Amino acid-deriving chiral polymers with potential for biotechnological applications

Introduction Polyamidoamines (PAAs) represent a well-known family of soluble synthetic functional polymers with a recognized potential for biotechnological applications. PAAs are obtained by stepwise Michael-type polyaddition of prim-monoamines or sec-diamines with bisacrylamides. The reaction occurs at pH = 8-9 aqueous solution and at room temperature, with the involved amine groups preserving their amine character. By using α-amino acids as monomers, poly(amidoaminoacid)s (PAACs) are obtained. PAACs maintain the chirality and the amphoteric properties of the starting monomers. Material and Methods (D)-,(L)- and (D,L)-Arginine were added to a suspension of N,N’-methylenebisacrylamide and lithium hydroxide in water under vigorous stirring. The reaction mixture was heated to 50°C for 5 days then acidified to pH = 4. To evaluate acid-base properties, 0.1 M NaCl solutions of ARGO7 isomers were titrated with 0.1 M NaOH and then back-titrated with 0.1 M HCl at 25°C under inert atmosphere. Their secondary structure was investigated in aqueous solution by circular dichroism (CD) spectroscopy at 25°C and pH ranging from 3.0 to 10.0. Results Amphoteric polyamidoaminoacids isomers were obtained with 58% yield, Mn = 8500 and PDI = 1.08. Their β corrected pKa1 (COOH) and pKa2 (main chain tert-amine) values were evaluated exploiting the modified Henderson-Hasselbach equation. L-ARGO7 speciation curves show an isolectric point of ≈10 and +0.25 positive net average charges per repeating unit at pH 7.4, resulting in a highly citycompatible polymer with an IC50 ≥ 8 mg/mL. Cell internalization in Balb/3T3 cells, also highlight its preferential localization in the perinuclear region. Circular dichroism spectra show broad signals in the 250-340 nm region, but none at pH = 5. Discussion (D)-,(L)- and (D,L)-ARGO7 were obtained with yield and Mn comparable to previous Ca2+ catalyzed synthesis. Their pKa1 values resembled that of arginine, whereas pKa2 decreased by two units due to the acrylamide electron withdrawing effect. Both β constants exhibit deviations from ideal behaviour, more pronounced for the carboxyl group. D- and L-ARGO7 gave mirror imaging CD spectra consistent with pH-dependent conformation transitions. These properties, together with the high cytocompatibility and a possible stereochemical-governed intracellular localization, may affect their interactions with biological structures as proteins and living cells.