Self-Structuring in Water of Polyamidoamino Acids with Hydrophobic Side Chains Deriving from Natural α-Amino Acids

This paper reports on synthesis, acid-base properties and self-structuring in water of chiral polyamidoamino acids (PAACs) obtained by polyaddition of N,N′-methylenebisacrylamide with l-alanine, l-valine and l-leucine (M-l-Ala, M-l-Val, M-l-Leu) with potential for selective interactions with biomolecules. The polymers maintained the acid-base properties of amino acids. In water, the circular dichroism spectra of PAACs revealed pH-dependent structuring in the range 3–11 and in the wavelength interval 200–280 nm. Taking as reference the values at pH 3, the differential molar ellipticities were plotted in the pH interval 3–11. Sigmoidal curves were obtained presenting inflection points at pH 8.1, 6.8 and 7.3 for M-l-Ala, M-l-Val and M-l-Leu, respectively, corresponding to the amine half-ionization. Theoretical modeling showed that PAACs assumed stable folded conformations. Intramolecular interactions led to transoid arrangements of the main chain reminiscent of protein hairpin motif. Oligomers with ten repeat units had simulated gyration radii consistent with the hydrodynamic radii obtained by dynamic light scattering.


S2
. 1 (Fig. S3). The titration of the tert-amine group started around pH 5, that is, after complete deprotonation of the carboxyl group. Deprotonation of the ammonium ion took place between the first and second inflections. Therefore, the half-neutralization point was evaluated as the mid-point between them. In forward titrations, the starting pH was 1.8, that is, at incomplete COOH deprotonation. Therefore, in order to determine the midpoint of the COOH deprotonation tract, the starting of COOH deprotonation was extrapolated by considering that COOH and tert-amine groups were in equal numbers, hence COOH deprotonation needed the same titrant volume as that used for the ammonium ion deprotonation.

M-L-Leu
β parameter determination. The β parameters of the generalized Henderson-Hasselbalch equation (Eq. S1a) were determined for both pK a1 (side -COOH) and pK a2 (chain tert-amine) to ascertain the presence of interactions between ionizable groups on adjacent monomeric units. The β parameters were determined by firstly selecting the specific buffer region intervals marked by each pKa. The dissociation degree, α, was then calculated in each zone as the ratio between the reacted moles and the total amount of moles necessary to reach complete neutralization. β Values were finally obtained from Eq. S1b (corresponding to Eq. 2 in the main text) as the slope of the pH versus -log((1-S6 α)/α) curve (Fig. S4). Points near inflections deviated from ideality and were not considered. Fig. S4 shows the β-corrected pK a values in the chosen α intervals.
Determination of simulated titration curves. Simulated titration curves were determined following the De Levie approach [1] in order to iteratively refine pKa and β values to achieve the best fitting to the experimental data.
• Initial conditions: (Eq. S6) Ionic strength: • Charge balance: where (Eq. S9a-e): Combining all former conditions, the following solving equation, representing the whole forward titration curve, was obtained in terms of VT as a function of pH: where: The best fitting simulated titration curves, obtained from Eq. S10 in the buffer regions relative to both side -COOH and tert-amine groups by using the iteratively refined pKa and β values reported in Table 1  Eq. S12a = = Eq. S12b

= =
Eq. S12c With D and y as previously described, and where the K a1 and K a2 values were corrected for β 1 and β 2 .
S9 S10 Figure S3. Forward and backward titration curves referred to the 1 st experiment of Table S1. c) Figure S6. Comparison of the optimized geometry of M-L-Ala at pH 1 obtained in implicit water using the solvent dielectric constant (before simulations in explicit water) using the two simulation strategies described in the main text.
In green the conformation obtained at 300 K, superimposed to the final geometry eventually obtained after the MD runs for 500 ps at 500 K, then for 500 ps at 400 K and finally 500 ps at 300 K (color codes: C atoms dark grey; H atoms light gray; N atoms blue; O atoms red) is shown. In the upper part of the figure, the superposition of the whole molecules is shown in CPK representation. The lower part reports the conformations of the main chain with no H atoms that were eventually achieved by the two strategies at 300 K viewed along three orthogonal directions.

Simulation method
The simulation method and the adopted strategy exactly matched those described in ref.
[20] of the main text and are briefly summarized here. The InsightII/Discover 2000 [2] package of programs was used adopting the consistent valence force field CVFF [3].
The molecules, comprising 10 repeat units, were prepared using the available templates assuming the appropriate charges at different pH values in order to have a positive (L + ), a null (L 0 ) and a negative (L -) charge per repeat unit.
Starting from a fully extended chain, after an initial geometry optimization, the molecules were subjected to MD runs of 5 ns in an effective medium with a distancedependent dielectric constant, and then of 0.5 ns in explicit water, adopting a cubic cell with a local density of 1 g cm -3 , with final geometry optimizations. Equilibration of the system was monitored through the time change of the total and potential energy together with its components (such as the Coulomb and the van der Waals and the torsional components), and of the end-to-end distance. The dynamic equations were integrated using the Verlet algorithm with a time step of 1 fs at a temperature of 300 K, controlled through the Berendsen thermostat, and the instantaneous coordinates were periodically saved for further analysis. All the energy minimizations where carried out with the conjugate gradient method up to an energy gradient lower than 4×10 -3 kJ mol -1 Å -1 .