Molecular characterization of L-phenylalanine terminated poly(L-lactide) conjugates

Abstract

Peptide–polymer conjugates made of poly(L-lactide) and L-phenylalanine or L,L-diphenylalanine (F–PLA and FF–PLA, respectively) have been synthesized by the ring-opening polymerization of L-lactide using the peptide fragment as an initiator. The structure of the conjugates was confirmed by ¹H NMR, FT-IR, GPC, UV-Vis and CD. Molecular dynamics simulations have been used to identify both the conformational preferences of the FF–PLA conjugate in solution and the potential intramolecular interactions between the peptide and polymer blocks, while TD-DFT calculations have been applied to model the electronic transitions observed by the UV-Vis absorption spectroscopy. Results show that the polymer fragment prefers a random coil or a mix of helix/strand while the peptide fragment tends to have folded and helical conformations. Although the degree of interaction between the two fragments is slightly higher than that reported for other peptide–polymer conjugates, it is small enough to suggest that FF–PLA is a potential candidate to aggregate forming peptide-guided organizations via self-assembly. On the other hand, quantum mechanical calculations have allowed us to identify the π → π* transition, which is typically observed in helical peptides and proteins, as well as the n → π* transition along the N–C–O backbone.

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Introduction

Polymer bioconjugates result from the combination of a segment of biological origin with a synthetic polymer block,¹ the term peptide–polymer conjugate being frequently used in the specific case of integrated peptides. This type of hybrid macromolecules combines unique properties that come from the precise chemical structure and functionality of peptides and the stability, functions and processability of synthetic polymers. In spite of this, for a few decades research on peptide–polymer conjugates has been focused on the combination of peptides with poly(ethylene oxide) (PEO).^2–6 However, with the recent explosion in the fields of nanotechnology and biotechnology, the design and fabrication of peptide–polymer conjugates have become an expanding field of interdisciplinary research in the last few years.^7–11

The fabrication of well-defined peptide–polymer conjugates is usually performed using one of the following approaches:^12,13 (1) coupling the peptide segments to pre-formed synthetic molecules through one or more reactive sites; (2) growing the polymer from the peptide segment; (3) assembling the peptide on an already formed polymer; and (4) polymerizing short peptides with a polymerizable functionality. In all cases, these approaches are expected to provide hybrid molecules able to adopt 3D organizations, which should be defined by the peptide secondary structure.¹⁴ Some of these approaches have been recently used to prepare peptide–PEO conjugates based on phenylalanine (F) homopeptides (e.g. FFFF–PEO,^15,16 KLVFF–PEO,¹⁷ FFKLVFF–PEO¹⁸ and F–ω-hydroxyl-PEO¹⁹).

In this work we present the preparation and characterization at the molecular level of the peptide–polymer conjugate resulting from the combination of FF and poly(L-lactide) (PLA), hereafter denoted FF–PLA. Conjugates based on PLA, which is a biodegradable material obtained from renewable resources, are expected to present important medical and biotechnological applications because of their interesting properties. PLA can be obtained by polycondensation from lactic acid or by ring-opening polymerization (ROP) of its cyclic dimer, lactide. Lactic acid is a new renewable resource derived from the starch of either corn or sugar beets, which is fermented to form glucose and then converted into lactic acid. Although the variety of stereoisomeric polymers that can be obtained from lactic acid or lactide polymerization are complete as single-use packaging materials, films and other commodity uses, they find their most outstanding use in biomedical devices in which they slowly hydrolyze back to lactic acid and re-enter the Krebs cycle. Also, these polymers have been employed as scaffolds for tissue engineering and in drug delivery systems. Excellent reviews of many chemical and practical aspects of PLA have been published.^20–22

In a recent study, the synthesis of L-phenylalanine-terminated PLA, F–PLA, was described using a three-step procedure.²³ First, the hydroxyl-terminated PLA was synthesized through the ROP of L-lactide initiated by n-butanol under the presence of tin(II) ethylhexanoate. Subsequently, the complete capping of the hydroxyl end group of PLA with Boc-L-phenylalanine was achieved by using a mixed anhydride of Boc-L-phenylalanine under the catalysis of 4-(1-pyrrolidinyl) pyridine. Finally, the free amino end group was obtained by removal of the t-butoxycarbonyl group through trifluoroacetic acid treatment under anhydrous conditions. Unfortunately, this product was not characterized but used as macroinitiator for the synthesis of poly(L-lactide)-b-poly(L-lysine) block copolymers. More recently, this strategy has been also used to prepare other block copolymers, as for example those derived from poly(ester amide) and polyester.²⁴

This work is focused on the synthesis and basic characterization of thermal and electronic properties of F–PLA and FF–PLA. The process followed for this purpose is the contrary to that reported in ref. 23 and discussed above, since peptide fragments have been used as initiators of the polymerization reactions (i.e. the polymer has been grown from the peptide segment). Furthermore, the molecular structure of FF–PLA has been examined at the atomistic level using Molecular Dynamics (MD) simulations to determine the organization of the FF and PLA fragments as well as to ascertain the level of interactions among them. After this, the size of the model used for classical MD simulations has been conveniently reduced to investigate the electronic transitions provoked by the FF fragment through quantum mechanical calculations. More specifically, time-dependent density functional theory (TD-DFT) calculations have been applied to identify the electronic transitions detected by UV-Vis spectroscopy.

Methods

Materials

L-Phenylalanine (H–Phe–OH) and L,L-diphenylalanine (H–Phe–Phe–OH) were purchased from Bachem, stored at 0 °C and used as received. L-Lactide was purchased from Sigma Aldrich, stored at 8 °C and dried in vacuum before use. Solvents of analytical grade were purchased from Panreac and used as received. Commercial polylactide (PLA Polymer 3051D) was purchased from NatureWorks® (U.S.A) and characterized by a melt flow index of 10–25 g/10 min (measured at 210 °C with 2.16 Kg according to the D-1238 standard).

Measurements

Molecular weights and polydispersity index (PDI) were estimated by gel permeation chromatography (GPC) using a liquid chromatograph (Shimadzu, model LC-8A) equipped with an Empower computer program (Waters). A PL HFIP gel column (Polymer Lab) and a refractive index detector (Shimadzu RID-10A) were employed. The polymer was dissolved and eluted in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at a flow rate of 0.5 mL min⁻¹ (injected volume 100 μL, sample concentration 3 mg mL⁻¹). The number and weight average molecular weights were calculated using polymethyl methacrylate standards.

¹H NMR spectra were recorded with a Bruker AMX-300 spectrometer operating at 300.1 MHz. Chemical shifts were calibrated using tetramethylsilane as an internal standard and CDCl₃ (δ(¹H) = 7.24 ppm) was used as the solvent.

Infrared absorption spectra were recorded with a Fourier Transform FTIR 4100 Jasco spectrometer in a 4000–600 cm⁻¹ range. A Specac-Teknokroma model MKII Golden Gate attenuated total reflection (ATR) set-up was also employed.

Calorimetric data were obtained by differential scanning calorimetry with a TA Instruments Q100 series equipped with a refrigerated cooling system (RCS). Experiments were conducted with around 5 mg of sample under a flow of dry nitrogen and at a heating rate of 20 °C min⁻¹. Samples were previously quenched from the melt state at the maximum rate allowed by the equipment. Calibration was performed with indium.

Circular dichroism (CD) spectra were measured in a 1 mm path length using a Jasco J-815 spectropolarimeter (Jasco, Japan) and analyzed with the supplied Spectra-Manager software. The temperature was kept constant at 20 °C using a temperature controlled water bath. The samples were dissolved in HFIP.

Ultraviolet spectra were recorded with a Shimadzu UV-3600 ultraviolet-visible spectrometer from polymer solutions in HFIP.

Preparation of the glassware

In order to assure that ROP of lactide was only initiated by amino acids, reaction tubes were always silanized. Glassware and magnetic stirrers were always stored at 140 °C before use.

ROP of L-lactide initiated by L-phenyalanine or L,L-diphenylalanine

1.5 g of dried L-lactide and the H–Phe–OH or H–Phe–Phe–OH initiators (Fig. 1) were introduced in a silanized reaction tube. The lactide/initiator molar ratio was always 50 : 1. Reaction was performed under magnetic stirring in an oil bath at a temperature of 135–140 °C. The reaction mixture was firstly kept under a nitrogen stream for ten minutes and then the tube was sealed. Polymerization process was monitored by ¹H NMR to confirm the total lactide conversion. After the required time (ca. 240 h for both H–Phe–OH and H–Phe–Phe–OH initiated reactions), reaction tubes were cooled in ice to stop polymerization. The obtained pale solids were repeatedly washed with methanol and recovered by centrifugation. Finally, polymers were purified by re-precipitation with methanol from chloroform solutions.

Fig. 1 Synthesis of F–PLA and FF–PLA conjugates initiated by L-phenylalanine (H–Phe–OH) and L,L-diphenylalanine (H–Phe–Phe–OH).

Molecular dynamics simulations

The polymeric moiety of the conjugate molecule was a 40 residues-long tail of PLA linked to the C-terminus of a diphenylalanine peptide. The initial system was built using AMBER's LEAP utility.²⁵ The initial conformation was solvated in HFIP. The force field parameters for the solvent were taken from Fioroni et al.²⁶ and the AMBER GAFF force field.²⁷ The PLA residues were parameterized using the AMBER ff03 force field.²⁸ 14 Cl⁻ and 15 Na⁺ ions were added to neutralize the charge of the system. The initial system contained 48 430 atoms and was placed in an orthorhombic box of dimensions 62 × 104 × 112 ų.

All MD simulations described in this work were performed by using AMBER ff03 potential²⁸ and the NAMD computer package.²⁹ The Berendsen thermostat-barostat³⁰ was used to control temperature and pressure, with a relaxation time of 800 ps. The SHAKE³¹ algorithm was applied to bonds involving hydrogen atoms. The integration time step was set to 2 fs. The cutoff distance for nonbonding interactions was 14 Å. Particle Mesh of Ewald (PME)³² was applied for computing electrostatic interactions beyond the cutoff distance. The real space term was determined by the van der Waals cut off (14 Å), whereas the reciprocal term was estimated by interpolation of the effective charge into a charge mesh with a grid thickness of one point per cubic Å. Periodic boundary conditions were applied in all simulations in this paper using the nearest image convention.

The equilibration protocol consisted of the following: initially, the potential energy of the system was minimized for 2000 steps using steepest descent algorithm. Next, the system was heated up to 500 K during 500 ps in a NVT-MD simulation. After this, 500 ps at 298 K of NVT-MD were run. Finally, 500 ps of NPT-MD were run for density relaxation with a pressure of 1 atm. The box size was optimized until the density of the system reached approximately 1.6 g cm⁻³, which is the density of the HFIP solvent. The production runs were carried out in constant volume.

The conformational exploration was performed using a Simulated Annealing/Molecular Dynamics (SA-MD) protocol. This strategy used the final outputs of the density relaxation, heated them to 900 K and cooled them to 300 K over 10 ns, saving a snapshot every 2 ps (5000 per cycle). The potential energy of the obtained structures was relaxed through 500 steps of conjugate gradient minimization in order to resolve minor clashes and reach a nearby local minimum. The five lowest potential energy conformations were selected as starting points for the next round. The protocol was run for 8 rounds. Each round produced 3250 snapshots, a total of 26 000 structures being obtained.

In recent years some powerful and advanced sampling methods has been tested,³³ even though these are significantly demanding in terms of computer resources. In this work the choice of the SA-MD methodology is based on the fact that it has proven useful for conformational exploration of flexible molecules in solution,^34,35 for which sophisticated methods are not required. These advantages include the ability of the method to overcome high potential energy barriers and to proficiently explore flat potential energy surfaces compared to plain MD, which is highly dependent on the initial geometry. To make sure that the sampling was sufficient, we followed a conformational clustering protocol.^34,35 After each round the conformations were clustered using the VMD clustering protocol³⁶ with a 6.5 Å root-mean square-deviation (RMSD) threshold on the C-alpha atoms. The VMD clustering protocol uses the Quality Threshold method that determines the number of clusters in advance. We determined the number of clusters per round to be the smallest number that assigns a cluster to at least 90% of the conformations. Fig. 2 shows the number of clusters as a function of the number of SA-MD rounds. As seen, the number of clusters stabilized on approximately 30 clusters after 8 rounds.

Fig. 2 Variation of the number of clusters against the number of SA-MD rounds.

Quantum mechanical calculations

Density functional theory (DFT) methods have been used to reduce the size of the molecular model used to simulate the conjugate by classical MD. Thus, the reduced model should capture the essential trends associated with the electronic transitions provoked by the FF fragment and detected by UV-Vis spectroscopy. DFT calculations were performed on molecular systems formed by the FF fragment attached to a PLA oligomer containing n repeat units (FF–nPLA). The WB97XD functional, which includes empirical dispersion to describe π–π interactions,³⁷ combined with 6-31G(d)³⁸ basis set (BS) were used for this purpose. The carboxylic acid of the C-terminus has been considered in the deionized state as a negatively charged carboxylate group. All geometry geometric optimizations were carried out using the Gaussian 09 package of programs.³⁹

Subsequently, the electronic absorption spectrum was computed for the FF–4PLA, in order to compare to the experimental UV-Vis data and help interpreting them. It is well-known that such simulations can be sensitive to the selected level of approximation⁴⁰ and several DFT functionals as well as Pople's BS have consequently been tested on the above-optimized structure to ensure the simulations reliability. On the BS side, we considered extending the basis set in terms of zeta, polarization and diffusion functions, while on the DFT side, various blends of exchange–correlation formalisms were assessed with respect to the stability of respective results.

Results and discussion

Synthesis and characterization

The selected lactide/initiator ratio (i.e. 50 : 1) allowed us to obtain peptide–polymer conjugates with a relatively high molecular weight. Specifically, number average molecular weights of 49 000 g mol⁻¹ and 66 000 g mol⁻¹ and polydispersity indices of 1.41 and 1.48 were attained for samples coming from H–Phe–OH (F–PLA) and H–Phe–Phe–OH (FF–PLA), respectively.

¹H NMR spectra were monitored during ROP to assure a total conversion since signals attributed to the lactide monomer (i.e. 5.37 and 1.68 ppm) were clearly differentiated from those corresponding to the polymer in the conjugate (Fig. 3). Long reaction times were needed for both polymerizations, with the ROP reaction being slightly faster when H–Phe–Phe–OH was employed (e.g. the time necessary to achieve a conversion of 70% increased from 80 to 160 h when H–Phe–OH was used). PLA in conjugates were obtained with a yield close to 70% after purification by re-precipitation when the reaction was performed for 240 h, a reaction time that assured a practically complete conversion without any sign of thermal degradation.

Fig. 3 ¹H NMR spectra of FF–PLA obtained by ROP using L,L-diphenylalanine (H–Phe–Phe–OH) as initiator and after 240 h of reaction. Insets show representative signals after the indicated reaction times as well as the aromatic and the methine protons of the phenylalanine and lactoyl terminal groups of the polymer recovered by reprecipitation. Signals associated to a 10₇ helical conformation are indicated by dashed garnet circles.

¹H NMR spectra of synthesized conjugates showed the typical signals of PLA: a quadruplet at 5.19, 5.17, 5.15 and 5.13 ppm corresponding to the proton of the methine unit, and the doublet at 1.76 and 1.73 ppm corresponding to the protons of methyl groups. Furthermore, small signals attributed to the terminal lactoyl unit (4.4–4.3 ppm) and also to the phenylalanine terminal units (aromatic protons at 7.3–7.2 ppm) could also be detected as shown in the insets of Fig. 3.

FTIR spectra of synthesized samples were similar and showed the typical absorption bands of PLA^41–43 (Fig. S1†). Thus, the most intense signals were observed at 1749 cm⁻¹ ν(C=O); 1452 cm⁻¹ δ_as (CH₃); 1361 cm⁻¹ δ(CH); 1183 cm⁻¹ ν_as(C–O–C) + r_as(CH₃) for the crystalline phase; 1128 cm⁻¹ r_s(CH₃); 1085 cm⁻¹ ν_s(C–O–C); and 1043 cm⁻¹ ν(C–CH₃).

Polymers in the synthesized conjugates are semicrystalline, as shown in Fig. S2† where the DSC heating scan of a representative melt quenched sample (i.e. FF–PLA) is displayed. Typical features such as a glass transition with a significant relaxation peak, an exothermic cold crystallization peak and a complex melting peak close to 140 °C were detected. In fact, a degree of crystallinity of 30–33% could be estimated for both F–PLA and FF–PLA samples from the melting enthalpy (106 J g⁻¹) for a 100% crystalline sample.⁴⁴ Small signals of FTIR spectra observed at 1209 cm⁻¹ (ν_as (C–O–C) + r_as(CH₃) for a 10₇ helical conformation) and 921 cm⁻¹ (coupling of the C–C stretching and CH₃ rocking mode for long segments with a 10₇ helical conformation) were in agreement with a preferred molecular conformation of PLA.^{41–43,45,46}

UV-Vis spectra clearly demonstrated that ROP was effectively initiated by the phenylalanine derivatives since typical absorption peaks associated with the aromatic rings could always be observed. Thus, Fig. 4 shows that the characteristic five peaks at 246, 250, 256, 262 and 266 nm of the H–Phe–Phe–OH compound were still detected in the spectra of the synthesized FF–PLA samples. As expected, the considered peaks were more significant when polymerization was initiated by the dipeptide. It is also remarkable that commercial PLA did not show any significant absorption in the region comprised between 246 and 266 nm, and consequently absorbance peaks of synthesized PLA samples should be unambiguously assigned to linked phenylalanine units.

Fig. 4 UV-Vis absorption spectra of 1,1,1,3,3,3-hexafluoroisopropanol solutions of H–Phe–Phe–OH (a), F–PLA (b) and FF–PLA (c). For the sake of completeness, the spectrum of commercially available PLA (d) is also plotted.

We used CD spectroscopy to characterize the secondary structure of PLA, H–Phe–OH, H–Phe–Phe–OH, F–PLA and FF–PLA dissolved in HFIP (Fig. 5). Amorphous and crystalline PLA dissolved in HFIP exhibited a positive CD band at 210 nm similar to that registered in acetonitrile solution.⁴⁷ The CD spectra of H–Phe–OH and H–Phe–Phe–OH show one positive band at wavelength of 226 nm and 237 nm, respectively, and several negative several dichroic bands, the ones centered at 222 and 208 nm being typically attributed to n–π* and parallel-polarized π–π* excitonic transitions in peptides. These two negative bands disappear upon the conjugation of these peptides to PLA (Fig. 5, inset). A positive band at 230 nm is observed for F–PLA, this position being very similar to that registered for H–Phe–OH (Δ = 4 nm). For FF–PLA the positive band is centered at 227 nm, reflecting that the difference with respect to the individual peptide (Δ = 10 nm) is slightly higher than for F–PLA. Although CD spectra cannot provide high resolution information on the secondary structure, these results suggest that the interactions between the peptide and polymer blocks are practically inexistent in F–PLA and weak in FF–PLA.

Fig. 5 CD spectra in HFIP of commercial PLA at 0.05 mg mL⁻¹ (i), H–Phe–OH at 5 mg mL⁻¹ (b), F–PLA at 5 mg mL⁻¹ (c), H–Phe–Phe–OH at 5 mg mL⁻¹ (d) and FF–PLA at 2.5 mg mL⁻¹ (e).

Conformational preferences in solution

The SA-MD protocol was used to study the conformational preferences of FF–PLA system. Conformations were clustered using a geometry-based clustering method that measures the pairwise distances between conformations when projected onto a lower-dimensional space and filters outliers.⁴⁸ The analysis, which was done on conformations within the 10% lowest energy range to filter out noise, resulted in 13 representative clusters after retaining only clusters with at least 10 members, involving 301 conformations after the removal of outliers.

The average number of structures per cluster is 23 ± 16. The lowest energy conformations in each cluster were within 26 kcal mol⁻¹ compared to the lowest energy structure detected. Fig. 6 shows a histogram of the energy distribution of all the 10% low-energy conformations in the clusters. The energy units are relative to the lowest energy conformation. As seen, the energies distribute between 0 kcal mol⁻¹ (above the lowest energy conformation) and about 50 kcal mol⁻¹, with a peak around 30–40 kcal mol⁻¹ above the minimum. The energy distribution can give indications about the conformational accessibility of the system. This is based on the fact that the probability of a state to exist at a certain temperature can be expressed as a function of the energy difference between that state and the lowest energy state through a Maxwell–Boltzmann distribution. Moreover, a small number of states within an energy range indicates fewer thermodynamically accessible structures at a given temperature and consequently low conformational variability in terms of energy. In this case, as also shown below, the system exhibits some structural variability.

Fig. 6 Energy distribution between the cluster members derived from SA-MD on FF–PLA. The number on the x-axis is the energy relative to the lowest energy conformation, where 0 represents the lowest energy conformation.

Fig. 7a shows the Ramachandran plot for flexible backbone dihedral angles of the 10% low-energy conformations of the PLA in FF–PLA, which have been denoted ξ₁ and ξ₂ (Fig. 7b). As seen, the majority of the conformations exhibit a random coil or a mix of helix/strand. Since we used the L-lactide configuration, the Ramachandran plot of PLA is a mirror image of the typical L-amino acid plot. The ξ₁–ξ₂ distribution is generally compatible with the allowed regions. Approximately 13% of the dihedral angles are in the helical region (ξ₁ between 35° and 90°, and ξ₂ between 15° and 70°) and 8% in the extended/semi-extended region (ξ₁ between 90° and 180°, and ξ₂ between −90° and −180°). Fig. 7c shows the values of the dihedrals associated with the ester bond of the PLA block, which have been denoted as ω (Fig. 7b). As expected, ω distributes mainly around ±180°. Indeed, only 9.6% of the ω angles are in the range of −150° to 150°, and only about 1% of the ω angles are between −120° and 120°.

Fig. 7 (a) Ramachandran plot showing the distribution of the ξ₁–ξ₂ backbone dihedral angles in PLA block of the 2600 lowest energy conformations found for FF–PLA in HFIP solution. (b) Scheme displaying the labels used to identify the backbone dihedral angles. (c) Distribution of the backbone dihedral angle associated to the ester bond (ω) in the PLA block and (d) Ramachandran plot showing the distribution of the φ–ψ backbone dihedral angles in the FF block of the 2600 lowest energy conformations found for FF–PLA in HFIP solution.

Fig. 7d represents the Ramachandran plot for flexible backbone dihedral angles of the 10% low-energy conformations of the peptide fragment (φ and ψ in Fig. 7b) in FF–PLA. The analysis has been focused on the second phenylalanine residue since definition of the ψ dihedral is not possible for the first one. As can be seen, the most populated φ–ψ distributions correspond to folded and helical regions, even though regions associated with extended and semiextended conformations are also occasionally visited. The conformational preferences of the N-acetyl-N′-methylamide derivative of FF were explored in a recent study by combining a building procedure with quantum mechanical calculations at the B3LYP/6-31G(d) level.⁴⁸ The 100 initial geometrical arrangements for FF, which were constructed using a combinatorial growth of the peptide chain by considering 10 minimum energy conformations for F, converged over a total of 78 minimum energy structures. However, clustering analysis converged 78 such minimized structures into 20 different conformations. For each cluster the conformational characteristics of the backbone were practically identical, differences being only found in the arrangements of the side groups. Among the 20 basic organizations, the conformation that presented the lowest energy corresponded to a double γ-turn (also denoted 2.2₇ ribbon), which was the outcome of replicating the most favored arrangement of F. This regular organization has only been observed in short peptides.⁴⁹ In the three following organizations each phenylalanine residue adopted a different conformation. Thus, the second organization presented a folded structure that was the outcome of forming an intramolecular hydrogen bond between the C=O and NH moieties of the blocking groups. This particular arrangement was associated with a β-turn motif or with an embryonic helical organization that would render a 3₁₀ helix if the peptide could be prolonged beyond the two residues of phenylalanine. The third organization resulted from the combination of distorted extended and γ-turn motifs while the fourth showed a hydrogen bond associated with the γ-turn motif of the second phenylalanine residue and an inter-residue N–H⋯π interaction. The rest of the organizations resulted from repetition of two extended or semi-extended conformations, from the combination of the latter conformation with a folded pattern, or from the repetition of two helical conformations. Details about all such structures were provided in ref. 48. It is worth noting that the conformational profile predicted by quantum mechanical calculation for the N-acetyl-N′-methylamide derivative of FF is fully consistent with the conformational preferences displayed in Fig. 7d.

The crystal structure of H–Phe–Phe–OH was reported by Görbitz more than one decade ago.⁵⁰ Görbitz discovered that this small peptide crystallizes with hydrogen bonded head-to-tail chains forming hydrophilic channels embedded in a hydrophobic matrix created by the peptide side chains. The sequence formed by two consecutive phenylalanine residues was proposed to be an attractive model for membrane channels due to the substantial size of the hydrophilic channels. Interestingly, the conformation adopted by the unblocked peptide in the solid state, which was identified in ref. 48 as a minimum energy conformation, is one of the visited conformations depicted in Fig. 7d. Indeed, an overall comparison of Fig. 7d with the conformational profile of N-acetyl-N′-methylamide derivative of FF⁴⁸ suggests that the influence of the polymer on the peptide fragment conformation in the conjugate is relatively small.

Representative low-energy conformations obtained for FF–PLA by our search and clustering process are shown in Fig. 8a. As seen, the low-energy conformations are partially-folded, mostly with a small hydrophobic core, and exhibit a mix of secondary structure and random coil conformations, in accordance with the dihedral angle analysis shown above. Fig. 8b shows the pairwise RMSD between the energy minima, evidencing values ranging from 2 to 12 Å. This corresponds to a wide distribution of conformations. The RMSD was calculated for all but the hydrogen atoms.

Fig. 8 (a) Representatives of the low-energy conformational clusters for FF–PLA in HFIP solution. Hydrogen atoms were omitted for clarity. (b) Pairwise RMSD between the 13 low-energy cluster centers identified for FF–PLA in HFIP solution. See color bar for values.

Fig. 9a and b shows the radius of gyration (R_g) and the end-to-end distance (d_e–e), respectively, for each PLA block of the low-energy conformations, measured between the O1 atom of the first repeat unit and the C-terminus. The average R_g and d_e–e were 20.1 ± 2.5 Å and 52.9 ± 8.0 Å, respectively. This result indicates that most of the conformations adopted by the PLA block in HFIP solution were generally rather compact with respect to the size of the extended polymer, which is expected considering its hydrophobic nature.

Fig. 9 (a) Radius of gyration (R_g) and (b) end-to-end distance (d_e–e) of the low-energy conformations. (c) Representative interactions between FF and PLA blocks.

Analysis of the interactions between the FF and PLA blocks in the conjugate reveals that the non-terminal phenylalanine residue was frequently involved (∼30% of low energy conformations) in a hydrogen bond with the main chain of the PLA block (i.e. usually with the repeat unit immediately after it). Another hydrogen bond between the main chain of the non-terminal phenylalanine residue and the second PLA repeat unit was observed in approximately 13% of the low-energy conformations while the interaction between the C-terminus and the second PLA repeat unit appears in approximately 17% of the conformations (see Fig. 9c). However, no other significant inter-block secondary interaction (i.e. with a population higher than 5%) was detected suggesting that FF and PLA tend to organize independently. Although the interactions between FF and PLA fragments are scarce, these are more frequent than those recently observed for FF–PEG, where PEG refers to poly(ethylene glycol), in which the two counterparts remained independent, preserving their inner physical properties.⁵¹

Results obtained in this section suggest that FF–PLA possesses an enormous potential to form self-assemblies for biomedical applications via peptide-guided organization. The self-assembling capabilities of FF are well known. For example, Boc–FF⁵² and Fmoc–FF⁵³ [where Boc and Fmoc refer to tert-butyloxycarbonyl and N-(fluorenyl-9-methoxycarbonyl, respectively)] organize in microspheres and nanofibrils, respectively. While FF is able to assemble into peptide nanotubes,⁵⁴ disassembly can be achieved depending on the solution that surrounds the structure. No major changes are expected in the ability of FF to assemble, as their interactions with the polymer fragment are scarce. We are aware that the high crystallinity of PLA can interfere with the FF assembly since it is well known that simultaneous polymer crystallization and peptide organization become less likely.⁵⁵ However, the synthetic procedure used in this work allows controlling the molecular weight of PLA, the crystallinity of PLA decreasing with the molecular weight. Our research is currently oriented towards the self-assembly of FF–PLA using polymer fragments of low molecular weight.

Selection of reduced models

The most stable structures of the 6 (from a total of 13) representative clusters of lower energy conformations derived from MD simulations of FF–PLA in HFIP (see previous section) were used to construct the starting coordinates of FF–nPLA models, where n refers to the number of repeat units in the PLA block. Initially, reduced models were constructed considering n = 16, the rest of repeat units being removed. After geometry optimization at the UWB97XD/6-31+G(d,p) level, the inter-block secondary interactions and flexible backbone dihedral angles of the resulting reduced models were compared with those of original structures derived from MD simulations. As all inter-block interactions were retained and both the maximum and average deviations between the flexible backbone dihedral angles for each pair of structures (σ_max and σ_av, respectively) were lower than selected threshold values (i.e. σ_max = 60° and σ_av = 45°, respectively), new reduced models were constructed with n = 10 and subsequently re-optimized. As occurred for FF–16PLA, the agreement between the six optimized and the six MD structures was very high for FF–10PLA. Following the same strategy, FF–4PLA models were built. Analysis of the results indicated that 4 is smallest number of repeat units required to fulfill the imposed criteria (i.e. preservation of inter-block interactions and small deviations of the backbone dihedral angles, σ_max < 60° and σ_av < 45°). Thus, calculations on FF–2PLA led to very important conformational distortions in the backbone as well as to the loss of some inter-block interactions.

Fig. 10a represents the variation of σ_av with n for FF–nPLA models. As expected, σ_av increases with decreasing n. More specifically, σ_av ranges from 9.7° to 22.0° and from 15.6° to 40.6° for n = 16 and 4, respectively. Although in some cases the reduction of n represents a significant increment of σ_av (e.g. for models #3 and #6 the value of σ_av increases from 11.7° to 20.6° and from 15.9° to 35.4°, respectively, when n decreases from 10 to 4), the computational effort required to carry out TD-DFT calculations on FF–nPLA decreases by a huge amount with such reduction. Fig. 10b depicts the variation of the relative energy (ΔE) with n for the six calculated models. Although the energy gap (i.e. ΔE of the most unfavored model) decreases from 31.2 to 14.4 kcal mol⁻¹ when n decreases from 16 to 4, the ΔE order remains practically identical. Thus, the most remarkable change occurs for models #1, #2 and #3, which are practically isoenergetic when n = 4 while they are separated by an energy gap of 5.4 kcal mol⁻¹ when n = 16. On the other hand, it is worth noting that σ_av is 15.6°, 17.3° and 29.6° for the three more stable models of FF–4PLA. Fig. 10c represents more stable reduced models, which are within an ΔE interval of 0.2 kcal mol⁻¹ (Fig. 10b), evidencing that the inter-block interaction between FF and PLA is preserved after reduction of the number of polymer repeat units from 40 to 4.

Fig. 10 (a) Variation of the average deviation between the flexible backbone dihedral angles of FF–PLA structures derived from classical MD simulations and the FF–nPLA reduced models derived from DFT calculations (σ_av) with the number of PLA repeat units (n). (b) Variation of the relative energy (ΔE) with n for FF–nPLA DFT reduced models. (c) Atomistic picture of the three more stable reduced models obtained for FF–4PLA (ΔE ≤ 0.2 kcal mol⁻¹). The inter-block hydrogen bonding distance (dashed line) is indicated in Å.

Typically for FF–4PLA models, three more or less pronounced stabilizing interactions appear in the structures: (i) a hydrogen bond between the C=O and N–H, as classically observed in a β-turn; (ii) a quadruple interaction between both phenyl groups. Several recent experimental studies have emphasized the importance of aromatic interactions in stabilizing helices and beta-hairpins in designed synthetic peptides;^56,57 (iii) A C–H⋯π interaction between the methyl and one of the phenyl groups. Such interaction is a weak attractive force occurring between CH groups and π-systems. They are known to make important contributions to bio-molecular structure and function.^58–60 The presence of such weak interactions between the FF and PLA blocks is fully consistent with the CD spectra displayed in Fig. 5.

Electronic transitions

Fig. 11 shows the UV-Vis spectra of FF–PLA recorded in diluted HFIP solution (0.01 mg mL⁻¹). As was mentioned above, the absorption bands comprised between 245 and 270 nm clearly correspond to the diphenylalanine block. Deconvolution of the UV-Vis spectra is essential to obtain information about the specific transitions that contribute to each observed peak and also to compare the calculated spectra with the experimental ones (see below). Results from the deconvolution are included in Fig. 11. Satisfactory statistical parameters (i.e. correlation coefficient, R² > 0.997, and standard error, s.e. < 0.001) were obtained by considering five peaks centered on 246, 251, 257, 263 and 269 nm for the bands at λ comprised between 245 and 270 nm, and three peaks centered at 276, 288 and 303 nm for the broad band at λ > 270 nm.

Fig. 11 Absorption (absorbance expressed within an arbitrary unit) spectrum of FF–PLA. Solid black and grey lines represent the experimentally recorded and simulated spectra, respectively. The curves (dashed black lines) and wavelength (in nm) at the maxima resulting from the deconvolution process are also shown.

The simulation of the electronic excitations has been carried out for the most stable conformer identified for the FF–PLA tetramer at the structural study stage, namely FF4–PLA, by using the well-established TD-DFT computational scheme. The same WB97XD level of approximation was first selected, and the accuracy of the spectrum was then increased by enlarging the original BS, i.e. Pople's 6-311+G(d,p) was used, and by considering a Polarizable Continuum Model (PCM) to take the solvation effects into account. Doing so allowed keeping in the limits of accepted precision for excited states calculation in this upper part of the UV-Vis absorption spectra.

Indeed, as depicted in Fig. 12, FF4–PLA UV spectrum nicely reproduces the shape of the observed FF–PLA absorption spectrum with a consistent shift of ∼35 nm with respect to the de-convoluted experimental data (see Fig. 11). That is, we have the five excitation wavelengths computed (A to E in Fig. 12) at 208, 215, 222, 229 and 232 nm for observed peaks (1 to 5 in Fig. 12) at 246, 251, 257, 263 and 268 nm, with well-reproduced intervals between the spectral lines.

Fig. 12 Comparison of the experimental (dash line and numbered peaks) FF–PLA and calculated (plain line and labeled peaks) FF–4PLA spectra. The last one has been simulated by convoluting the TD-DFT transitions with a 0.2 eV empirical bandwidth while the corresponding heights are proportional to the respective oscillator strengths. See text for discussion.

The topology of the molecular orbitals involved in these transitions has also been investigated, and two major contributions have been highlighted (Fig. 13). First, the typical π → π* arising in this part of the spectrum were indeed observed. Second, a n → π* transition can be identified along the N–C–O backbone, corresponding to one of the two essential transitions observed in a peptide bond. Consistently with Bulheller et al.,⁶¹ the non-bonding orbital is centered about one of the lone pairs of the oxygen in CO while the anti-bonding orbital lies along the peptide bond. In an asymmetric molecule such a transition can show very low total absorption of light but high optical activity, as observed for helical polypeptides and proteins. Besides, n → π* interactions between carbonyl groups have been shown to play a role in protein folding,⁶² while carbonyl–carbonyl interactions between adjacent backbone amides have indeed been implicated in the conformational stability of proteins.

Fig. 13 A typical π → π* transition calculated at 208 nm; a similar π → π* transition in one ring is only observed at 231 nm. (b) A n → π* character is clearly identified along the N–C–O frame in a transition at 222 nm.

Conclusions

F- and FF-terminated PLA conjugates were prepared by ROP of L-lactide, using the peptide as initiator. Because of its simplicity, this process presents advantages with respect to the three-step route recently reported.²³ The structure of the two peptide–polymer conjugates was confirmed by ¹H NMR, IR, GPC and UV-Vis.

MD simulations of FF–PLA in HFIP revealed that the peptide fragment retains the intrinsic conformational preferences of diphenylalanine. This feature is consistent with the relatively scarce interactions found between FF and PLA blocks, which essentially consist of hydrogen bonds between the non-terminal phenylalanine residue and the L-lactide unit immediately after it. Thus, PLA tend to organize independently, which is a prerequisite for the formation of peptide-guided assemblies, corroborating the interpretation of CD spectra. However, in order to promote the formation of nanostructures through FF-guided assemblies, the crystallinity of PLA should be reduced by controlling the molecular weight during the ROP. Thus, future investigations on FF–PLA should be focused on exploiting the conjugate by avoiding the competition between the intrinsic tendency of PLA to crystallize and the intrinsic tendency of FF to organize in nanostructures.

Finally, TD-DFT calculations were achieved to model the electronic transitions observed by the UV-Vis absorption spectroscopy of the most stable FF–PLA conformation. They confirm the five excitations experimentally observed, and thanks to the involved molecular orbital topology, we were able to assign them to the usual π → π* transitions, as well as to the n → π* transition along the N–C–O backbone.

Acknowledgements

This work was supported by BMICINN-FEDER funds (MAT2012-34498 and MAT2012-36205), and by the Generalitat de Catalunya (2009SGR925, 2009SGR1208 and XRQTC). Authors are indebted to the Centre de Supercomputació de Catalunya (CESCA) for computational facilities. Support for the research of C.A. was received through the “ICREA Academia”. We thank Dr Guillermo Revilla-Lopez for his help with the computational simulations. The calculations were carried out in part on the supercomputing facilities in the College of Science and Mathematics at UMass Boston. This research used resources of the “Plateforme Technologique de Calcul Intensif (PTCI)” located at the University of Namur, Belgium, which is supported by the Belgian National Fund for Scientific Research (F.R.S.-FNRS). Catherine Michaux and Eric A. Perpète also thank the Belgian National Fund for Scientific Research for their associate and senior research associate positions, respectively.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra01534g

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