Red‐light Emitting Orthogonally Trireactive Gold Nanoclusters for the Synthesis of Multifunctionalized Nanomaterials

Abstract

For the development of highly multifunctionalized nanomaterials, the introduction of functional molecules on gold nanoclusters containing thiols preinstalled with connecting groupsconstitutes a promising approach. However, the uniform introduction of multiple connecting groups while avoiding side reactions is a challenging task. Herein, the synthesis of gold nanoclusters (ca. 1 nm) coated with thiol peptides bearing azido, amino, and aminooxy groups is reported. These nanoclusters emit red‐light before and after the functionalization, enabling application to cell imaging. A detailed structure analysis using transmission electron microscope and X‐ray absorption spectroscopy reveals the formation of Au _n (SR) _m nanoclusters as promising motifs for red‐light emission. The sequential modification of the trireactive nanoclusters with RGD (Arg‐Gly‐Asp) peptides, metal chelators, and anticancer drugs via [3+2] cycloaddition, oximation, and amidation reactions at each functional group furnish red‐light‐emissive nanomaterials exhibiting remarkable toxicity against A549 human lung cancer cells. Integration of the multiligation chemistry and gold nanocluster engineering pave the way toward the development of advanced multifunctional nanomaterials for biological applications.

Gold nanoclusters (ca. 1 nm) coated with thiol peptides containing azido, amino, and aminooxy groups are synthesized. These gold nanoclusters adopted Au _n (SR) _m nanocluster compositions, which are promising for red‐light emission. Sequential modification of gold nanoclusters with RGD peptides, metal chelators, and anticancer drugs via [3+2] cycloaddition, oximation, and amidation yielded red‐light‐emissive nanomaterials with anti‐cancer activity.

1. Introduction

Small gold nanoclusters with a size of ≈1–2 nm exhibit high chemical and photostability, moderate excretion from the body due to their small size, and low toxicity, which render them suitable for applications in cell imaging, drug delivery, and medical diagnosis.^{[ 1} , ² , ³ , ^{4 ]} Recently, there has been a growing interest in nanomaterials with multiple functionalities from a multimodality perspective, which has prompted extensive research on the development of efficient synthetic methods for the even incorporation of multiple functional molecules onto nanomaterials.^{[ 5} , ⁶ , ^{7 ]}

The reduction of a gold source such as HAuCl₄ in the presence of thiol ligands is one of the most widely used methods for the synthesis of small gold nanoclusters.^{[ 8 ]} To avoid side reactions elicited by the strong Lewis acidity of HAuCl₄ and the reductant, thiols preinstalled with connecting groups that tolerate these conditions are introduced onto gold nanoclusters, enabling the subsequent introduction of functional molecules (Figure 1A).^{[ 9} , ¹⁰ , ^{11 ]} Although multireactive gold nanoclusters have been prepared employing multiple types of thiols with a connecting group, a mixture of inseparable disproportionately functionalized nanoclusters is generally obtained (Figure 1B).

Figure 1.

A) Introduction of functional molecules on gold nanoclusters via modification of connecting groups. B) Random introduction of different types of connecting groups on gold nanoclusters. C) Our previous work: synthesis of bisreactive gold nanoclusters and their difunctionalization.^{[ 12 ]} D) This work: synthesis of red‐light‐emitting trireactive gold nanoclusters and their trifunctionalization.

To achieve a uniform introduction of multiple connecting groups onto gold nanoclusters, we previously developed a novel method based on click chemistry (Figure 1C). Using sodium phenoxide as the reductant of HAuCl₄ and a thiol peptide bearing azido and amino groups, we synthesized bisreactive gold nanoclusters (2.6 nm).^{[ 12 ]} This method effectively suppressed the undesired reduction of the azido group to an amino group, enabling the uniform introduction of orthogonally functionalizable azido and amino groups on the surface of gold nanoclusters. In this study, as part of our ongoing research on the development of highly multifunctionalized nanomaterials, we synthesized trireactive gold nanoclusters employing a thiol unit with an aminooxy group as the third type of connecting group in addition to azido and amino groups (Figure 1D). The sequential reaction at each connecting group enabled the efficient synthesis of trifunctionalized gold nanoclusters that were smaller in size (ca. 1 nm) and exhibited red‐light‐emission property, which was not observed in our previously developed bisreactive gold nanoclusters (2.6 nm), rendering them suitable for cell imaging. Furthermore, the trifunctionalized gold nanoclusters demonstrated anticancer activity.

2. Results and Discussion

Aminooxy groups are known to react bioorthogonally with carbonyl compounds such as aldehydes to form oximes^{[ 13} , ^{14 ]} in the presence of azido and amino groups.^{[ 15 ]} Therefore, we selected the aminooxy group as the third type of connecting group in addition to the amino and azido groups, and designed the thiol‐containing peptide 1 as the ligand of gold nanoclusters (Scheme 1 ). After preparing precursor 2 using a solid‐phase synthesis method, cleavage from the resin and global deprotection were conducted via treatment with trifluoroacetic acid and water, affording 1 in 39% yield. In this step, using dithiothreitol as a trityl group trapping agent effectively avoided the reduction of the azido group.^{[ 16 ]} Using sodium phenoxide as a reductant, trifunctionalizable gold nanoclusters AuNc1 were synthesized from 1.^{[ 12 ]} The presence of azido groups in AuNc1 was confirmed via infrared (IR) absorption spectroscopy; the characteristic stretching vibration band of the azido group was observed at 2113 cm⁻¹ in the corresponding spectrum (Figure S1A, Supporting Information).

Scheme 1.

Synthesis of thiol unit 1 and trireactive gold nanoclusters AuNc1.

Transmission electron microscopy (TEM) measurements revealed that AuNc1 was ≈1.0 ± 0.3 nm in diameter (Figure 2A), suggesting the presence of diverse gold nanoclusters with sizes between Au₁₅ and Au₁₄₄, with Au₂₅ likely being the median.^{[ 17 ] 1}H NMR and inductively coupled plasma atomic emission spectroscopy analyses indicated that AuNc1 contained ≈645 nmol mg⁻¹ of ligand 1, corresponding to the average number of 6.9 ligands per AuNc1 particle (Figure S2 and Table S1, Supporting Information). This number of ligands was smaller than that of gold nanoclusters protected with glutathione (MW = 307.3), which contain 10–39 ligands,^{[ 18} , ^{19 ]} probably due to the relative bulkiness of 1 (MW = 665.8). Interestingly, AuNc1 emitted red‐light (λ_em = 630 nm), while a featureless absorption band was observed ≈300–500 nm (Figure 2B). AuNc1 could be excited by 488 nm light, rendering it suitable for cell imaging (vide infra, quantum yield (QY) = 0.80%). When the reaction time for the synthesis was extended from 24 to 48 h, nonemissive AuNcS1 with a larger metallic core of 2.4 ± 0.6 nm and ≈333 Au atoms^{[ 17 ]} was obtained, according to the TEM analysis (Scheme S1 and Figure S3, Supporting Information). These characteristics are in good agreement with those of our previously reported bisreactive gold nanoclusters, indicating that a shorter reaction time is conducive to obtain smaller AuNc1 clusters with emission property.^{[ 12 ]}

Figure 2.

A) TEM image and particle size distribution of AuNc1. B) UV–vis absorption, emission, and excitation spectra of AuNc1 (0.1 mg mL⁻¹) in sodium phosphate buffer (pH 7.4). The optical light path for the UV–vis measurments was 1 cm. Excitation wavelength of the emission spectrum was 405 nm. The emission wavelength for the excitation spectrum was 630 nm.

Au _n (SR) _m clusters protected with glutathione (AuNcS2) are known to exhibit similar light‐emission properties and UV–vis absorption features to AuNc1, suggesting a Au _n (SR) _m composition for AuNc1 (Figure S4, Supporting Information).^{[ 20} , ^{21 ]} To gain more insight into the composition of AuNc1, X‐ray absorption spectroscopy (XAS) measurements were conducted. Au L₃‐edge XAS experiments were performed for AuNc1 and AuNcS2 ^{[ 20 ]} in water at room temperature, and the corresponding X‐ray absorption near edge structure (XANES) spectra were obtained (Figure 3A). The peak intensity at the white line (11924 eV), which corresponds to the electric dipole transition from 2p_3/2 to 5d orbitals, decreased compared with that of Au(OH)₃, and the XANES spectrum resembled that of Au foil after treatment with sodium phenoxide, indicating the clean formation of low‐valent Au species in AuNc1. The white line intensity of AuNc1 was slightly higher than that of Au foil, and a featureless XANES post‐edge region was observed (Figure S5, Supporting Information), which is in good agreement with the reported XANES features of Au _n (SR) _m nanoclusters.^{[ 22 ]} We further analyzed the extended X‐ray absorption fine structure (EXAFS) of AuNc1. The Fourier transform of the EXAFS oscillation of AuNc1 showed an intense peak at the radial structure function of 2.0 Å, which can be assigned to Au–S scattering (Figure 3B). A fitting analysis for AuNc1 confirmed the presence of Au–Au scattering in addition to the Au–S scattering with interatomic distances of 2.90 Å (Au–Au) and 2.32 Å (Au–S), respectively (Table S2 and Figure S6, Supporting Information). This result also suggested the formation of Au _n (SR) _m nanoclusters, which is consistent with the UV–vis absorption spectroscopy and XANES results. In addition, the coordination number (CN) of Au–S and Au–Au was determined to be 2.1 and 0.7, respectively. On the basis of an EXAFS analysis of Au₂₂(SR)₁₈ nanoclusters having red‐light‐emission property, Zhang, Jian, and Xie et al. suggested a Au₈ core protected by two trimeric and two tetrameric staple motifs as a reasonable structure.^{[ 23 ]} Given the reported CN values of Au₂₂(SR)₁₈ (CN_Au–S = 1.7 and CN_Au–Au = 0.7), we propose a similar structure consisting of a small Au(0) core with long staple motifs for AuNc1, which is likely responsible for the red‐light‐emission properties. They also reported that oligomeric Au(I)–thiolate complexes exhibit emission in ethanol‐rich solvent systems.^{[ 20 ]} These oligomeric Au(I)–thiolate complexes display characteristic absorption bands at 330 and 375 nm. In contrast, AuNc1 did not show such absorption bands, thereby ruling out the possibility of these oligomeric structures (Figure 2B).

Figure 3.

Au L₃‐edge XAS analysis. A) XANES spectra. B) k ³‐weighted FT‐EXAFS. AuNc1 (red line), AuNcS2 (blue line), Au foil (black line), and Au(OH)₃ (black dotted line) are shown.

Next, we explored the reactivity of the azido groups in AuNc1. Almost all azido groups of AuNc1 reacted with N‐propargyltrifluoroacetamide (3) via a copper‐catalyzed [3+2] azide–alkyne cycloaddition (CuAAC) reaction (Scheme 2 ),^{[ 24} , ²⁵ , ^{26 ]} as was evidenced by the disappearance of the azido signal in the IR spectrum of AuNc2 (Figure S1B, Supporting Information). ¹⁹F NMR measurements indicated that AuNc2 contained an average of 7.3 CF₃ groups per particle, and the azido groups were converted with 96% efficiency (Figure S7 and Table S1, Supporting Information). The amount of CF₃ groups (7.3) in AuNc2 introduced by the click reaction of azido groups in AuNc1 and the amount of thiol ligands (6.9) in AuNc1 were nearly equal indicating that little or no reduction of azido groups proceeded during AuNc1 synthesis (Scheme 1; Table S1, Supporting Information). Similarly, the azido groups of AuNc1 were almost entirely consumed by reacting with alkyne 4 bearing a cyclic RGD (Arg‐Gly‐Asp) peptide motif^{[ 27} , ^{28 ]} to afford AuNc3 (Scheme 2; Figure S1C, Supporting Information). These results indicate the high reactivity of AuNc1 in the click reaction. Almost no change in emission QY was observed in AuNc3 modified via click reaction (QY = 0.84%).

Scheme 2.

Derivatization of AuNc1 by CuAAC.

To prepare nanoclusters with high emission efficiency, we conducted oximation reactions between the aminooxy groups on the gold nanoclusters and rhodin g7 (5), a chlorin derivative containing an aldehyde group (Scheme 3 ). AuNc1 and AuNc3 were conjugated with rhodin g7 to yield AuNc4 and AuNc5, respectively (Scheme 3). UV–vis absorption measurments quantified that AuNc4 and AuNc5 contained average 6.7 and 6.2 rhodin g7 molecules per particle, respectively (Figure S8, Supporting Information). These values are close to the average ligand number of AuNc1 (6.9), suggesting that the oximation reaction proceeded with high efficiency. Both AuNc4 and AuNc5 exhibited red‐fluorescence from rhodin g7 (Figure 4 ; Figure S8, Supporting Information), making these nanoclusters suitable for cell experiments (vide infra).

Scheme 3.

Derivatization of AuNc1 and AuNc3 by oximation reactions.

Figure 4.

UV–vis absorption, emission, and excitation spectra of AuNc4 A) and AuNc5 B) in sodium phosphate buffer (pH 7.4). The optical light path for the UV–vis measurments was 1 cm. Excitation wavelength of the emission spectrum was 426 nm. The emission wavelength for the excitation spectrum was 700 nm.

We then investigated whether these red‐light‐emitting gold nanoclusters could be applied to cell imaging. Human lung cancer A549 cells and human lung normal fibroblasts WI‐38 cells were treated with AuNc1 and AuNc3, respectively, and excited with 488 nm laser under confocal microscopy (Figure 5 ). Clear cell uptake was observed in A549 cancer cells (Figure 5A), while minimal uptakes of both AuNc1 and AuNc3 were detected in WI‐38 cells (Figure 5B). These findings suggest that these gold nanoclusters are potentially applicable for targeted cell imaging, although their low emission efficiency is a limitation. To further quantify the cell uptake of the gold nanoclusters and minimize the impact of cellular autofluorescence (Figure S9, Supporting Information), flow‐cytometry analysis was performed using AuNc4 and AuNc5 conjugated with rhodin g7 (Figure 6 ). Both AuNc4 and AuNc5 were internalized by A549 cells, regardless of the presence of the cyclic RGD motif (Figure 6A). Moreover, the addition of cyclic RGD peptide 4 did not significantly inhibit the uptake of AuNc5, indicating that these gold nanoclusters possess an inherent ability to internalize into A549 cells. In fact, cell uptake of gold nanoclusters in the relavent‐size range has been reported in the literature, which is in consistence with the present results.^{[ 26 ]} Slight uptakes of AuNc4 and AuNc5 by WI‐38 normal cells were also observed, suggesting a degree of selectivity toward cancer cells (Figure 6B).

Figure 5.

Confocal microscopy images of A549 A) and WI‐38 B) cells incubated with AuNc1 (50 µg mL⁻¹) or AuNc3 (50 µg mL⁻¹) for 24 h. Blank indicates cells untreated with nanoclusters. Gold nanoclusters were excited at 488 nm with aligned laser intensity. Red‐light‐emissions were observed at 570–620 and 663–738 nm, respectively. White scale bars indicate 50 µm.

Figure 6.

Flow‐cytometry histograms of A549 A) and WI‐38 B) cells pretreated with AuNc4 (20 µg mL⁻¹) or AuNc5 (20 µg mL⁻¹) for 24 h. Blank indicates cells untreated with nanoclusters. Cyclic RGD peptide 4 (10 µm) was added for blocking experiments. Gold nanoclusters were excited with a 638 nm laser, and fluorescence was detected at 663–677 nm. Each histogram was generated from 10⁴ cell counts.

To showcase the usefulness of the trireactive gold nanoclusters for developing multifunctional nanomaterials, we subjected AuNc1 to multiple modifications. Valine (Val)–citrulline (Cit)–monomethyl auristatin E (MMAE) is used as the payload of antibody–drug conjugates (ADCs) by releasing the anticancer drug MMAE through caspase‐mediated hydrolysis of the Val–Cit sequence in cancer cells.^{[ 29 ]} However, Val–Cit–MMAE exhibits high hydrophobicity, which impairs the medicinal effect of ADCs.^{[ 30 ]} In fact, when we introduced this unit onto our previously reported bisreactive gold nanoclusters, insoluble precipitates formed, likely due to the hydrophobicity of the MMAE‐containing moiety (Scheme S2, Supporting Information). To address this issue, we used the present trireactive gold nanoclusters for introducing a 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) derivative with a hydrophilic tetracarboxylic acid moiety in advance (Scheme 4 ). Thus, oximation of AuNc3 bearing a cyclic RGD peptide was conducted via the reaction with DOTA‐aldehyde 6, yielding AuNc6. Subsequently, the reaction of AuNc6 with the N‐hydroxysuccinimide ester derivative of Val–Cit–MMAE 7 afforded AuNc7 (Scheme 4), which retained the red‐light emission properties (λ_em = 620 nm, Figure S4, Supporting Information). A TEM analysis revealed that the cluster sizes of AuNc6 and AuNc7 were 1.2 ± 0.3 and 1.1 ± 0.2 nm, respectively, which were similar to that of AuNc1 (Figure S10E,F, Supporting Information). Fortunately, the preintroduction of the DOTA unit conferred water solubility to AuNc7. These results illustrate the synthetic utility of trireactive AuNc1 for developing multifunctional nanomaterials.

Scheme 4.

Further derivatization of AuNc3 with DOTA and MMAE units.

Finally, the toxicity of the gold nanoclusters toward cancerous A549 cells was evaluated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazol‐3‐ium bromide (MTT) assay. AuNc7 bearing MMAE units exhibited remarkable anticancer activity in a concentration‐dependent manner, whereas AuNc1 and AuNc3 lacking MMAE units showed negligible toxicity (Figure 7A). These results indicate that our trireactive gold nanoclusters served as useful platforms for the development of bioactive multifunctional nanomaterials. Unfortunately, AuNc7 also exhibited substantial toxicity toward normal WI‐38 cells, likely attributable to the inherent property of gold nanoclusters being internalized by the cells (Figure 7B). Employing more specific targeting molecules such as antibodies and peptides, which enhace selectivity to cancer cells, is expected to realize the clinical applications of these nanomaterials.

Figure 7.

Cytotoxicity of gold nanoclusters AuNc7 (red circle), AuNc1 (green rhombus), and AuNc3 (blue triangle) on A549 A) and WI‐38 cells B). The average and standard deviation of at least five independent experiments are shown.

3. Conclusion

In summary, we synthesized trireactive gold nanoclusters (AuNc1) bearing azido, amino, and aminooxy groups as orthogonally reactive connecting groups. A structural analysis via TEM, NMR, and XAS revealed that AuNc1 were ca. 1 nm in size. These clusters possess molecular‐like properties with red‐light‐emission, rendering them potentially applicable for cell imaging. Furthermore, AuNc1 could be easily multifunctionalized into bioactive nanomaterials via click, oximation, and amidation reactions. The DOTA unit was introduced to improve the water solubility of the material modified with a hydrophobic drug. Moreover, AuNc1 hold potential for applications in theranostics, such as targeted imaging and α therapy, by using a suitable nuclide. Beyond the life sciences, the gold nanoclusters developed in this study hold promise for diverse applications as novel nanomaterials in environmental, energy, and other fields.

Conflict of Interest

The authors declare no conflict of interest.

Supporting information

Supporting Information

Watanabe K., Uetake Y., Hata M., Kuwano A., Yamamoto R., Yamamoto Y., Kodera M., Kitagishi H., Niwa T., Hosoya T., Red‐light Emitting Orthogonally Trireactive Gold Nanoclusters for the Synthesis of Multifunctionalized Nanomaterials. Small 2025, 21, 2408747. 10.1002/smll.202408747

Data Availability Statement

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