In the format provided by the authors and unedited.

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Supplementary information for:

Controlled membrane translocation provides a mechanism for signal transduction and amplification

Matthew J. Langton,a Flore Keymeulen,a Maria Ciaccia,a Nicholas H. Williams b and Christopher A. Hunter *a aDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom;

bDepartment of Chemistry, University of Sheffield, Sheffield S3 7HF, UK

Contents:

Experimental procedures 2

Synthetic procedures 4

Transmembrane signalling experiments – additional data 9

References 13

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2678

NATURE CHEMISTRY | www.nature.com/naturechemistry 1

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1. Experimental procedures 1H-NMR and 13C-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 Companion) using pre-packed cartridges of silica (50µm PuriFlash® Column). GPC purification of the vesicles was carried out using GE Healthcare PD-10 desalting columns prepacked with Sephadex G-25 medium. Fluorescence spectroscopic data were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent). pH measurements were conducted using a Mettler-Toledo “Seven Compact” pH meter equipped with an “In-lab Micro” electrode. Vesicles were prepared as described below using Avestin “LiposoFast” extruder apparatus, equipped with polycarbonate membranes with 200 nm pores.

Materials

All reagents and solvents were used without further purification. Lipids were purchased from Sigma Aldrich and used without further purification.

Fluorescence experiments

Fluorescence excitation experiments were recorded using the following parameters: emission wavelength = 510 nm, excitation range 380−480 nm, recorded at 2 minute intervals. For experiments in which vesicles contained 250 µM solution of ester 2, the excitation and emission slits were set at 5 nm. For control experiments in which ester 2 was added to the extra-vesicle solution, the excitation and emission slits were set at 2.5 nm.

Dynamic Light Scattering (DLS) Experiments

DLS experiments were undertaken using a Malvern Zetasizer Nano S90 apparatus, equipped with a 4 mW HeNe laser source (633 nm). The sample was placed in a thermostat-controlled cell-holder maintained at 25 °C and the scattered light collected at an angle of 173°. Unimodal intensity weighted distributions of the hydrodynamic diameters were obtained and the averaged values of five measurements reported (see Supplementary Figure 5). General procedure for vesicles preparation

To a round bottom flask was added chloroform/ethanol solutions of the lipids (1,2-Dioleoyl-sn-glycero-3-phosphocholine DOPC and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOPE, in a 3:2 ratio) and a chloroform solution of 1 in order to obtain final concentrations of 2 mM and 50 µM for the lipids and 1, respectively. The solvent was removed in vacuo and dried under high vacuum for 1 h. A 100 mM solution of HEPES buffer at pH 6.8 (2.5 mL) was added to the flask containing the lipids, and sonicated for 1 min. The suspension was subjected to 5 cycles of freeze-thaw using liquid nitrogen. To the suspension was added stock solutions of ester 2 and zinc chloride as appropriate to reach final concentrations of 250 µM. The suspension was extruded for 19 times through a polycarbonate filter with 200 nm pores in an Avestin Lipofast apparatus, and then the vesicles were separated by the bulk solution using prepacked GPC columns eluting with a 100 mM NaCl solution at pH 6.8.

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Imaging of Vesicles by Total Internal Reflection Microscopy

Borosilicate glass coverslips (VWR international, Ø 50 mm) were cleaned using an argon plasma cleaner (PDC-002, Harrick Plasma) for 30 min to remove any fluorescent residues. Multi-well slide chambers (Grace Bio-Labs CultureWell chambered coverglass 50 well) were separated from original coverglass and affixed to the cleaned coverslides. Wells were coated with 10 µL of poly-L-lysine (70 000–150 000 molecular weight, Sigma-Aldrich) for 30 minutes, before being washed three times with 100 mM NaCl solution. 10 µL of each sample was then adsorbed to the cover slides for 15 minutes before imaging.

The samples were imaged using a home-built (Klenerman lab, Cambridge, UK) total internal reflection fluorescence microscope as described previously.1 The total internal reflection mode restricts the fluorescence illumination to within 200 nm from the sample slide. A 405 nm laser (Oxxius LaserBoxx) was aligned and directed parallel to the optical axis at the edge of a 1.49 NA TIRF objective (APON60XO TIRF, Olympus), mounted on an inverted Nikon Eclipse Ti microscope. Fluorescence was collected by the same objective and separated from the returning TIRF beam by a dichroic (ZT 405/532 rpc, AHF analysentechnik AG), and passed through an appropriate emission filter (FF03-525/50-25, Semrock). The control of the hardware was performed using custom-written scripts (bean-shell) for MicroManager (NIH). The images were recorded on an EMCCD camera (Evolve 512 Delta, Photometrics) operating in frame transfer mode (EMGain of 4.4 e–/ADU and 250 ADU/photon). Each pixel was 240 nm in length. Images were recorded for 30 frames with an exposure time of 50 ms and averaged using ImageJ (NIH) software. The intensity of 405 nm illumination was 100 µW at the top of the objective in epifluorescence.

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2. Synthetic procedures

Supplementary Scheme 1. Synthesis of ester 2

Supplementary Scheme 2. Synthesis of transducer 1

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Substrate 2

Compound 2 was prepared as previously described.2 Briefly, 8-hydroxypyrene-1,3,6-trisulfonate trisodium salt 3 (1.30 g, 2.50 mmol) and sodium acetate (25 mg, 0.30 mmol) were dissolved in acetic anhydride (15 mL), and the mixture was stirred at reflux for 48 h. After this time, the mixture was allowed to cool to room temperature and it was diluted with tetrahydrofuran containing 10% (v/v) of acetic acid (15 mL). The solid was collected by vacuum filtration and washed with cold acetone (3 × 15 mL) and diethylether (2 × 15 mL), to afford the product as a pale brown solid (1.20 g, 84%) and characterised by comparison with data from the literature. 1H NMR (400 MHz, D2O) δ (ppm): 9.26 (s, 1H), 9.21 (d, 1H, 3J = 10.0 Hz), 9.15 (d, 1H, 3J = 10.0 Hz), 9.10 (d, 1H, 3J = 10.0 Hz), 8.49 (s, 1H), 8.39 (d, 1H, 3J = 10.0 Hz), 2.63 (s, 3H).

SI-1

A solution of 2,6-diacetylpyridine (300 mg, 1.84 mmol), hydroxylamine hydrochloride (84 mg, 1.2 mmol) and sodium acetate (12 mg, 0.15 mmol) in water (10 mL) was refluxed for 1 h and then stirred at rt for 15 h. The white solid formed was filtered, washed with water and purified by flash chromatography (SiO2, 0-20% ethyl acetate in 40-60 Pet. ether) to give the product as a white solid (212 mg, 65%). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.07-8.02 (m, 3H), 7.84 (t, 3J = 8.0 Hz, 1H), 2.79 (s, 3H), 2.46 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 200.4, 157.0, 153.6, 152.8, 137.3, 123.8, 123.8, 121.6, 25.8, 10.4. HRMS calc. for C9H9NO2-H+: 164.0712; found: 164.0719.

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SI-2

To a solution of lithocholic acid (500 mg, 1.33 mmol) in 1,4-dioxane (12 mL) at 10 °C was added triethylamine (0.19 mL, 1.33 mmol). Ethyl chloroformate (0.125 mL, 1.33 mmol) was then added and the mixture was stirred at 10 °C for 10 min. Morpholine (0.71 mL, 8.2 mmol) was then added, and the mixture refluxed for 18 h. The reaction mixture was poured into saturated NaHCO3(aq) (30 mL) and extracted with DCM (3 × 15 mL). The organic layers were dried over MgSO4 and evaporated under reduced pressure to give a white solid, which was purified by flash chromatography (SiO2, 0-40% gradient of ethyl acetate in dichloromethane). The product was isolated as a white solid (290 mg, 49%). 1H NMR (400 MHz, CDCl3) δ (ppm): 3.68-3.58 (m, 7H), 3.45 (t, 3J = 5.0 Hz, 2H), 2.39-2.32 (m, 1H), 2.23-2.16 (m, 1H), 1.97-0.91 (m, 33H), 0.64 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ (ppm): 172.5, 72.0, 67.1, 66.8, 56.6, 56.1, 46.2, 42.9, 42.2, 42.0, 40.6, 40.3, 36.6, 36.0, 35.8, 35.5, 34.7, 31.5, 30.7, 30.2, 28.4, 27.3, 26.6, 24.4, 23.5, 21.0, 18.6, 12.2. HRMS calc. for C28H47NO3-H+: 446.3634; found: 446.3617.

SI-3

BH3.THF (1 M, 10 mL, 2.25 mmol) was added dropwise to solid SI-2 (200 mg, 0.45 mmol) and

mixture was refluxed for 2 h. The solution was cooled to rt and stirred for a further 15 h, after which the reaction was quenched with methanol (15 mL). The solvent was evaporated, the crude purified by column chromatography (SiO2, 0-60% ethyl acetate gradient in 40-60 Pet ether) to afford the product as a white solid (174 mg, 89%). 1H NMR (400 MHz, CDCl3) δ (ppm): 4.24-4.18 (m, 2H), 3.70 (dt, 3J = 12.5 Hz, 4J = 3.5 Hz, 2H), 3.65-3.57 (m, 1H), 2.98-2.94 (m, 2H), 2.79-2.64 (m, 4H), 2.03-0.91 (m, 35H), 0.64 (s, 3H). 13C-NMR (100 MHz, CDCl3) δ (ppm): 72.0, 62.2, 58.0, 58.0, 56.6, 56.3, 42.9, 42.2, 40.6, 40.3, 36.6, 36.0, 35.8, 35.5, 34.7, 33.6, 30.7, 28.5, 27.3, 26.6, 24.4, 23.5, 21.0, 19.4, 18.8, 12.2. HRMS calc. for C28H49NO2-H+: 432.3842; found: 432.3821.

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SI-4

To a solution of SI-3 (65 mg, 0.13 mmol), 4-dimethylaminopyridine (1.0 mg, 0.01 mmol) and triethylamine (35 mg, 0.19 mmol) in DCM (10 mL) at 0°C was added chloroacetylchloride (22 mg, 0.19 mmol) in DCM (5 mL) dropwise. The reaction was allowed to warm to rt and stirred for a further 16 h. The reaction was washed with water (2 × 5 mL), dried over MgSO4, filtered and the solvents removed in vacuo. The crude was purified by column chromatography (SiO2, 0-15% gradient of MeOH in DCM) to afford the product as a white solid (62 mg, 95%). 1H NMR (400 MHz, CDCl3) δ (ppm): 4.81 (m, 1H), 4.04 (m, 2H), 2.88 (m, 2H), 1.98-1.69 (m, 8H), 1.58 (m, 2H), 1.48-1.39 (m, 8H), 1.29-1.00 (m, 10H), 0.94 (m, 6H), 0.64 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 166.8, 77.3, 76.7, 63.8, 58.3, 56.4, 56.0, 51.8, 42.7, 41.9, 41.3, 40.4, 40.1, 35.7, 35.4, 34.9, 34.6, 32.9, 32.0, 28.3, 27.0, 26.5, 26.3, 24.1, 23.3, 20.8, 20.1, 18.5, 12.0. HRMS calc. for C30H50ClO3-H+: 508.3557; found: 508.3565.

SI-5

To a solution of mono-oxime SI-1 (16 mg, 0.089 mmol) in DMF (5 mL) at 0°C was added potassium carbonate (82 mg, 0.59 mmol). After warming to rt the mixture was stirred for 1h, before cooling to 0°C and adding a solution of SI-4 (30 mg, 0.059 mmol) in DMF (5 mL). The reaction was warmed to rt and stirred for a further 16 h, before diluting with DCM (50 mL) and washing with brine (2 × 10 mL). The organic layer was dried over MgSO4, filtered and the solvent removed in vacuo. The crude was purified by column chromatography (SiO2, 0-10% gradient of MeOH in DCM) to afford the product as a white solid (25 mg, 65%). 1H NMR (400 MHz, CDCl3) δ (ppm): 8.09 (d, 3J = 7.9 Hz, 1H), 8.01 (d, 3J = 7.9 Hz, 1H), 7.81 (t, 3J = 7.9 Hz, 1H), 4.86 (m, 1H), 3.75 (m, 4H), 2.76 (s, 3H), 2.49 (s, 3H), 2.47 (app br. s, 4H), 2.31 (2H, m), 1.99-0.99 (m, 30H), 0.93 (m, 8H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 200.3, 200.1,169.3, 157.1, 153.1, 152.6, 136.9, 124.1, 121.5, 75.3, 71.3, 67.0, 59.7, 56.5, 56.2, 53.8, 42.7, 41.9, 40.5, 40.2, 35.8, 35.7, 35.0, 34.6, 33.6, 32.2, 29.7, 28.3, 27.0, 26.6, 26.3, 25.7, 24.2, 23.3, 23.1, 20.8, 18.7, 12.0, 11.2. HRMS calc. for C39H59N3O5-H+: 650.4533; found: 650.4545.

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Transducer 1

To a solution of SI-5 (17 mg, 0.026 mmol) in 1:1 CHCl3/ethanol (5 mL) was added a solution of hydroxylamine hydrochloride (1.8 mg, 0.026 mmol) and sodium acetate (1.1 mg, 0.013 mmol) in H2O (0.5 mL) and the reaction heated at 60°C for 16 h. The reaction was diluted with CHCl3 (10 mL), and washed with water (2 × 5 mL). The organic layer was dried over MgSO4, filtered and the solvents removed in vacuo to yield the product as a white solid (25 mg, 99%). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.90 (m, 2H), 7.65 (t, 3J = 7.9 Hz, 1H), 4.77 (m, 3H), 3.84 (m, 4H), 2.62 (br. m, 4H), 2.48 (s, 3H), 2.41 (s, 3H), 1.85-0.77 (m, 40H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 170.0, 157.7, 155.7, 154.0, 152.5, 136.2, 119.9, 119.6, 75.1, 71.2, 66.4, 60.2, 58.5, 56.2, 54.6, 53.8, 53.6, 42.5, 41.6, 40.3, 39.9, 35.6, 34.8, 34.5, 33.6, 32.0, 29.7, 28.4, 28.0, 26.9, 26.3, 26.2, 24.2, 23.3, 20.8, 18.9, 18.4, 12.0, 11.1, 10.0. HRMS calc. for C39H60N4O5-H+: 665.4642; found: 665.4628.

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3. Transmembrane signalling experiments – additional data

Ratiometric analysis of the excitation spectrum of 3 (Supplementary Figure 1) enables the in-situ determination of the vesicle internal solution pH at the end-point of the signalling experiment, using the ratio of the emission intensity measured at 510 nm arising from excitation at 460 and 405 nm, respectively, in conjunction with the calibration curve (Supplementary Figure 2).3 Raising the pH of the external solution from pH 6.8 to pH 9.0 resulted in only a slight increase in lumen pH, from 6.8 to 7.0. Control experiments in which substrate 2 was added to the outside of vesicles containing transducer 1, at both pH 6.8 and pH 7.0, revealed that the transducer is inactive towards the ester hydrolysis reaction at these pH values (Supplementary Figure 4), and thus the 0.2 pH unit increase in lumen pH during the signalling experiment does not account for the catalyst activation.

Supplementary Figure 1. Change in excitation spectra of the vesicle suspension with time during the signalling experiment. (a) Change in relative fluorescence emission intensity at 510 nm, exciting at 415 nm. The external solution was raised to pH 9 at t = 20 min (arrow). (b) Excitation spectra of the vesicle suspension (λem = 510 nm) at (i) t = 0 and (ii) t = 250 min. The ratio of the peaks at 460 nm and 405 nm was used to determine the pH of the solution inside the vesicles using the calibration curve in Supplementary Figure 2. At t = 250, I460/I405 = 0.7, corresponding to a final lumen pH of 7.0.

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Supplementary Figure 2. pH as a function of fluorescence response for HPTS (3). The ratio of the fluorescence emission intensity at 510 nm from excitation at 460 nm and 405 nm (I460/I405) was used to determine the pH of the solution inside the vesicles. A solution of vesicles formed from 3:2 DOPC/DOPE lipids and 2.5% 1, encapsulating 250 µM 3 and 250 µM ZnCl2 in 100 mM HEPES buffer was lysed with Triton X-100, and the pH adjusted by adding small aliquots of dilute NaOH(aq). After each addition, the pH of the solution was measured using a pH electrode and the excitation spectrum recorded. Top: fluorescence excitation spectra (excitation between 380 – 480 nm, emission at 510 nm) recorded between pH 6.5 and 9.3 (isosbestic point at 415 nm). Bottom: pH vs I460/I405. The data was fitted to a Henderson-Hasselbach type equation (Supplementary Equation 1, r2 = 0.995), to obtain the calibration curve, using the following parameters: a = 4.03; b = 7.81; where x = I460/I405.

SupplementaryEquation1: 𝒑𝑯 = 𝒃 − 𝒍𝒐𝒈 𝒂!𝒙𝒙

0

50

100

150

200

250

300

350

380 400 420 440 460 480

I / a

.u.

λ / nm

6 6.5

7 7.5

8 8.5

9 9.5 10

0 0.5 1 1.5 2 2.5 3 3.5 4

pH

I460/I405

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Supplementary Figure 3. Control experiment with substrate 2 outside of vesicles containing transducer 1. Transducer 1 is inactive at pH 6.8 (black) and pH 7.0 (grey), demonstrating that small drifts in pH around 7 are not responsible for catalyst activation. Experiment conducted with 250 µM ZnCl2 and 250 µM substrate 2 (added at t = 0) in 100 mM HEPES at pH 6.8 or 7.0 on the exterior of 200 nm DOPC/DOPE vesicles (2 mM lipids, 2.5 mol% 1) vesicles containing 100 mM HEPES buffer at pH 6.8. Plot shows relative fluorescence emission intensity at 510 nm, exciting at 415 nm vs. time. The intensity is normalised to the excitation intensity corresponding to 250 µM 3.

The signalling process can be switched reversibly by changing the pH of the external solution (main text, Fig 4b). To demonstrate that the rate of ester hydrolysis remains constant in two successive “ON” states, we carried out two parallel switching experiments (Supplementary Figure 4). A single batch of vesicles was split into two cuvettes. In the first, the external pH was raised to pH 9.0, and the reaction was monitored by fluorescence spectroscopy as previously described (Supplementary Figure 4a). In the second, the external solution was raised to pH 9.0, and then lowered back to pH 7.0 after 60 minutes. After a further 60 minutes the pH was raised to pH 9.0, and the fluorescence emission was recorded (Supplementary Figure 4b, time scale off-set to correspond to (a)). This data was consistent with that obtained at pH 9.0 without a subsequent switching cycle (Supplementary Figure 4 and Supplementary Table 1, pH 9.0), indicating that there is no rearrangement or degradation of the transducer during the switching experiment.

Supplementary Figure 4. Catalyst switching by reciprocating transmembrane motion. Fluorescence emission intensity at 510 nm, exciting at 415 nm (points), fitted to a pseudo-first order rate equation (black line). (a) For a single ON stroke (green, kobs = 6.3 × 10-3 min-1); (b) for a second ON stroke after a cycle of 60 min ON followed by 60 min OFF (orange). Orange data off-set by −60 mins, and fitted to obtain kobs = 6.2 × 10-3 min-1.

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Supplementary Table 1. Kinetic data for signalling experiments

pH (outside) k (10-3 min-1)

9.0 6.3 ± 0.5

8.0 3.7 ± 0.8

7.0 0.6 ± 0.1

Kinetic data obtained by fitting to a pseudo-first order rate equation. Rate constants obtained from an average of three separate experiments, and errors given at 2σ. Vesicle composition and experimental conditions as described above.

Vesicle volume

The average volume of a single vesicle can be easily estimated through simple geometrical calculations, using an average diameter of 200 nm and a membrane thickness of 4 nm: Vves = 4 × 10-21

m3. For a 2 mM vesicle suspension (with respect to lipid concentration), using an average area of 0.6 nm2 per lipid,4 the ratio of the total internal volumes of the vesicles (Vin) to external volume (of the cuvette, Vout) is estimated to beVin

Vout= 0.02

Supplementary Figure 5. Dynamic Light Scattering (DLS). Left: DLS distributions for five measurements of DOPC/DOPE vesicles prepared by passing through 200 nm membranes as described above. Right: average vesicles dimensions determined by DLS and the corresponding poly-dispersity index.

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4. References

1. Jönsson, P. et al. Hydrodynamic trapping of molecules in lipid bilayers. Proc. Natl. Acad. Sci. 109, 10328–

10333 (2012).

2. Wang, P. S. P., Nguyen, J. B. & Schepartz, A. Design and High-Resolution Structure of a β3-Peptide Bundle

Catalyst. J. Am. Chem. Soc. 136, 6810–6813 (2014).

3. Clement, N. R. & Gould, J. M. Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous

hydrogen ion concentration in phospholipid vesicles. Biochemistry (Mosc.) 20, 1534–1538 (1981).

4. Kučerka, N., Nieh, M.-P. & Katsaras, J. Fluid phase lipid areas and bilayer thicknesses of commonly used

phosphatidylcholines as a function of temperature. Biochim. Biophys. Acta BBA - Biomembr. 1808, 2761–

2771 (2011).