Methods

2.3.1 Benzamidine-conjugated Thiophene (Ligand) Synthesis Protocol

2-(thiophen-3-yl)ethyl 4-cyanobenzoate (1): 4-Cyanobenzioc acid (1.2 equiv,

2.36 g, 16.04 mmol) and dimethylaminopyridine (0.1 equiv, 0.195 g, 1.6 mmol) were placed in a 100 ml flask, sealed and degassed for 15 min. To that, added 3-thiophene ethanol (1.0 equiv, 1.75 g, 13.36 mmol) followed by addition of dichloromethane (30 ml) and N,N-dimethylformamide (10 ml) under argon. After the reaction mixture was stirred at 0 °C for 20 min under argon N,N'-dicyclohexylcarbodiimide (1.2 equiv, 3.30 g, 16.04 mmol) in 10 ml dichloromethane was added dropwise for 15 min to the mixture. The reaction mixture was then allowed to stir at 0 °C for 72 h. After completion of the reaction, the reaction mixture was allowed to stand overnight at 0

°C to precipitate the urea byproduct, which was filtered and evaporated the solvent to obtain crude product. The obtained crude product was partitioned between water and ethyl acetate and extracted five times with ethyl acetate. The combined organic layers were dried over anhydrous MgSO₄. The solvent was evaporated under reduced pressure and then the residue was purified by column chromatography on silica gel (eluents: ethyl acetate and hexane) to yield 1 (3.20 g, 93 %). ¹H NMR (400 MHz, DMSO-d6): d = 2.12 (t, 2H, -CH2), 4.50–4.53 (t, 2H, -OCH2), 7.09–7.11 (d, 1H, thiophene ring), 7.33–7.34 (d, 1H, thiophene ring), 7.48–7.49 (m, 1H, thiophene ring), 8.00–8.02 (t, 2H, aromatic ring), 8.06–8.11 (t, 2H, aromatic ring).

(Z)-2-(Thiophen-3-yl)ethyl 4-(N'-acetoxycarbamimidoyl)benzoate (2): To a solution of 2-(thiophen-3-yl)ethyl 4-cyanobenzoate (1.0equiv, 2.0 g, 7.77 mmol), hydroxylamine hydrochloride (1.5 equiv, 0.809 g, 11.65 mmol) in methanol (20 ml) was added diisopropylethylamine (2 ml) under argon. The reaction mixture was

refluxed at 65 °C for 6 hours and was then allowed to stir at 25 °C for 25 h.

Evaporated the solvent under reduced pressure to obtain the benzamidoxime residue.

To this, added acetic acid (20 ml) followed by addition of acetic anhydride (3.0 equiv, 2.3 g, 23.31 mmol). The reaction mixture was then stirred at 25 °C for 1 h. After completion of the reaction, evaporated the solvent under reduced pressure and obtained a white residue. The residue was re-dispersed in 70 ml of ethyl acetate and washed three times with brine solution (150 ml) and one time with Millipore water.

The organic layer was dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure and then the residue was purified by column chromatography on silica gel (eluents: ethyl acetate and hexane) and SEC (eluent: chloroform) to yield 2 (3.40 g, 75 %). ¹H NMR (400 MHz, CDCl3-d1): d = 2.24 (s, 3H, -COCH3), 3.10–

3.14 (t, 2H, -CH2), 4.52–4.55 (t, 2H, -OCH2), 5.18 (s, 1H, -NH2), 7.01–7.02 (d, 1H, thiophene ring), 7.07–7.08 (d, 1H, thiophene ring), 7.26–7.29 (m, 1H, thiophene ring), 7.74–7.77 (m, 2H, aromatic ring), 8.02–8.04 (m, 2H, aromatic ring).

2-(Thiophen-3-yl)ethyl 4-carbamimidoylbenzoate hydrochloride (3): To a solution of (Z)-2-(thiophen-3-yl)ethyl 4-(N'-acetoxycarbamimidoyl)benzoate (2) (1.0 equiv, 0.5 g, 1.61 mmol) in acetic acid (2.4 ml) added potassium formate (15.0 equiv, 2.03 g, 24.19 mmol) in methanol (7 ml) and stirred at 25 °C for 15 min. To this, added 10 % Pd/C (0.1 equiv, 0.074 g, 0.16 mmol). The reaction mixture was then stirred at 25 °C for 18 h. After completion of the reaction, the mixture was filtered, washed extensively with ethanol. Evaporated the solvent under reduced pressure to obtain hydrogenated residue. The residue was re-dispersed in ethanol (40 ml), added 1 M hydrochloric acid (7 equiv, 11.6 ml, 11.6 mmol) and stirred for 15 min, filtered and washed extensively with ethanol. Evaporated the filtrate under reduced pressure and

dried the residue under vacuum for 24 h to obtain 3 (0.26 g, 51.5 %). ¹H NMR (400 MHz, CDCl3-d1): d = 3.12–3.14 (t, 2H, -CH2), 4.55–4.57 (t, 2H, -OCH2), 5.18 (s, 1H, -NH2), 7.05–7.07 (d, 1H, thiophene ring), 7.18–7.20 (d, 1H, thiophene ring), 7.33–

7.36 (m, 1H, thiophene ring), 7.87–7.91 (m, 2H, aromatic ring), 8.16–8.19 (m, 2H, aromatic ring).

2.3.2 Characterization of Ligand

The synthesized compounds were identified by ¹H NMR spectroscopy (AL400, JEOL Ltd., Tokyo, Japan), and HR-MS analysis. The compounds were purified by preparative SEC (column: Shodex K-5001, Showa Denko K. K., Tokyo, Japan) using chloroform as the eluent.

2.3.3 Computational Simulations Software

The initial structure of benzamidinium-trypsin complex for further simulation was taken from Protein Data Bank (PDB structure 3atl). All the simulations were performed using a Molsoft L.L.C., ICM simulation software, in which all atoms are considered as ball and stick type atoms. Docking simulation time is about 1 h.

2.3.4 Isothermal Titration Calorimetry (ITC) Measurement

Titration experiments were performed with an iTC200 isothermal titration calorimeter (Microcal, Inc., Northampton, MA, USA). 50 The instrument was calibrated using EDTA-CaCl₂ standard solution. 10 mM of CaCl₂ in a 50 mM of tris buffer (pH 8) was used as a buffer medium to prepare ligand and protein solutions in all the experiments. The concentration range of trypsin in the cell was between 0.05 and 0.10 mM and the concentration range of benzamidine-conjugated thiophene

(ligand) in the syringe was between 2 and 15 mM. The ligand and trypsin solutions were degassed (~ 10 min) prior to use. To perform controlled experiments, the concentration of 0.05 mM trypsin and 2 mM benzamidine hydrochloride was used.

During the titration experiments, a stirring speed of between 500 and 1000 r.p.m. was maintained in the cell to stir the trypsin solution. Ligand solution in the syringe was titrated into cell after a stable baseline had been achieved. The injection sequence consisted of an initial injection of 0.4 µl to prevent artifacts arising from the filling of the syringe, followed by injections of 2.0 ml each at specified intervals keeping a maximum number of injections to 19. Cell temperatures used in the experiments were 20, 25, and 30 °C.

All other measurement parameters were as follows:

Reference power = 5 and 10 µCal/sec Feedback parameters = high

Initial delay before staring the measurement = 60 sec Injection duration of first injection = 0.8 sec

Injection duration from second injection = 4 sec Injection intervals (spacing) = 180 to 720 sec Filter period = 5 sec

Obtained data were analyzed using Origin software (Microcal, Inc.) assuming a single binding site and fitting to a single-site binding isotherm that yields enthalpy of binding (ΔH), binding constant (K), Gibbs energy of binding (ΔG), and entropy of binding (ΔS).

2.3.5 Solution Preparation for Electrochemical Analysis

Firstly, 100 mM tris buffer having pH 8 was prepared and the pH of the buffer was altered by the addition of diluted hydrochloric and or diluted sodium hydroxide, accordingly. 100 mM of Potassium ferricyanide (C₆N₆FeK₃) electrolyte solution was prepared (329.3 mg) in 10 ml of tris buffer solution at pH 7 and diluted the mother electrolyte solution to 1 mM or 10 mM by the addition of tris buffer. Different concentrations of ligand between 0.161 and 6.45 mM, and different concentrations of trypsin between 0.1 µM and 100 µM were freshly prepared in tris buffer at pH 8, respectively.

2.3.6 Experimental Setup for Electro-chemical Polymerization

Electro-chemical experiments were carried out using a Cyclic Voltammetry (700C, ALS/CH instruments, Inc., TX, USA). The gold electrode (AUE Gold electrode, OD (outer diameter): 6 mm, ID (inner diameter): 3 mm; manufactured by ALS Co., Ltd, Tokyo, Japan and distributed by BAS Inc., Tokyo, Japan) was used as the working electrode, an Ag/AgCl electrode (RE-1S, manufactured by ALS Co., Ltd, Tokyo, Japan and distributed by BAS Inc., Tokyo, Japan) was used as reference electrode, and a platinum wire (Pt, 1 mm diameter, manufactured by ALS Co., Ltd, Tokyo, Japan and distributed by BAS Inc., Tokyo, Japan) was used as the counter electrode.

Prior to each experiment, the gold electrode was manually polished with a 6 µm polishing diamond solution on a polishing paper for a period of 60 s and washed thoroughly with milli-Q water, followed by polishing with a 0.05 µm alumina polishing slurry on a polishing paper for a period of 60 s and washed thoroughly with

milli-Q water, and the electrode. Ag/AgCl electrode and Pt wire were briefly washed with milli-Q water and wiped the remaining water prior to each experiment.

To perform the electrochemical measurements, the electrodes were setup, keeping the distance among the working, counter and reference electrodes approximately to 1 mm. Then 15 µl of analyte solution was dropped on the gold electrode, ensuring that the entire surface of the working electrode was covered with the analyte and the reference and counter electrodes were in contact with the analyte.

A range of sweeping potentials between −0.3 and 0.8 V were applied for electro-chemical measurement of electrolyte keeping the starting potential of 0 V and a scanning speed of 100 mV s⁻¹. To check the electro-chemical polymerization efficiency of BeT-1, a range of sweeping potentials between −0.4 and 1.6 V and a range of scanning cycles between four and ten were applied keeping the starting potential of 0 V and a scanning speed of 100 mV s⁻¹.

After the electro-chemical polymerization of analyte, the gold electrode was gently washed with milli-Q water. The counter and reference electrodes were gently washed with milli-Q water. After drying all the electrodes in air, the electrodes were setup again, and then an electrolyte solution was dropped on the gold electrode to check the resultant current by applying optimized sweeping potentials.

2.3.7 FT-IR Spectroscopy Measurement

Attenuated total reflectance fourier transform infrared spectroscopy measurements (ATR-FTIR) were carried out on a FTIR spectrometer (FT/IR-4100, JASCO). In the instrument is equipped with a ZnSe single crystal prism with refractive index of 2.4. Gold-coated silicon chip (size: 5mm x 10 mm) was used to measure the FTIR spectra of benzamidine-conjugated thiophene ligand or after

electro-polymerization of the same ligand. As a background measurement, FTIR spectra of a bare gold-coated silicon chip was recorded and used as baseline in all the measurements. To check the ligand FTIR spectra, the ligand (1 mg) was initially dissolved in methanol (1 ml) solvent. Few microliters of the solution was drop-casted onto a gold-coated silicon chip and dried over N2 gas for about 10 min to evaporate the methanol solvent. To check the FTIR spectra of electro-polymer, the ligand (0.1 mg) was dissolved in 50 mM tris buffer (1 ml) at pH 8 and electro-polymerized onto a gold-coated silicon chip using cyclic voltammetry and switching the potential between -0.2 and 1.6 V with 100 mV s^-1 scan rate. Then, ligand or electro-polymer deposited chip was carefully placed on prism and recorded the FTIR spectra between 4500 and 600 cm^-1 range.