Controlled Synthesis of Au25 Superatom Using a Dendrimer Template

Superatoms are promising materials for their potential in elemental substitution and as new building blocks. Thus far, various synthesis methods of thiol-protected Au clusters including an Au25 superatom have been investigated. However, previously reported methods were mainly depending on the thermodynamic stability of the aimed clusters. In this report, a synthesis method for thiol-protected Au clusters using a dendrimers template is proposed. In this method, the number of Au atoms was controlled by the stepwise complexation feature of a phenylazomethine dendrimer. Therefore, synthesis speed was increased compared with the case without the dendrimer template. Hybridization for the Au25 superatoms was also achieved using the complexation control of metals.


Introduction
Some clusters consisting of a few to several dozen metal atoms can provide discrete atom-like electron states according to DFT calculations with the Jellium model [1,2]. Metal clusters with this property, called "superatoms," are attracting attention. Among them, a halogen superatom of Al 13 − [3] and an alkali-metal superatom of Cu 8 − /Ag 8 − [4] are wellknown examples. In the latter case, electronegativity lower than the alkali metal elements (Li, Na, K, Rb, or Cs) is proposed, demonstrating a super-alkali superatom. Therefore, the development of synthesis methods for such superatoms has been a significant target of recent research.
Among these superatoms, Au clusters have been focused on as useful candidates for superatoms that can be synthesized not only in the gas phase but also in the solution phase [5,6]. Since the synthesis of thiol-protected Au clusters was reported by Brust and Schiffrin et al. [5], lots of research  has investigated them. Especially, Au 25 SR 18 [30,31], Au 38 SR 24 [6,32,33] and Au 144 SR 60 [6,34,35] (R: hydrocarbons) are studied as superatomic clusters. They were purified and characterized by single crystal analysis [30,31]. In addition, the introduction of different elements, such as Pd, Pt, Cu, Ag, Cd, and Hg [23,24,27,28,[36][37][38][39] into Au clusters has been reported. The mixing has been carried out by metal-exchanging. However, the synthesis of these materials often requires low-temperature conditions [30,40] or an etching reaction for several days [41] to increase the synthetic yields.

Controlled Assembly of AuCl 3 on TPM G4
The TPM G4 has tetraphenylmethane in the core and a fourth-generation imine skeleton in the branches, with higher electron density in the inner layers ( Figure S1). In order to use the TPM G4 as a precise template, metal salts and the imine parts of the TPM G4 are required to be connected by a 1:1 ratio. Based on the previous reports [65][66][67][68][69], the mixed solution of chloroform:acetonitrile = 1:1 was used as a solvent for the metal assembly. The stepwise coordination fashion between the dendrimer and AuCl 3 was confirmed by UV-Vis titration ( Figure S2). Isosbestic points, according to the number of imines at each layer of the TPM G4, were observed during the addition of the acetonitrile solution of AuCl 3 . The result indicates its 1:1 complexation. The observed isosbestic points at 336.5 nm (0~4 equiv.), 344.0 nm (4~12 equiv.), 332.0 nm (12~28 equiv.) and 328.0 nm (28~60 equiv.) indicate the stepwise coordination of the inner layer imines as shown in the Figure S2. These results indicate that the TPM G4 can be used as an accurate template for Au clusters.
The Au 25 PET 18 superatom (PET: 2-phenylethanethiol: SCH 2 CH 2 Ph) was synthesized by adding thiol-ligand solution at the same time as the reduction of AuCl 3 in the TPM G4 ( Figure 1). Though this method is similar to our previously reported methods [62,67,70,72], it is different due to the addition of the thiol ligands to protect the surface of Au clusters. In addition, the solvent of NaBH 4 was changed from methanol to ice-cold water. When NaBH 4 /methanol solution was used, Au 25 PET 18 was not obtained. After the reactions, the solution was stirred for 3 h. Then the solvent was removed using an evaporator after filtration. The obtained dark-brown solid was purified by washing with a water/methanol mixture, and the Au clusters were extracted with acetonitrile. The obtained solution was subjected to MALDI-TOF-MS measurement in linear mode ( Figure 2a). As a result, the peak corresponding to Au 25 PET 18 (m/z = 7390.73) was detected with the fragment peak of [Au 21 PET 14 ] + (m/z = 6057.25) [73]. The calculated exact mass [74] of Au 25 PET 18 (m/z = 7391.93) confirms the formation of the aimed superatom. UV-vis spectra of the synthesized Au 25 PET 18 ( Figure 2b) showed broad absorption from 800 nm to 600 nm and 450 nm indicating the formation of Au 25 PET 18 [40,[75][76][77]. The expected peak around 400 nm was unclear. This may be due to the overlap of the dendrimer absorption peaks with the peak top around 320 nm.
Molecules 2022, 27, x FOR PEER REVIEW 2 of the most famous dendrimers is the polyamidoamine (PAMAM) dendrimer, which is lim ited in accuracy for metal assembly due to its random complexation fashion. In contras phenylazomethine dendrimers enable atomicity control for cluster synthesis [59][60][61][62][63][64][65][66][67][68][69] Some clusters prepared by this method are found to exhibit high catalytic activities d pending on the number of atoms. In addition, the specific high activity of a Pt17 catalyst also reported [70]. Superatom synthesis using this phenylazomethine dendrimer has als been reported. The synthesis and properties of Al13 − [71] and Ga13 − [72] in the dendrim were also demonstrated. In this study, we investigated dendrimer-templating synthes for the thiol-protected Au25 superatom. As the template, a phenylazomethine dendrim with a tetraphenyl methane core part (TPM G4) was used. Due to the metal-complexatio feature of the TPM G4, one atom blending was also demonstrated.

Controlled Assembly of AuCl3 on TPM G4
The TPM G4 has tetraphenylmethane in the core and a fourth-generation imine ske eton in the branches, with higher electron density in the inner layers ( Figure S1). In ord to use the TPM G4 as a precise template, metal salts and the imine parts of the TPM G are required to be connected by a 1:1 ratio. Based on the previous reports [65][66][67][68][69], th mixed solution of chloroform:acetonitrile = 1:1 was used as a solvent for the metal assem bly. The stepwise coordination fashion between the dendrimer and AuCl3 was confirme by UV-Vis titration ( Figure S2). Isosbestic points, according to the number of imines each layer of the TPM G4, were observed during the addition of the acetonitrile solutio of AuCl3. The result indicates its 1:1 complexation. The observed isosbestic points at 336 nm (0~4 equiv.), 344.0 nm (4~12 equiv.), 332.0 nm (12~28 equiv.) and 328.0 nm (28~6 equiv.) indicate the stepwise coordination of the inner layer imines as shown in the Figu S2. These results indicate that the TPM G4 can be used as an accurate template for A clusters.
The Au25PET18 superatom (PET: 2-phenylethanethiol: SCH2CH2Ph) was synthesize by adding thiol-ligand solution at the same time as the reduction of AuCl3 in the TPM G ( Figure 1). Though this method is similar to our previously reported method [62,67,70,72], it is different due to the addition of the thiol ligands to protect the surface Au clusters. In addition, the solvent of NaBH4 was changed from methanol to ice-co water. When NaBH4/methanol solution was used, Au25PET18 was not obtained. After th reactions, the solution was stirred for 3 h. Then the solvent was removed using an evap rator after filtration. The obtained dark-brown solid was purified by washing with a w ter/methanol mixture, and the Au clusters were extracted with acetonitrile. The obtaine solution was subjected to MALDI-TOF-MS measurement in linear mode ( Figure 2a). As result, the peak corresponding to Au25PET18 (m/z = 7390.73) was detected with the fra ment peak of [Au21PET14] + (m/z = 6057.25) [73]. The calculated exact mass [74] of Au25PET (m/z = 7391.93) confirms the formation of the aimed superatom. UV-vis spectra of the syn thesized Au25PET18 (Figure 2b) showed broad absorption from 800 nm to 600 nm and 45 nm indicating the formation of Au25PET18 [40,[75][76][77]. The expected peak around 400 n was unclear. This may be due to the overlap of the dendrimer absorption peaks with th peak top around 320 nm.   Size distribution and elemental analysis of the synthesized clusters were performed by STEM/EDS measurements (Figures 2c and S3). The estimated size was 1.2 ± 0.3 nm, close to the longest interatomic distance of 11.99 Å observed by single-crystal analysis reported in the previous paper [31]. EDS mapping also indicates that Au and S are present in the same particle. XPS measurement was conducted with tetraoctylammonium bromide (TOAB) which was added to the solution before casting on highly oriented pyrolytic graphene (HOPG) ( Figure S4). The observed peaks of Au 4f, which show higher binding energy than that of bulk Au, suggested the successful formation of the Au25 superatom [29].

Comparison with Existing Methods
In the previous cases, thiol protected Au clusters were generally synthesized by reducing multimeric Au-thiol complexes [30,40,41]. In these methods, only the most stable species were obtained by the etching process after forming large Au clusters. In contrast, the Au cluster was synthesized directly through the collecting process of AuCl3 to the dendrimer in this method ( Figure 1). Therefore, the cluster formation speed is different from the previous cases [41]. MALDI-TOF-MS spectra confirmed the direct synthesis process (Figures 3a and S5). The intense peak corresponding to Au25PET18 (m/z = 7390.73) was generated within 15 min without peaks of large Au clusters. This result suggests that the assembly process on the TPM G4 is directly responsible for the Au25 nucleation. After one day of reaction, the main peak was still detectable; however, the peaks of degraded clusters also appeared. Therefore, it was appropriate to control the reaction time within a few hours of synthesis using the TPM G4. Size distribution and elemental analysis of the synthesized clusters were performed by STEM/EDS measurements (Figures 2c and S3). The estimated size was 1.2 ± 0.3 nm, close to the longest interatomic distance of 11.99 Å observed by single-crystal analysis reported in the previous paper [31]. EDS mapping also indicates that Au and S are present in the same particle. XPS measurement was conducted with tetraoctylammonium bromide (TOAB) which was added to the solution before casting on highly oriented pyrolytic graphene (HOPG) ( Figure S4). The observed peaks of Au 4f, which show higher binding energy than that of bulk Au, suggested the successful formation of the Au 25 superatom [29].

Comparison with Existing Methods
In the previous cases, thiol protected Au clusters were generally synthesized by reducing multimeric Au-thiol complexes [30,40,41]. In these methods, only the most stable species were obtained by the etching process after forming large Au clusters. In contrast, the Au cluster was synthesized directly through the collecting process of AuCl 3 to the dendrimer in this method ( Figure 1). Therefore, the cluster formation speed is different from the previous cases [41]. MALDI-TOF-MS spectra confirmed the direct synthesis process (Figures 3a and S5). The intense peak corresponding to Au 25 PET 18 (m/z = 7390.73) was generated within 15 min without peaks of large Au clusters. This result suggests that the assembly process on the TPM G4 is directly responsible for the Au 25 nucleation. After one day of reaction, the main peak was still detectable; however, the peaks of degraded clusters also appeared. Therefore, it was appropriate to control the reaction time within a few hours of synthesis using the TPM G4. The same synthetic procedure was carried out without the dendrimer (Figur This method also shows the formation of Au25PET18 after a reaction time of about 3 (Figures 3b and S7). This reaction time is considered to be derived from the etchin cess of the nanoparticles to the Au25 superatom by the ligands.
These differences in the formation process can be seen in the appearance of the tions ( Figure S8). When the dendrimer (TPM G4) was used, the color of the so changed from red (0 min) [41] (the early stage of Au25PET18 synthesis) to brown (a min) (color of Au25PET18). The color was maintained for 12 h. On the other hand, case without the TPM G4, the solution turned black at 0 min immediately after the r tion and remained grayish at 15 min. After 11 h, the formation of the Au25PET18 wa gested by the color of the solution; however, the solution contained a black precip indicating the formation of large particles. In the case of the dendrimer (Figure 3a), s peaks with small intensity are also observed in the range of m/z = 3000-7500 at 15 min reduction. These peaks are considered to be the small Au-thiol multimer and decom products of the TPM G4 ( Figure S9).

Synthesis of MAu24PET18 Using the Dendrimer TPM G4 as a Template
The advantage of the TPM G4 includes metal blending ability. Here, we investi the blending of metals using the TPM G4. In the case of previous methods, the intr tion of other metals using the ligand effect was reported [20,24,27,28,[36][37][38][39]78]. The b ing of metals was conducted using the complexation process (Figure 4a). In this synt one equivalent of Pd(CH3CN)4(BF4)2 and 24 equivalents of AuCl3 were coordinated dendrimer, and the thiol was added simultaneously with the reduction (Figure 4a formation of PdAu24PET18 was confirmed in the MALDI-TOF-MS spectrum (Figur The structure of the PdAu24PET18 was previously proposed as a Pd encapsulated stru ( Figure S10). The MALDI-TOF-MS spectra show the formation of not only PdAu2 but also Au25PET18. In fact, Au25PET18 was observed as a strong peak, and the inten The same synthetic procedure was carried out without the dendrimer ( Figure S6). This method also shows the formation of Au 25 PET 18 after a reaction time of about 30 min (Figures 3b and S7). This reaction time is considered to be derived from the etching process of the nanoparticles to the Au 25 superatom by the ligands.
These differences in the formation process can be seen in the appearance of the solutions ( Figure S8). When the dendrimer (TPM G4) was used, the color of the solution changed from red (0 min) [41] (the early stage of Au 25 PET 18 synthesis) to brown (after 2 min) (color of Au 25 PET 18 ). The color was maintained for 12 h. On the other hand, in the case without the TPM G4, the solution turned black at 0 min immediately after the reduction and remained grayish at 15 min. After 11 h, the formation of the Au 25 PET 18 was suggested by the color of the solution; however, the solution contained a black precipitate, indicating the formation of large particles. In the case of the dendrimer (Figure 3a), several peaks with small intensity are also observed in the range of m/z = 3000-7500 at 15 min after reduction. These peaks are considered to be the small Au-thiol multimer and decomposed products of the TPM G4 ( Figure S9).

Synthesis of MAu 24 PET 18 Using the Dendrimer TPM G4 as a Template
The advantage of the TPM G4 includes metal blending ability. Here, we investigated the blending of metals using the TPM G4. In the case of previous methods, the introduction of other metals using the ligand effect was reported [20,24,27,28,[36][37][38][39]78]. The blending of metals was conducted using the complexation process (Figure 4a). In this synthesis, one equivalent of Pd(CH 3 CN) 4 (BF 4 ) 2 and 24 equivalents of AuCl 3 were coordinated to the dendrimer, and the thiol was added simultaneously with the reduction (Figure 4a). The formation of PdAu 24 PET 18 was confirmed in the MALDI-TOF-MS spectrum (Figure 4b).
The structure of the PdAu 24 PET 18 was previously proposed as a Pd encapsulated structure ( Figure S10). The MALDI-TOF-MS spectra show the formation of not only PdAu 24 PET 18 but also Au 25 PET 18 . In fact, Au 25 PET 18 was observed as a strong peak, and the intensity of PdAu 24 PET 18 was about 1/13 of that of Au 25 PET 18 This result suggests the partial reconstruction of the stable Au 25 PET 18 structure. STEM/EDS analysis of the prepared particles confirmed the presence of Pd and Au on the same particle ( Figure S11).
PdAu24PET18 was about 1/13 of that of Au25PET18 This result suggests the partial reconstruction of the stable Au25PET18 structure. STEM/EDS analysis of the prepared particles confirmed the presence of Pd and Au on the same particle ( Figure S11). In the case of Pt introduction, the presence of Pt and Au on the same particle was confirmed by STEM/EDS analysis. However, the MALDI-TOF-MS spectra of the sample are not useful, unfortunately ( Figure S12c). The observed peak at m/z = 7392 is close to that of Au25PET18; therefore, we could not determine the formation of PtAu24PET18 in the MS spectra. In this case, the comparison of the MS spectra is difficult since the masses of PtAu24PET18 (m/z = 7390.93) and Au25PET18 (m/z = 7391.93) are very close ( Figure S14).

Assembly Accumulation of AuCl3 on Dendrimer TPM G4
Solution preparation was performed in a glove box under a nitrogen atmosphere. First, AuCl3 (22.7 mg, 74.7 µmol) was dissolved in acetonitrile to prepare a 2.99 mM solution. The dendrimer TPM G4 (0.380 mg, 34.4 nmol) was dissolved in chloroform and acetonitrile was added to prepare a 3.43 µM chloroform:acetonitrile = 1:1 solution. The complexation of AuCl3 and the TPM G4 was monitored by a UV-Vis spectrometer.

Synthesis of Au25PET18 Using Dendrimers
AuCl3 acetonitrile solution (10.4 mM, 25.0 mL) and the TPM G4 chloroform:acetonitrile = 1:1 solution (44.9 µM, 10.0mL) were prepared in a glove box under a nitrogen at- In the case of Pt introduction, the presence of Pt and Au on the same particle was confirmed by STEM/EDS analysis. However, the MALDI-TOF-MS spectra of the sample are not useful, unfortunately ( Figure S12c). The observed peak at m/z = 7392 is close to that of Au 25 PET 18 ; therefore, we could not determine the formation of PtAu 24 PET 18 in the MS spectra. In this case, the comparison of the MS spectra is difficult since the masses of PtAu 24 PET 18 (m/z = 7390.93) and Au 25 PET 18 (m/z = 7391.93) are very close ( Figure S14).

Assembly Accumulation of AuCl 3 on Dendrimer TPM G4
Solution preparation was performed in a glove box under a nitrogen atmosphere. First, AuCl 3 (22.7 mg, 74.7 µmol) was dissolved in acetonitrile to prepare a 2.99 mM solution. The dendrimer TPM G4 (0.380 mg, 34.4 nmol) was dissolved in chloroform and acetonitrile was added to prepare a 3.43 µM chloroform:acetonitrile = 1:1 solution. The complexation of AuCl 3 and the TPM G4 was monitored by a UV-Vis spectrometer. were added for reduction and thiol protection. The amount of PET (Au:PET = 1:6) was determined based on a previous report [44]. The reaction was monitored by MALDI-TOF-MS.

Synthesis of Au 25 PET 18 Using Dendrimers
After the addition of the reducing agent, the organic layer of the reaction solution was filtered. Then, the solution was concentrated using an evaporator. The obtained samples were washed several times with water/methanol. For STEM measurements, 1 µL of the sample solution (acetonitrile) was drop-casted onto a TEM grid (grid with carbon support film Super Hi-Res Carbon SHR-C075 STEM Cu75P grid, Okenshoji Co., Ltd., Tokyo, Japan). After the casting, the TEM grids were vacuum dried overnight. For XPS measurements, TOAB was added to the sample solution at 1.2 equivalents of Au. The solution was cast onto HOPG (490HP-AB HOPG SPI-3(ZYH) Grade 5 × 5 × 1 mm, Alliance Biosystems).
In this method, Au 25 PET 18 can be synthesized by using a TPM G4 solution with concentrations of 9.29 µM-3.04 mM. One equivalent of the metal salt of Pd or Pt was added to the dendrimer solution under atmospheric conditions and stirred for 5 min. Then, 24 equivalents of Au metal salt were added, and the mixture was stirred for 1 h under a light shield. After 1 h, ice-cold water containing NaBH 4 (54.9 mg, 1.45 mmol) and 2-phenylethanethiol (90 µL, 671 µmol) were added simultaneously to the dendrimer complex solution. The amount of 2-phenylethanethiol was determined based on a previous report [44]. The reaction was monitored by MALDI measurements.

Characterization
The UV-vis absorption spectra were obtained by Shimadzu UV-3150 and UV-3600 spectrometers. MALDI-TOF-MS spectra were obtained by Bruker microflex-YI, ultrafleXtreme. DCTB was used as the matrix, and toluene or chloroform was used as the solvent at a concentration of 20 mg/mL. The samples were mixed in 1:20 volume ratios. STEM measurements were carried out by a JEOL JEM-ARM200F equipped with an EDS analyzer (acceleration voltage: 80 kV). XPS measurements were carried out by ULVAC-PHI ESCA 1700R. The X-ray source was monochrome Al Kα (1486.7 eV). The neutralizer's emission current was 1 µA.

Conclusions
Dendrimer template synthesis of a Au 25 superatom was developed with thiol protection. Unlike conventional synthetic methods, the conditions for synthesis are unique in that they do not require special conditions (e.g., low temperature reaction, a reaction in two separated aqueous and organic layers, or a long reaction time in the order of several days). The reduction and thiol coordination are performed for AuCl 3 in the TPM G4, resulting in a short-time synthesis. The addition of different elements using this method was also demonstrated by using Pd or Pt atoms.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27113398/s1, Figure S1. Schematic diagram of the structure and coordination of the dendrimer (TPM G4) used in this study. Figure S2.  Figure S5. MALDI-TOF-MS spectra after 2 h of the Au 25 PET 18 synthesis reaction with TPM G4. Figure S6. Synthetic procedure of Au 25 PET 18 cluster not using TPM G4. Note: main product is Au nanoparticle. The concentration of AuCl 3 was the same as when synthesized using TPM G4. In the presence of dendrimers, the equivalent of 25 equivalents of AuCl 3 is 45 µM. Figure S7. MALDI-TOF-MS spectra after 2 h of the Au 25 PET 18 synthesis reaction without TPM G4. Figure S8. Comparison during synthesis of Au 25 PET 18 with and without TPM G4. A significant color change is observed immediately after reduction. Figure S9. Image of the effect of dendrimer during Au 25 PET 18 synthesis and its main and byproducts. Figure S10. The image of the stable structures of PdAu 24 PET 18 (note: draw a figure with reference to the previous report [76]). Figure S11.