Supramolecular peptide nanotubes as artificial enzymes for catalysing ester hydrolysis

Peptide-based artificial enzymes are attracting significant interest because of their remarkable resemblance in both composition and structure to native enzymes. Herein, we report the construction of histidine-containing cyclic peptide-based supramolecular polymeric nanotubes to function as artificial enzymes for ester hydrolysis. The optimized catalyst shows a ca. 70-fold increase in reaction rate compared to the un-catalysed reaction when using 4-nitrophenyl acetate as a model substrate. Furthermore, the amphiphilic nature of the supramolecular catalysts enables an enhanced catalytic activity towards hydrophobic substrates. By incorporating an internal hydrophobic region within the self-assembled polymeric nanotube, we achieve a 55.4-fold acceleration in hydrolysis rate towards a more hydrophobic substrate, 4-nitrophenyl butyrate. This study introduces supramolecular peptide nanotubes as an innovative class of supramolecular scaffolds for fabricating artificial enzymes with better structural and chemical stability, catalysing not only ester hydrolysis, but also a broader spectrum of catalytic reactions.


c. Synthesis of CP-His-PEG
CP-His-NH 2 (10.0 mg, 0.0078 mmol) and CH 3 O-PEG-NHS (59.0 mg, 0.0112 mmol) were dissolved in 1.0 mL anhydrous DMF, with the addition of NMM (2.4 mg, 0.0235 mmol).The reaction was left for 24 h.Then the DMF solution was precipitated in cold diethyl ether.The precipitate was collected using centrifugation and dried under N 2 .The resulting solid was then redissolved in 2 mL DCM and 10 mL diethyl ether was added dropwise to obtain precipitate.This process was repeated twice.Finally, CP-His-PEG was obtained by drying under vacuum as an off-white solid (yield: 30.0 mg).

b. Synthesis of Boc-His(Trt)-CP-PEG
Boc-His(Trt)-CP-N 3 (14.0mg, 0.0088 mmol) and CH 3 O-PEG-BCN (68.8 mg, 0.0132 mmol) were dissolved in 1.5 mL DMF.The reaction was left for 3 days.Then the DMF solution was precipitated in cold diethyl ether.The precipitate was collected using centrifugation and dried under N 2 .The resulting solid was then redissolved in 2 mL DCM and 10 mL diethyl ether was added dropwise to obtain precipitate.This process was repeated twice.Finally, Boc-His(Trt)-CP-PEG was obtained by drying under vacuum as an off-white solid (yield: 40.0 mg).

b. Synthesis of His 2 -CP-N 3
Removal of the protecting groups were achieved by adding a mixture of TFA 1 mL, TIPS 50 µL and DI water 50 µL to Boc-His(Trt) 2 -CP-N 3 (24.0mg, 0.012 mmol) and stirring for 3 h.The resulting solution was then precipitated in cold diethyl ether and washed twice to give an off-white powder as

Synthesis of OCT-His-CP-PEG a. Synthesis of OCT-His(Trt)-OH
OCT-His(Trt)-OH was prepared via solid phase peptide synthesis (SPPS) on a Prelude Automated Peptide SynthesizerTM (Protein Technologies Inc.) using 2-chlorotrityl chloride resin as the solid support.Fmoc-His(Trt)-OH was coupled to the resin using DIPEA (4 eq.) in DMF, followed by capping of unreacted resin sites using a solution of MeOH:DIPEA:DCM (7:1:2, v/v/v).Deprotection of the Fmoc group of the amino acids was done using 20% piperidine in DMF.Subsequently octanoic acid was coupled under the condition of octanoic acid (5 eq.), HCTU (5 eq.) and NMM (10 eq.) in DMF.In

c. Synthesis of OCT-His(Trt)-CP-PEG
OCT-His(Trt)-CP-N 3 (19.0mg, 0.012 mmol) and CH 3 O-PEG-BCN (91.9 mg, 0.018 mmol) were dissolved in 1.5 mL DMF.The reaction was left for 3 days.Then the DMF solution was precipitated in cold diethyl ether.The precipitate was collected using centrifugation and dried under N 2 .The resulting solid was then redissolved in 2 mL DCM and 10 mL diethyl ether was added dropwise to obtain precipitate.This process was repeated twice.The obtained solid was dissolved in DI water and lyophilized to give an off-white powder as OCT-His(Trt)-CP-PEG (yield: 55.0 mg).

d. Synthesis of OCT-His-CP-PEG
OCT-His(Trt)-CP-PEG (40.0 mg) was dissolved in 0.5 mL DCM, with the addition of 0.5 mL TFA.
The reaction was left for 3 h.Then the solution was precipitated in cold diethyl ether and washed twice.

S2. Self-assembling behaviour of CP-His-PEG, His-CP-PEG, His 2 -CP-PEG, and OCT-His-CP-PEG
The self-assembly of the conjugates was realized simply by dissolving them into PBS buffer (pH=7.4) at different concentrations.For SAXS measurement, 5 mg mL -1 solutions were used.For SLS measurement, solutions of 3 different concentrations (1, 2, 3 mg mL -1 ) were used.Table S2 N agg values of CP-His-PEG, His-CP-PEG, His 2 -CP-PEG and OCT-His-CP-PEG obtained from SLS data.

S3. Determination of the catalytic activity towards PNPA or PNPB hydrolysis a. Determination of the extinction coefficient of 4-nitrophenol in PBS buffer
A series of the hydrolysis product 4-nitrophenol solutions in PBS buffer (pH=7.4) with different concentrations were prepared (25, 50, 100, 250, 500 µM).The UV-vis spectra were then measured using a quartz cuvette with a path length of 2 mm.After plotting the absorbance at 400 nm against 4nitrophenol concentration, a linear fitting was carried out to obtain the slope value as (0.00208±0.00003)µM -1 .The extinction coefficient of 4-nitrophenol at 400 nm in PBS buffer was calculated according to Beer-Lambert law, , which gives a value of 10400 M -1 cm -1 .
Plot of absorbance at 400 nm vs. 4-nitrophenol concentration in PBS buffer.

b. Kinetics experiment
The catalytic activities of the histidine-containing compounds were determined using either 4nitrophenyl acetate (PNPA) or 4-nitrophenyl butyrate (PNPB) as the substrate.The hydrolytic product is 4-nitrophenol with an absorption band peaked at 400 nm.The reaction rate was determined by monitoring the change of absorbance at 400 nm over time, which was subsequently converted into concentration using the measured extinction coefficient of 4-nitrophenol.Stock solutions of the histidine-containing compounds were prepared in PBS buffer, and the stock solution of PNPA or PNPB was prepared in DMSO.PNPA or PNPB hydrolysis was carried out in a quartz cuvette containing a certain amount of catalyst and 1 mM PNPA or PNPB with a total volume of 500 µL at a controlled temperature.
The initial hydrolytic rates were calculated by a linear fitting of the evolution of absorbance at 400 nm over reaction time at the very beginning of the reaction, assuming the substrate concentration held approximately constant.

c. Determination of apparent activation energy (E a )
The hydrolysis of PNPA or PNPB was monitored at different temperatures (25, 40, 60, and 80 °C) at His-CP-PEG concentrations of 0, 40, and 80 µM, respectively, while PNPA or PNPB concentration was kept at 1 mM.The initial hydrolytic rates were calculated by a linear fitting of the evolution of absorbance at 400 nm over reaction time at the very beginning of the reaction, assuming the substrate concentration held approximately constant.By fitting the evolution of initial hydrolytic rates as a function of reaction temperatures using Arrhenius equation, E a related to the PNPA or PNPB hydrolysis could be obtained.
, which could be transformed to A linear fitting is conducted between ln(v 0 ) and 1/T, which gives the slope as -E a /R.

Figure
Figure S1 (a) HPLC spectra of CP-His-NH 2 and CP-His-PEG monitored by a UV detector at 280 nm; (b) GPC traces of CH 3 O-PEG-NHS and CP-His-PEG.

c.
Synthesis of His-CP-PEGBoc-His(Trt)-CP-PEG (35.0 mg) was dissolved in 0.5 mL DCM, with the addition of 0.5 mL TFA.The reaction was left for 3 h.Then the solution was precipitated in cold diethyl ether and washed twice.The obtained solid was dissolved in DI water and lyophilized to give an off-white powder as His-CP-PEG (yield: 28.0 mg).

His 2 -
CP-N 3 (yield: 15.0 mg).MS (ESI-ToF) (m/z): [M+H] + 1323.8 (calculated: 1323.8).c.Synthesis of His 2 -CP-PEGHis 2 -CP-N 3 (10.0mg, 0.0076 mmol) and CH 3 O-PEG-BCN (59.0 mg, 0.0113 mmol) were dissolved in 1.5 mL DMF.The reaction was left for 3 days.Then the DMF solution was precipitated in cold diethyl ether.The precipitate was collected using centrifugation and dried under N 2 .The resulting solid was then redissolved in 2 mL DCM and 10 mL diethyl ether was added dropwise to obtain precipitate.This process was repeated twice.The obtained solid was dissolved in DI water and lyophilized to give an off-white powder as His 2 -CP-PEG (yield: 30.0 mg).

Figure
Figure S3 (a) HPLC spectra of His 2 -CP-N 3 and His 2 -CP-PEG monitored by a UV detector at 220 nm and 280 nm; (b) GPC traces of CH 3 O-PEG-BCN and His 2 -CP-PEG.
The obtained solid was dissolved in DI water and lyophilized to give an off-white powder as OCT-His-CP-PEG (yield: 35.0 mg).

Figure
Figure S7 (a) HPLC spectra of OCT-His(Trt)-CP-PEG and OCT-His-CP-PEG monitored by a UV detector at 280 nm; (b) GPC traces of CH 3 O-PEG-BCN and OCT-His(Trt)-CP-PEG.
SasView software was used to fit the SAXS data, using a core-shell cylinder model.SLD values were calculated based on the molecular structure of the conjugate and solvent.The radius of the core value was fixed at 5 Å, representing the radius of the cyclic peptide itself.

Figure
Figure S9 SLS data of CP-His-PEG, His-CP-PEG, His 2 -CP-PEG and OCT-His-CP-PEG measured in water.

Table S1 Fitting
parameters using a core-shell cylinder model by SasView software.CP-

Table S3 E
a values obtained by fitting the evolution of initial hydrolytic rates as a function of reaction temperatures using Arrhenius equation.