Artificial transmembrane signal transduction mediated by dynamic covalent chemistry

Reversible formation of covalent adducts between a thiol and a membrane-anchored Michael acceptor has been used to control the activation of a caged enzyme encapsulated inside vesicles. A peptide substrate and papain, caged as the mixed disulfide with methane thiol, were encapsulated inside vesicles, which contained Michael acceptors embedded in the lipid bilayer. In the absence of the Michael acceptor, addition of thiols to the external aqueous solution did not activate the enzyme to any significant extent. In the presence of the Michael acceptor, addition of benzyl thiol led to uncaging of the enzyme and hydrolysis of the peptide substrate to generate a fluorescence output signal. A charged thiol used as the input signal did not activate the enzyme. A Michael acceptor with a polar head group that cannot cross the lipid bilayer was just as effective at delivering benzyl thiol to the inner compartment of the vesicles as a non-polar Michael acceptor that can diffuse across the bilayer. The concentration dependence of the output signal suggests that the mechanism of signal transduction is based on increasing the local concentration of thiol present in the vesicles by the formation of Michael adducts. An interesting feature of this system is that enzyme activation is transient, which means that sequential addition of aliquots of thiol can be used to repeatedly generate an output signal.

1 H NMR and 13 C NMR spectra were recorded on a 700 and 400 MHz Bruker spectrometer. Chemical shifts are reported as δ values in ppm. All the 1 H NMR spectra were referenced to residual isotopic impurity of CDCl3 (7.26 ppm) and DMSO-d6 (2.50 ppm). 13 C NMR spectra (100 MHz) were referenced to the CDCl3 peak (77.0 ppm) and DMSO-d6 (39.5 ppm). The following abbreviations are used in reporting the multiplicity for NMR resonances: s=single, d=doublet, t= triplet, and m=multiplet. The NMR data were processed using MestReNova 10.0.2. High resolution electrospray ionization mass spectrometry (HRMS-ESI) was performed on Waters LCT Premier TOF Spectrometer or by the Mass Spectrometry Service at the Department of Chemistry. The LCMS analysis of samples was performed using Waters Acquity H-class UPLC coupled with a single quadrupole Waters SQD2. ACQUITY UPLC BEH C8 Column, 130Å, 1.7 µm, 2.1 mm X 50 mm was used as the UPLC column. The conditions of the UPLC method are as follows: Solvent A: Water +0.1% Formic acid; Solvent B: Acetonitrile +0.1% Formic acid; Gradient of 0-4 minutes 5% -100%B + 1 minute 100% B with re-equilibration time of 2 minutes. Flow rate: 0.4 ml/min; Column temperature of 40 °C. Melting point measurements were performed on Mettler Toledo MP90. Infrared (IR) spectra were recorded on Bruker Alpha. FTIR Spectrometer with single reflection diamond Platinum ATR. UV-Vis spectra were recorded using a Cary 60 (Agilent Technologies) in Hellma Analytics Suprasil quartz cuvettes. Fluorescence measurement were performed using a micro-plate reader BMG CLARIOstar-430-0157; Software version 5.01 R2. The volume in each well was 150µL and the gain for the fluorescence emission of the coumarine dye fluorescence emission intensity at 440 nm (λexc = 365 nm) was 1200 for each measurement. All the measurement were repeated from three different preparations in duplicate. Fluorescence intensity was recorded every 2 minutes. pH measurements were conducted using a Mettler-Toledo "Seven Compact" pH meter equipped with an "Inlab Micro" electrode. GPC purification of vesicles was carried out using Sephadex G-100 with a manual column. Vesicles were prepared using Avestin "LiposoFast" extruder apparatus, equipped with polycarbonate membranes with 200 nm pores. Lipids were purchased from Sigma Aldrich and used without further purification. All the reagents and solvents were purchased from Sigma Aldrich and Acros and used without further purification. Deactivated Papain-SSMe was purchased from Thermo Fisher Scientific (Life Technologies) as part of the Thiol and Sulfide Quantitation kit T6060. Synthesis and Characterization

2.1
Intermediate 1 S1 was synthesized according to the procedure reported in C. Bravin

Loaded vesicles with 2 for UV-Vis titration and binding constant determination
A vesicle suspension was prepared in a LoB Eppendorf microcentrifuge tube by adding 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) in chloroform and receptor 2 from a methanol solution. The solvent was evaporated under nitrogen flow for 30 minutes until a dry lipid film was obtained. The lipid film was rehydrated with buffer (50 mM HEPES, 100 mM NaCl, pH=7.2) and sonicated for one minute. The solution was subjected to 4 freeze-thaw cycles using liquid nitrogen and a 40 o C water bath. Subsequently the mixture was extruded 19 times through a 400 nm pore polycarbonate filter in an Avestin Lipofast apparatus. The extruded vesicles were eluted with buffer over a manual size-exclusion column (0.5 g Sephadex G-100, equilibrated with buffer. The vesicles were obtained with a final concentration of 0.50 mM with a 10% loading of 2 (0.05 mM). The loading of receptor 2 in the vesicles was confirmed by UV-Vis and HPLC.

UV-Vis spectra of thiol addition in Vesicles and Methanol
Scheme S1 Addition of thiols 3a,3b to 2 leads to formation of the corresponding Michael adducts 4a,4b.
Kinetics of thiols 3a,3b addition to acceptor 2 embedded in vesicles were monitored with UV-Vis spectroscopy.

Vesicles
To vesicles formed by DOPC (0.5 mM, HEPES buffer, pH=7.2) loaded with receptor 2 (10% loading, 0.05 mM) were added different equivalent of thiols 3a,3b in buffer from stock solutions at 5 mM concentration. UV-Vis spectra of kinetic experiments related to the addition of several equivalents of each thiol 3a,3b to the system is reported in Figure S7.

Methanol
To 2 (0.075 mM) were added different equivalent of thiols 3a,3b from 20 mM methanol solution. UV-Vis spectra of kinetic experiments related to the addition of several equivalent of each thiol 3a,3b to the system is reported in Figure S8.

Kinetic profile of the 2 adduct formation and binding constant determination
The formation of the adducts was monitored taking into account that the absorbance of 1 at 300 nm is quenched after addition of thiols, 1 adopting the same procedure as reported in our previous work. 2 In Table S1-S2 are reported the concentration values for each species at the equilibrium when the adducts were formed in vesicles. In Table S3-S4 are reported the concentration values when the adducts were formed in methanol. In the last column of each table are reported the binding constant calculated at each equilibration point. The average binding constant value for each system is given with the related error and they are reported in Table S5 for both systems. The kinetic trend for the formation of adducts 3a,3b at different equivalents of thiols added with respect to 2 are reported using vesicles (Figure S10-S14) and in methanol solution (Figure S15-S19).

Purification of enzyme and peptide encapsulated vesicles
The HPLC traces of the fraction obtained after the GPC column confirms the presence of the Michael acceptors 1 or 2 together with the peptide substrate in the vesicles fraction respect to vesicles containing only peptide and enzyme. The other fractions collected show that the non-embedded compounds are retained in the column and the peptide is eluted after several fractions ( Figure S13).

Signalling experiment controls
Signalling experiment were performed using a microplate reader using the parameter as described in the general method section. The data were normalized for the maximum fluorescence intensity of the reader.

Assessment of vesicles leakage upon thiols addition
Addition of thiols to vesicles loaded only with enzyme does not produce a significant change in fluorescence intensity if the substrate is present externally ( Figure S14 for receptor 1, Figure S15 for receptor 2), confirming the system stability upon thiol addition. After the addition of a detergent the vesicles leak, therefore the active enzyme can interact with the substrate in solution.  Sodium Cholate R-SH S16

Reactivity between different thiols and enzyme
The thiols were reacted with the protected S-SMe papain. Addition of thiol at different concentration shows that the enzyme displays the same reactivity for the thiols ( Figure S17). In Figure S16 is reported the variation of fluorescence intensity after 350 min at different thiol concentration. In addition, benzyl thiol induce a deactivation of the enzyme respect to the other thiols ( Figure S16a). This is showed by the fluorescence intensity plateau that is reached around after 100 min. after the thiol addition. This evidence is supported also in section S6.3.       Fitting of the experimental data is reported as Figure 4 in the manuscript.

Papain deactivation
In order to establish the deactivation of papain due to benzyl thiol, a series of experiment have been devised. These experiments address the origin of the observed plateau for the fluorescence intensity when the enzyme is reacted with benzyl thiol.
The side reactivity of the generated coumarin derivative was proved by reacting 7-Amino-4-methylcoumarin with the thiol series at different concentration and monitoring the fluorescence emission. No significant changes in the fluorescent intensity were detected, therefore the plateau observed cannot be addressed to side reactivity of the fluorescent product with the thiols, especially benzyl thiol. The plateau observed in the case of benzyl thiol during signalling experiment has not been attributed to thiol oxidation. As shown in Figure S30 by adding the enzyme at different time interval to a solution containing the thiol, the fluorescence intensity observed is similar, hence concluding that minor eventual oxidation of the thiol is not the cause for the enzyme to stop working. In the experiment showed in Figure S31, the addition of 3a in a competition experiment with thiol 3b to papain display the previously observed plateau effect. In this case, after a first addition of thiol 3b after 20 min. (black arrow) a second addition of thiol (red arrow): 3a (red line) or 3b (black line) led to different outcomes. For 3a, after an initial burst the reaction stops after 100 min reaching a plateau, hence demonstrating that thiol 3a reversibly inhibits the enzyme. As comparison for 3b, the further addition of this thiol increase the enzymatic activity.