Solid Phase Supported “Click”-Chemistry Approach for the Preparation of Water Soluble Gold Nanoparticle Dimers – Supplemental Material
M. Homberger†,*, S. Schmid †, J. Timper†, U. Simon†
† Institute of Inorganic Chemistry, RWTH Aachen University and JARA-Fit
Landoltweg 1, 52074 Aachen (Germany)
*To whom correspondence should be addressed. E-mail: . Telephone: 0049 (0)241 80 99683, Fax: 0049 (0)241 80 9903
Experimental Part
Materials: TBTA, N-(3-dimethylamino-propyl)-N’-ethylcarbodiimide hydrochloride (EDC), diisopropylethylamine (DIPEA), prop-2-yn-1-amine, lipoic acid (lip), HAuCl4, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), aminopropylbromide hydrobromide, NaN3, in DMSO were purchase from Aldrich. 3-azidopropan-1-amine was purchased from Sigma and amino-propyl-tritethoxy silane (APTES) was obtained from Acros.
Instrumentation: NMR: Bruker Avance II 400, SEM: Leo Supra 35VP.
Synthesis of lipYne (5-(1,2-dithiolan-3-yl)-N-(prop-2-yn-1-yl)pentanamide)
557mg (2.9mmol) EDC are suspended under argon in 6ml of dry dichloromethane. Upon addition of 376mg (2.9mmol) DIPEA the EDC is dissolved. After stirring for 10min. at room temperature the solution is cooled down to 0°C. 187mg (3.4mmol) prop-2-yn-1-amine is added and the solution is stirred for additional 10min. at 0°C. 500mg (2.4mmol) lip is dissolved in 5ml dry dichloromethane and are added dropwise within 5min. to the reaction mixture. The yellow reaction mixture is allowed to come to room temperature.
After 12h 139mg (0.73mmol) EDC and 40mg (0.73mmol) prop-2-yn-1-amine are added and the reaction mixture is stirred for additional 3h.
After evaporation of the solvent the product is obtained as a yellow oil, which is dissolved in dichloromethane and purified by chromatography (DCM:MeOH 10:1) yielding 390mgmg (1.6mmol, 78 %) of lipYne as a light yellow solid.
1H-NMR (CDCl3, 400 MHz): δ (ppm) = 1.37 – 1.45 (m, 2 H), 1.56 – 1.67 (m, 4 H), 1.80 – 1.89 (m, 1 H), 2.13 – 2.17 (m, 3 H), 2.35 – 2.43 (m, 1 H), 3.02 – 3.15 (m, 2 H), 3.49 – 3.52 (m, 1 H), 3.98 – 4.00 (m, 2 H), 5.62 (s, br., 1 H).
13C-NMR (CDCl3, 100 MHz): δ (ppm) = 25.18 (CH2), 28.83 (CH2), 29.18 (CH2), 34.60 (CH2), 36.12 (CH2), 38.48 (CH2), 40.23 (CH2), 56.38 (CH), 71.62 (CH), 79.58 (C), 172.26 (C).
EA: calc. C: 54.28%, H: 7.04%, N: 5.75% exp. C: 54.43; H: 6.87%, N: 5.63%.
IR: ν = 3270, 3229, 3068, 2935, 2853, 2681, 2465, 2117, 1696, 1636, 1542, 1446, 1325, 1247, 1138, 1028, 929, 697.
Synthesis of lipAz (N-(3-azidopropyl)-5-(1,2-dithiolan-3-yl)pentanamide)
3-azidopropan-1-amine
10 g (46mmol) aminopropylbromide hydrobromide are suspended in 30 ml of water. 9.7 g (151mmol) NaN3 are dissolved in 45 ml water and added to the hydrobromide solution. This reaction mixture is heated under reflux. After 16 hours the solvent is evaporated until a white precipitate occurs. The reaction mixture is dispersed with 150 ml of diethyl ether at – 10 °C and 10 g of NaOH are added. The reaction mixture is allowed to come to room temperature. The crude product is extracted three times with 45 ml diethylether. The combined organic phases are dried over NaSO4. After filtration, evaporation of the solvent and crystallization 980mgmg (9.8mmol, 21 %) of a light yellow solid are obtained.
N-(3-azidopropyl)-5-(1,2-dithiolan-3-yl)pentanamide
557mgmg (2.9mmol) EDC are suspended under argon in 6 ml of dry dichloromethane. Upon addition of 376mgmg (2.9mmol) DIPEA the EDC is dissolved and after 10 min. stirring at room temperature the solution is cooled down to 0 °C. 340mgmg (3.4mmol) 3-azidopropan-1-amine dissolved in 2 ml dry DCM is added. Now 500mgmg (2.4mmol) lipoic acid are dissolved in 5 ml dry dichloromethane and are added dropwise within 5 min. to the reaction mixture. After 12 hours 139mgmg (0.73mmol) EDC and 72mgmg (0.73mmol) 3-azidopropan-1-amine are added and this reaction mixture is stirred for further 3 h.
After evaporation of the solvent a yellow oil is obtained which is purified by chromatography (DCM:MeOH 10:1). After evaporation of the solvent 500mgmg (1.7mmol, 72 %) of lipAz are obtained as a yellow solid.
1H-NMR (CDCl3, 400 MHz): δ (ppm) = 1.36 – 1.43 (m, 2 H), 1.56 – 1.67 (m, 4 H), 1.70 – 1.76 (m, 2 H), 1.82 – 1.87 (m, 1H), 2.10 – 2.14 (m, 2 H), 2.37 – 2.42 (m, 1H), 3.02 – 3.12 (m, 2 H), 3.25 – 3.33 (m, 3 H), 3.49 – 3.52 (m, 1 H), 5. 68 (s, br, 1 H).
13C-NMR (CDCl3, 100 MHz): δ (ppm) = 25.4 (CH2), 28.9 (CH2), 30.3 (CH2), 34.7 (CH2), 36.4 (CH2), 37.2 (CH2), 38.5 (CH2), 43.1 (CH2), 49.4 (CH2), 56.4 (CH), 172.9 (C).
EA: calc. C: 45.81%, H: 6.99%, N: 19.42%; exp. C: 46.09%, H: 6.01%, N: 18.95%.
IR: ν = 3297, 3079, 2930, 2859, 2097, 1645, 1550, 1437, 1367, 1260, 1131, 1050, 928.
15nm AuNP@citrate
AuNP@citrate stabilized AuNPs are prepared according to literature. [1]
The obtained colloid solution is characterized by AAS, UV-vis, DLS and STEM measurements.
AAS: 78.87mg/l Au
UV-Vis: λmax = 519nm
DLS: d = 18.6 nm
STEM: 15.7nm ±1.5nm
15nm AuNP@lip/lipAz
2 ml of the citrate capped 15nm AuNPs solution are mixed with 1 ml of a 5:1 mixture of lip and liAz in carbonate buffer (2mmol/l) and stirred at room temperature over night. After 14 h the red solution is centrifuged (13.000 rpm, 20min.) and the solid residue is dispersed in 1 ml of carbonate buffer solution (10mmol/l, pH = 9.9), yielding a red solution. The obtained solution is characterized by UV-vis, DLS and STEM.
UV-Vis: λmax = 524nm, DLS: d = 28nm, STEM: 15.5nm ±0.9nm
15 nm AuNP@lip/lipYne
2ml of the citrate capped 15nm AuNPs solution are mixed with 1ml of a 5:1 mixture of lip/lipYne in carbonate buffer (2mmol/l) and stirred at room temperature over night. After 14h the red solution is centrifuged (13.000rpm, 20min.) and the solid residue is dispersed in 1ml of carbonate buffer solution (10mmol/l, pH = 9.9).
UV-Vis: λmax = 523nm, DLS: d = 28nm, STEM: 13.4nm ±2.8nm
CuAAC in solution
150μl of a solution of sodium ascorbate in water (2mmol/l), 50μl of a solution of TBTA in DMSO (2mmol/l) and 50μl of a solution of CuSO4 in ultrapure water (2mmol/l) are mixed at room temperature. To this clear solution 1250 μl of a solution containing the AuNP@lip/lipAz in carbonate buffer (2mmol/l) are added. Immediately afterwards, 500μl of the solution of AuNP@lip/lipYne in carbonate buffer is added and the reaction mixture is allowed to stay at room temperature for 6h. Within this time UV-Vis measurements are performed to control the progress of the reaction.
Silanization of silicon wafers
Silicon wafers were cleaned in isopropanol/KOH, thoroughly washed with ultrapure water, refluxed in H2O2/NH3/H2O (1:1:3) for 1h and finally washed with ultrapure water again. Activation was achieved by immersion of the substrates into methanol/HCl (1:1) for 30min. Afterwards the substrates were washed three times and dried over night at 60°C. The dried substrates were silanized with 5% APTES (amino-propyl-tritethoxy silane) in ethanolic solution for 1h, washed 3 times with ethanol by sonication and dried for 3h at 120°C.
Solid phase supported CuAAC
Immobilization and ligand exchange
1ml of the solution containing AuNP@citrate is diluted with 1ml of ultrapure water. 1ml of this solution is incubated with the silanized silicon wafer. After one hour the wafer is thoroughly rinsed with ultrapure water and dried with N2. The substrates are then incubated at room temperature with a mixture of 1ml carbonate buffer (10mM, pH9.9) and 10µl of a solution containing lip (0.17mmol, 34.5mg) and lipYne (0.03mmol, 8.7mg) in 100ml carbonate buffer (10mM)). After 3h the substrates are thoroughly washed with ultrapure water and dried with N2.
CuAAC
Solid phase supported “click”-reaction has been performed by varying the catalyst to reactants ratio. For this the following set of experiments has been performed.
Exp-eriment / molar amount of catalyst / Total catalyst volume added/µl / AuNP@lip/lipAz Volume added/µlA / CuSO4 : 1·10-4mmol
Sodium Ascorbat : 2·10-5mmol
TBTA : 2.5·10-4mmol / 185 / 500
B / CuSO4 : 2·10-4mmol
Sodium Ascorbat : 4·10-5mmol
TBTA : 5·10-4mmol / 370 / 1000 (of a 1/1 dilution)
C / CuSO4 : 3·10-4mmol
Sodium Ascorbat : 6·10-5mmol
TBTA : 7.5·10-4mmol / 555 / 1500 (of a 1/2 dilution)
D / CuSO4 : 4·10-4mmol
Sodium Ascorbat : 8·10-5mmol
TBTA : 1·10-3mmol / 740 / 2000 (of a 1/3 dilution)
General procedure:
The catalyst solution is performed by mixing the respective volume of sodium ascorbate solution (2mmol/l in H2O), TBTA-solution (2mmol/l in DMSO) and CuSO4-solution (2mmol/l in H2O). The catalyst solution is combined with the solution of lip/lipAz functionalized AuNPs. The functionalized substrates from the immobilization step are incubated with the catalyst/AuNP solution for 5h at room temperature. Afterward the catalyst/AuNP solution is removed and the substrates are thoroughly washed several time with ultrapure water and dried with N2. The thus prepared substrates are directly transferred into the vacuum sample chamber of SEM for characterization (Table 2)
Table 1: SEM-Measurements of reference experiments
/ Reference:AuNP@citrate immobilized on aminosilanized Si-substrate, after ligand exchange (lip/lipAz) and incubating with lipalkin AuNP (without cat)
Overall structure number: 2074
Single Particles: 1994
Dimers: 57 (3%)
Trimers: 16
Tetramers: 7
Higher aggregates: 5
Table 2: SEM measurements.
Experiment A:
Overall structure number: 2657
Single Particles: 2300
Dimers: 311 (12%)
Trimers: 30
Tetramers: 12
Higher aggregates: 4 /
Experiment B:
Overall structure number: 2881
Single Particles: 2346
Dimers: 454 (16%)
Trimers: 64
Tetramers: 14
Higher aggregates: 3
Experiment C:
Overall structure number: 1961
Single Particles: 1710
Dimers: 226 (12%)
Trimers: 20
Tetramers: 3
Higher aggregates: 2 / Experiment D:
Overall structure number: 1630
Single Particles: 1171
Dimers: 376 (23%)
Trimers: 58
Tetramers: 12
Higher aggregates: 3
In order to investigate the dissolution feasibility of the dimer structures from the solid phase support, sample D was dipped into carbonate buffer solution, 50µl of an ethanolic solution containing lip (2mmol/L) as additional stabilizing ligand is added and treated with ultra sound. Within a few seconds the aqueous phase turned red, indicating dissolution of the AuNP structures from the surface. This phase was investigated by TEM measurements.
References
[1] Turkevich, J.; Stevenson, P. C.; Hillier, J. J. Phys. Chem. 1953, 57, 670– 673.
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