pH-Responsive Self-Assembly of Designer Aromatic Peptide Amphiphiles and Enzymatic Post-Modification of Assembled Structures

Supramolecular fibrous materials in biological systems play important structural and functional roles, and therefore, there is a growing interest in synthetic materials that mimic such fibrils, especially those bearing enzymatic reactivity. In this study, we investigated the self-assembly and enzymatic post-modification of short aromatic peptide amphiphiles (PAs), Fmoc-LnQG (n = 2 or 3), which contain an LQG recognition unit for microbial transglutaminase (MTG). These aromatic PAs self-assemble into fibrous structures via π-π stacking interactions between the Fmoc groups and hydrogen bonds between the peptides. The intermolecular interactions and morphologies of the assemblies were influenced by the solution pH because of the change in the ionization states of the C-terminal carboxy group of the peptides. Moreover, MTG-catalyzed post-modification of a small fluorescent molecule bearing an amine group also showed pH dependency, where the enzymatic reaction rate was increased at higher pH, which may be because of the higher nucleophilicity of the amine group and the electrostatic interaction between MTG and the self-assembled Fmoc-LnQG. Finally, the accumulation of the fluorescent molecule on these assembled materials was directly observed by confocal fluorescence images. Our study provides a method to accumulate functional molecules on supramolecular structures enzymatically with the morphology control.


Characterization of Fmoc-LnQG peptides.
The synthesized Fmoc-LnQG peptides were analyzed by MALDI TOF MS and HPLC as shown in Figure S1. The major peaks found in MALDI TOF MS spectra corresponded to the sodium adducts (m/z 674.094 and 787.024 for Fmoc-L2QG and Fmoc-L3QG, respectively) and the potassium adducts (m/z 690.094 and 803.010 for Fmoc-L2QG and Fmoc-L3QG, respectively). The purities of Fmoc-L2QG and Fmoc-L3QG were 98.7 % and 98.1 %, respectively, by HPLC analysis.

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Scheme S1. Chemical structures, formula, and molecular weights of Fmoc-LnQG peptides and their conjugate with OG. Figure S4. MALDI TOF MS spectra of Fmoc-L2QG (a-d) and Fmoc-L3QG (e-h) after the MTG reaction with OG at (a,e) pH 5, (b,f) pH 6, (c,g) pH 7 and (d,h) pH 8. MS peaks for Fmoc-LnQG peptide-OG conjugates were detected except for Fmoc-L3QG at pH 5 (e) and 6 (f), where reaction rates were too low to be detected by MALDI TOF MS.

Evaluation of MTG specific activity at various pH.
The specific activity of MTG was evaluated by a standard hydroxamate method [R1] using 10 mM (Figure S5a

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of enzyme that produces 1 mol of hydroxamate per minute as the active unit of MTG, 1 U, and the relative specific activity of MTG was defined as the relative activity when the activity at pH 5 was set to 1. The specific activity at pH 6 and 7 using 200 mM buffer failed to be measured because precipitation was formed under these conditions (Figure S5b). Figure S5. Relative specific activity of MTG measured using 10 mM (a) or 200 mM (b) buffers at various pH values.

MTG reaction of Fmoc-LnQG peptides with TAMRA cadaverine and Sulforhodamine cadaverine.
A reaction sample for each self-assembled PA was prepared in 10 mM buffer at each pH ([Fmoc-L2QG] = 2.0 mM, [Fmoc-L3QG] = 1.0 mM) and MTG reaction was performed under the same conditions as OG except that TAMRA cadaverine (Figure S6a,b) or Sulforhodamine cadaverine (Figure S6c,d) was used instead of OG. The reaction proceeded at 25°C for 2 h, and the reaction was stopped by adding NEM to inactivate MTG. HPLC analysis (column: Inertsil ODS-3; eluent conditions: 0.1% TFA water/ACN from 60/40 to 20/80 linear gradient; flow rate: 1 mL/min) was conducted to evaluate the enzymatic reaction rate at each pH by using the value of an absorption (583 nm) from TAMRA cadaverine or Sulforhodamine cadaverine.

MTG reaction rate of Z-QG with Ac-Lys-OH.
Z-QG (30 mM) were prepared using 200 mM buffers at pH 5-8. Ac-Lys-OH (100 mM) and MTG (0.3 U/mL) were added, and MTG reaction was proceeded at 37°C. Figure S8. Initial reaction rates of Z-QG.

Titration curves of Fmoc-LnQG assemblies to determine apparent pKa values.
One milliliter of 2.5 mM Fmoc-L2QG or 1.5 mM Fmoc-L3QG solution was prepared using ultrapure water. Sodium hydroxide (0.01 M) was added to the above Fmoc-LnQG solutions until the solution pH reached to 10.0. The solutions were vortexed until the peptides were fully dissolved. The titration experiments were performed by adding small volumes of 0.01 M HCl.