina (50 μm RediSep® Rf Column). GPC purification of the vesicles was carried out using GE Healthcare PD-10 desalting columns prepacked with Sephadex® G-25 medium. Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) in Hellma® Analytics Suprasil® quartz cuvettes. pH measurements were conducted using a Mettler-Toledo SevenCompactTM pH meter equipped with an InLab® Micro electrode. Vesicles were assembled in Eppendorf® polypropylene ProteinLoBind® polypropylene microcentrifuge tube and extruded as described below using Avanti® Polar Lipids extruder kits, equipped with Avestin® LiposoFast Liposome Factory 200 nm polycarbonate membranes with GE Healthcare Whatman® 10 mm polyester filter support. Solutions or vesicles suspensions were transferred using Eppendorf Multipette® Xstream Pippette with Combitips Advanced® or Hamilton Microliter™syringes. All reagents and solvents were used without further purification. Chemicals were purchased from Sigma-Aldrich® and used without further purification. 4 (13.2 mg, 0.018 mmol) was dissolved in dry dichloromethane (1 ml) and cooled to 0 o C. Trifluoroacetic acid (1 ml) was added and the reaction was left to warm up to room temperature and left to stir for 30 min. The solvents were removed under vacuo and the obtained slurry was dissolved in DCM (10 ml) and washed with a saturated solution of NaHCO3 (3x10 ml). The organic phase was dried over MgSO4 and the solvent removed in vacuo to afford the afforded the product as a white solid. (10 mg, 77%).

S7
A solution of compound 10 (70 mg, 0.22 mmol) and sodium methoxide (12 mg, 0.22 mmol) in methanol (1 mL) was stirred at room temperature for 1.5 h. The reaction mixture was diluted with methanol (5 mL) and neutralized with Amberlyst-15 resin. The solution was poured into diethyl ether (50 mL), and the precipitate was filtered and dried to give the product as a white amorphous solid (34 mg, 77%).

Ester Hydrolysis Experiments
Fluorescence spectroscopic data were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent). Fluorescence excitation experiments were recorded using the following parameters: emission wavelength = 510 nm, excitation range 380−480 nm, recorded at 4 minute intervals, with the excitation and emission slits set at 5 nm. All experiments were repeated a minimum of two times on independently prepared samples to confirm reproducibility. At the end of the experiment, 10 μL of 1M NaOH was added to hydrolyze all of the remaining Ac-HPTS substrate, and the emission measured at this end point was used to normalize the data. Figure S6. Time dependent normalized fluorescence intensity traces (Em=510 nm, Ex=415 nm) for Ac-HPTS hydrolysis. In bulk activity experiment: at t=0 min, 100 µM 5 was added to a solution containing 25 µM Ac-HPTS, 250 µM CdSO4 or 250 µM ZnCl2 in 50 mM MES buffer, pH=6.6, 100 mM NaCl, 0.05% Triton-x100. As a control, no CdSO4 was used.

Dose response experiments:
Vesicles were prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC chloroform solution). For vesicles containing the molecules, the corresponding amount was added to the lipid solution. The solvent was evaporated under a stream of nitrogen and the residue dried for 2h under high vacuum. The resulting thin film was hydrated in 500 µL of buffer solution (50 mM MES, 100 mM NaCl, pH=6.6) containing 250 µM Ac-HPTS. After hydration, the suspension was subjected to five freeze-thaw cycles (liquid nitrogen, water at room temperature). The obtained suspension was extruded 21 times through a 200 nm polycarbonate membrane. The vesicle suspension was separated from any extravesicular content by size exclusion chromatography using prepacked Sephadex G-25M columns (prequilibrated with buffer solution, as mobile phase). The obtained solution was diluted to the final concentration of 1 mM and used as stock solution within the day. 500 μL of stock vesicle solution (DOPC/5 97.5/2.5) was placed into a quartz fluorimetric cell. At t=30 min, CdSO4 was injected from an aqueous stock solution at the corresponding concentration. At the end of the experiment, 10 μL of 5% Triton X-100 and 1M NaOH was added to lyse the vesicles and hydrolyze all of the remaining Ac-HPTS substrate. The emission measured at this end point was used to normalize the data taking into account of the dilution factor.
Hydrazone activity experiments: 500 μL of stock vesicle solution (DOPC/5 97.5/2.5) was placed into a quartz fluorimetric cell. At t=0 min, compounds were injected from a stock solution in DMSO. At t=30 min, 1 mM CdSO4 was injected S15 from an aqueous stock. At the end of the experiment, 10 μL of 5% Triton X-100 and 1M NaOH was added to lyse the vesicles and hydrolyze all of the remaining Ac-HPT substrate. The emission measured at this end point was used to normalize the data taking into account of the dilution factor. Figure S7. Effect of addition of 6 on the activity of membrane compound 4. Red data: at t=30 min (2), 1 mM CdSO4 was added to a 1 mM vesicle solution containing 2.5 mol% 4. Green data: at t=0 min (1), 1 mM of 6 was added to a 1 mM vesicle solution containing 2.5 mol% 4, and at t=30 min (2), 1 mM CdSO4 was added.

ON-OFF switching experiments:
500 μL of stock vesicle solution (DOPC/5 97.5/2.5) was placed into a quartz fluorimetric cell. At t=30 min, 1 mM 6 was injected from a stock solution in DMSO. At t=60 min, 1 mM CdSO4 was injected. At t=75 min, 2 mM EDTA was injected. At t=130 min, 2 mM CdSO4 was injected. At the end of the experiment, 10 μL of 5% Triton X-100 and 1M NaOH was added to lyse the vesicles and hydrolyze all of the remaining Ac-HPTS substrate. The emission measured at this end point was used to normalize the data taking into account of the dilution factor. S16

Cadmium Transport Experiments
Vesicles were prepared from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC chloroform solution). The solvent was evaporated under a stream of nitrogen and the residue dried for 2h under high vacuum. The resulting thin film was hydrated in 500 µL of buffer solution (10 mM MES, 100 mM CdSO4, pH=6.6) containing 1 mM HPTS. After hydration, the suspension was subjected to five freeze-thaw cycles (liquid nitrogen, water at room temperature). The obtained suspension was extruded 21 times through a 200 nm polycarbonate membrane. The vesicle suspension was separated from any extravesicular content by size exclusion chromatography using prepacked Sephadex G-25M columns (prequilibrated with buffer solution, as mobile phase). The obtained solution was diluted to the final concentration of 0.1 mM and used as stock solution within the day. 500 μL of stock vesicle solution (DOPC) was placed into a quartz fluorimetric cell. Prior to the experiment, 5mol% of compound 5 or compound 5 and 1 mM 2-formylpyridine were injected and left to equilibrate for 30 mins. (formation of the conjugate was verified by LCMS-UV). At t = 1 min, a corresponding volume of aqueous H2SO4 (0.1 M) was injected in order to decrease the pH of the bulk solution to 6.1 (volume of needed H2SO4 solution was predetermined for each batch of vesicles using a SevenCompact pH/Ion S220 pH meter) The emission of HPTS at 510 nm was monitored at two excitation wavelengths (405 and 460 nm) simultaneously. Change in fluorescence was registered for 5 mins after which the vesicles are lysed with 10 μL 5% aqueous Triton X-100 solution to equilibrate the intra and extravesicular solution. Figure S8. Comparison of the effect 5 (blue), the hydrazone conjugate formed by 5 and 6 (yellow) and of DMSO (red) on cadmium transport across vesicle bilayer membranes. DOPC vesicles containing 1 mM HPTS were prepared at pH 6.6 and suspended in 10 mM MES buffer with 100 mM CdSO4 and DMSO (5 µl), 5 (5 mol% relative to lipids) or the hydrazone conjugate formed by 5 and 6 (5 mol% relative to lipids) were added. At t = 1 min., H2SO4 was added to lower the external pH to 6.1, and the local pH inside the vesicles was monitored using the ratio of the fluorescence emission at 510 nm due to the phenol (λex = 405 nm) and phenolate (λex = 460 nm) forms of HPTS. At t = 6 min., the vesicles were lysed by addition of Triton-x100 to obtain the HPTS emission at a pH of 6.1.