Preparation of Cu Nanocomposites from EDA, DETA, and Jeffamine Cored PAMAM Dendrimers with TRIS and Carboxyl Surface Functional Groups

This study presents the synthesis and UV-Vis characterization of Cu nanocomposites from ethylenediamine (EDA) (E), diethylenetriamine (DETA) (D), and Jeffamine® T-403 (P) cored PAMAM dendrimers (PAMAMs) with TRIS and carboxyl surface functional groups. Cu-PAMAM dendrimer encapsulated nanoparticles (Cu-DENs) were characterized by UV-Vis spectroscopy. Disappearance of the 680 nm d-d transition and 270–300 nm ligand to metal charge transfer (LMCT) peaks and the formation of monotically increasing exponential band were used as the evidence of the successful synthesis of Cu-DENs in addition to immediate color change of dendrimer-metal mixture solutions from blue to golden brown by reduction. Synthesized Cu-DENs could be facilitated as novel alternatives to the existing nanomaterials used in a wide range of applications involving bio and chemical sensors, catalysis, hydrogenations, oxidations, semiconductors, noble metals, magnetic dendrimer nanocomposites, environmental cleanup and many others.


Introduction
Dendrimers are a new class of polymeric materials and cascade molecules with their spherical three-dimensional (3D) morphology, typically symmetric around a core. There has been a growing interest toward dendrimers due to their controllable structures, globular shapes and definite monodisperse nanosizes. 1 In particular, controllable synthesis of dendrimers allows them to be architecturally designed with the desired physical and chemical properties for the desired applications. 2 Design variables of dendrimers are a core, repeating branches emanating from the core, and surface-groups. Selectivity on these variables makes dendrimers proper host and candidate for guest molecules so that various metal nanoparticles have been successfully stabilized using dendrimers as templates with tunable solubility and surface reactivity. 3 Dendrimer encapsulated nanoparticles (DENs) are used as templates to control size, stability and solubility of nanoparticles in the range of 1 nm to up to 4-5 nm. 4 Crooks et al. 4 announced dendrimers as good hosting metal nanoparticles because of their uniform structures leading to well-defined nanoparticles, availability to participate in catalytic reactions, resistance to agglomeration, and selectivity to control encapsulation of small substrate molecules.
Modification of surface-groups of dendrimers is an important process to gain the desired solubility. TRIS is the abbreviation of the common known organic compound, tris(hydroxymethyl) aminomethane, with the chemical formula of (HOCH 2 ) 3 CNH 2 . It is widely used in biochemistry, molecular biology, and is highly water-soluble. Surface modified dendrimers with TRIS are highly water soluble. 22 In our recent study, we have shown the microwave-assisted fast, facile and one-pot synthesis of a series of TRIS-terminated poly(amidoamine) PAMAM (PAMAM-TRIS) dendrimers with ethylenediamine (E), diethylenetriamine (D), and Jeffamine (P) core. 23 Here, we have introduced the synthesis of generation-3 (G3) and -4 (G4) E, D, and P cored PAMAM dendrimers (PAMAMs) with TRIS and carboxyl end groups, and later used these templates for the preparation of Cu nanocomposites. Indeed, this study is the first study presenting the synthesis and UV-Vis characterization of G3 and G4, D and P cored PAMAM-COOH dendrimers templates, and Cu-PAMAM-TRIS and Cu-PAMAM-COOH nanocomposites. Synthesized Cu-DENs can take a potential use in the future studies with a wide range of applications.

1. Materials and Apparatus
Jeffamine ® T403 Mn 440, diethylenetriamine (DE-TA), methyl acrylate, ethylenediamine (EDA), methanol, n-butanol, tris(hydroxymethyl) aminomethane (TRIS), NaOH, 37% HCl, NaH 2 PO 4 , NaCl, potassium hydrogen phthalate (KHP), CuSO 4 . 5 H 2 O, and NaBH 4, were supplied from Merck. All solutions were prepared by 18.2 MΩ Millipore Milli-Q deionized water. NaOH solutions were used as titrant after standardized with a primary grade KHP. Standardized HCl against KHP was used as excess acid in order to adjust initial pH of dendrimer solutions. Dendrimer solutions were stored at 4 °C. Unless otherwise stated all the chemicals were in analytical grade and used without further purification.

Synthesis of PAMAMs
PAMAM-TRIS (Cn.TRIS) dendrimers were synthesized according to our recent study. 23 C refers to dendrimer core, which are ethylenediamine (E), dietilentriamine (D), and Jeffamine (P) while n is the generation number. PAMAM-COOH dendrimers (Cn.COOH) were prepared by the slight modification of literature procedure, 24 and our previous study. 25 During the synthesis of PAMAM-COOH dendrimers, briefly, a methanolic solution of esterterminated PAMAM dendrimers (PAMAM-OCH 3 ) (Cn.5) dendrimers was mixed with 1.5 M equiv. of NaOH per terminal ester. The final mixture was stirred for 24 h. Excess amount of solvent was removed under vacuum at bath temperature 65 °C. The remaining oil was dissolved in methanol and again evaporated in vacuo. Drying under vacuum resulted in a white powder product. Yields were 100%. The detailed experimental, characterization data were given in the supporting information (Table S1). Synthesized G3-G4 E, D and P cored PAMAM-COOH, and PAMAM-TRIS dendrimers were used for the preparation of dendrimer encapsulated Cu nanomaterials (Cu-DENs).

3. Spectroscopic Titrations of PAMAMs with Cu 2+ Ions and Synthesis of Cu(II)-PAMAM Dendrimer Complexes
The maximum number of Cu 2+ ions, PAMAMs can complex and to be used in the synthesis of Cu-DENs, were determined via spectroscopic titrations. We have shown the microwave-assisted synthesis, characterization and Cu(II) intra-dendrimer complexes of E, D, and P cored G3, G4 PAMAM-TRIS dendrimers in our recent study. 23 By using the same procedure, we have performed the spectroscopic titrations of E, D, and P cored G3, G4 PA-MAM-COOH dendrimers and their Cu(II) complexes. Concentrations used to titrate aqueous solutions of PA-MAM-COOH dendrimers with Cu 2+ ions were given in Table 1.

4. Preparation of Cu-DENs Using PAMAM-COOH and PAMAM-TRIS Dendrimer Templates
Syntheses of Cu-DENs were adapted from the literature. 10,26 In general, 10.0 mL of 5.0 μM aqueous dendrimer solutions were prepared. This concentration was kept constant for all dendrimer solutions. The pH of the aqueous dendrimer solution was adjusted to pH ∼8. Correct amount of aqueous CuSO 4 (pH ∼4.62) ( Table 1) was added to each dendrimer solution. The final pH of the solu-tion was about pH ∼5. The solution was than stirred for 30 minutes to allow the complexation of Cu 2+ ions with the inner tertiary amines of dendrimers. Afterwards, chemical reduction of TRIS and carboxyl terminated Cu 2+ loaded Cu 2+ /dendrimers with 20 molar excess NaBH 4 in 0.10 M NaOH was resulted in intradendrimer Cu-DENs. Then, pH was adjusted to pH ∼8 at where Cu-DENs are most stable 26 with a negligible amount of dropwise addition of 0.01-0.10 M HCl and NaOH solutions. The formation of Cu-DENs was monitored with UV-Vis spectroscopy.

1. Synthesis of PAMAMs
Microwave-assisted synthesis of PAMAM-TRIS dendrimers with E, D and P core from half generation PAMAM-OCH 3 dendrimers, were performed according to our previous studies 27 and, 23 respectively. On the other hand, PAMAM-COOH dendrimers were prepared by slight modification of the literature procedure 24 and our recent study. 25 The results were in good agreement with the literature. 23,27 General procedures for the preparation and characterization PAMAM-COOH dendrimers were given in the supplementary material. Molecular weights of the synthesized dendrimers were in the ranges of 2584 and 13647 g/mol. The prepared dendrimers were stored in methanolic solution and stored at ± 4 °C. Some selected properties and characterization data of dendrimers were presented in Table 2.

2. Characterization of PAMAMs
Synthesized water-soluble dendrimers could easily be characterized via 1 H NMR and 13 C NMR. Figure 1 show the sample 1 H NMR spectrum monitoring of the conversion of D3.5 to D4.COOH, and D4.TRIS. All the expected signals are at the correct intensities and places. The resonances from methyl ester at 3.78 ppm are no longer visible and confirm the complete conversion of the ester groups to acids (D4.COOH, middle in Figure 1) while formation of a new singlet at 3.71 ppm, resulting from re-   Figure 1). Moreover, formation of the 181.61, 181.46, 174.86 ppm peaks corresponding to acids and interior amides prove the fully conversion of D3.5 to D4.COOH. Likewise, 1 H NMR, the strong resonances corresponding to the methyl groups of terminal methyl group at 173.13(C=O) and 51.87(COOCH 3 ) ppm in the 13 C NMR are no more available (see supplementary file characterization data). Similar spectral changes were observed during the synthesis of E, D, and P cored water-soluble dendrimers. In 13 C NMR of TRIS-terminated conversions, the formation of 56.67(NHCR 3 ) and 63.6 (CH 2 OH) ppm bands were indicated the fully formation of TRIS-terminated PAMAMs. 23 Therefore, 1 H NMR and 13 C NMR spectroscopy evaluations prove a good purity.
In ATR spectroscopy, the disappearance of esteric peak at 1730 cm -1 and formation of two asymmetrical stretching at 1650-1550 cm -1 and a weaker, symmetrical band near 1400 cm -1 also prove the complete conversion of ester terminated D3.5 to D4.COOH ( Figure  2). Similar spectral deconvolutions were observed during the synthesis of E, D, and P cored water-soluble dendrimers.

Spectroscopic Titrations of TRIS and Carboxyl-Terminated PAMAMs
In order to learn about Cu 2+ binding capacity of different generation TRIS and carboxyl-terminated E, D and P cored PAMAMs (Table 2), spectroscopic titration studies were performed. Figure 3 shows the sample spectros-copic titration spectra of carboxyl-terminated E4.COOH, D4.COOH, and P4.COOH dendrimers. Investigation of the Figure 3 reveals that absorbances of Cu 2+ /DEN complexes increases with the addition of the increasing amount of Cu 2+ .
In their former studies, Crooks et al. 10,26 declared that the number of tertiary amines are important to determine the exact number of the maximum metal loading capacity of PAMAMs. In our intra-dendrimer complexation studies, we have observed a sudden deep-blue color change with the addition Cu 2+ ions over PAMAM-TRIS and PAMAM-COOH dendrimer solutions. We have characterized this color change by UV-Vis spectroscopy, and observed a common broad absorption band at λmax = 680 nm for E, D, and P cored PAMAM-TRIS and PA-MAM-COOH dendrimers (Figure 3). This band (d-d transition) indicates the formation of tetradentate (1:4 dendrimer to tertiary amine molar ratio) complexes between the tertiary amine groups of PAMAMs and Cu 2+ ions. 23,28 Figure 4 shows the spectroscopic titration curves of E4.COOH, D4.COOH and P4.COOH dendrimers with Cu 2+ ions. The experimental end points derived from these curves were presented in Table 3 regarding absorbance at λmax = 680 nm versus Cu 2+ /DENs molar ratio plots. Indeed, maximum molar excess of Cu 2+ ions that can be loaded to dendrimers were calculated from these plots. Results showed that PAMAM-COOH dendrimers used in this study absorb the number of Cu 2+ ions equivalent the number of tertiary amine numbers, and the structure of these dendrimers are at the desired monodipersity similar to those of PAMAM-TRIS dendrimers, we reported in our recent study. 23

4. Synthesis and UV-Vis Characterization of Cu-DENs
Most common procedure for the synthesis of DENs involves first the determination of the appropriate number of metal ion ratio to be complexed with dendrimer solutions under pH control, and then this is followed by the reduction of metal ions inside the dendrimer by an appropriate reducing agent. 10 Sodium borohydride (NaBH 4 ) is used as the reducing agent. Appropriate chemical excess of NaBH 4 leads to zerovalent intradendrimer metal nanoparticles. Immediate change in metal ion dendrimer aqueous solution color indicates the reduction. In the case of Cu, LMCT peak at around 300 nm arising from Cu-DENs are replaced with a monotically increasing absorbance band toward shorter wavelength. This indicates the formation of small nanoparticles. In addition, an absence of plasman peak at around 570-590 nm proves that Cu clusters are smaller than 4 nm. 29,30 Transition d-d complex band resulting from the coordination of internal amine groups of ethanol amine-terminated PAMAMs were reported at λmax at 605 nm. 10 This band reported in the range of 600-800 nm depending on surface modification with TRIS. 28 Syntheses of Cu-DENs were adapted from the literature. 10,26 After the number of Cu 2+ ions that can be loaded to E, D and P cored PAMAM-TRIS dendrimers were determined from binding studies reported in our recent study, 23 similar synthetic procedures were applied for the synthesis Cu(II) intra-dendrimer complexes of E, D, and P cored PAMAM-COOH dendrimers.
A straightforward evidence for the reduction of Cu 2+ ions into dendrimer cavity comes from the immediate change in solution color from blue to golden brown as being parallel to literature 10,26 (Figure 6). Upon the addition of appropriate molar ratio of calculated CuSO 4 solutions to the aqueous dendrimer solutions, strong bands at around 270-280 nm for carboxyl ( Figure 7) and 280-300 nm for TRIS-terminated ( Figure 8) PAMAMs were occurred, respectively. These bands are assigned to LMCT bands. [31][32][33] In addition, d-d copper transition band at around 680 nm for all Cu 2+ /DEN complex solutions were observed (Figure 7, 8). This band could not be observable for low concentrations. After the addition of excess reducing agent NaBH 4 , LMCT peak at around 300 nm and d-d transition band at 680 nm in all Cu 2+ /DENs were immediately disappeared and replaced with a nearly exponen-   tial monotically increasing spectrum towards to shorter wavelengths (Figure 7, 8). Moreover, the size of Cu clusters can be understood from the absence of a Mie plasman peak at around 570 nm in UV-Vis spectra (Figure 8).
The absence of this band indicates that Cu clusters within the dendrimer cavity are smaller than 5 nm. 10 Results of the UV characterizations are in good agreement with the previously reported Cu-DENs. 10

Conclusions
In this study, TRIS and Carboxyl surface modified E, D and P cored G3-G4 PAMAM dendrimers were synthesized as macromolecular templates, and used in the synthesis of Cu-DENs. In particular, G3 and G4, D and P cored PAMAM-COOH dendrimer templates, and Cu-PA-MAM-TRIS and Cu-PAMAM-COOH nanocomposites were prepared and characterized for the first time.
Investigations of the maximum metal loading capacity of PAMAM-TRIS and PAMAM-COOH dendrimers were determined by the spectroscopic titrations with Cu 2+ ions and optimum molar ratios were calculated. The most sparingly evidence of the successfully synthesis of Cu-DENs came from the color change from blue to golden brown. UV-Vis evidence of this color change was the disappearance of d-d complexation band at ∼680 nm and formation of monotically increasing exponential absorption band towards to low wavelengths. The size of Cu clusters can be understood from the absence of a Mie plasmon peak at around 570 nm in UV-Vis spectra. The absence of this band indicates that Cu clusters are smaller than 5 nm. 10 All of these observations indicated the succesfull synthesis of Cu-DENs. Catalytic properties of these novel series of Cu-DENs might be interesting for catalytic evaluation in organic synthesis, and in a wide range of future studies.