d- and l-Arginine-Derived ... - ACS Publications

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Self-Ordering Secondary Structure of D- and L‑Arginine-Derived Polyamidoamino Acids Amedea Manfredi,† Nicolò Mauro,‡ Alessio Terenzi,‡,¶ Jenny Alongi,† Federica Lazzari,† Fabio Ganazzoli,§ Giuseppina Raffaini,*,§ Elisabetta Ranucci,*,† and Paolo Ferruti*,† †

Dipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università degli Studi di Palermo, via Archirafi 32, 90100 Palermo, Italy § Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, via L. Mancinelli, 7, 20131 Milano, Italy ‡

S Supporting Information *

ABSTRACT: This paper reports on synthesis, acid−base properties and pH-dependent structuring in water of D-, Land D,L-ARGO7, bioinspired polymers obtained by polyaddition of the corresponding arginine stereoisomers with N,N′methylenebis(acrylamide). The circular dichroism spectra of Dand L-ARGO7 showed a peak at 228 nm and quickly and reversibly responded to pH changes, but were nearly unaffected by temperature, ionic strength, and denaturating agents. Theoretical modeling studies of L-ARGO7 showed that it assumed a folded structure. Intramolecular interactions led to transoid arrangements of the main chain reminiscent of the protein hairpin motif. Torsion angles showed a quite similar distribution at pH 6 and 14 consistent with the similarity of the CD spectra from pH 6 upward.

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incorporating chiral side chains. PAACs are an off-spring of polyamidoamines (PAAs)14 and as PAAs maintain the essentials of the starting monomers, in this case chirality and amphoteric properties, being in that distinct from polypeptides and polypeptoids. The polyaddition of D-, L-, and D,L-arginine with N,N′-methylenebis(acrylamide) was performed in water at 50 °C and pH > 9 (Scheme 1). The concurrent PAA hydrolysis

hiral biomolecules are central to all fundamental recognition and replication functions in biological systems. Chirality also governs biomaterial−cell interactions.1,2 As a consequence, the design of chiral synthetic polymers with the capability for self-assembly into stable secondary structures and specific biorecognition represents an attracting biomimetic approach.3,4 Polypeptides5 and polypeptoids6 are well-known bioinspired polymers based on α-amino acids and Nsubstituted glycines derivatives, respectively. In these polymers, the amino acid-deriving moieties lack the intrinsic amphoteric properties of amino acids. Different from polypeptides, polypeptoids are deprived of main-chain chirality7 and are chiral only if chiral are their N-substituents. Chiral polypeptides, their hybrids with synthetic polymers, for example, poly(acrylic acid),8 and polypeptoids9 may structure in solution. Other chiral polymers, in which polyvinyl-,10 polyolefin-,11 or polyacetylene12 chains carry α-amino acid pendants linked by amide bonds, behave similarly. Recently, the synthesis of the bioinspired L-ARGO7 polymer was obtained by the Michael-type polyaddition of L-arginine with N,N′-methylenebis(acrylamide).13 L-ARGO7 proved endowed with cell permeation ability and minimal cytotoxicity. Here we present the synthesis, acid−base properties, experimental, and theoretical modeling studies of the pHdependent structuring in water of L-ARGO7 and its chiral and racemic stereoisomers, namely, D- and D,L-ARGO7. These polymers represent the first examples of polyamidoamino acids (PAACs) based on α-amino acids other than glycine © XXXX American Chemical Society

Scheme 1. Synthesis of ARGO7 Polymers

previously found in polyadditions with simple amines run at >40 °C14 was not observed. Fairly high molecular weight ARGO7 polymers were obtained without added catalysts (see SI for the synthetic details and Table S1 for molecular weights). ARGO7 polymers are amphoteric with pH-dependent physicochemical and biological properties. Their protonation Received: July 7, 2017 Accepted: August 21, 2017

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DOI: 10.1021/acsmacrolett.7b00492 ACS Macro Lett. 2017, 6, 987−991

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ACS Macro Letters constants were determined using the generalized HendersonHasselbach eq (eq 1): 1−α pH = pK a − β log (1) α where Ka is the apparent weak acidic dissociation constant being pH-determining in the buffer titration zone considered; α is the acid dissociation degree; and β is the Katchalsky and Spitnik parameter15 accounting for possible interactions between ionizable groups on repeat units being spatially or topologically adjacent. pKa’s and β parameters were determined by the potentiometric titration and data treatment procedures described in SI (including Figures S2−S4 and Tables S2−S5). The values of pKa1 (side −COOH), pKa2 (chain tert-amine), β1, β2 of D-, L-, and D,L-ARGO7 are reported in Table 1. In all cases, Table 1. pKa and β Values of ARGO7 Isomersa in the α Range 0.2−0.8 (β1) and 0.1−0.9 (β2) isomer D-ARGO7 L-ARGO7 D,L-ARGO7

a b

pKa1b

β1b

pKa2c

β2c

2.24 ± 0.10 2.31 ± 0.02 2.34 ± 0.06

0.60 ± 0.06 0.60 ± 0.13 0.57 ± 0.07

6.41 ± 0.07 6.43 ± 0.06 6.39 ± 0.02

1.12 ± 0.02 1.14 ± 0.06 1.25 ± 0.01

The pKa of the guanidine group (≥12.3) was not determined. Carboxyl group. cAmine group.

the isoelectric point (IP) was 9.7. Comparing these sets of data allowed ascertaining that no significant chirality-dependent differences could be observed among ARGO7 isomers. Each ARGO7 repeat unit could exist in four ionization states whose relative distributions vs pH were calculated with the pKa and β values of Table 1 following the method reported in SI. LARGO7 speciation diagrams are reported in Figure S5. The circular dichroism (CD) spectra of D- and L-ARGO7 at 25 °C in 0.1 M NaCl and in the 2.1−12.1 pH range exhibited patterns and intensities consistent with ordered secondary structures (Figure 1 for L- and Figure S6 for D-ARGO7). D,LARGO7 spectra were flat (Figure S7). At pH > 5, the molar ellipticity curves of L-ARGO7 (referred to the repeat unit) showed a maximum around 228 nm, whose value increased with pH up to pH ∼ 8.1, and then remained approximately constant. Plotting the differential molar ellipticity at 228 nm, calculated taking as reference values at pH 2 versus pH in the 2.0−12.1 pH range, mirror-image sigmoidal curves were obtained for the two antipodes (Figure 1b for L- and Figure S8 for D-ARGO7) presenting three noticeable points, in the case of L-ARGO7 the lower plateau at pH 2.0−5.5, the inflection point at pH ∼ 6 and the upper plateau from pH 7.5 upward. The speciation diagrams (Figure S5) explained these results: the strongly acidic and basic carboxyl- and guanidine groups were mostly ionized throughout the pH range considered, whereas the weakly basic chain tert-amine groups were mostly ionized at pH < 7, but poorly ionized at pH > 7. Hence, ARGO7 was electrically balanced at basic pH’s up to 12, but carried supernumerary positive charges at acidic pH’s. Noticeably, L-ARGO7 pH-induced conformational changes were fully reversible (Figure 1c). The dependence of L-ARGO7 spectral patterns on temperature, ionic strength, polymer concentration, weight-average molecular weight (Mw), and presence of guanidinium chloride and urea was studied at 228 nm and pH ∼ 8.3. The resultant spectra were remarkably insensitive to these parameters. In particular, changing the temperature in the 5−70 °C interval

Figure 1. L-ARGO7 CD spectra: pH dependence (a); differential molar ellipticity at 228 nm (b); reversibility with respect to a pH change (c).

and back to 25 °C produced only a modest and reversible effect (Figure S9), indicating conformational thermodynamic stability. Equally modest was the effect of the ionic strength (up to 2 M NaClaq) and of the denaturating agents, both guanidinium chloride and urea (2 M; Figure S10a), indicating that electrostatic interactions and hydrogen bonding were not the main modes of structure formation. Varying concentration in the 0.15−1.0 mM range did not alter the CD pattern (Figure S10b). Even the Mw, in the range 2.5−10.5 kDa, had no influence on CD spectra (Figure S11). No extensive aggregations were revealed by dynamic light scattering (DLS) analysis carried out following the method reported in SI in the 2−12 pH range at 1 mg mL−1 (Figure 2). The hydrodynamic radius, Rh, did not significantly differ within a wide pH range. 988


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μ was lowest at pH 1, where all units were positively charged (Figure S5) and the dipole vectors pointed to opposite directions mutually compensating to some extent. Conversely, μ was highest at pH 6, where many units were electrically balanced, and moderately decreased at pH 14, where most units were negatively charged, and the local dipoles largely depended on the CO groups (Figure 3c). The compact structure of LARGO7 was fully consistent with the experimental results of Figure 2 for large Mw chains, taking into account the different molecular masses, and of the different calculated quantities (i.e., Rg and R vs the hydrodynamic radius Rh), ruling out an extended conformation. Such a feature was allowed both by the main chain flexibility, and by the topological separation among the charged groups. The molecular conformation was then dictated by the intramolecular interactions mainly of electrostatic nature due to the local dipoles, and to a few (up to 10) intramolecular hydrogen bonds within the main chain, within the side groups, and among them. Moreover, the small fluctuations of the end-to-end distance within the MD runs (see also the animations in the SI) further confirmed the rigidity of the equilibrium conformation. The intramolecular interactions led to a few folded local structures defined by the polymer main chain reminiscent of the hairpin conformation22−24 (a secondary structural motif of proteins which might eventually lead to a β-sheet structure), as shown for instance in Figure 3a. The hairpin conformation was best seen at pH 1 in water thanks to the almost flat geometry locally taken by large parts of the main chain, but short and more numerous such hairpins were present in three dimensions at different pH’s. This structural motif required two roughly straight and antiparallel strands, and was characterized by a typical distribution of torsional angles around all the bonds of the main chain, defined by a sequence of four consecutive atoms. These torsion angles tended to be found in a transoid arrangement peaked around ±180° (see Figure 3a), while the values in the ±60° to ±120° range allowed for the strand turns and the random conformations. A similar distribution was found at pH 14 and at pH 6, so that the number of such angles, comprised within a range of 30° around the ±180° values, was quite the same, while at pH 1 this distribution was much more concentrated around ±180° than at larger pH values, indicating longer antiparallel strands (Figure 3a). The extreme pH values of the simulation studies (1 and 14) were not considered in the CD experimental studies, being limited to the pH range 2−12.1 because of PAAC chains stability problems. The only modest difference in torsion angle distribution at pH 14 compared to pH 6 was consistent with the inflection at pH ∼ 6 and the flattening from pH ∼ 8 upward of the molar ellipticity curves versus pH at 228 nm (Figure 1b). In conclusion, this paper reports on synthesis and acid−base properties of D-, L-, and D,L-ARGO7 and provides insights into the pH-dependent self-assembly in water of the chiral isomers. ARGO7 polymers are the first examples of PAACs, bioinspired polymers obtained by polyaddition with bis(acrylamide)s of natural α-amino acids (apart from glycine) and their stereoisomers. Different from polypeptides and polypeptoids, PAACs maintain both chirality and amphoteric properties of α-amino acids. D- and L-ARGO7 showed CD spectra with a peak centered at 228 nm, indicating ordered secondary structures. Plotting Δθ at 228 nm vs pH gave sigmoidal curves in mirrorimage relationship with inflection point at pH ∼ 6. The pHinduced conformational changes were quickly and fully

Figure 2. pH dependence of L-ARGO7 particle size from DLS.

The molecular dynamics (MD) simulations were carried out for L-ARGO7 with 10 repeat units (number-average molecular weight, Mn = 3280) at different pH values, hence, at different charges (Table 2), using a previously adopted Simulation Table 2. Properties of Simulated L-ARGO7 at Different pH Values in Water at the End of the Molecular Dynamics Run pH

charge (e)

Rga (nm)

Rb (nm)

Sc (nm2)

μd (D)

1 6 14

+20 +10 −10

1.03 0.92 0.90

2.51 1.32 1.84

26.2 20.7 21.4

26.40 45.51 37.73

a

Gyration radius. bEnd-to-end distance. cSurface area accessible to the solvent. dDipole moment. The standard deviation is in all cases less than 2% of the calculated values.

Method,16−18 largely validated19 (see SI for more details about the adopted software, force field, and the chosen methodology). Starting from a fully elongated chain, the simulation runs were carried out first in an effective dielectric medium, and then in explicit water. All simulations showed that L-ARGO7 never displayed typical polyelectrolyte extended conformations. Conversely, at all pH’s, regardless of the overall charge, simulations yielded coiled structures, as shown by the gyration radius (Rg) and by the end-to-end distance (R) defined as the distance between the outermost N atoms along the main chain (Table 2 for results in water and Table S6 for the effective dielectric medium). These coiled structures were quickly achieved in the MD runs, with small fluctuations after equilibration, as shown by the end-to-end distance values in the effective dielectric medium (Figure S12), with only minor changes in water. The molecular compactness at different pH’s was also shown by the main-chain conformations (Figure 3a), and best characterized by the Rg values (Table 2), much smaller than those of the fully elongated chain R (9 nm) and that of the contour length (∼11 nm), due to a slight bend of the repeat units. Moreover, Rg showed little dependence on the overall charge, due to the intramolecular conformational preferences and the local interactions. The simulations in water showed slightly larger sizes than in the dielectric medium (Table S6), particularly for the chain with the largest total charge (pH 1), due to the explicit hydration. Furthermore, in water, Rg was only slightly larger at pH 1 than at other pH values, in spite of the larger overall charge. The same trend was shown by the solvent accessible surface (Figure 3b), defined as the surface accessible to a 1.4 Å radius spherical probe (roughly the size of a water molecule), and by the end-to-end distance R. Moreover, the dipole moment (μ) variation versus pH (Table 2) matched the pH dependence of the CD spectra (Figure 1a).20,21 In fact, 989


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Figure 3. (a) Main chain molecular conformations in water at the end of the MD runs (see also the animation in the SI) at pH 1, 6, and 14 and torsion angle distributions around the main chain bonds: the histogram are shown with a binning of 30°. Color codes: C atoms dark gray; H atoms light gray; N atoms blue; O atoms red. (b) Solvent accessible surface area in water. The surface area is in dark gray near C atoms, in light gray near H atoms, in blue near N atoms and in red near O atoms. (c) Dipole moments in water. Color codes are the same as in panel (a).

reversible. The conformations were little affected by temperature, ionic strength and denaturating agents. Theoretical modeling of L-ARGO7 indicated that it assumed a folded structure, with a slightly larger Rg for the largest chain positive charge at pH 1. The intramolecular interactions led to transoid arrangements of the main chain reminiscent of the protein hairpin motif, with longer antiparallel strands at pH 1. Torsion angles showed a quite similar distribution at pH 14 and 6 consistent with the similarity of the CD spectra above pH 6. Finally, as regards perspectives, L-ARGO7 proved highly cytobiocompatible and was internalized in cells.13 Polymers containing chiral α-amino acid moieties as side-substituents showed different interactions with organic structures.25 It is not unreasonable to presume that D-, L-, and (D,L)-ARGO7 may show chirality-dependent interactions with subcellular components, opening the way for intracellular selective drug targeting.

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Detailed experimental description, molecular dynamics simulations details, supporting figures and tables (PDF). Molecular dynamics simulation (AVI). Molecular dynamics simulation (AVI). Molecular dynamics simulation (AVI). Molecular dynamics simulation (AVI). Molecular dynamics simulation (AVI). Molecular dynamics simulation (AVI).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: giuseppina.raff[email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

ASSOCIATED CONTENT

Amedea Manfredi: 0000-0002-4243-7054 Nicolò Mauro: 0000-0003-0246-3474 Jenny Alongi: 0000-0002-1912-5191 Fabio Ganazzoli: 0000-0002-9762-4535 Paolo Ferruti: 0000-0002-5404-440X

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00492. 990


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(17) Raffaini, G.; Ganazzoli, F. Surface Hydration of Polymeric (bio)materials: A Molecular Dynamics Simulation Study. J. Biomed. Mater. Res., Part A 2010, 92, 1382−1391. (18) Raffaini, G.; Ganazzoli, F. Protein Adsorption On Biomaterial And Nanomaterial Surfaces: A Molecular Modeling Approach to Study Non-Covalent Interactions. J. Appl. Biomater. Biomech. 2010, 8, 135− 145. (19) Raffaini, G.; Ganazzoli, F.; Malpezzi, L.; Fuganti, C.; Fronza, G.; Panzeri, W.; Mele, A. Validating a strategy for molecular dynamics simulations of cyclodextrin inclusion complexes through single-crystal X-ray and NMR experimental data: a case study. J. Phys. Chem. B 2009, 113, 9110−9122. (20) Besley, N. A.; Hirst, J. D. Theoretical Studies toward Quantitative Protein Circular Dichroism Calculations. J. Am. Chem. Soc. 1999, 121, 9636−9644. (21) Rosenfeld, L. Quantenmechanische Theorie der natürlichen optischen Aktivität von Flüssigkeiten und Gasen. Z. Phys. 1929, 52, 161−174. (22) de Alba, E.; Jiménez, M. A.; Rico, M. Turn Residue Sequence Determines β-Hairpin Conformation in Designed Peptides. J. Am. Chem. Soc. 1997, 119, 175−183. (23) Mei, C. G.; Jahr, N.; Singer, D.; Berger, S. Hairpin Conformation of An 11-mer Peptide. Bioorg. Med. Chem. 2011, 19, 3497−3501. (24) Mondal, S.; Varenik, M.; Bloch, D. N.; Atsmon-Raz, Y.; Jacoby, G.; Adler-Abramovich, L.; Shimon, L. J. W.; Beck, R.; Miller, Y.; Regev, O.; Gazit, E. A Minimal Length Rigid Helical Peptide Motif Allows Rational Design of Modular Surfactants. Nat. Commun. 2017, 8, 14018. (25) Wang, X.; Gan, H.; Sun, T.; Su, B.; Fuchs, H.; Vestweber, D.; Butz, S. Stereochemistry triggered differential cell behaviours on chiral polymer surfaces. Soft Matter 2010, 6, 3851−3855.

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University of Vienna, Institute of Inorganic Chemistry, Waehringerstrasse 42, A-1090 Vienna, Austria.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A. T. has received funding from the Mahlke-Obermann Stiftung and the European Union’s Seventh Framework Programme (grant agreement no. 609431).

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REFERENCES

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