Binding of G-quadruplex DNA and serum albumins by synthetic non-proteinogenic amino acids: Implications for c-Myc-related anticancer activity and drug delivery

Abstract

This study delves into the impact of two synthetic non-natural amino acids, 7 and 8, on the structural dynamics of serum albumin and their potential significance in anticancer drug delivery systems. Crucially, the dihydrofuran-containing compound 7 has been identified to bind to the G-quadruplex (G4) DNA sequence 22-mer Pu22, a mimic of the proto-oncogene c-Myc, as ascertained by circular dichroism (CD) and UV spectroscopy. Our docking studies suggest that 7 binds to the G4 structure from the side of the G-quartet in a quasi-parallel manner, engaging in ten intermolecular interactions, including hydrogen bonds, π-lone pair and π-alkyl interactions. Notably, one interaction involves the heterocyclic ring of the compound. Compound 7 emerges as a notable structure modulator, showcasing a significant enhancement in protein α helix formation, as observed in a serum albumin binding CD experiment, and the capability to form supramolecular networks, as evidenced by dynamic light scattering (DLS) and Scanning Electron Microscopy (SEM), with the added benefit of encapsulating the natural anticancer drug curcumin within its self-assemblies. Toxicity assessments on human fibroblast cell lines demonstrate that both compounds are non-toxic, highlighting their biocompatibility and potential for safe biomedical applications. Interestingly, the triazole-based compound 8 induces distinctive structural changes in serum albumins, elucidated through CD and UV spectra using bovine serum albumin (BSA) as a model albumin target.

Graphical abstract

Roviello and colleagues explore synthetic non-natural amino acids as platforms for drug delivery and potential anticancer agents in this work. Compound 7 binds to the c-Myc G-quadruplex DNA and forms aggregates capable of encapsulating the anticancer drug curcumin. Through synthetic modifications, these compounds could lead to both novel anticancer drugs and targeted delivery systems.

Introduction

Unusual amino acids serve as fundamental building blocks in modern medicinal chemistry.^1,2 Their distinctive three-dimensional structure, characterized by a chiral central core and one or two potentially diverse side chains, is endowed with a high degree of functionality. This unique configuration renders them invaluable as starting materials for synthesizing complex molecules, integral components for structure-activity relationship (SAR) strategies, key elements in the development of peptidomimetic drugs,³ and promising candidates as standalone drugs.¹ A notable subset of unusual amino acids incorporates 2-aminobutyric acid, often utilized as a substitute for Ser/Cys in novel drug development. This substitution enhances the hydrophobicity and metabolic stability of the resultant drug. A noteworthy example is birinapant, a second-generation bivalent antagonist of the inhibitor of apoptosis proteins currently undergoing clinical development for cancer treatment.⁴ Concurrently, heterocyclic compounds play significant roles in pharmaceutical chemistry, contributing to various biochemical functions and serving as the core structures in diverse medicinal compounds. Drugs containing heterocyclic core skeletons, such as antifungal, anti-inflammatory, anti-bacterial, antiviral, antioxidant, anti-allergic, anticancer, and antihypertensive agents, are widespread.⁵ In the realm of therapeutic agents, nitrogen-producing heterocycles, particularly triazole rings,⁶ are ubiquitous and feature prominently in a broad spectrum of pharmaceutical drugs, demonstrating antimicrobial,⁷ anticancer,⁸ antihypertensive,⁹ antidiabetic,¹⁰ antidepressant,¹⁰ and other relevant properties. Chirality is a prevalent feature in pharmaceuticals, with the majority of drugs containing chiral molecules as active pharmaceutical ingredients, and the behavior of individual enantiomers of these substances can differ under physiological conditions, underscoring the importance of enantioselective synthesis in the pharmaceutical field.¹¹ This also highlights the pivotal role of enantioselective synthesis in the pharmaceutical industry, emphasizing its significance.^12,26 In this context, metal complexes of Schiff bases derived from tridentate ligands and amino acids have emerged in the last decades as leading tools for the asymmetric synthesis of unusual amino acids.^13,27 This approach contributes to the synthesis of chiral molecules, advancing the pharmaceutical industry’s search for novel and effective therapeutic agents, with growing interest in the asymmetric synthesis of unusual amino acids highlighting its importance and providing structurally diverse, enantiomerically enriched derivatives for biological studies. This research direction holds promise for advancing our understanding of the intricate interplay between amino acid structure and biological activity, opening avenues for innovative drug discovery and biomaterial design. The present investigation explores different properties of non-natural amino acids 7 and 8, delving into their impact on quadruple helical DNA and protein structural properties and their potential as integral components in anticancer drug delivery systems (Figure 1).

Figure 1.

Structural representation of the synthetic amino acids, i.e., compounds 7 and 8, assayed in the present work

Remarkably, serum albumin’s role as a versatile pharmaceutical carrier, combined with the distinctive properties of synthetic amino acids, forms the basis for advanced drug delivery strategies. Overall, our study employs a comprehensive approach, integrating UV analysis, fluorescence studies, dynamic light scattering (DLS), circular dichroism (CD), molecular docking, and cell vitality assays, aiming to unravel the structural changes in biomacromolecules induced by compounds 7 and 8 and explore their applications in tailored drug delivery strategies. The investigation extends beyond structural elucidation into biocompatibility and drug encapsulation, and the toxicity profiles of compounds 7 and 8 on human fibroblast cell lines establish their non-toxic nature, affirming their potential suitability for biomedical applications. Furthermore, the capability of compound 7 to form supramolecular networks with curcumin encapsulation introduces a novel dimension to drug delivery applications, amplifying the potential impact of synthetic amino acids in the field. In fact, in the pursuit of transformative drug delivery systems, the integration of artificial amino acids assumes paramount importance, enriching our comprehension of DNA-drug and protein-drug interactions and pioneering the design of advanced pharmaceutical delivery platforms. The intersection of synthetic non-natural amino acids with the herein reported biomolecular targets represents a frontier where scientific innovation converges with therapeutic applications, promising a paradigm shift in precision medicine and personalized therapeutics.

The c-Myc proto-oncogene plays a critical role in cell growth, proliferation, and apoptosis, making it a prominent target in cancer therapy.^14,15 Overexpression of c-Myc is associated with various cancers, including breast, colon, and lung cancer, and is linked to poor prognosis. Recent advances have unequivocally demonstrated the presence and functional significance of G-quadruplexes (G4s) in human cells, underscoring their role as critical targets for small-molecule cancer therapies. Notably, G4 structures are four-stranded DNA motifs that can regulate gene transcription, and stabilizing these structures with small molecules can inhibit the transcription of oncogenes, thereby suppressing tumor growth. In more detail, targeting the c-Myc promoter region, which contains a G4 structure, has emerged as a promising strategy for modulating c-Myc expression, with these findings establishing a foundational framework for the development of an advanced generation of intelligent, supramolecular G4 binders with exceptional sensing and stability attributes for future anticancer therapies.^15,16

Results

Synthesis of compounds 7 and 8

The chiral Ni^II complexes of Schiff’s base of dehydroamino acids with chiral auxiliaries (S)-2-N-(N′-benzylprolyl)aminobenzophenone, Ni^II-(S)-BPB-Δ-Ala (1) and Ni^II-(S)-BPB-Δ-Aba (2), were synthesized according to a procedure reported previously.^17,18 The addition of heterocyclic nucleophile 3 to the chiral dehydroalanine complex Ni^II-(S)-BPB-Δ-Ala (1), carried out in dimethylformamide in the presence of KOH, led to the complex 5 (Figure 2) with good to very good diastereoselectivities.

Figure 2.

Synthetic route to compounds 7 and 8

The asymmetric addition of the heterocyclic nucleophile 4 to the C=C bond of the chiral dehydroaminobutyric acid complex Ni^II-(S)-BPB-Δ-Aba (2) proceeded in CH₃CN in the presence of K₂CO₃ at 50°C–60°C (Figure 2). The reactions afforded a mixture of diastereomeric complexes with a large excess of the diastereomer having the (2S,3S) configuration at the newly generated chiral center (6). The reactions were monitored by TLC (SiO₂, CHCl₃/CH₃COCH₃, 3:1) by following the disappearance of the traces of the initial complexes 1 and 2 and the establishment of a thermodynamic equilibrium between the diastereoisomers formed from the addition products. The target α-amino acids 7 and 8 were isolated, respectively, from the mixture of the diastereomeric complexes 5 and 6 after acidic hydrolysis and ion-exchange demineralization (Figure 2). Amino acids 7 and 8 were, thus, obtained after crystallization from a water-ethanol (1:1) mixture, and their structure and absolute configuration were determined using physicochemical analysis methods. The chiral auxiliary (S)-BPB was recovered in a quantitative yield (>90%) without any loss of its initial enantiomeric excess and could be reused. All synthesized compounds were characterized by ¹H- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis (Figures S1–S10). In the following sections, we will present the results of various experiments, including protein molecular docking studies (Figure S11), scanning electron microscopy (SEM) imaging of compound 7 in the presence of curcumin (Figure S12), UV binding assays with compound 8 (Figure S13), and cytotoxicity tests on MDA-MB cells (Figure S14). Additionally, we also performed further analyses, such as DLS for compound 7 aggregates (Table S1), molecular docking interactions of compound 7 with Pu22 DNA (Tables S2 and S3).

UV studies of compounds 7 and 8

To gain further insight into the properties of compounds 7 and 8, UV-visible (UV-vis) and fluorescence spectroscopies were employed to examine their concentration-dependent behavior and the potential for self-assembly (Figures 3A and 3B). The UV-vis spectra of the compounds 7 and 8 were recorded at concentrations ranging from 16.0 μM to approximately 1.0 mM (Figure 3A), with compound 7 displaying a broad band centered at 230 nm that increased proportionally with concentration (Figure 3A, left), while compound 8 showed a band centered at 255 nm (Figure 3A, right).

Figure 3.

Spectroscopic, DLS, and SEM analyses of synthetic amino acid samples

(A and B) UV (A) and fluorescence (B) analysis of compounds 7 (left) and 8 (right). Different colors in the UV datapoints plotted in the insets stand for experimental (black) and theoretical (red) data. Data are shown as mean ± SD from two independent experiments).

(C) DLS analysis (left) and SEM images (right) of compound 7 (1 mM, in 25 mM sodium phosphate [pH 7.4]).

When plotting the UV absorption at 230 and 255 nm for compound 7 and compound 8, respectively, as a function of concentration, a linear behavior was observed in the range of 16–125 μM, with a negative deviation at higher concentrations for both compounds, which is typical in aggregate formation. However, the deviation from linearity was more pronounced for compound 7, suggesting the presence of a higher percentage of aggregates at the concentrations explored for this amino acid derivative. This evidence was further supported by DLS analyses. Specifically, the DLS experiment was conducted on a solution of compound 7 at a concentration of 1.0 mM and 25°C, allowing the determination of average particle size, homogeneity, and stability. Immediately after dissolution, the DLS profile for nanoaggregates of compound 7 revealed a single population with a narrow distribution and a mean hydrodynamic diameter of approximately 1,000 nm (Figure 3C, left). Particle size homogeneity was further confirmed by the relatively low polydispersity index (PdI) values (Table S1). In contrast, the DLS profile for the nanoaggregates of compound 8 could not be analyzed due to a wide particle size distribution (data not shown). To further investigate the self-assembly of the compounds, we monitored their intrinsic fluorescence (Figure 3B).

The intrinsic fluorescence of aromatic rings in natural compounds, such as tryptophan, has been extensively studied and shown to be attenuated by aromatic stacking interactions of indole rings during self-assembly. Therefore, this analysis is crucial for determining whether self-assembly is driven by the stacking of aromatic rings. In the case of compound 8, as the concentration increased, the fluorescence intensity at 285 nm (λ_ex = 255 nm) steadily increased (Figure 3B, right), which suggests a low presence of self-assembling aggregates. On the contrary, for compound 7, an attenuation of the fluorescence intensity was observed as the concentration increased (Figure 3B, left), therefore suggesting a stacking phenomenon of the aromatic rings of compound 7.

The morphological features of the molecular self-assemblies of compound 7 were also examined by SEM, using a drop-casting method to apply the solution onto glass slides (see the materials and methods section). The images clearly show that compound 7 forms well-defined structures, predominantly spherical in shape (Figure 3C, right), with a size comparable to that observed in the DLS experiments. This morphology is likely due to π-stacking interactions. The resulting exclusion of water around the aliphatic/aromatic units is expected to facilitate nanoaggregate bending, leading to vesicle formation, which is consistent with our fluorescence and UV-vis data.

Entrapment of curcumin by amino-acid-based self-assemblies

One of the most exciting applications of nanostructures is their potential to deliver bioactive molecules/drugs used as therapeutics. To this end, we sought to monitor the binding capabilities of nanostructures generated with a high concentration of compound 7 by loading them with curcumin, which was used as a model for a hydrophobic drug.¹⁹ In this context, curcumin loading in aggregates of compound 7 was investigated through UV-vis spectrophotometric titrations. Notably, a progressive decrease in the intensity of the curcumin band at 426 nm (n→π∗ transition) was observed as the concentration of the compound 7 increased, when added to a fixed concentration of curcumin solution (Figure 4A).

Figure 4.

Curcumin binding assay performed on compound 7

(A) UV changes upon progressive addition of compound 7, demonstrating the ability of its molecular aggregates to encapsulate curcumin. Decrease in absorption at 426 nm (%) vs. concentration is shown as an inset (data are shown as mean ± SD from two independent experiments) (B) Curcumin loading efficiency into aggregates of compound 7; standard curve and corresponding linear fit shown as an inset (data are shown as mean ± SD from two independent experiments). (C) Heat-triggered curcumin release from compound 7 aggregates at a compound/curcumin ratio of 1:0.1.

This behavior suggested that interactions of various nature were established between the drug and the nanoaggregates as already reported for similar systems.¹⁹ The encapsulation efficiency of the curcumin-loaded aggregates was determined by measuring the amount of curcumin remaining in the supernatant through fluorescence emission analysis at λ = 540 nm (excitation at λ = 450 nm). The curcumin-loaded aggregates of compound 7 were formed by mixing both solutions in specific mole ratios to determine the optimal ratio for maximum encapsulation efficiency. The concentration of compound 7 was kept constant, while the concentration of curcumin was varied, resulting in mole ratios of compound 7 to curcumin of 1:0.025, 1:0.05, 1:0.1, and 1:0.5. The optimal curcumin loading efficiency, achieved at a 1:0.1 ratio, was determined to be 32% ± 2% (Figure 4B). The morphological features of these aggregates were also examined by SEM analysis, revealing a conservation of the spherical shape of the aggregates but with a slight enlargement of their size (Figure S12).

Moreover, the release of curcumin from the compound 7 aggregates at a 1:0.1 ratio was also investigated. To this end, curcumin-loaded aggregates were incubated at 50°C, and after 16 h, the solution was centrifuged. Subsequently, the supernatant was analyzed by measuring the fluorescence emission in the range of 490–700 nm (λ_exc = 450 nm). Notably, as shown in Figure 4C, a fluorescence recovery was observed compared to the signal recorded before treatment, indicating the breakage of the aggregates and the release of entrapped curcumin molecules.

In contrast, the aggregates formed by compound 8 did not produce any change in the absorption intensity of curcumin, indicating the absence of entrapment (Figure S13). Furthermore, we explored the ability of the synthetic amino acids to bind serum albumins, which is an important aspect in view of their usage as drugs for the transport in vivo,²⁰ and performed CD and UV studies using bovine serum albumin (BSA) as a serum albumin model (Figure 5).

Figure 5.

CD and UV binding studies on compounds 7  and 8  with 9.5 μM BSA (in 1× PBS [pH 7.4])

The experiments related to compound 7 on the left and those related to compound 8 on the right of the figure.

By analyzing the CD spectra reported in Figure 5, it emerged that both molecules interact with BSA, with compound 7 leading to a higher structural formation in BSA (enhanced α helix, decreased random coil, Table 1). On the other hand, compound 8 provokes an evident structural loss of serum albumin, as evidenced by both CD and UV spectra and CD deconvolution (Table 1).

Table 1.

CD deconvolution of CD spectra of BSA in the presence of 7 and 8

Compound	Δα(x-BSA) (%)	Δβ-sheet(x-BSA) (%)	ΔRandom coil(x-BSA) (%)
7	65.6−57.1 = +8.5	6.8−11.9 = −5.1	27.6−31.0 = −3.4
8	75.1−84.2 = −9.1	1.3 + 1.8 = +3.1	23.6−17.6 = +6.0

Variations (Δ) of α, β-sheet and random rates are reported and given as percentages.

HDOCK molecular docking studies allowed us to gain insights into the mechanism by which the compounds bind to BSA. Remarkably, considering the top-1 poses for the complexes, each ligand binds to different subdomains of BSA, with 7 interacting with chain A, whereas 8 binds to chain B of the protein (Figure 6).

Figure 6.

BSA molecular docking and cellular studies

(Top) Three-dimensional (3D) views of the top-ranked poses for the complexes formed by BSA with 7 (left) and 8 (right). Note how the chain of the protein involved with the interaction predicted for the ligands is indicated as A (chain A, orange) or B (chain B, blue). For clarity, the ligand within each complex structure is highlighted in yellow and enclosed within a yellow rectangle. Bottom: viability studies performed on human normal cells (NHDFs) in the presence of compounds 7 (orange) and 8 (gray). Cells were treated with compounds 7 and 8 at different concentrations for 24 h. Cell viability was measured by the crystal violet assay, and values were expressed as a percentage with respect to untreated cells. Each value expresses an average ± SD of two separate experiments performed in triplicate.

Following the above studies, we aimed to assess some biological properties of compounds 7 and 8 by performing cellular viability assays. Remarkably, our cellular studies (Figure 6, bottom) showed no significant changes in human cell viability in the presence of 7 and 8, denoting an absence of any significant toxicity of the tested compounds on normal human dermal fibroblasts (NHDFs). Moreover, we explored the binding of our two synthetic non-proteinogenic amino acids to Pu22, a 22-mer mimic of the proto-oncogene c-Myc. In more detail, CD and UV spectroscopy were employed to ascertain the interaction with the DNA target (Figure 7A). Although the spectral changes were subtle, they were still significant, with compound 7 binding effectively to the Pu22 sequence, altering the CD profile and reducing UV absorbance at 250 nm, indicative of changes in the secondary structure of the G4. In contrast, compound 8 did not affect the CD profile or UV absorbance at all, suggesting no significant binding interaction (data not shown).

Figure 7.

Binding of compound 7 to the parallel G4 structure of the 22-mer mimic of c-Myc DNA Pu22

(A) Interaction with Pu22 (2.5 μM in 1× PBS [pH 7.4]), as revealed by CD and UV spectroscopy. Insets display decreased CD intensity and UV absorbance upon compound 7 binding. (B) Molecular docking studies depicting the binding of compound 7 with Pu22: two 3D views show the top-1 pose (model 1) predicted by HDOCK for the most stable amino acid-G4 DNA complex. The ligand is highlighted in yellow for clarity.

Molecular docking studies using HDOCK^21,22 with the model of the c-Myc Pu22 parallel G4 structure (PDB: 6AU4) provided insights into the binding mechanism (Figure 7B). Compound 7 interacts with the G4 structure from the side of the guanine-quartet in a quasi-parallel manner, positioning its five-membered ring above the upper G-quartet (Figure 7B) and forming ten diverse intermolecular interactions, including hydrogen bonds and π-lone pair and π-alkyl interactions (Table S2). Notably, one interaction involves the heterocyclic ring of compound 7.

In the molecular docking analysis using HDOCK, the involvement of guanine residues from the Pu22 DNA in binding to compound 7 is notable (Table S3), and across all three models (1, 2, and 3), guanine residues (DG) are consistently implicated in the receptor-ligand interface, with four DG residues appearing in each model. Specifically focusing on model 1, which represents the top-ranked docking pose described in this study, DG residues play a critical role in interacting with 7. DG residues at positions 6, 10, 15, and 19, as well as DT 20 and DA 21, form hydrogen bonds and other intermolecular interactions with 7, with bond distances starting from 2.531 Å. These interactions are crucial for stabilizing the binding conformation between 7 and the Pu22 DNA, potentially influencing the modulation of the c-Myc G4 structure, which is relevant in anticancer therapeutic strategies.

In other terms, these findings underscore the potential of compound 7 to modulate biological pathways associated with the stabilization of the c-Myc oncogene promoter, further highlighting its potential anticancer activity. In other terms, the ability of compound 7 to bind to the c-Myc G4 structure suggests its suitability for developing novel anticancer systems. This, combined with its curcumin transporting ability as well as the non-toxic profile on human fibroblast cell lines, underscores its promise for biomedical applications.

Discussion

Overall, the experimental landscape unfolds a complex interplay of structural dynamics and functional attributes influenced by compounds 7 and 8 on serum albumin, with compound 7’s significant increase of α helix formation, coupled with the formation of supramolecular networks, positioning it as a potent candidate for drug delivery systems. Remarkably, this distinctive capability is reinforced by its demonstrated proficiency in encapsulating curcumin. Also, compound 8 introduces distinctive BSA structural changes, elucidated through CD and UV spectra, with the molecular docking revealing unique subdomain interactions, that provide intricate details of the modulatory effects of this synthetic non-natural amino acid on serum albumin. Moreover, the conducted DNA-binding study underscores the significant potential of synthetic non-proteinogenic amino acids in modulating c-Myc proto-oncogene expression through their interaction with the c-Myc DNA G4 structure. CD and UV spectroscopy revealed that compound 7 effectively binds to the Pu22 sequence, a 22-mer mimic of c-Myc DNA, inducing alterations in the CD profile and reducing UV absorbance at 250 nm, indicative of slight but significant structural changes in the G4. These findings suggest that compound 7 may affect the c-Myc G4 structure, a critical regulator of gene transcription, which may lead to the modulation of its oncogene activity associated with various cancers, including breast, colon, and lung cancer. Molecular docking studies using HDOCK provided mechanistic insights into how 7 interacts with the c-Myc parallel G4 structure (using the model with PDB: 6AU4). Notably, compound 7 binds from the side of the guanine-quartet in a quasi-parallel manner, positioning its five-membered ring above the upper G-quartet and participating in ten diverse intermolecular interactions, including hydrogen bonds, π-lone pair and π-alkyl interactions. This mode of binding highlights compound 7’s potential to modulate biological pathways involved in c-Myc oncogene promoter stabilization, thus emphasizing its intrinsic anticancer activity. Furthermore, self-assembling and serum albumin binding studies have shown the profound impact of compound 7, marked by enhanced BSA α helix formation and the establishment of supramolecular networks, positioning it as a transformative element in drug delivery systems. Moreover, the distinct BSA structural changes induced by compound 8, as revealed by CD and UV spectra, provide valuable insights into the diverse modulatory effects of synthetic amino acids on proteins. Crucially, the non-toxic nature of compounds 7 and 8 on human fibroblast cell lines revealed by our cellular studies positions them as promising candidates for biomedical applications. This, coupled with the ability of compound 7 to form aggregates capable of encapsulating curcumin and bind the c-Myc G4 structure, opens promising horizons for drug delivery strategies, promising efficient biomedical applications of this synthetic amino acid. Our SEM images clearly indicate that compound 7 forms well-defined structures that are predominantly spherical in shape (Figure 3C, right), with a size comparable to that observed in DLS experiments. This morphology is likely due to π-stacking interactions. The resulting exclusion of water around the aliphatic/aromatic units is expected to facilitate the bending of the nanoaggregates, leading to vesicle formation, as previously reported in other similar systems^23,24 and consistent with our fluorescence and UV-vis data. Remarkably, the self-assembled aggregates of compound 7 undergo disassembly upon exposure to heat, determining the curcumin release, as we observed during our fluorescence studies at 50°C. This mechanism could be tuned for controlled drug delivery, enabling release of the therapeutic cargo under specific conditions. In addition, we performed preliminary anticancer tests on MDA-MB cells. However, we did not observe significant anticancer effects (Figure S14), likely due to the compounds' tendency to aggregate under the explored experimental conditions. Despite this, we plan to modify the structure of compound 7 to enhance its anticancer potential, and the binding of compound 7 to the c-Myc G4 structure offers promising prospects in this regard, possibly by incorporating the synthetic amino acid structure into short peptides as seen in recent literature examples.²⁵

In conclusion, compound 7 demonstrates promising binding to oncogenic DNA G4 structures and a favorable safety profile in human fibroblasts, while its ability to form heat-responsive aggregates for controlled release of the model drug curcumin highlights its potential for targeted drug delivery applications.

Materials and methods

Synthetic procedures

The silica gel L-40/100 was purchased from Merck (Germany), while Ni(NO₃)₂⋅6H₂O, K₂CO₃, (СH₂O)n, СHCl₃, (CH₃CO)₂O, CH₃COOH, CH₃COCH₃, CH₃CN, CH₃OH, Na₂CO₃, NH₄OH, HCl, KOH, C₂H₅OH, DMF, and 2-aminobenzophenone were purchased from Aldrich (USA). The nucleophilic reagents were synthesized in the department of Organic Chemistry of YSU. All used solvents were freshly distilled. ¹Н- and ¹³C-NMR spectra (Figures S1–S8) were recorded on a Mercury-300 Varian (300 MHz). The optical rotation was measured on a PerkinElmer 341 polarimeter. The melting point values were measured with a Melting Point Stuart SMP30 apparatus. Electrospray ionization (ESI) MS analysis (Figures S9 and S10): sample analysis was performed using a Prominence I LC-2030C 3D Plus instrument from Shimadzu, Kyoto, Japan.

General method for the synthesis of complex 5

To 0.81 g (1.6 mmol) of complex 1 in 5 mL of DMF was added 0.26 g (4.8 mmol) of KOH and 1.02 g (4.8 mmol) of the nucleophile 3. The reaction mixture was stirred at room temperature or at 25°C under argon atmosphere. The course of the reaction was monitored by TCL (SiO₂, CH₃COCH₃/CHCl₃, 1:3) by following the disappearance of the initial complex 1. Upon completion of the reaction, the mixture was neutralized by CH₃COOH and poured into stirred distilled water. After 15 min, it was filtered and dried in air.

Mp 101°C–103°C; [α]_D²⁰ = +1,555° (C = 0.1, CH₃OH); ¹H-NMR (CDCl₃), δ, ppm: 1.32 (3H, s, CH₃); 1.49 (3H, s, CH₃); 1.96–2.24 (2H, m, γ,δ-Hα); 2.45–2.66 (2H, m); 2.69–2.83 (2H, m); 3.48 (1H, dd, J = 10.9, 5.7, α-4 Pro); 3.51–3.59 (5H, m); 3.59 (1H, d, J = 2.6, CH₂ Phe); 3.79 (2H, t, J = 4.9, OCH₂); 3.82 (1H, dd, J = 9.5, 3.7, CHCH₂); 4.42 (1H, d, J = 12.6, CH₂Ph); 6.61–6.70 (2H, m, Ar); 7.08–7.24 (4H, m, Ar); 7.34–7.40 (2H, m, Ar); 7.44–7.55 (3H, m, Ar); 8.05–8.10 (2H, m, ortho-Ph); 8.19 (1H, dd, J = 8.7, 1.0, C₆H₄); 8.55 (1H, b.t, J = 5.6, NH).

13С-NMR (CDCl₃): 23.9 (CH₃); 23.9 (γ-CH₂ Pro); 24.3 (CH₃); 30.8 (β-CH₂ Pro); 33.7 (CH₂); 42.5 (NCH₂); 57.3 (δ-CH₂ Pro); 62.5 (OCH₂); 63.2 (CH₂ Ph); 69.7 (CH); 70.4 (α-CH Pro); 87.1 (CMe₂); 117.8; 120.8 (CH); 123.8 (CH); 126.2; 127.0 (CH); 128.0 (CH); 128.8 (CH); 128.9 (α-CH); 129.0 (CH); 129.3 (CH); 129.8 (CH); 131.6 (α-CH); 132.4 (CH); 133.3; 133.4 (CH); 133.6; 142.5; 161.6; 171.1; 171.2; 178.7; 180.5; 182.3.

Isolation of the target amino acid (S)-2-amino-4-(4-((hydroxyethyl)carbamoyl)-2,2-dimethyl-5-oxo-2,5-dihydrofuran-3-yl)butanoic acid (7)

Decomposition of the diastereomeric complex 5 and isolation of the target β-substituted α-amino acid 7 was carried out according to the earlier developed procedure.¹³

Mp 245°C–247°С; [α]_D²⁰ = 17.1° (C = 0.5, 6N HCl); ¹H-NMR (DMSO/CCl₄ 1/3 +CF₃COOD) δ, ppm: 1.13 (3H, m, CH₃); 1.54 (3H, m, CH₃); 4.45 (1H, dd, J₁ = 7.8, J₂ = 5.3, CH₂); 4.57 (1H, dd, J₁ = 14.2, J₂ = 7.8, CH₂); 4.78 (1H, dd, J₁ = 14.2, J₂ = 5.3, CH₂); 4.88 (2Н, dt, J₁ = 5.3, J₂ = 1.6, NCH₂); 5.15 (1H, dq, J₁ = 17.0, J₂ = 1.6, -CH₂); 5.17 (1Н, dq, J₁ = 10.6, J₂ = 1.6, -CH₂); 5.88 (1Н, ddt, J₁ = 17.0, J₂ = 10.6, J₃ = 5.3, CH₂).

13С-NMR (DMSO/CCl₄ 1/3 +CF₃COOD) ppm: 22.5 (CH₂), 23.96 (CH₃), 23.99 (CH₃), 41.1 (NCH₂), 59.6 (OCH₂), 86.5 (CMe₂), 118.4, 160.1, 160.2, 170.2, 179.8.

General method for the synthesis of complex 6

To 2.62 g (5 mmol) of complex 2 in 20 mL of MeCN were added with stirring 1.38 g (10 mmol) of K₂CO₃ and 1.5 g (7.5 mmol) of nucleophile 4 at 50°C–60°C. The reaction was monitored by TLC (SiO₂, CHCl₃/Me₂CO [3:1]) following the disappearance of the spot on the initial complex 2. Upon completion of the reaction, the mixture was filtered, the K₂CO₃ precipitate washed with CH₃CN, and the solution evaporated to dryness. A small part of the dry residue (0.1 g) was chromatographed on SiO₂ (CHCl₃/Me₂CO, 3:1, 20 × 20 cm) to isolate the pure diastereomeric complex 6-addition product, the structure and absolute configuration of which were clarified by the methods of spectroscopic analysis.

Mp 191°C–193°C; [α]_D²⁰ = +3.30 (с = 0.1, CH₃OH); ¹H NMR (CDCl₃), δ, ppm: 1.01 (3H, d, J = 6.7, CH₃ i-But); 1.02 (3H, d, J = 6.7, CH₃ i-But); 1.02 (3H, t, J = 7.3, CH₃ Pr); 1.20 (3H, d, J = 7.2, CH₃CH); 1.72–2.03 (5H, m); 2.16 (1H, m, CH, i-But); 2.33–2.47 (1H, m); 2.53 (2H, d, J = 7.1, CH₂ i-But), 2.69–2.80 (1H, m); 2.87–3.02 (1H, m); 3.36 (1H, dd, J = 10.4, 6.5, α-H Pro); 3.33–3.40 (1H, m); 3.61 (1H, d, J = 12.7, CH₂ Ph); 3.85 (1H, ddd, J = 13.6, 9.7, 5.8, NCH₂); 4.11–4.23 (1H, m); 4.15 (1H, d, J = 4.0, NCHCHCH₃); 4.43 (1H, d, J = 12.7, CH₂Ph); 5.54 (1H, qd, J = 7.2, 4.0, CHCH₃); 6.66 (1H, ddd, J = 8.3, 6.4, 1.2, H-4 C₆H₄); 6.69 (1H, dd, J = 8.3, 2.4, H-4 C₆H₄); 7.15 (1H, ddd, J = 8.7, 6.4, 2.4, H-5 C₆H₄); 7.17 (1H, tt, J = 7.4, 1.2, H-4 Ph); 7.26–7.34 (3H, m, Ar); 7.49–7.59 (4H, m, Ar), 7.90–7.94 (2H, m, H-2, 2′-Ph); 8.43 (1H, dd, J = 8.7, 1.2, H-6 C₆H₄):

13С NMR (CDCl₃) δ, ppm: 11.5 (CH₃); 17.2 (CH₃); 21.9 (CH₂); 22.5 and 22.7 (Me₂); 23.5 (γ-CH₂ Pro); 26.6 (CH But); 30.7 (β-CH₂ Pro); 34.5 (CH₂); 46.4 (CH₂); 57.2 (CHMe); 55.5 (δ-CH₂ Pro); 62.8 (CH₂Ph); 70.2 (α-CH Pro); 73.3 (NCHCH); 120.5 (C-4 C₆H₄), 123.5 (C-6 C₆H₄); 126.2; 127.5 (CH); 128.9 (3,3′-CH Ph); 128.9 (CH); 129.1 (CH); 129.1 (CH); 129.2 (CH); 129.8 (CH); 131.8 (2,2′-CH Ph); 132.7 (3-CH C₆H₄); 133.0; 133.9 (5-CH C₆H₄); 134.6; 143.4; 150.3; 168.8; 172.5; 175.6; 180.1:

Isolation of the target amino acid (2S,3S)-2-amino-3-(3-isobutyl-4-propyl-5-thioxo-4,5-dihydro-1 H-1,2,4-triazol-1-yl)butanoic acid (8)

The decomposition of the diastereomeric complex 6 and the isolation of the target amino acid 8 were carried out according to the earlier developed procedure.¹³

Mp 197°C–199°C; [α]_D²⁰ = −0.4167 (c = 6, HCl); ¹H-NMR՝ (DMSO/CCl₄ 1/3 +CF₃COOD) δ, ppm: 0.95 (3H, t, J = 7.4); 0.98 (3H, d, J = 6.6); 0.99 (3H, d, J = 6.6); 1.49 (1H, d, J = 7.0); 1.66–1.77 (2H, m); 2.02–2.16 m; 2.49 (1H, d, J = 7.2); 3.77–3.92 (2H, m); 4.19 (1H, d, J = 6.1); 5.36 (1H, qd, J = 7.0, 6.1):

¹³C NMR՝ (DMSO/CCl₄ 1/3 +CF₃COOD) ppm: 11.0 (CH₃); 14.7 (CH₃); 21.3 (CH₂); 22.3 (Me₂); 26.2 (CH); 33.7 (CH₂); 45.6 (CH₂); 52.2 (CH); 54.6 (CH); 150.7; 166.4; 168.2.

UV analysis

UV spectra were recorded using a JASCO J-815 CD spectropolarimeter (Jasco, Easton, MD, USA). In more detail, high-tension voltage (HT(V)) spectra were first obtained from the CD instrument, where the data were converted from HT voltage to UV absorbance. The UV spectra shown in Figures 3 and 5 were then normalized between 0 and 1 for further analysis. For compound 7, solutions in the 0.016–1.0 mM concentration range were prepared, while for compound 8, solutions in the 0.016–0.5 mM concentration range were used (25 mM sodium phosphate [pH 7.4]). UV experiments were performed in duplicate, and all spectra were corrected for background by subtracting the proper blank. UV-vis spectroscopy was also used to assess the entrapment of curcumin in the assemblies of compound 7. For this purpose, a curcumin solution (10 μM in 25 mM sodium phosphate buffer [pH 7.4]) was prepared and left to sonicate for several minutes. Then, the solution was analyzed using UV-vis spectroscopy to observe a characteristic peak at 426 nm. Subsequently, a concentrated solution of compound 7 was gradually added to the curcumin solution, and any changes in the intensity of the peak located at 426 nm were recorded after 10 min of incubation.

Fluorescence analysis

Fluorescence experiments were performed on a FluoroMax-4 Horiba Scientific spectrofluorometer. Samples of compound 7 and compound 8 were prepared in the 0.015–1.0 mM concentration range (25 mM sodium phosphate [pH 7.4]). Fluorescence spectra of compound 7 were recorded in the 250–320 nm (λ_em) range upon excitation at 230 nm (λ_ex) using 5 nm slit bandwidths for both excitation and emission. Fluorescence spectra of compound 8 were recorded in the 270–400 nm (λ_em) range upon excitation at 255 nm (λ_ex) All fluorescence experiments were performed in duplicate.

The encapsulation efficiency of curcumin was evaluated by indirectly calculating the amount of curcumin incorporated into the self-assembled aggregates of compound 7 using fluorescence techniques. Aggregates were formed by mixing solutions of compound 7 and curcumin, both dissolved in 25 mM sodium phosphate buffer at pH 7.4, in various mole ratios. The concentration of compound 7 was kept constant at 1.0 mM, while the curcumin concentration was varied to achieve mole ratios of 1:0.025, 1:0.05, 1:0.1, and 1:0.5 in order to determine the optimal ratio for maximum encapsulation. The mixture was allowed to incubate overnight at 25°C. Following this, the mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. The amount of curcumin within the supernatant was quantified by measuring the fluorescence emission at 540 nm (excitation at 450 nm), which corresponds to the peak emission of standard curcumin, and calculated by the calibration curve of standard curcumin. The amount of curcumin trapped in the aggregates was determined by subtracting the quantity in the supernatant from the total quantity used during the preparation. Fluorescence intensities were averaged from two separate experiments.

To prepare the standard curve, a curcumin stock solution was made at a concentration of 0.1 mg/mL in 25 mM sodium phosphate buffer at pH 7.4. From this, serial dilutions were made to prepare solutions with concentration ranging from 0.025 to 0.1 mg/mL. The fluorescence emission at 540 nm (excitation at 450 nm) was measured for each concentration. A standard curve was then plotted by correlating the signal intensity with the curcumin concentration.

The loading efficiency was calculated using the following formula:

loading efficiency (%) = (amount of curcumin encapsulated/curcumin added) × 100 = [((curcumin added – free “unentrapped curcumin”))/curcumin added] × 100. (Equation 1)

Additionally, the release of curcumin from compound 7 aggregates was evaluated. Curcumin-loaded aggregates (in a 1:0.1 compound 7:curcumin ratio) were incubated in a 1 mL buffer solution at 50°C. After 16 h, the mixture was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. The fluorescence emission of the supernatant was measured in the range of 490–700 nm (excitation at 450 nm), with fluorescence intensities normalized and background intensities subtracted. The results from two separate experiments were averaged.

DLS

DLS measurements were carried out on a Zetasizer Nano ZS (Malvern Instrument) using 12 mm square polystyrene cuvettes (DTS0012, Malvern Instrument). The solutions of compounds 7 and 8 were prepared at a 1.0 mM concentration in 25 mM sodium phosphate (pH 7.4). All the analyses were performed in triplicate at room temperature with a scattering angle of 173° and an equilibration time of 60 s.

SEM

The morphological characterization of our samples was carried out by SEM analysis collecting images of the compound 7 solution (1.0 mM) in the dried state on an field-emission (FE)-SEM Ultra Plus (Zeiss) microscope at 5 kV. For sample preparation, 20.0 μL (diluted in phosphate buffer [pH 7.4], 25 mM) of the sample was deposited on a thin glass slide, air dried for 16 h at room temperature, and then sputtered with a 10-nm-thick gold layer. After these steps, images of the samples were acquired.

CD analysis

CD spectra were recorded using the same JASCO J-815 CD spectropolarimeter. Solutions of compound 7 were prepared in the 0.016–1.0 mM concentration range, and solutions of compound 8 were prepared in the 0.016–0.5 mM concentration range similarly to how was previously reported in this section. The CD experiments were conducted in duplicate using 30 equiv of ligand relative to BSA and 5 equiv of ligand relative to G4 DNA in a 1× PBS solution at 22°C and pH 7.4. The optical path length (b) was 2 × 0.4375 cm, and a Hellma-238-QS tandem quartz cell (Hellma Italia S.r.l., Milano, Italy) was used. Each spectrum was obtained by averaging three scans. The spectrum of the complex solution, obtained after mixing the ligand and biomacromolecule solutions, is shown in blue in the figures containing both the CD and UV spectra.

CD spectra deconvolution

CD spectra deconvolution was performed to unravel the BSA secondary structure components influenced by compounds 7 and 8 on serum albumin. To deconvolute the CD spectra, CD (mdeg) and wavelength (nm) data were input into the CD3 program (http://lucianoabriata.altervista.org/jsinscience/cd/cd3.html, accessed August 8, 2024). For protein structure analysis, only data with positive coefficient values were used, and the "fit alpha, beta, coil" option was selected.

Molecular docking

Molecular docking studies were undertaken to reveal subdomain interactions between compounds 7 and 7 and biomolecules. For the docking of BSA with compounds 7 and 8, the protein structure with PDB: 4F5S was used. The ligand structures for compounds 7 and 8 were initially energy minimized by editing them in Molview (http://molview.org, accessed on August 8, 2024) and performing energy minimization. The minimized structures were saved as .mol files, visualized in Discovery Studio (Dassault Systèmes Corporate, Waltham, MA, USA, v.2021), and then saved as .pdb files (Molview → energy minimization → Mol → Pdb). Docking was conducted using the HDOCK server (http://hdock.phys.hust.edu.cn, accessed on August 8, 2024), with compounds 7 and 8 as ligands and the protein as the target (Figure S11). A similar docking procedure was applied using G4 DNA as the target, with the c-Myc Pu22 parallel G4 structure (PDB: 6AU4) as the model. The top-ranked poses for each complex were analyzed to identify specific binding regions within the target, providing insights into the structural changes involved.

Cellular assays

Viability assays were conducted to assess the impact of synthetic non-natural amino acids on NHDFs and human breast adenocarcinoma (MDA-MB). Cells were grown in the presence and absence of the compounds, and viability was assayed to evaluate their biocompatibility. NHDFs were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine serum (FBS), 1 μg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, and 10 μg/mL heparin. MDA-MBs were maintained in DMEM supplemented with 10% FBS and 2 mM L-glutamine. Media and supplements were supplied by Thermo Fisher Scientific (Milano, Italy). Total cell counts and viability were determined by trypan blue exclusion using a LUNA-II automated cell counter (Logos Biosystems, Anyang, South Korea). For the cytotoxicity assay, 4 × 10³ and 5 × 10³ cells were seeded for cancer and normal cells, respectively, in 50 μL medium per well in 96-well flat-bottom microplates and incubated overnight to allow cell adhesion at 37°C in a 5% CO₂ humidified atmosphere. Subsequently, the culture medium was removed, and the cells were incubated with 100 μL of growth medium with different concentration of compounds for 24 h. Compounds 7 and 8 were solubilized in water and ethanol at 8.75 and 4.12 mM, respectively. Cell viability was determined by the crystal violet assay. The amount of dye taken up was quantified with a plate reader (Multiskan Fc 10094, Thermo) at 595 nm. Data were represented as the percentage of proliferating cells versus the control (vehicle-treated cells) and are expressed as means ± SD of two independent experiments performed in triplicate.

Data availability

All of the data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

G.N.R. and H.S. would like to thank Italian National Research Council (CNR) and Armenian Higher Education and Science Committee of the Ministry of Education and Science (MESRA) for their support through their collaboration (the Armenian-Italian bilateral research project 25SC-CNR-1D007, CNR/MESRA (Armenia) scientific cooperation [CNR protocol no. 2605 - 8/01/25]. This work was also supported by the Higher Education and Science Committee of RA in the frame of research project no. 21T-1D057.

Author contributions

G.N.R., A.S., and H.S. conceived, designed, and supervised the study; H.S., S.P., L.S., and G.M. performed the synthesis; R.P., G.N.R., and P.L.S. performed the analysis and data interpretation; G.N.R. wrote the manuscript; and H.S., R.P., and P.L.S. commented on and revised the manuscript.

Declaration of interests

The authors declare no conflicts of interest related to this work.