Dual-Targeting Gold Nanoparticles: Simultaneous Decoration with Ligands for Co-Transporters SGLT-1 and B⁰AT1

Abstract

Sodium–glucose co-transporter 1 (SGLT1) and sodium-dependent neutral amino acid transporter (B⁰AT1) are mainly expressed on the membrane of enterocytes, a type of epithelial cell found in the intestines. In addition to their physiological role in the absorption of nutrients, a protective role in the integrity of the intestinal barrier has been established. The natural ligands of SGLT1 (d-glucose) and of B⁰AT1 (l-glutamine) can trigger a protective anti-inflammatory effect on the intestinal epithelium. The literature suggests the activation of common intracellular pathways upon engagement of the two transporters, whose functional forms are composed of oligomers or clusters. Simultaneous activation of these two co-transporters could lead to a potential multitarget and synergistic anti-inflammatory effect. Therefore, nanoplatforms containing multiple copies of the ligands could represent chemical tools to study the potential simultaneous activation of the two co-transporters. For these reasons, in this study, a set of different gold nanoparticles decorated with derivatives of d-glucose and of l-glutamine were designed and prepared. In particular, the synthesis of suitable sulfur-ending functionalized ligand derivatives, including a C-glucoside derivative, their anchoring to gold nanoparticles and their physical–chemical characterization have been carried out. The obtained nanostructures could represent promising multifunctional platforms for further investigation of the existence of possible multitarget and synergistic effects toward the two co-transporters SGLT1 and B⁰AT1.

1. Introduction

The key role of the intestine in the maintenance of body homeostasis as well as in the regulation and control of the immune response to pathogens is nowadays an unquestionable scientific fact, supported by many relevant studies in the field of immunology and pathophysiology [1,2,3,4,5,6]. Several studies published in the last two decades have demonstrated that d-glucose, administered at high concentrations in vitro to intestinal epithelial cells (IECs) and in vivo in mice (oral administration), has a protective effect against cell/tissue damages caused by pathogens expressing lipopolysaccharides (LPSs) [7,8]. These studies propose that the mechanism of the glucose-mediated cytoprotective effect depends on increased glucose uptake by enterocytes, mediated by sodium–glucose co-transporter 1 (SGLT1). The unidirectional translocation of glucose from the intestinal lumen to the cytoplasm of IECs, mediated by this transmembrane protein, represents one of the principal mechanisms for the absorption of glucose occurring on the brush border membrane in the gut [9,10,11].

More recently, other works have outlined the protective effect of SGLT1 engagement with high oral doses of d-glucose and non-metabolizable 3-O-methyl-d-glucopyranose (3OMG) against damage induced by Toll-like receptor (TLR) ligands in intestinal epithelial cells in a murine model of septic shock and in liver injury and death induced by LPS or by an overdose of acetaminophen [12,13]. Glucose- and 3OMG-induced downregulation of systemic inflammatory cytokine production and enhanced anti-inflammatory cytokine production were observed. However, the substantial quantities of d-glucose and 3OMG required to elicit anti-inflammatory effects pose a significant limitation for potential therapeutic applications. In a previous study, we identified a glucose derivative, C-glucoside 1 (Figure 1), which exerts anti-inflammatory activity at a molar concentration five orders of magnitude lower than that of d-glucose [14]. Furthermore, we observed a significative protective role for C-glucoside 1 in intestinal mucosa in chemotherapy-induced mucositis [15]. Our previous results suggested that C-glucoside 1 mediates a protective activity, similar to that observed for d-glucose, due to its agonistic interaction with SGLT1. This binding produces an activated form of the transporter, which activates an intracellular signaling pathway that ultimately results in protective and anti-inflammatory effects.

On the other hand, l-glutamine (Figure 1) is the most abundant amino acid in the total body free amino acid pool, and recently, its role in nutrition is receiving growing interest. l-glutamine is a major energy source for the intestinal epithelium, and it is involved in the maintenance of intestinal epithelial homeostasis. In fact, l-glutamine represents an important supplement in diets within therapeutic supportive care protocols; its enrichment in parenteral hyperalimentation can decrease villus atrophy associated with exclusive parenteral feeding [16,17,18]. The protective anti-inflammatory effects of glutamine are also well established [19,20]. For example, diet supplementation with glutamine may protect the intestinal tract from radiation and some chemotherapeutic agents. In animal models of gastrointestinal mucositis, oral glutamine treatment mitigates mucosal damage and enhances intestinal recovery after chemotherapy-induced injury [21,22,23,24]. For example, studies have shown that orally administering l-glutamine can prevent gut mucosal injuries caused by cisplatin in rats. Additionally, this drug upregulates l-glutamine transport in human intestinal epithelial cells, potentially leading to increased intracellular levels of glutathione (GSH) [25,26].

Similarly to d-glucose, the absorption of l-glutamine by the intestinal epithelium is mediated by several transport systems, among which sodium–glutamine cotransporter 1 (B⁰AT1) is the main transmembrane protein that mediates the absorption of l-glutamine by villus enterocytes [27]. One of the critical events compromising the biological functions of the intestinal epithelium—such as nutrient absorption and the barrier effect against pathogen invasion—is the disruption of the correct permeability due to alterations in the junctional systems of epithelial cells in response to various forms of damage [28,29]. The fundamental link between SGLT1 activation and l-glutamine uptake is the ability to modulate tight junctions activating intracellular signaling pathways involved in junctional open–close mechanisms and remodeling [30,31,32,33,34]. Thus, the simultaneous activation of both receptors could be of great relevance, as it could lead to a multitarget and synergistic protective effect.

Moreover, both SGLT1 and B⁰AT1 co-transporters have been described to be involved in anti-inflammatory and anti-apoptotic signaling in the repair of plasma membrane integrity and tight junction integrity, thus representing potential molecular targets for a therapeutic approach aimed at the prevention of intestinal barrier damages and/or its recovery [35].

Several reports have suggested that functional SGLT1 is an oligomeric protein resulting from the homodimerization of two identical subunits [36,37,38,39]. The evidence also suggests that the B⁰AT1 transporter is mainly assembled as a cluster [40], and the B⁰AT1 transporter localized in the small intestine displays a low-affinity constant for the amino acid substrate [40]. These findings suggest that the study of multitarget and multivalent effects of the ligand C-glycoside 1 or l-glutamine could lead to a significant increase in the activity.

Multivalent binding is a commonly observed process in biological systems, in particular during cellular recognition events and in signal transduction pathways. Multivalent interactions are significantly stronger than the individual binding of an equal amount of monovalent ligands to a multivalent receptor [41,42]. Receptor dimerization/clustering is a well-known and ubiquitous phenomenon.

Gold nanoparticles (AuNPs) are one of the most used nanoplatforms for the study of multivalency phenomena, owing to their good stability, unique optical features, and ease and versatility in decoration/functionalization with multiple copies of thiol-bearing ligands on the gold surface [43,44,45,46,47,48]. In particular, Glyco-Gold Nanoparticles (GAuNPs), i.e., AuNPs decorated with carbohydrates, are receiving increasing attention thanks to their marked potentialities in biomedicine [49], for the study of carbohydrate-mediated interactions involved in biochemical and physiological processes [50,51], for diagnostics [52,53], for the investigation of multivalency effects of sugars [54,55], including the modulation of enzyme activity [51,56], and as promising platforms for vaccine development [57,58,59]. Furthermore, the simultaneous introduction of different thiol ligands on the same gold nanoplatform paves the way for “multifunctional” nanosystems [55,57].

Therefore, we reasoned that GAuNPs displaying multiple copies of SGLT1 and B⁰AT1 ligands could be suitable nanotools for modulating the activity of these co-receptors. In the present article, AuNPs decorated with thiol-ending d-glucose derivatives (a β-O-alkylglucose derivative and C-glucoside 1) and/or l-glutamine derivatives were prepared and fully characterized. These nanosized tools could be used for future studies aimed at evaluating the multivalent and synergistic effects on the target proteins SGLT1 and B⁰AT1 (Figure 2).

2. Materials and Methods

2.1. General Remarks

All commercial chemicals were purchased from Merck and Alfa Aesar. All chemicals were used without further purification. Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck©, Darmstadt, Germany) with detection under UV light when possible, or by charring with a solution of (NH₄)₆Mo₇O₂₄ (21 g), Ce(SO₄)₂ (1 g), concentrated H₂SO₄ (31 mL) in water (500 mL) or with an ethanol solution of ninhydrin. Flash-column chromatography was performed on silica gel 230–400 mesh (Merck) or on an Isolera Flash Chromatography System (Biotage Sweden AB™, Uppsala, Sweden). ¹H and ¹³C NMR spectra were recorded at 25 °C, unless otherwise stated, with a Varian Mercury 400 MHz instrument (Varian Inc., Palo Alto, CA, USA), on a Bruker DRX-300 spectrometer and on Bruker AvanceTM NEO 400 MHz (Bruker©, Billerica, MA, USA). Chemical shift assignments, reported in parts per million, were referenced to the corresponding solvent peaks. Mass spectra were recorded on an ABSciex 2000 QTRAP LC/MS/MS system with an ESI source (ABSciex©, Framingham, MA, USA). TEM examination was carried out at 200 KeV with a Philips CM200 microscope (Philips©, Amsterdam, The Netherlands). UV spectra were obtained with a UV/vis Perkin-Elmer Lambda 12 spectrophotometer (Perkin-Elmer©, Waltham, MA, USA). Infrared spectroscopy analyses were performed in ATR mode using a Jasco© FT/IR-4600 system ((Jasco Europe©, Cremella, Italy).

2.2. Synthesis of Ligands

2.2.1. Synthesis of C-Glucoside Derivative 2

For compounds 6 and 7, synthetic procedures and characterizations are already reported in [60,61].

2.2.2. 2,3,4-Tri-O-benzyl-6-O-acetyl-α-C-allyl-glucopyranoside 8

A mixture of Ac₂O/trifluoroacetic acid 4:1 (70 mL), prepared at 0 °C in argon atmosphere, was added via a double-tip needle to a round-bottomed flask containing 2 g (3.55 mmol) of 2,3,4,6-tetra-O-benzyl-α-d-glucopyranose 7. The solution was stirred vigorously at 0 °C and the reaction followed by TLC (Hexane/EtOAc 8.5:1.5). After 1.5 h, no more starting compound was present, and the solution was gently poured into ice water and stirred for 10 min. The aqueous solution was extracted with EtOAc (3×), the organic phase was then washed once with a sodium hydrogen carbonate saturated solution and twice with distilled water. The organic phase was dried over sodium sulfate, filtered and concentrated. The resulting crude was purified by flash chromatography (silica gel, eluent: Hexane/EtOAc 9:1), affording compound 8 (1.61 g, 3.12 mmol, 88% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.43–7.24 (m, 15H, CH Ar), 5.79 (ddt, J = 17.2, 10.2, 6.9 Hz, 1H, H2′), 5.18–5.05 (m, 2H, H3′a,b), 4.98 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.89 (d, J = 10.7 Hz, 1H, CH₂Ph), 4.83 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.72 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.65 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.57 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.24 (d, J = 3.5 Hz, 2H, H6a,b), 4.11 (dt, J = 9.3, 5.9 Hz, 1H, H1, H1), 3.85 (t, J = 9.0 Hz, 1H, H3), 3.76 (dd, J = 9.4, 5.8 Hz, 1H, H2), 3.70 (dt, J = 9.8, 3.5 Hz, 1H, H5), 3.48 (dd, J = 9.8, 8.7 Hz, 1H, H4), 2.50 (t, J = 8.2 Hz, 2H, H1′a,b) and 2.04 (s, 3H, CH₃CO).

¹³C NMR (101 MHz, CDCl₃) δ 170.92 (CH₃CO), 138.59 (Cq Ar), 138.15 (Cq Ar), 137.83 (Cq Ar), 134.40 (C2′), 128.80, 128.65, 128.59, 128.58, 128.49, 128.29, 128.11, 128.05, 128.00, 127.95 and 127.85 (C Ar × 15), 117.24 (C3′), 82.37 (C3), 80.04 (C2), 77.89 (C4), 75.65 and 75.21 (CH₂Ph), 73.68 (C1), 73.23 (CH₂Ph), 69.68 (C5), 63.62 (C6), 29.89 (C1′) and 21.00 (CH₃CO).

MS: m/z = 517.6 [M+H]⁺.

2.2.3. 2-(2,3,4-Tri-O-benzyl-6-acetyl-α-D-glucopyranosyl)-ethanal 9

To a solution of 8 (3.53 g, 6.84 mmol) in CH₂Cl₂ (100 mL, 0.07 M) at −78 °C, O₃ was bubbled until a pale blue color appeared. The reaction was followed by TLC (Hexane/EtOAc 8:2). At the end of the reaction, the excess of O₃ was removed by purging the mixture with a stream of argon at −78 °C and then Ph₃P (17.1 mmol, 2.5 eq) was added. The mixture was stirred for 24 h at room temperature and then concentrated by rotary evaporation. The product was purified from the crude reaction by flash chromatography (silica gel, eluent: Hexane/EtOAc 8:2 to 7:3), affording aldehyde 9 (2.92 g, 5.63 mmol, 82% yield).

¹H NMR (400 MHz, CDCl₃) δ 9.72 (s, 1H, H2′ (CHO)), 7.43–7.24 (m, 15H, CH Ar), 4.93 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.87 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.83 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.75–4.66 (m, 2H, CH₂Ph, H1), 4.63 (d, J = 11.5 Hz, 1H, CH₂Ph), 4.58 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.30–4.18 (m, 2H, H6), 3.82–3.74 (m, 2H, H2, H3), 3.74–3.63 (m, 1H, H5), 3.53–3.43 (m, 1H, H4), 2.89 (dd, J = 15.8, 5.0 Hz, 1H, H1′a), 2.74 (ddd, J = 16.2, 8.6, 2.8 Hz, 1H, H1′b) and 2.05 (s, 3H, CH₃CO).

¹³C NMR (101 MHz, CDCl₃) δ 199.49 (C2′ (CHO)), 170.83 (CH₃CO), 138.29, 137.67 and 137.65 (Cq Ar), 128.66, 128.64, 128.61, 128.20, 128.18, 128.14, 127.99 and 127.94 (C Ar), 81.90 (C2), 78.88 (C3), 77.30 (C4), 75.54 (CH₂Ph), 75.07 (CH₂Ph), 73.67 (CH₂Ph), 70.95 (C5), 69.58 (C1), 63.20 (C6), 41.26 (C1′) and 20.95 (CH₃CO).

MS: m/z = 519.6 [M+H]⁺.

2.2.4. 2-(2,3,4-Tri-O-benzyl-6-acetyl-α-D-glucopyranosyl)-ethanol 10

Compound 9 (2800 mg, 5.39 mmol) was dissolved in dry MeOH under argon. The solution was then cooled to 0 °C and NaBH₄ (2.16 mmol) was added in three portions. The reaction was stirred at 0 °C and followed by TLC (Hexane/EtOAc 6:4). After 4 h, glacial acetic acid was added at 0 °C until pH = 3. The reaction was concentrated, and the residue was diluted in dichloromethane, then washed with a HCl 1 N solution (1×) and twice with brine (2×). The organic phase was dried over Na₂SO₄, filtered and evaporated. The crude was purified by flash chromatography (silica gel, eluent: Hexane/EtOAc 5.5:4.5 + 1% MeOH), yielding compound 10 (2672 mg, 5.14 mmol, 95% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.40–7.26 (m, 15H, m, CH Ar), 4.95 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.88 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.81 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.73 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.64 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.59 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.29 (dd, J = 11.7, 2.0 Hz, 1H, H6a), 4.26–4.14 (m, 2H, H1, H2), 3.87–3.75 (m, 4H, H2′a,b, H3, H5), 3.72 (dd, J = 9.2, 5.8 Hz, 1H, H6b), 3.43 (t, J = 9.0 Hz, 1H, H4), 2.44 (s, 1H, OH), 2.10–1.98 (m, 4H, CH₃CO, H1′a) and 1.98–1.86 (m, 1H, H1′b).

¹³C NMR (101 MHz, CDCl₃) δ 170.86 (CH₃CO), 138.41, 137.92 and 137.74 (Cq Ar), 128.59, 128.57, 128.55, 128.13, 128.06, 128.03, 128.00, 127.98 and 127.85 (C Ar), 81.87 (C2), 79.46 (C3), 77.82 (C4), 75.49 (CH₂Ph), 75.00 (CH₂Ph), 73.37 (C5), 73.33 (CH₂Ph), 70.30 (C1), 63.77 (C6), 60.81 (C2′), 27.88 (C1′) and 20.91 (CH₃CO).

MS: m/z = 521.4 [M+H]⁺, 543.5 [M+Na]⁺.

2.2.5. 2-(2,3,4-Tri-O-benzyl-6-acetyl-α-D-glucopyranosyl)-1-azidoethane 11

Alcohol 10 (2598 mg, 5 mmol) was dissolved in dry THF under argon. Ph₃P (15 mmol) was added and the solution cooled to 0 °C. Diisopropyl azodicarboxylate (DIAD, 15 mmol) and, after 10 min, diphenylphosphoryl azide ((PhO)₂PON₃, 16 mmol) were added dropwise; the reaction was then raised and stirred at room temperature. The disappearance of the starting compound was followed by TLC (Hexane/EtOAc 7:3); after 2 h, the crude was concentrated and purified by flash chromatography (silica gel, eluent: Hexane/EtOAc 9:1). A total of 2446 mg (4,48 mmol, 90% yield) of azide 11 was obtained.

¹H NMR (400 MHz, CDCl₃) δ 7.38–7.26 (m, 15H, CH Ar), 4.94 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.86 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.80 (d, J = 10.8 Hz, 1H, CH₂Ph), 4.71 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.62 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.56 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.23 (d, J = 3.4 Hz, 2H, H6a,b), 4.16–4.07 (m, 1H, H1), 3.82–3.70 (m, 2H, H2, H3), 3.68–3.60 (m, 1H, H5), 3.50–3.27 (m, 3H, H4, H2′a,b), 2.05 (s, 3H, CH₃CO) and 1.96 (dd, J = 13.7, 6.5 Hz, 2H, H1′a,b).

¹³C NMR (101 MHz, CDCl₃) δ 170.90 (CH₃CO), 138.45, 137.95 and 137.76 (Cq Ar), 128.66, 128.61, 128.23, 128.13, 128.06 and 127.92 (C Ar), 82.07 (C3), 79.48 (C2), 77.71 (C4), 75.60 (CH₂Ph), 75.17 (CH₂Ph), 73.38 (CH₂Ph), 71.34 (C1), 70.19 (C5), 63.64 (C6), 47.96 (C2′), 24.64 (C1′) and 21.93 (CH₃CO).

MS: m/z 546.5 = [M+H]⁺.

2.2.6. 2-(2,3,4-Tri-O-benzyl-α-D-glucopyranosyl)-1-azidoethane 12

Pure azido derivative 11 (2420 mg, 4.44 mmol) was dissolved in MeOH (25 mL, ≈0.2 M) and 2 mL of a methanolic solution of sodium methoxide (2.2 M) was added. The reaction was stirred at room temperature for 12 h. After the disappearance of the starting compound, checked by TLC (Hexane/EtOAc 7:3), an amount of acidic resin (Amberlite IRA-120 H⁺) was added to the reaction. The resin was filtered off and the solution was evaporated to dryness, affording compound 12 (2116 mg, 4.21 mmol, 95% yield), which did not require further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.41–7.27 (m, 15H, CH Ar), 4.94 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.88 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.82 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.72 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.67–4.60 (m, 2H, CH₂Ph), 4.11 (dd, J = 13.2, 7.2 Hz, 1H, H1), 3.83–3.74 (m, 2H, H6a, H3), 3.74–3.64 (m, 2H, H2, H6b), 3.56–3.45 (m, 2H, H4, H5), 3.45–3.37 (m, 1H, H2′a), 3.37–3.27 (m, 1H, H2′b), 1.98 (dd, J = 13.8, 7.0 Hz, 2H, H1′a,b) and 1.87 (s, 1H, OH).

¹³C NMR (101 MHz, CDCl₃) δ 138.60, 138.05, 138.02 (Cq Ar), 128.65, 128.57, 128.17, 128.09, 128.04, 128.01 and 127.84 (C Ar), 82.11 (C3), 79.69 (C2), 77.85 (C4), 75.55 (CH₂Ph), 75.22 (CH₂Ph), 73.44 (CH₂Ph), 72.14 (C5), 71.38 (C1), 62.34 (C6), 48.04 (C2′) and 24.71 (C1′).

MS: m/z = 504.4 [M+H]⁺, 526.3 [M+Na]⁺.

2.2.7. 2-(2-(2-(Pent-4-en-1-yloxy)ethoxy)ethoxy)ethan-1-ol 13

Compound 13 was prepared according to a procedure described in [62].

A solution of 1006 mL of NaOH 50% (12.6 mmol) was added to 10 g of triethyleneglycol at 100 °C. The reaction was stirred for 30 min at 100 °C and 5-bromo-1-pentene (12.6 mmol) was added and the reaction stirred at the same temperature for 24 h. The reaction was followed by TLC (CH₂Cl₂/MeOH 9:1). After 24 h, the reaction was diluted with water and extracted six times with EtOAc. The organic phases were combined, dried over Na₂SO₄ and evaporated; the crude was purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH 11:1). A total of 2039 g (9.34 mmol, 74% yield) of monopentenylated triethylenglycol 13 was obtained.

¹H NMR (400 MHz, CDCl₃) δ 5.75 (ddt, J = 16.9, 10.1, 6.6 Hz, 1H, H4), 4.96 (dd, J = 17.2, 1.6 Hz, 1H, H5a), 4.90 (d, J = 10.2 Hz, 1H, H5b), 4.08 (t, J = 5.1 Hz, 1H, OH), 3.66 (d, J = 3.7 Hz, 2H, -CH₂CH₂O), 3.63–3.57 (m, 6H, -CH₂CH₂O), 3.57–3.50 (m, 4H, -CH₂CH₂O), 3.42 (t, J = 6.7 Hz, 2H, H1a,b), 2.05 (q, J = 7.3 Hz, 2H, H3a,b) and 1.68–1.57 (m, 2H, H2a,b).

¹³C NMR (101 MHz, CDCl₃) δ 138.15 (C4), 114.84 (C5), 72.73, 70.80, 70.53, 70.44, 70.12 and 69.94 (-CH₂CH₂O), 61.49 (C1), 30.15 (C3) and 28.61 (C2).

MS: m/z = 219.2 [M+H]⁺.

2.2.8. 2-(2-(2-(Pent-4-en-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate 14

To a solution of compound 13 (1.62 g, 7421 mmol) and DMAP (0.148 mmol) in CH₂Cl₂ (40 mL, ≈0.2 M), p-toluenesulphonyl chloride (14.84 mmol) and Et₃N (18.55 mmol) were added. The reaction was stirred at r.t. and followed by TLC (CH₂Cl₂/MeOH 9.5:0.5). After 10 h, TLC indicated the disappearance of the starting material, and 40 mL of water was added. The organic phase was separated and the aqueous phase was extracted thrice with CH₂Cl₂. The combined organic phases were dried with Na₂SO₄ and evaporated. The crude was purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH 9.9:0.1), affording tosylate 14 (1976 g, 5.3 mmol, 72% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.79 (d, J = 8.2 Hz, 2H, CH Ar), 7.33 (d, J = 8.0 Hz, 2H, CH Ar), 5.80 (ddt, J = 16.8, 10.1, 6.6 Hz, 1H, H4), 5.00 (dd, J = 17.1, 1.5 Hz, 1H, H5a), 4.94 (d, J = 9.4 Hz, 1H, H5b), 4.20–4.09 (m, 2H, -CH₂CH₂OTs), 3.72–3.64 (m, 2H, CH₂CH₂OTs), 3.64–3.52 (m, 8H, -CH₂CH₂O-), 3.45 (t, J = 6.7 Hz, 2H, H1a,b), 2.44 (s, 3H, CH₃PhSO₃), 2.10 (q, J = 7.2 Hz, 2H, H3a,b) and 1.72–1.60 (m, 2H, H2a,b).

¹³C NMR (101 MHz, CDCl₃) δ 144.90 (Cq Ar), 138.36 (C4), 133.04 (Cq Ar), 129.92 and 128.08 (C Ar), 114.82 (C5), 70.86, 70.81, 70.74, 70.64, 70.17, 69.36 and 68.77 (CH₂CH₂O-), 30.32 (C3), 28.84 (C2) and 21.77 (CH₃PhSO₃).

MS: m/z = 373.4 [M+H]⁺.

2.2.9. 2-(2,3,4-Tri-O-benzyl-6-(2-(2-(2-(pent-4-en-1-yloxy)ethoxy)ethoxy)ethyl)-α-D-glucopyranosyl)-1-azidoethane 15

Compound 12 (1024 mg, 2036 mmol) was dissolved in dry DMF (20 mL, 0.1 M) under argon and 3054 mmol of NaH (60% dispersion in mineral oil) was added at room temperature; after 10 min, a solution of tosylate 14 (3.9 mmol) in 5 mL of dry DMF was added dropwise. The reaction was stirred at r.t. under argon atmosphere and followed by TLC (Hexane/EtOAc 7:3). After 12 h, the reaction was quenched by adding some drops of MeOH; the mixture was diluted with water and extracted three times with EtOAc. The organic phase was dried over sodium sulfate, filtered and evaporated and the crude purified by flash chromatography (silica gel, eluent Hexane/EtOAc 7:3), affording 1065 g (1.7 mmol, 83% yield) of compound 15.

¹H NMR (400 MHz, CDCl₃) δ 7.35–7.22 (m, 15H, CH Ar), 5.79 (ddt, J = 16.9, 10.1, 6.6 Hz, 1H, H10), 4.99 (dd, J = 17.1, 1.6 Hz, 1H, H11a), 4.93 (d, J = 12.0 Hz, 1H, H11b), 4.90 (d, J = 11.1 Hz, 1H, CH₂Ph), 4.84 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.79 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.69 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.63 (d, J = 11.0 Hz, 1H, CH₂Ph), 4.59 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.15–4.05 (m, 1H, H1), 3.75–3.70 (m, 2H, H2, H3), 3.68–3.54 (m, 13H, H4, -CH₂CH₂O-), 3.54–3.47 (m, 3H, H5, H6a,b), 3.42 (t, J = 6.7 Hz, 2H, H7a,b), 3.39–3.25 (m, 2H, H2′a,b), 2.08 (dd, J = 14.2, 7.3 Hz, 2H, H9a,b), 1.99–1.89 (m, 2H, H1′a,b) and 1.70–1.60 (m, 2H, H8a,b).

¹³C NMR (101 MHz, CDCl₃) δ 138.70 (Cq Ar), 138.39 (C10), 138.34 (Cq Ar), 138.09 (Cq Ar), 128.58, 128.55, 128.50, 128.04, 128.01, 127.98, 127.88 and 127.74 (C Ar), 114.80 (C11), 82.31 (C3), 79.63 (C2), 77.98 (C4), 75.59 (CH₂Ph), 75.16 (CH₂Ph), 73.37 (CH₂Ph), 71.67 (C1), 71.43 (C5), 71.10, 70.78, 70.73, 70.67, 70.59, 70.29 and 70.15 (C6, CH₂CH₂O-), 48.02 (C2′), 30.32 (C9), 28.84 (C8) and 24.47 (C1′).

MS: m/z = 704.7 [M+H]⁺.

2.2.10. 2-(2,3,4-Tri-O-benzyl-6-(1-thioacetylpentyl-(5-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)))-α-D-glucopyranosyl)-1-azidoethane 16

Compound 15 (384 mg, 0.545 mmol) was dissolved in CH₂Cl₂ (5.4 mL, 0.1 M) and the solution transferred into a sealed glass vial; thioacetic acid (0.656 mmol) and DPAP (0.055 mmol) were added and the reaction was stirred at r.t. under UV light exposure at 365 nm (using a UVA lamp of 4 W located 2 cm away from the glass vial). The progress of the reaction was followed by TLC (Hexane/EtOAc 7:3). After 2 h, TLC indicated the disappearance of the starting material; the crude was directly subjected to flash chromatography (silica gel, eluent Hexane/EtOAc 7:3), which afforded pure thioester 16 (357 mg, 0.46 mmol, 85% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.36–7.27 (m, 15H, CH Ar), 4.91 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.85 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.81 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.70 (d, J = 11.6 Hz, 1H, CH₂Ph), 4.67–4.58 (m, 2H, CH₂Ph), 4.16–4.07 (m, 1H, H1), 3.75–3.71 (m, 2H, H2, H3), 3.69–3.47 (m, 16H, H4, H5, H6a,b, CH₂CH₂O-), 3.44–3.27 (m, 4H, H7a,b, H2′a,b), 2.85 (t, J = 7.3 Hz, 2H, H11a,b), 2.31 (s, 3H, CH₃C(O)S), 2.00–1.90 (m, 2H, H1′a,b), 1.62–1.52 (m, 4H, H10a,b, H8a,b) and 1.46–1.34 (m, 2H, H9a,b).

¹³C NMR (101 MHz, CDCl₃) δ 196.10 (CH₃C(O)S), 138.73, 138.37 and 138.12 (Cq Ar), 128.61, 128.58, 128.52, 128.07, 128.03, 128.01, 127.91 and 127.76 (C Ar), 82.34 (C3), 79.67 (C2), 78.02 (C4), 75.61 (CH₂Ph), 75.19 (CH₂Ph), 73.40 (CH₂Ph), 71.71 (C1), 71.46 (C5), 71.22, 71.12, 70.75, 70.70, 70.61, 70.34 and 70.18 (C6 sugar, CH₂CH₂O-), 48.06 (C2′), 30.79 (CH₃C(O)S), 29.49 (C8), 29.23 (C10), 29.16 (C11), 25.49 (C9) and 24.51 (C1′).

MS: m/z = 780.6 [M+H]⁺, 752.6 [M-N₂+H]⁺, 802.6 [M+Na]⁺, 818.6 [M+K]⁺.

2.2.11. 2-(2,3,4-Tri-O-benzyl-6-(1-thio-(5-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)))-α-D-glucopyranosyl)-1-azidoethane (dimer) 17

Pure thioester 16 (38 mg, 0.049 mmol) was dissolved in MeOH (1 mL, 0.05 M) and 1.3 mg (0.024 mmol) of solid sodium methylate was added. The reaction was stirred at r.t. whilst exposed to air to promote the oxidation of the thiol group into a disulfide bond. After 24 h, TLC (Hexane/EtOAc 6.5:3.5) indicated the completion of the reaction. The reaction mixture was concentrated to dryness; ¹H-NMR analysis of the crude sample indicated the complete removal of the acetyl group and the formation of a disulfide bond (triplet signal at 2.65 ppm). The residue was re-suspended in CH₂Cl₂, and the undissolved material was filtered off and discarded; the filtrate was concentrated to dryness and directly used for the next reaction.

¹H NMR (crude sample) (500 MHz, CDCl₃) δ 7.37–7.24 (m, 15H, CH Ar), 4.94–4.90 (m, 1H, CH₂Ph), 4.85 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.81 (d, J = 10.9 Hz, 1H, CH₂Ph), 4.72–4.68 (m, 1H, CH₂Ph), 4.65–4.59 (m, 2H, CH₂Ph), 4.12 (dt, J = 9.8, 5.0 Hz, 1H, H1), 3.73 (dd, J = 8.5, 6.0 Hz, 2H, H2, H3), 3.70–3.49 (m, 16H, CH₂CH₂O-, H4, H5, H6a,b), 3.44–3.29 (m, 4H, H7a,b, H2′a,b), 2.71–2.62 (t, 2H, J = 7.3 Hz, H11a,b), 2.01–1.89 (m, 2H, H1′a,b), 1.68 (dt, J = 15.0, 7.5 Hz, 2H, H10a,b), 1.62–1.52 (m, 2H, H8a,b) and 1.47–1.37 (m, 2H, H9a,b).

2.2.12. 2-(6-(1-Thio-(5-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)))-α-D-glucopyranosyl)-1-aminoethane (dimer) 18

Compound 18 was prepared following a Birch reduction protocol on protected derivative 17, according to a procedure described in [63], with slight modifications. Some drops of liquid ammonia were collected in a two-necked round-bottomed flask cooled to −78 °C and a small piece of sodium (approx. 20–30 mg) was added. The solution turned blue immediately, and after 10 min, a dry THF solution (1,4 mL) of the crude compound 17 was added. The reaction was stirred at −78 °C for 15 min, then powdered NH₄Cl (≈200 mg) was added and the reaction was warmed to r.t. The solvent was evaporated and the crude was subjected to reverse phase C18 chromatography (gradient eluent: H₂O–MeOH); a total of 14 mg (0.032 mmol as monomer, 65% yield over two steps) of 18 was obtained.

¹H NMR (500 MHz, CD₃OD) δ 4.11 (ddd, J = 11.6, 5.1, 2.7 Hz, 1H, H1), 3.79–3.58 (m, 18H, H2, H3, H4, H5, H6a,b, CH₂CH₂O-), 3.51 (t, J = 6.6 Hz, 2H, H7a,b), 3.23–3.18 (m, 1H, H2′a), 3.17–3.10 (m, 1H, H2′b), 2.74–2.67 (m, 2H, H11a,b), 2.15–2.05 (m, 1H, H1′a), 1.99–1.90 (m, 1H, H1′b), 1.76–1.68 (m, 2H, H10a,b), 1.66–1.57 (m, 2H, H8a,b) and 1.51–1.43 (m, 2H, H9a,b).

¹³C NMR (101 MHz, CD₃OD) δ 76.10 (C1), 74.44, 71.87, 71.23, 70.73, 70.63, 70.60, 70.32 and 70.17 (C3, C2, C4, C5, C6, C7, CH₂CH₂O- × 6), 39.63 (C11), 38.06 (C2′), 29.45 (C8), 29.21 (C10), 25.63 (C9) and 24.20 (C1′).

MS: m/z = 882.2 [M+H]⁺.

2.2.13. 5-(N,N-Dimethylamino)-N-(2-(6-(1-thio-(5-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)))-α-D-glucopyranosyl)-naphthalene-1-sulfonamide (dimer) 2

Compound 18 (10 mg, 0.023 mmol as monomer) was dissolved in MeOH; Et₃N (10 µL) and dansyl chloride (16 mg, 0.06 mmol) were added and the reaction was stirred at r.t. The reaction was followed by TLC (EtOAc/MeOH 9:1 and EtOAc/MeOH/H₂O/AcOH 5:5:1:1). After 10 h, the crude was purified by normal phase flash chromatography (silica gel, eluent: EtOAc/MeOH 8:2) and then with reverse phase C18 Cartridge, affording 6 mg (0.009 mmol, 39% yield) of compound 2.

¹H NMR (500 MHz, CD₃OD) δ 8.54 (t, J = 14.1 Hz, 1H, CH Ar), 8.36 (d, J = 8.7 Hz, 1H, CH Ar), 8.21 (dd, J = 7.3, 1.1 Hz, 1H, CH Ar), 7.59 (t, J = 7.7 Hz, 2H, CH Ar), 7.27 (d, J = 7.3 Hz, 1H, CH Ar), 3.86–3.77 (m, 1H, H1), 3.68–3.63 (m, 2H, H6a, CH₂CH₂O-), 3.62–3.55 (m, 10H, CH₂CH₂O-), 3.55–3.51 (m, 2H, H6b, CH₂CH₂O-), 3.49–3.42 (m, 3H, H2, H7a,b), 3.39–3.34 (m, 2H, H3, H5), 3.18–3.11 (m, 1H, H4), 3.00–2.91 (m, 2H, H2′a,b), 2.88 (s, 6H, (CH₃)₂N-), 2.65 (t, J = 7.2 Hz, 2H, H11a,b), 1.77–1.71 (m, 2H, H1′a,b), 1.69–1.62 (m, 2H, H10a,b), 1.60–1.51 (m, 2H, H8a,b) and 1.47–1.39 (m, 4H, H9a,b).

¹³C NMR (101 MHz, CD₃OD) δ 152.4 (CqAr), 135.2 (CqAr), 131.18 (CH Ar), 130.9 (CqAr), 130.32 (CH Ar), 129.12 (CH Ar), 124.39 (CH Ar), 120.60 (CH Ar), 116.43 (CH Ar), 114.2 (CqAr), 75.23, 75.05, 73.52, 72.56 and 72.17 (C1, C2, C3, C4, C5), 72.93, 72.13, 71.92, 71.52, 71.50 and 71.30 (C7, CH₂CH₂O- x 6), 57.46 (C6), 45.85 (CH₃)₂N-), 39.82 (C11), 41.30 (C2′), 29.78 (C8), 29.45 (C10), 26.12 (C1′) and 25.23 (C9).

MS: m/z = 1348.8 [M+H]⁺, 1370.7 [M+Na]⁺.

2.3. Synthesis of L-Glutamine Derivative 4 and 5

2.3.1. 5-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)pent-1-ene 19

To a solution of tosylate 14 (1976 mg, 5.3 mmol) in dry DMF (35 mL), 15.9 mmol of NaN₃ was added. The reaction was stirred at r.t. for 2 h and then at 80 °C for 10 h. The reaction was followed by TLC (Hexane/EtOAc 8:2). At the end of the reaction, the suspension was diluted with water and extracted with EtOAc (3×). The organic phases were combined, dried over sodium sulphate and evaporated. The residue was purified by flash chromatography (silica gel, eluent: Hexane/EtOAc 8:2), affording azide 19 (1012 mg, 4.16 mmol, 78% yield).

¹H NMR (400 MHz, CDCl₃) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H, H4), 5.01 (dd, J = 17.1, 1.6 Hz, 1H, H5a), 4.95 (d, J = 10.2 Hz, 1H, H5b), 3.72–3.61 (m, 8H, -OCH₂CH₂-), 3.61–3.54 (m, 2H, -OCH₂CH₂-), 3.46 (t, J = 6.7 Hz, 2H, H1a,b), 3.38 (t, J = 5.0 Hz, 2H, -OCH₂CH₂N₃), 2.10 (q, J = 7.3 Hz, 2H, H3a,b) and 1.73–1.61 (m, 2H, H2a,b).

¹³C NMR (101 MHz, CDCl₃) δ 138.40 (C4), 114.80 (C5), 70.82, 70.81, 70.77, 70.21 and 70.15 (-OCH₂CH₂-), 50.78 (-OCH₂CH₂N₃), 30.34 (C3) and 28.86 (C2).

MS: m/z = 244.2 [M+H]⁺.

2.3.2. 2-(2-(2-(Pent-4-en-1-yloxy)ethoxy)ethoxy)ethan-1-amine 20

Compound 19 (1153 mg, 4.73 mmol) was dissolved in THF (30 mL) and Ph₃P (5.68 mmol) was added. The reaction was stirred at r.t. for 2 h, then 5.67 mmol of distilled water was added and the reaction was heated to 60 °C and stirred for 30 min, following the disappearance of the starting material observed by TLC (Hexane/EtOAc 8:2 and CH₂Cl₂/MeOH 9:1). The solvent was then evaporated and the crude purified by flash chromatography (silica gel, eluent: CH₂Cl₂/MeOH 9:1 to CH₂Cl₂/MeOH/NH₃ (aq) 7:3:1), affording amine 20 (0.951 g, 4.4 mmol, 93% yield).

¹H NMR (400 MHz, CD₃OD) δ 5.83 (ddt, J = 13.5, 10.1, 6.7 Hz, 1H, H4), 5.02 (dd, J = 17.1, 1.1 Hz, 1H, H5a), 4.95 (d, J = 10.1 Hz, 1H, H5b), 3.69–3.53 (m, 12H, -OCH₂CH₂-), 3.52–3.45 (m, 2H, -OCH₂CH₂NH₂), 2.12 (dd, J = 14.4, 7.2 Hz, 2H, H3a,b) and 1.72–1.58 (m, 2H, H2a,b).

¹³C NMR (101 MHz, CD₃OD) δ 139.44 (C4), 115.23 (C5), 73.67, 71.56, 71.50, 71.26, 71.12 and 70.39 (C1, -OCH₂CH₂-), 41.42 (-OCH₂CH₂NH₂), 31.33 (C3) and 30.03 (C2).

MS: m/z = 218.3 [M+H]⁺.

2.3.3. (9H-Fluoren-9-yl)methyl-(S)-(3,7-dioxo-1,1,1-triphenyl-11,14,17-trioxa-2,8-diazadocos-21-en-6-yl) carbamate 21

Compound 20 (133 mg, 0.612 mmol) was dissolved in dry CH₂Cl₂ (6 mL, 0.1 M) in argon atmosphere. The solution was cooled to 0 °C and Fmoc-L-Gln(Trt)-OH (0.64 mmol), HOBt (0.918 mmol) and EDC (0.918 mmol) were added. The reaction was warmed to r.t. and stirred under argon. After 15 h, TLC (CH₂Cl₂/MeOH 9.5:0.5 and CH₂Cl₂/MeOH/NH₃ (aq) 9:1:0.1) indicated the absence of the starting compound; the reaction was diluted with CH₂Cl₂ and washed with water (3×). The organic phase was collected, dried over sodium sulphate, filtered and evaporated. The crude was purified by flash chromatography on Biotage System (silica gel, gradient CH₂Cl₂ 100%–CH₂Cl₂/MeOH 9.3:0.7). A total of 390 mg (0.48 mmol) of compound 21 was obtained (79% yield).

¹H NMR (500 MHz, CDCl₃) δ 7.80 (d, J = 7.5 Hz, 2H, CH Ar Fmoc), 7.63 (d, J = 7.4 Hz, 2H CH Ar Fmoc), 7.43 (t, J = 7.4 Hz, 2H CH Ar Fmoc), 7.38–7.20 (m, 17H, CH Ar Fmoc, CH Ar Trt), 7.11 (s, 1H, CONHTrt), 6.84 (s, 1H, -NHCH₂CH₂O-), 6.04 (d, J = 6.7 Hz, 1H, CONH Fmoc), 5.83 (ddt, J = 16.9, 10.2, 6.6 Hz, 1H, H15), 5.04 (ddd, J = 17.1, 3.3, 1.5 Hz, 1H, H16a), 5.01–4.96 (m, 1H, H16b), 4.41 (d, J = 7.1 Hz, 2H, NHCOCH₂-Fmoc), 4.24 (t, J = 7.0 Hz, 1H, CH Fmoc), 4.19–4.10 (m, 1H, H4), 3.64–3.51 (m, 10H, H7, H8, H9, H10, H11), 3.49–3.40 (m, 4H, H12a,b, H6a,b), 2.58–2.49 (m, 1H, H2a), 2.49–2.39 (m, 1H, H2b), 2.18–2.07 (m, 3H, H14a,b, H3a), 2.03–1.95 (m, 1H, H3b) and 1.73–1.65 (m, 2H, H13a,b).

¹³C NMR (101 MHz, CDCl₃) δ 171.80 (C5), 171.32 (C1), 156.23 (OCONH Fmoc), 144.48 (Cq Ar Trt), 143.80 (Cq Ar Fmoc), 143.68 (Cq Ar Fmoc), 141.17 (Cq Ar Fmoc), 141.14 (Cq Ar Fmoc), 138.11 (C15), 128.64 (C Ar Trt, C Ar Fmoc), 127.82 (C Ar Trt, C Ar Fmoc), 127.62 (C Ar Trt, C Ar Fmoc), 127.01 (C Ar Trt, C Ar Fmoc), 126.88 (C Ar Trt, C Ar Fmoc), 125.10 (C Ar Fmoc), 119.87 (C Ar Fmoc), 114.73 (C16), 70.56, 70.48, 70.37, 70.34, 70.07 and 69.90 (C7, C8, C9, C10, C11, Cq Trt), 69.30 (C12), 66.76 (NHCOCH₂-Fmoc), 54.06 (C4), 47.06 (CH Fmoc), 39.20 (C6), 33.17 (C2), 30.11 (C14), 29.33 (C3) and 28.59 (C13).

MS: m/z = 810.9 [M+H]⁺.

2.3.4. (S)-2-amino-N1-(2-(2-(2-(pent-4-en-1-yloxy)ethoxy)ethoxy)ethyl)pentanediamide 22

Compound 21 (148 mg, 0.18 mmol) was dissolved in 1.8 mL of CH₂Cl₂ (0.1 M) and 0.4 mL of TFA was added. After 6 h, TLC (CH₂Cl₂/MeOH 9.5:0.5) showed no more starting compound (Trt deprotection) and the reaction was neutralized to pH 7 with NH₃(aq). The solvent was evaporated and the residue was dissolved in DMF (8 mL). Piperidine was added (2 mL) and the reaction was stirred at r.t. After 12 h, TLC showed the formation of a more polar compound (Fmoc removal); the reaction was then concentrated and the residue purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH/NH₃ (aq) 8.5:1.5:0.1). A total of 60 mg (0.17 mmol) of deprotected compound 22 was obtained (96% yield).

¹H NMR (500 MHz, CD₃OD) δ 5.83 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H, H15), 5.02 (ddd, J = 17.1, 3.5, 1.6 Hz, 1H, H16a), 4.95 (ddt, J = 10.2, 2.2, 1.2 Hz, 1H, H16b), 3.67–3.60 (m, 6H, -CH₂CH₂O-), 3.60–3.54 (m, 4H, -CH₂CH₂O-), 3.48 (t, J = 6.5 Hz, 2H, H12a,b), 3.46–3.33 (m, 3H, H4, H6a,b), 2.28 (t, J = 7.8 Hz, 2H, H2a,b), 2.15–2.09 (m, 2H, H14a,b), 1.99–1.90 (m, 1H, H3a), 1.88–1.79 (m, 1H, H3b) and 1.70–1.62 (m, 2H, H13a,b).

¹³C NMR (101 MHz, CD₃OD) δ 173.20 (C5), 172.90 (C1), 138.78 (C15), 115.91 (C16), 70.65, 70.62, 70.38, 70.34, 70.12 and 69.85 (C7, C8, C9, C10, C11), 69.45 (C12), 54.06 (C4), 40.20 (C6), 32.17 (C2), 30.53 (C14), 29.26 (C3) and 28.87 (C13).

MS: m/z = 346.4 [M+H]⁺, 368.4 [M+Na]⁺.

2.3.5. (S)-S-(1,4-Diamino-1,5-dioxo-9,12,15-trioxa-6-azaicosan-20-yl)-ethanethioate 23

Compound 22 (60 mg, 0.174 mmol) was dissolved in MeOH (1.7 mL, 0.1 M). Thioacetic acid (0.23 mmol) and DPAP (0.035 mmol) were added to this solution and the reaction was stirred at r.t. under UV light exposure at 365 nm. After 2 h, TLC (CH₂Cl₂/MeOH/NH₃ (aq) 7:3:0.2) indicated the complete conversion of the starting compound; the reaction was concentrated and the residue was purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH/NH₃ (aq) 8.5:1.5:0.1). A total of 29 mg (0.07 mmol) of thioester 23 was obtained (47% yield).

¹H NMR (500 MHz, CD₃OD) δ 4.32 (dd, J = 8.4, 5.7 Hz, 1H, H4), 3.66–3.60 (m, 6H, -CH₂CH₂O-), 3.60–3.53 (m, 4H, -CH₂CH₂O-), 3.47 (t, J = 6.5 Hz, 2H, H12a,b), 3.40 (t, J = 5.4 Hz, 1H, H6a), 3.36 (t, J = 5.5 Hz, 1H, H6b), 2.87 (t, J = 7.3 Hz, 2H, H16a,b), 2.34–2.26 (m, 5H, CH₃COSCH₂, H2a,b), 2.11–2.01 (m, 1H, H3a), 1.95–1.86 (m, 1H, H3b), 1.63–1.55 (m, 4H, H13a,b, H15a,b) and 1.47–1.39 (m, 2H, H14a,b).

¹³C NMR (101 MHz, CD₃OD) δ 197.53 (CH₃C(O)S-), 173.91 (C5), 173.30 (C1), 72.04, 71.62, 71.56, 71.28, 71.14 and 70.44 (-CH₂CH₂O-), 54.38 (C4), 40.39 (C6), 32.56 (C2), 30.62 (C13), 30.52 (CH₃C(O)S-), 30.17 (C15), 29.77 (C16), 29.10 (C3) and 26.39 (C14).

MS: m/z = 422.6 [M+H]⁺, 444.5 [M+Na]⁺.

2.3.6. (S)-2-Amino-N1-(2-(2-(2-((5-mercaptopentyl)oxy)ethoxy)ethoxy)ethyl)pentanediamide (dimer) 4

Compound 23 (10 mg, 0.0237 mmol) was dissolved in 0.6 mL of MeOH (previously degassed under a stream of argon) and 0.06 mmol of sodium methoxide was added. The reaction was stirred at r.t., and after 24 h, a sample of the crude was analyzed by ¹H-NMR to check the complete thioester deprotection. The spectrum clearly indicated the disappearance of the triplet at 2.9 ppm (-CH₂-SAc) and of the singlet at 2.3 ppm (CH₃C(O)S-). A new triplet at 2.65 ppm was present, indicating the formation of a disulfide bond. The reaction was then acidified with HCl 1 N (final pH = 6) and the solvent evaporated. The crude was directly used for the preparation of the nanoparticles.

¹H NMR (500 MHz, CD₃OD) δ 4.31 (dd, J = 8.6, 5.6 Hz, 1H, H4), 3.68–3.58 (m, 8H, -CH₂CH₂O-), 3.56 (t, J = 5.5 Hz, 2H, -CH₂CH₂O-), 3.49 (t, J = 6.5 Hz, 2H, H12a,b), 3.44–3.33 (m, 2H, H6a,b), 2.70 (t, J = 7.2 Hz, 2H, H16a,b), 2.30 (t, J = 7.6 Hz, 2H, H2a,b), 2.11–2.03 (m, 1H, H3a), 1.96–1.90 (m, 1H, 3b), 1.71 (dt, J = 14.7, 7.3 Hz, 2H, H15a,b), 1.65–1.56 (m, 2H, H13a,b) and 1.54–1.43 (m, 2H, H14a,b).

¹³C NMR (101 MHz, CDCl₃) δ 174.01 (C5), 173.33 (C1), 72.13, 71.50, 71.47, 71.24, 71.08 and 70.55 (-CH₂CH₂O-), 54.49 (C4), 40.33 (C6), 39.54 (C16), 32.55 (C2), 30.26 (C13), 30.00 (C15), 29.01 (C3) and 26.03 (C14).

MS: m/z = 758.1 [M+H]⁺.

2.3.7. Tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-5-oxo-9,12,15-trioxa-6-azaicos-19-enoate 24

Boc-l-glutamic acid 1-tert-butyl ester (149 mg, 0.49 mmol) and compound 20 (0.58 mmol) were dissolved in CH₂Cl₂ (4.9 mL, 0.1 M) under argon and 0.98 mmol of Et₃N was added; the solution was stirred at r.t. for 15 min. The temperature was set to 0 °C and HOBt (0.73 mmol) and DCC (0.73 mmol) were added. The reaction was stirred at r.t. until TLC (CH₂Cl₂/MeOH 9.5:0.5) indicated the disappearance of the starting material. The reaction was recovered as described for compound 22 and the product was purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH 9.8:0.2), affording compound 24 (207 mg, 0.47 mmol, 95% yield).

¹H NMR (400 MHz, CDCl₃) δ 6.44 (s, 1H, -NHCH₂CH₂O-), 5.80 (td, J = 16.9, 6.7 Hz, 1H, H15), 5.26 (d, J = 7.6 Hz, 1H, NHBoc), 5.01 (d, J = 17.2 Hz, 1H, H16a), 4.95 (d, J = 10.2 Hz, 1H, H16b), 4.14 (s, 1H, H2), 3.67–3.52 (m, 10H, -CH₂CH₂O-), 3.51–3.36 (m, 4H, H12a,b, H6a,b), 2.30–2.20 (m, 2H, H4a,b), 2.18–2.00 (m, 3H, H14a,b, H3a), 1.95–1.81 (m, 1H, H3b), 1.74–1.62 (m, 2H, H13a,b) and 1.50–1.37 (m, 18H, (CH₃)₃ -Boc, -tBu).

¹³C NMR (101 MHz, CDCl₃) δ 172.20 (C5), 171.57 (C1), 155.81 (OCONH Boc), 138.25 (C15), 114.87 (C16), 82.14 (Cq OtBu), 79.78 (Cq Boc), 70.77, 70.59, 70.33, 70.12 and 69.87 (C7-C12), 53.71 (C2), 39.37 (C6), 32.70 (C4), 30.27 (C14), 29.14 (C3), 28.78 (C13), and 28.40 and 28.05 ((CH₃)₃ OtBu, Boc).

MS: m/z = 503.4 [M+H]⁺.

2.3.8. (S)-2-Amino-5-oxo-9,12,15-trioxa-6-azaicos-19-enoic acid 25

Protected L-glutamine derivative 24 (246 mg, 0.49 mmol) was dissolved in a mixture of CH₂Cl₂/trifluoroacetic acid (12 mL/3 mL) and stirred at r.t. The reaction was followed by TLC (CH₂Cl₂/MeOH/NH₃ (aq) 7:3:0.3), and after 2 h, the solution was neutralized with NH₃ (aq). The crude was concentrated and the product purified by flash chromatography (silica gel, eluent: CH₂Cl₂/MeOH/NH₃ (aq) 7:3:0.2), yielding 52 mg (0.15 mmol, 30% yield) of compound 25.

¹H NMR (400 MHz, CD₃OD) δ 5.83 (ddt, J = 17.0, 10.1, 6.8 Hz, 1H, H15), 5.07–4.96 (m, 2H, H16a,b), 3.67–3.52 (m, 11H, H2, H7a,b, H-8a,b, H9a,b, H10a,b, H11a,b), 3.49 (t, J = 6.5 Hz, 2H, H12a,b), 3.37 (t, J = 5.3 Hz, 2H, H6a,b), 2.46 (t, J = 7.1 Hz, 2H, H4a,b), 2.20–2.03 (m, 4H, H14a,b, H3a,b) and 1.72–1.62 (m, 2H, H13a,b).

¹³C NMR (101 MHz, CD₃OD) δ 174.87 (C1), 173.28 (C5), 139.43 (C15), 115.24 (C16), 71.57, 71.51, 71.24, 71.08 and 70.47 (C7-C12), 55.10 (C2), 40.47 (C6), 32.91 (C4), 31.34 (C14), 29.98 (C13) and 27.79 (C3).

MS: m/z = 347.3 [M+H]⁺.

2.3.9. (S)-22-Amino-2,19-dioxo-9,12,15-trioxa-3-thia-18-azatricosan-23-oic acid 26

Compound 25 (50 mg, 0.144 mmol) was dissolved in MeOH (1.5 mL, 0.1 M). Thioacetic acid (0.19 mmol) and DPAP (0.03 mmol) were added to this solution and the reaction was stirred at r.t. under UV light exposure at 365 nm. After 2 h, TLC (CH₂Cl₂/MeOH/NH₃ (aq) 7:3:0.2) indicated the complete conversion of the starting compound; the reaction was concentrated and the residue was purified by flash chromatography (silica gel, eluent CH₂Cl₂/MeOH/NH₃ (aq) 7:3:0.2). A total of 32 mg (0.075 mmol) of thioester 26 was obtained (53% yield).

¹H NMR (500 MHz, CD₃OD) δ 3.66–3.60 (m, 7H, -CH₂CH₂O-, H2), 3.60–3.57 (m, 2H, -CH₂CH₂O-), 3.55 (t, J = 5.4 Hz, 2H, H7), 3.48 (t, J = 6.5 Hz, 2H, H12), 3.37 (t, J = 5.5 Hz, 2H, H6), 2.87 (t, J = 7.3 Hz, 2H, H16a,b), 2.45 (t, J = 7.1 Hz, 2H, H4a,b), 2.30 (s, 3H, CH₃C(O)S), 2.18–2.04 (m, 2H, H3a,b), 1.64–1.55 (m, 4H, H15a,b, H13a,b) and 1.48–1.40 (m, 2H, H14a,b).

¹³C NMR (101 MHz, CD₃OD) δ 197.51 (CH₃C(O)S), 190.57 and 175.09 (C1), 173.52 (C5), 72.02, 71.57, 71.52, 71.25, 71.09 and 70.50 (-CH₂CH₂O-), 55.63 (C2), 40.47 (C6), 33.07 (C4), 30.60 (C13), 30.53 (CH₃C(O)S), 30.14 (C15), 29.76 (C16), 27.97 (C3) and 26.36 (C14).

MS: m/z = 423.3 [M+H]⁺.

2.4. General Procedure for the Preparation of Ligand-Loaded Gold Nanoparticles

The thiol-ending ligand (3 eq. overall) was dissolved in MeOH at a final concentration of 12 mM. For nanoparticles decorated with different ligands, a suitable methanolic mixture was prepared, with a total final concentration of the ligands set to 12 mM (3 eq. overall). To this solution, 1 eq of a 25 mM aqueous solution of HAuCl₄ (hydrogen tetrachloroaurate) was added. After the addition, the reaction was stirred for 5 min at room temperature. During this step, Au(I) polymers were formed, and the reaction became turbid, with a pale-yellow color. Then, a 1 M water solution of NaBH₄ (27–33 eq) was added in four portions, ensuring vigorous stirring during the addition. The formation of a black precipitate indicated the generation of Au nanoparticles. The reaction was left for 2 h at 25° C with 180 r.p.m. stirring. The black precipitate (AuNPs) was washed several times with MeOH, with three cycles of vortexing and centrifugation (12,000 rpm, 2 min), removing the supernatant and adding fresh MeOH. In this step, the excess of dissolved ligands, not attached to the NPs, was removed. The washed residue containing AuNPs was diluted in HPLC-grade water and then purified by dialysis (Spectrum™ Spectra/Por™ Float-A-Lyzer™, 10,000 MWCO, 1–5 mL volume). The water of the dialysis bath was replaced with fresh HPLC-grade water thrice per day for three days. The final water dispersion of the functionalized AuNPs was freeze-dried, obtaining a dark-black powder. The prepared AuNPs were stored at 4 °C and then subjected to ¹H NMR, UV–Vis spectroscopy and TEM analysis. The ratio between the thiol ligands attached to the AuNPs was assessed, recording ¹H-NMR spectra of the initial mixture and of the methanolic supernatants after AuNP formation.

2.4.1. Preparation of AuNP1

A solution of 4.97 mg of GlcC₅SH (17.6 µmol) in 1.47 mL of HPLC-grade MeOH, 234.7 µL of a 25 mM aqueous solution of HAuCl4 and 197 µL of a 1 M aqueous solution of NaBH₄ were used to obtain 1.21 mg of AuNP1.

2.4.2. Preparation of AuNP2

A 2.3:1 mixture solution of GlcC₅SH (1.954 mg, 6.92 µmol) and compound 2 (2 mg, 2.97 µmol) was mixed with 132 µL of a 25 mM aqueous solution of HAuCl4 and with 99 µL of a 1 M aqueous solution of NaBH_4, affording 1.12 mg of AuNP2.

2.4.3. Preparation of AuNP3

A 1:1 mixture solution of GlcC₅SH (0.75 mg, 2.642 µmol) and L-glutamine derivative 4 (1 mg, 2.642 µmol) was mixed with 70.5 µL of a 25 mM aqueous solution of HAuCl4 and with 58 µL of a 1 M aqueous solution of NaBH_4, affording 0.49 mg of AuNP3.

2.4.4. Preparation of AuNP4

A 1.4:1:1.6 mixture solution of GlcC₅SH (0.55 mg, 1.947 µmol), L-glutamine derivative 4 (0.526 mg, 1.391 µmol) and compound 2 (1.5 mg, 2.226 µmol) was mixed with 74.18 µL of a 25 mM aqueous solution of HAuCl4 and with 65 µL of a 1 M aqueous solution of NaBH₄, affording 0.23 mg of AuNP4.

3. Results and Discussion

In the first phase of the present work, we designed a set of sulfur-functionalized derivatives of d-glucose, C-glucoside 1 and l-glutamine (Figure 3) in order to have the suitable compounds for the preparation of the corresponding gold nanoparticles (Figure 4) [54,64].

The role of the d-glucose derivative O-(5′-thiopentyl)-β-d-glucopyranoside (compound 3, GlcC₅SH), synthesized following a well-established protocol [65,66], was to improve the water dispersibility of the nanoparticles, since compound 1 showed low water solubility (approximately 0.5 mg/mL). Furthermore, GlcC₅SH was also selected as an “inner component” [51,66]. It should be noted that while ligands 2, 4 and 5 have a long and amphiphilic linker to impart flexibility and assist in the water dispersibility of the final AuNPs, GlcC₅SH has a short and hydrophobic linker to ensure the rigidity of the nanoparticle and allow the above-mentioned ligands to protrude from the gold surface. GlcC₅SH should thus assist the multivalent effect of the other thiol-ending ligands, rather than interfering with their biological effects, since SGLT1-mediated anti-inflammatory effects are observed only with huge amounts of orally administered d-glucose.

Firstly, we designed the synthetic route of the derivatives considering the attachment of a suitable spacer/linker bearing a terminal thiol group. This linker ensured the correct spatial separation between the core of the gold nanoparticle and the “active component” (C-glucoside 1 or l-glutamine), which is necessary for the interaction of the bioactive molecules with the target receptor. The linker that we chose was composed of a tri(ethyleneglycol) (TEG) chain and a five-carbon atom (C₅) spacer. Ethylene glycol polymers are extensively used in the pharmaceutical industry for their low toxicity, excellent solubility in aqueous media, resistance to non-specific protein adsorption and potential limited immunogenic and allergenic effects [67,68]. PEGylation is an important and emerging aspect in the drug delivery field; the conjugation of PEG to drugs has a positive impact on the pharmacokinetic and pharmacodynamic profile of the molecule to which it is conjugated. Moreover, polyethylene glycol (PEG) represents an inert polymer and is not metabolized. From a synthetic point of view, the insertion of a PEG chain on small molecules or drugs as well as biomacromolecules is now a relatively simple chemical transformation owing to numerous differently functionalized and commercially available PEG chains, and thanks to the development, over the last few decades, of many chemical (bio)orthogonal ligation strategies. For example, in a previous work, we developed a synthetic strategy to obtain heterobifunctionalized (poly)ethylene glycol chains for use as chemical tools in the modification of bioactive molecules and bio(macro)molecules [69]. The monofunctionalization of the triethyleneglycol linker with a pentenyl chain allowed for the introduction of a terminal double bond, which was exploited for the insertion of a sulfur-containing group, through a thiol–ene coupling (TEC) reaction [70]. Several synthetic procedures allow for the conversion of a terminal alkene into a thioether linkage, for example, using a Michael addition reaction between a thiol compound and a α,β-unsaturated carbonyl compound or exploiting a radical mechanism, as in the case of AIBN-catalyzed reactions. We decided to rely on a radical photoinitiator, called 2,2-dimethoxy-2-acetophenone (DPAP), which allows the thiol–ene reaction to occur in a faster and simpler fashion. This method permits the use of non-degassed solvents, and tolerates air atmosphere, moisture and even aqueous solvents. The reaction proceeded smoothly at room temperature; only a short (generally 30 min–1 h) UV light exposure at 365 nm was required (Scheme 1).

Concerning the compound 1 derivative, we decided to connect the linker to the hydroxyl group on the C-6 position of the pyranose ring for two main reasons: firstly, several works suggest that the C6 OH group of d-glucose is not crucial for binding to the SGLT1 interaction and translocation sites [71]; secondly, the primary hydroxyl group is the most easily functionalized among the hydroxyl groups of compound 1. Hence, to the best of our knowledge, the presence of a linear chain should not affect the biological activity of the molecule. For l-glutamine ligands, we designed two different derivatives bearing, respectively, the TEG-C5 chain on the nitrogen atom of the amide of lateral chain or connecting it directly to the Cα position of the amino acid, with the formation of a new amide group. We decided to synthesize two l-glutamine derivatives with TEG chains linked on two different positions of the aminoacidic structure, lacking information about the binding mode between L-glutamine and B⁰AT1 (i.e., which groups are involved in the binding). The preparation of both C-glucoside 1 and l-Gln derivatives proceeded to adopt a multistep synthesis, starting from commercially available O-Me-α-d-glucopyranoside and protected l-glutamine compounds, as described in Section 3.1.

Once the target ligand compounds were prepared, the experiment consisted of the preparation, purification and characterization of gold nanoparticles (Figure 4) loaded with compound 2, d-glucose derivative (GlcC₅SH) and l-glutamine derivatives 4 and 5, as described in Section 3.2.

3.1. Preparation of Ligands

Concerning the synthesis of compound 2 (i.e., the C-glucoside 1 bearing a suitable sulfur-ending linker), the following synthetic steps were carried out (see Scheme 2 which also includes the reaction conditions and yields of each step). Commercially available O-Me-α-d-glucopyranoside was subjected to a chemoselective protecting-group-free C-allylation according to the procedure described in [60] (compound 6). After perbenzylation (compound 7), a successive acetolysis reaction, using a mixture of trifluoroacetic acid and acetic anhydride, allowed for the regioselective replacement of the benzyl protecting group on the C-6 position with an acetyl ester (compound 8). The allyl handle was then manipulated by firstly oxidizing it into aldehyde 9, then reducing it to alcohol 10, and finally converting it into azidoethyl derivative 11 through a Mitsunobu-like reaction. The acetyl group was successively removed from the C6-OH position (compound 12), which was in turn alkylated with the electrophilic TEG-C5 chain bearing a p-toluensulphonate function as leaving group (compound 14). The tosylate linker 14 spacer was synthesized starting from triethyleneglycol, then monoalkylated with a 4-pentenyl chain (compound 13) and finally reacted with tosyl chloride. In our first planned synthesis, we subjected compound 15 to BCl₃-promoted benzyl ether deprotection, but we observed the simultaneous cleavage of the polyethyleneglycol chain. Hence, we decided firstly to subject the TEGylated sugar derivative to a radical-mediated reaction. Upon UV exposure at 365 nm and adding DPAP, a radical photoinitiator, the allyl group was converted into thioester 16. Successively, we attempted to use both catalytic hydrogenolysis and Birch conditions for the azide reduction and benzyl group cleavage, but surprisingly, in both cases, complete removal of the thioester group, generating a pentyl-ending chain, occurred. Indeed, the desulfurization of thioester by SET (single electron transfer) processes is described and rationalized in the literature [72]. Therefore, a change in strategy was needed, so we proceeded with deprotecting the thioester group and promoting its transformation into a disulfide bond to obtain a kind of “protected” thiol derivative (compound 17). Birch reduction of this compound led to the desired fully deprotected dimer 18, the two amine functionalities of which were transformed into dansyl-sulfonamides, affording the final dimer 2. This compound was used directly for the generation of the nanoparticles; it is possible to use both thiol-free and disulfide-bearing molecules, since the addition of an excess of NaBH₄ leads to the disulfide bridge reduction.

The synthesis of the l-glutamine derivatives 4 and 5 proceeded with the following steps (Scheme 3). The tosyl group on the C5-TEG chain of compound 14 was converted into the azide functionality (compound 19), which was reduced to amine derivative 20. A condensation between this compound and Fmoc-l-Gln-Trt-OH afforded the TEGylated derivative 21, which was subjected to protecting-group cleavage both in basic and acid conditions, affording the deprotected amino acid derivative 22. The thiol–ene coupling (TEC) with thioacetic acid gave thioester 23, which was then converted into the respective dimer 4 (the results was obtained from NMR analysis). The obtained disulfide was directly used for nanoparticle preparation. The condensation of amine 20 with Boc-l-Glu-O-tBu (Boc-l-glutamic acid 1-tert-butyl ester) gave intermediate 24, which was deprotected by acidic conditions. The successive TEC with thioacetic acid afforded thioester 26. Then, thioester deprotection with the methanolysis conditions used for compound 4 led to a complex mixture of side products, probably due to the competing deprotonation reaction of the α-carbon on the glutamine residue; this process did not occur for the first aminoacidic derivative since the racemizable carbon atom of the glutamine was located on the α position of the newly formed amide, and not on a carboxylic acid group as in 26. Hence, we decided to perform the preparation of the nanoparticles using the l-glutamine derivative 4.

3.2. Preparation of the Au Nanoparticles

After completing the synthesis of the ligands of interest, the preparation of four different gold nanoparticles (AuNP1–AuNP4, Figure 5) was carried out by using the disulfide ligands 2 and 4 and the d-glucose derivative O-(5′-thiopentyl)-β-d-glucopyranoside (GlcC₅SH, compound 3), synthesized according to [65,66].

The preparation of the gold nanoparticles involved a one-pot process, inspired by the methods developed in 1994 by Brust and Schriffin [73], and successively optimized by Penades and coworkers for the generation of GAuNPs [54]. The thiol-ending ligands were dissolved in MeOH, then added with a gold (III) salt, hydrogen tetrachloroaurate (HAuCl₄), to generate a kind of Au(I) polymer on which thiol compounds were grafted. The successive addition of a reducing agent, NaBH₄, provoked the further reduction from Au(I) to Au0, with the generation of the gold nanostructure decorated on its surface with the ligands of interest [64,66]. Experimental details and procedures for the preparation of the gold nanoparticles are described in the Materials and Methods section.

3.3. Nanoparticle Characterization

After dialysis and freeze-drying, the AuNPs were obtained as black powder and could be easily re-dispersed in water without flocculation.

The obtainment of decorated AuNPs with the synthesized ligands was preliminary assessed with infrared spectroscopy. In particular, FT-IR/ATR analyses on solid samples of AuNPs 1–4 were performed (Figure 6). The registered IR spectra on AuNP1–4 showed the presence of several bands associated with the functional groups of the decorating ligands, for example the stretching bands of sugar OH groups around 3300 cm⁻¹, the stretching bands of CH₂ of pentenyl chains at 2900 cm⁻¹, the C-O stretching band of ethylene glycol chains and of sugar rings in the region of 1015 cm⁻¹, as well as bands around 1650 cm⁻¹ (stretching of aromatics) and 1350 cm⁻¹ (OH bending). The IR spectra of AuNP3 and AuNP4 present a narrow band around 830 cm⁻¹ that can be associated with the bending vibrations of the amino group of the glutamine derivative grafted on AuNP 3 and 4.

Morphological characterization of AuNP1–4 was carried out by TEM (transmission electron microscopy) analysis. The measurement of the AuNPs’ size and their distribution was accomplished using ImageJ (version 1.54 bundled 64-bit) and Origin (version Pro 2023 64-bit SR1) software, adopting the procedure and the processing steps described recently by Zhang and Wang [74]. Except for the AuNP4 sample, for which we counted a little bit less than 100 nanoparticles, in AuNP1, 2 and 3, we counted more than 100 nanoparticles. The four analyzed dispersions revealed the presence of nanoparticles ranging between 2 nm and 6 nm in diameter (gold core), with those of 2 nm being the most representative one, as indicated in the diameter distribution histograms of

Table 1. TEM micrographs and size (diameter) distribution of the prepared nanoparticles.

Nanoparticle	TEM Micrograph	Diameter Distribution Histogram
AuNP1
AuNP2
AuNP3
AuNP4

.

In order to further confirm the mean size of the AuNPs, the AuNP2 sample was chosen as a model and was subjected to UV–VIS spectroscopy analysis (Figure 7). The nanoparticles were dissolved in water at two different dilutions (0.5 mg/mL and 0.25 mg/mL), and only at the higher concentration was it possible to notice a weak plasmon absorption band around 520 nm, which is correlated to the presence of few nanoparticles with a mean bigger core diameter.

According to the obtained results, AuNP2 and AuNP3 showed the best features in terms of final mass yield and size distribution. Moreover, AuNP2 and AuNP3 were the most representative model nanoparticles among the prepared ones, since they exposed the C-glucoside derivative 2 (AuNP2) or the L-glutamine derivative 4 (AuNP3), the two active anti-inflammatory ligands of interest of this work, and the D-glucose GlcC₅SH derivative, used for the modulation of the dispersibility of the nanoparticles.

For these reasons, we decided to deepen the physical–chemical characterization of AuNP2-3 by means of ¹H-NMR analysis, which allowed us to obtain more detailed structural information about the loading of the ligands on the AuNPs. The ¹H-NMR experiments were performed by dissolving the powder comprised of AuNP2 and AuNP3 in deuterium oxide; the obtained spectra show broad signals of the ligands, typical for this kind of nanosystem [64,66]. In particular, signals of d-glucose derivative GlcC₅SH, C-glucoside derivative 2 (in AuNP2) and L-glutamine derivative 4 (in AuNP3) grafted onto a nanosized entity are clearly visible in the respective ¹H-NMR spectra (Figure 8A). Before the preparation of the nanoparticles, an ¹H-NMR spectrum of a ligand-containing mixture was recorded to check the ligands’ ratio (Figure 8B); this was compared with the ¹H-NMR spectra recorded for the respective AuNPs, allowing for qualitative determination of the loading of the ligands on the NPs.

4. Conclusions

In this work, we presented the design, synthesis and characterization of gold nanoparticles decorated with different compounds, derivatives of d-glucose, C-glycoside 1 and l-glutamine. The last two represent active ligands of the co-transporters SGLT1 and B⁰AT1, with a protective role against inflammatory agents. The two active compounds were functionalized with a tri(ethylene glycol) chain and a five-carbon spacer with a terminal thiol group to ensure their attachment to the gold nanoparticles, exploiting the affinity between the gold atoms and the thiol groups, and to provide an adequate distance of these molecules from the core of each gold nanoparticle. A glucose derivative, with a pentyl chain bearing a terminal thiol group, was also included to improve the water solubility of the NPs. The obtained AuNPs represent a useful tool to assess the multivalent/synergic activities of the ligands and to unveil the biochemical and physiological mechanisms at the basis of the anti-inflammatory activity correlated with the two co-transporters SGLT1 and B⁰AT1.