Imaging C. Elegans with Thiolated Tryptophan-Based NIR Fluorescent Gold Nanoclusters

S1

Imaging C. elegans with thiolated tryptophan-based NIR fluorescent gold nanoclusters

Apurba Kr. Barman,1Amaresh Chaturbedi,2K. Subramaniam2 and Sandeep Verma1,3*

1Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur-208016 (UP), India, Fax: (+) 91-512-2597436, E-mail:

2Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur-208016 (UP), India

3DST Thematic Unit of Excellence on Soft Nanofabrication, Indian Institute of Technology Kanpur, Kanpur-208016 (UP), India

Table of Content:

1. Synthetic scheme for ligand 2S2

2.Synthetic scheme for ligand 3S3

4. Synthetic procedures for 1S4

5. Synthetic procedures for 2S4-S6

6.Synthetic procedures for 3S6-S9

7. Synthetic procedure for Au11(PPh3)8Cl3S10

8. Synthetic procedure for fluorescent AuNCsS10

9. Procedure for C. elegans imagingS11

10. HRMS of Au11(PPh3)8Cl3 S12

11.1H NMR and 13C NMR spectra of 2S13

12. HRMS of 2S14

13.1H NMR and 13C NMR spectra of 3S15

14. HRMS of 3S16

15. NMR spectra of AuNCsS17

16. MALDI mass spectra of 2-AuNCS18

17. MALDI mass spectra of 1-AuNCS20

18.MALDI mass spectra of 3-AuNCS22

Scheme S1: Synthetic scheme for ligand 2

Scheme S2: Synthetic scheme for ligand 3

Materials and methods: Dichloromethane (DCM), N, N-dimethylformamide (DMF), triethylamine and methanol were distilled following standard procedures prior to use. N, N’-dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole, L-tryptophan, 3-mercaptopropionic acid (3-MPA), HAuCl4, triphenylphosphine, absolute ethanol, DMSO, HPLC grade water were purchased from sd fine chem. limited, Spectrochem PVT. Ltd., Rankem Ltd. and used without further purification. Tris(2-aminomethyl)amine (Tren) was purchased from Sigma Aldrich. 1H and 13C NMR spectra were recorded on JEOL-JNM LAMBDA 400 model operating at 400 and 100 MHz, respectively and JEOL ECX-500 model operating at 500 MHz and 125 MHz, respectively. HRMS mass spectra were recorded at IIT Kanpur, India, on Waters, Q-Tof Premier Micromass HAB 213 mass spectrometer using capillary voltage of 2.6-3.2 kV.

Synthesis of (MPA-L-Trp-L-Trp)3tren (1):(Barman et al. 2010)

Synthesis of S-trityl-3-mercaptopropionicacid (A): 3-mercaptopropionicacid (5 g, 47 mmol) was dissolved in dry dichloromethane (DCM) (40 mL). Then triethylamine (6.6 mL, 33 mmol) was added and stirred. Solution of trityl chloride (13.2 g, 47 mmol) in DCM (30 mL) was added slowly to this solution. The reaction mixture was stirred for overnight at room temperature. Organic layer was washed with 1N aqueous HCl and brine solution and then dried over anhydrous sodium sulphate. The crude product obtained after evaporation was purified by silica gel column chromatography using dichloromethane and methanol (97:2) solvent system. Yield: 11.5 g (70%) Rf : 0.6 (5% methanol in dichloromethane) . 1H NMR (500 MHz, CDCl3, TMS) δ(ppm) : 1.24 (s, 1H); 2.21-224 (t, 2H, J=7.55Hz); 2.43-2.46 (t, 2H, J=7.25 Hz); 7.20-7.45 (m, 15H).

Synthesis of S-trityl-3-MPA-L-Trp-OMe (B): S-trityl-3-mercaptopropionic acid (A) (1.50 g, 4.30 mmol) and 1-hydroxybenzotriazole (0.72 g, 4.70 mmol) were dissolved in dry N, N-dimethylformamide (DMF) (10 mL) and DCM (10 mL), and reaction mixture was cooled to 0°C under nitrogen atmosphere. Solution of N, N′- dicyclohexylcarbodiimide (DCC) (0.97 g, 4.70 mmol) in DCM (5 mL) was added into the reaction mixture at 0°C under nitrogen atmosphere. The reaction mixture was stirred at 0°C for one-hour. After one-hour, hydrochloride salt of L-tryptophan methyl ester (1.10 g, 4.30 mmol) was added into the reaction mixture followed by the addition of triethylamine (1.20 mL, 8.6 mmol) and stirring was continued for overnight at room temperature. Next, the reaction mixture was filtered to remove N, N’-dicyclohexylurea and filtrate was concentrated in reduced pressure. The residue was dissolved in dichloromethane and organic layer was washed aqueous 10% NaHCO3 solution. The organic layer was then dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified through a silica gel column chromatography by using pet ether and ethyl acetate (70:30) as solvent system to give pure compound B as a white solid. Yield = 1.95 g (82%).Rf: 0.7 (5% methanol in dichloromethane).ESI-HRMS: [M+H]+ (C34H33N2O3S) Calculated = 549.2212, found = 549.2217.1H NMR (500 MHz, CDCl3, TMS), δ(ppm): 1.69-1.75 (1H, m), 1.90-1.96 (1H, m), 2.44-2.57 (2H, m), 3.27-3.29 (2H, m), 3.69 (3H, s), 4.83-4.87 (1H, dd, J=5.5 Hz, 13.15 Hz), 5.81-5.83 (1H, d, J=7.65 Hz), 6.92 (1H, s), 7.03-7.06 (1H, t, J=7.95 Hz), 7.13-7.31 (11H, m), 7.35-7.36 (6H, d, J=7.35 Hz), 7.44-7.46 (1H, d, J=7.95 Hz), 7.79 (1H, s). 13C NMR (125 MHz, CDCl3, TMS), δ(ppm): 27.5, 27.7, 35.6, 52.5, 52.8, 66.9, 109.8, 111.4, 118.6, 119.8, 122.3, 123.3, 126.8, 127.6, 128.1, 129.7, 136.1, 144.7, 170.8, 172.4.

Synthesis of S-trityl-3-MPA-L-Trp-OH (C): Compound B(1 g, 1.87 mmol) was dissolved in 10 mL of methanol. 1N aqueous NaOH solution (1.8 mL) was added and stirred for 4 hours at room temperature. Reaction mixture was then passed through strong cation exchange resin. Solvent was then evaporated to get the product as white solid. Yield= 925 mg (95%). Rf: 0.3 (10% methanol in dichloromethane).

Synthesis of (S-trityl-3-MPA-L-Trp)3tren (E):Synthesis of S-trityl-3-MPA-L-Trp-PNP (D):S-Trityl MPA-Trp-OH (C) (500 mg, 0.94 mmol) and p-nitrophenol (144 mg, 1.03 mmol) was dissolved indry dichloromethane (10 mL) and cooled to 0°C. A solution of DCC (214 mg, 1.03 mmol) in dichloromethane was added slowly and stirred for 1 hour at 0 °C temperature and then 3 hours at room temperature. Dicyclohexylurea was filtered off and organic layer was washed with 10% aqueous NaHCO3 solution and then with brine solution. The organic layer was then dried over anhydrous sodium sulphate and concentrated in reduced pressure. The product S-trityl MPA-L-Trp-OPNP (D)was obtained as a light yellow solid. Rf : 0.7 (5% methanol in dichloromethane).

This active ester (D) was dissolved in dry DMF (5 mL) at room temperature under nitrogen atmosphere. Tris-(2-aminoethyl)amine (tren) in (47 µL, 0.30 mmol) was added into the reaction mixture in portions under nitrigen atmosphere at room temperature. The reaction mixture was stirred for 10 hours. The reaction mixture was then concentrated under reduced pressure and dissolved in dichloromethane. Organic layer was washed with aqueous 1N HCl and 10% aqueous NaHCO3 solution and then dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified through silica gel column chromatography by by using dichloromethane and methanol (98:2) solvent system to give pure compound (E) as a white solid. Yield=340 mg, (65% in two steps). Rf: 0.6 (5% methanol in dichloromethane). ESI-HRMS: [M+H]+ (C105H103N10O6S3) Calculated = 1695.7224, found = 1695.7215.1H NMR (500 MHz, CDCl3, TMS), δ(ppm): 1.74-1.86 (6H, m), 1.91-1.99 (6H, m), 2.38-2.52 (6H, m), 2.71 (6H, s, brd), 3.07-3.15 (6H, m), 4.64-4.68 (3H, m), 6.94 (3H, s), 6.98-7.02 (3H, m), 7.09-7.21 (33H, m), 7.31-7.32 (18H, d, J=7.9 Hz), 7.50-7.52 (3H, d, J=7.65 Hz), 8.33 (s), 8.52 (s). 13C NMR (125 MHz, CDCl3, TMS), δ(ppm): 27.8, 28.1, 35.3, 37.8, 53.6, 54.3, 66.9, 110.0, 111.5, 118.7, 119.5, 123.7, 126.9, 127.7, 128.1, 129.7, 136.1, 144.7, 171.5, 171.9.

Synthesis of (MPA-L-Trp)3tren (2):Compound E (1696.19 200 mg, 0.12 mmol) was dissolved in dry dichloromethane (1 mL). Triethylsilane (57 µL, 0.36 mmol) was added to the solution and stirred for 5 min. Trifluoroacetic acid (1 mL) was then added and the reaction mixture was stirred for 1 hour. The solution was then concentrated under reduced pressure and diethyl ether was added. The precipitate formed was dried and dissolved in minimum volume of methanol and re-precipitated by adding diethyl ether, this process was repeated three times and the solid was dried in high vacuum pump. Yield=105 mg, (92%). Rf : 0.4 streak (10% methanol in dichloromethane). ESI-HRMS: [M+H]+ (C48H61N10O6S3) Calculated = 969.3938, found = 969.3937. 1H NMR (500 MHz, DMSO-d6, TMS), δ(ppm): 2.10-2.13 (3H, t, J=7.95 Hz), 2.34-2.38 (6H, m), 2.49-2.54 (6H, m), 2.88-2.93 (3H, t, J=9.15 Hz), 3.04-3.10 (9H, m), 3.47 (3H, s, brd), 4.43 (3H, s, brd), 6.91-6.94 (3H, t, J=6.75 Hz), 7.00-7.03 (3H, t, J=6.70 Hz), 7.11 (3H, s), 7.17-7.29 (3H, d, J=7.35 Hz), 7.52-7.53 (3H, d, J=7.05 Hz), 8.22 (6H, s), 10.80 (3H, s). 13C NMR (125 MHz, DMSO-d6, TMS), δ(ppm): 20.4, 28.1, 34.2, 52.1, 54.3, 110.5, 111.9, 118.8, 118.9, 121.5, 124.2, 127.7, 136.6, 171.1, 173.0.

Boc-Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH (F): Boc-L-Trp-OH(1 g, 3.28 mmol) was dissolved in 10 mL dry DMF in nitrogen atmosphere and cooled to 0°C. Sodium hydride (60% in paraffin oil, 315 mg, 7.87 mmol) was added and stirred for 0°C for 1 h. Ethyl bromoacetate (410 µL, 3.93 mmol) was then added and further stirred at room temperature for 4 h. Water (50 mL) was added and acidified to pH 2-3 with 1N aqueous HCl followed by extraction with ethyl acetate (3×50 mL). The organic layer was washed with water and dried in vacuum. The crude product was purified by silica gel column chromatography using DCM: methanol (97:3) as solvent system. Yield = 770 mg (60%). Rf : 0.6 (10% methanol in dichloromethane). ESI-HRMS: [M+Na]+, Calculated (C20H26N2NaO6) = 413.1689, Found = 413.1689. 1H NMR (500MHz, CDCl3, TMS, δ ppm): 1.21-1.24 (3H, t, J=6.85 Hz), 1.41 (9H, s), 3.26-3.35 (2H, m), 4.15-4.19 (2H, q, J=6.9 Hz), 4.63-4.65 (1H, m), 4.79 (2H, s), 5.10-5.11 (1H, d, J=7.35 Hz), 6.94 (1H, s), 7.08-7.11 (1H, m), 7.17-7.19 (2H, m), 7.58-7.60 (1H, d, J=8.25 Hz). 13C NMR (125MHz, CDCl3, TMS, δ ppm): 14.2, 28.4, 31.7, 47.8, 54.1, 61.8, 80.0, 109.1, 110.2, 119.4, 119.8, 122.3, 127.4, 128.7, 136.8, 155.6, 168.8, 175.4.

HCl salt of Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH (G): Boc-Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH(F)(700 mg 1.79 mmol) was stirred with 15 mL 1N HCl in ethyl acetate for 2 h. Solvent was evaporated and diethyl ether (30 mL) was then added. The residue was filtered and washed with diethyl ether to get pure product. Yield = 460 mg (79%). Rf : 0.6 (100% methanol, streak). ESI-HRMS: [M+H]+, Calculated (C15H19N2O4) = 291.1345, Found = 291.1346. 1H NMR (500MHz, CD3OD, TMS, δ ppm): 1.27-1.30 (3H, t, J=7.15 Hz), 3.49-3.53 (1H, dd, J=4.6 Hz, 15.2 Hz), 3.27-3.30 (1H, m), 4.21-4.25 (3H, m), 5.02 (2H, s), 7.10-7.13 (1H, t, J=6.85 Hz), 7.16 (1H, s), 7.18-7.21 (1H, t, J=8.05 Hz), 7.28-7.30 (1H, d, J=8.3 Hz), 7.63-7.65 (1H, d, J=7.7 Hz). 13C NMR (125MHz, CD3OD, TMS, δ ppm): 13.1, 26.1, 42.7, 53.3, 61.8, 107.7, 109.3, 118.3, 119.7, 122.3, 127.7, 128.4, 137.3, 170.6.

S-trityl-3-MPA-Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH (H): HCl. Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH (G) (350 mg, 1.07 mmol)was dissolved in 5 mL1, 4-dioxan. Sodium bicarbonate (180 mg, 2.14 mmol) was dissolved in 5 mL distilled water and added to the above solution and stirred for 5 min at room temperature. A solution of S-trityl-3-MPA-ONHS (477 mg, 1.07 mmol) in 5 mL 1,4-dioxan was then slowly added. The reaction mixture was stirred for another 6 h at room temperature. Solvent was then evaporated, residue was dissolved dichloromethane, and acidified with aqueous 1N HCl. Organic layer was then washed with water and dried over anhydrous sodium sulphate. The crude product was purified through silica gel column chromatography using DCM: methanol (96:4) as solvent system. Yield = 525 mg (78%). Rf : 0.4 (10% methanol in dichloromethane). ESI-HRMS: [M+Na]+, Calculated (C37H36N2NaO5S) = 643.2243, Found = 643.2244. 1H NMR (500MHz, CDCl3, TMS, δ ppm): 1.21-1.24 (3H, t, J=7.25 Hz), 1.75-1.81 (1H, m), 1.90-1.95 (1H, m), 2.43-2.47 (1H, m), 2.50-2.55 (1H, m), 3.25-3.34 (2H, m), 4.13-4.18 (2H, q, J=6.9 Hz), 4.60 (2H, d, J=1.9 HZ), 4.80-4.84 (1H, dd, J=5.43 Hz, 12.25 Hz), 5.91-5.92 (1H, d, J=7.25 Hz), 6.92 (1H, s), 6.99-7.02 (1H, t, J=8.0 Hz), 7.14-7.17 (6H, t, J=6.85 Hz), 7.20-7.23 (6H, t, J= 7.25 Hz), 7.26-7.30 (1H, m), 7.35-7.37 (3H, d, J=7.25 Hz), 7.41-7.44 (1H, t, J=8.4 Hz), 7.50-7.51 (1H, d, J=8.0 Hz). 13C NMR (125MHz, CDCl3, TMS, δ ppm): 14.2, 27.5, 29.7, 35.5, 47.6, 53.2, 61.8, 67.0, 10.9.1, 109.8, 119.1, 120.0, 122.4, 126.8, 127.6, 128.0, 128.6, 129.6, 136.7, 144.7, 168.9, 171.5, 174.7.

S-trityl-3-MPA-Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OPNP (I):S-trityl-3-MPA-Nind-(2-ethoxy-2-oxoethyl)-L-Trp-OH (H) (500 mg, 0.80 mmol) and p-nitrophenol (122 mg, 0.88 mmol) was dissolved indry DCM (10 mL) and cooled to 0°C. A solution of DCC (1836 mg, 0.88 mmol) in DCM was added slowly and stirred for 1 h at 0°C temperature and then 4 h at room temperature. Dicyclohexylurea was filtered off and organic layer was washed with 10% aqueous NaHCO3 solution and then with brine solution. The organic layer was then dried over anhydrous sodium sulphate and concentrated under reduced pressure. The product obtained was used in the subsequent step without further purification. Yield = 550 mg (92%). Rf : 0.7 (5% methanol in dichloromethane). ESI-HRMS: [M+Na]+, Calculated (C43H39N3NaO7S) = 764.2406, Found = 764.2409. 1H NMR (500MHz, CDCl3, TMS, δ ppm): 1.20-1.23 (3H, t, J=7.05 Hz), 1.91-1.94 (2H, t, J=7.05 Hz), 2.51-2.57 (2H, m), 3.33-3.37 (1H, dd, J=6.15 Hz, 15.0 Hz), 3.42-3.46 (1H, J=5.45 Hz, 14.95 Hz), 4.18-4.23 (2H, q, J=7.35 Hz), 4.68 (2H, s), 5.05-5.09 (1H, dd, J=5.8 Hz, 13.15 Hz), 5.99-6.01 (1H, d, J=7.3 Hz), 6.99-7.01 (3H, m), 7.14-7.15 (10H, m)7.35-7.37 (8H, d, J=7.65 Hz), 7.51-7.53 (1H, d, J=7.95 Hz), 8.12-8.14 (2H, d, J=9.15 Hz). 13C NMR (125MHz, CDCl3, TMS, δ ppm): 14.3, 25.8, 27.6, 35.5, 49.3, 53.3, 61.9, 67.0, 109.4, 119.1, 120.3, 122.5, 122.8, 125.2, 126.1, 126.8, 127.6, 128.0, 129.7, 136.9, 144.6, 145.5, 155.0, 168.6, 169.9, 171.2.

[S-trityl-3-MPA-Nind-(2-ethoxy-2-oxoethyl)-L-Trp]3tren (J):Compound I (530 mg, 0.72 mmol) was dissolved in dry DMF (5 mL) at room temperature under nitrogen atmosphere. Tris-(2-aminoethyl)amine (tren) (33µL, 0.22 mmol) was added into the reaction mixture in three portions over 3 h under nitrigen atmosphere at room temperature and further stirred for additional 10 h. The reaction mixture was then concentrated under reduced pressure. The crude compound was purified through silica gel column chromatography by using DCM: methanol (97:3) as solvent system to give pure compound. Yield = 280 mg, (60%). Rf : 0.4 (5% methanol in dichloromethane). ESI-HRMS: [M+H]+ Calculated (C117H121N10O12S3) = 1953.8328, Found = 1953.8322. 1H NMR (500MHz, CDCl3, TMS, δ ppm): 1.18-1.21 (9H), 1.69-1.87 (12H, m), 2.11-2.25 (6H, m), 2.34-2.46 (6H, m), 2.75-2.88 (3H, m), 3.08-3.19 (3H, m), 4.07-4.12 (6H), 4.55 (6H, s), 4.68-4.72 (3H, m), 6.87-7.09 (9H, m), 7.11-7.23 (36H, m), 7.29-7.37 (18H, m), 7.53-7.57 (3H, m). 13C NMR (125MHz, CDCl3, TMS, δ ppm): 14.2, 27.7, 29.8, 35.1, 38.0, 45.7, 47.5, 53.5, 61.6, 66.8, 108.9, 110.9, 114.4, 119.3, 119.7, 126.8, 128.0, 128.5, 129.6, 136.8, 144.7, 168.8, 171.3, 171.6.

[S-trityl-3-MPA-Nind-(carboxyethyl)-L-Trp]3tren (K): [S-trityl-3-MPA-Nind-(2-ethoxy-2-oxoethyl)-L-Trp]3tren (J) (250 mg, 0.13 mmol) was dissolved in 10 mL THF. Sodium hydroxide (16 mg, 0.40 mmol) dissolved in 2 mL water was added and stirred for overnight. The solution was then diluted with methanol and passed through strong cation exchange resin. Solvent was then evaporated to get pure product. Yield = 204 mg (85%). ESI-HRMS: [M+H]+ Calculated (C111H109N10O12S3) = 1869.7389, Found = 1869.7386. 1H NMR (500MHz, DMSO-d6, TMS, δ ppm): 1.70-1.73 (3H, m), 2.82-2.86 (3H, dd, J=8.4 Hz, 14.15 Hz), 2.11-2.17 (12H, m), 2.95-3.04 (9H, m), 3.52-3.57 (3H, m), 4.37-4.42 (3H, dd, J= 7.15 Hz, 15.85 Hz), 4.79 (6H, s), 6.91-6.94 (3H, t, J=7.65 Hz), 7.00 (3H, s), 7.02-7.05 (3H, t, J=7.3 Hz), 7.16-7.20 (12H, m), 7.23-7.27 (36H, m), 7.51-7.53 (3H, d, J=7.65 Hz), 8.04-8.04 (3H, d, J=6.1 Hz). 13C NMR (125MHz, DMSO-d6, TMS, δ ppm): 25.7, 27.7, 28.3, 34.3, 47.4, 54.0, 66.5, 67.6, 110.1, 110.7, 119.1, 121.7, 127.2, 128.3, 128.5, 129.6, 137.0, 145.0, 170.5, 171.0, 171.1.

[MPA-L-(Nind-carboxyethyl)-Trp]3tren (3): [S-trityl MPA-L-(Nind-carboxyethyl)-Trp]3tren (K) (180 mg, 0.09 mmol) was dissolved in dry dichloromethane (2 mL). Triethylsilane (46 µL, 0.27 mmol) was added to the solution and stirred for 2 min. Trifluoroacetic acid (2 mL) was then added and the reaction mixture was stirred for 1 h. The solution was then concentrated under reduced pressure and diethyl ether was added. The precipitate was dried and dissolved in minimum volume of methanol and re-precipitated by adding diethyl ether, this process was repeated three times and the solid was dried in high vacuum pump. Yield = 103 mg, (94%). Rf : 0.4 streak (10% methanol in dichloromethane). ESI-HRMS: [M+H]+ Calculated (C54H67N10O12S3) = 1143.4102, Found = 1143.4108. 1H NMR (500MHz, DMSO-d6, TMS, δ ppm): 1.19 (s, brd), 2.12-2.16 (3H, t, J=7.65 Hz), 2.35-2.38 (6H, t, J=6.1 Hz), 2.51-2.63 (9H, m), 2.87-2.92 (3H, dd, J=8.85 Hz, 13.7 Hz), 3.05-3.09 (3H, m), 3.25 (6H, brd, merged with DMSO), 4.42-4.47 (3H, m), 4.89 (6H, s), 6.96-6.99 (3H, t, J=7.35 Hz), 7.04-7.08 (6H, m), 7.26-7.28 (3H, d, J=7.9 Hz), 7.54-7.56 (3H, d, J=7.6 Hz), 8.02 (3H, s), 8.16 (3H, s, brd). 13C NMR (125MHz, DMSO-d6, TMS, δ ppm): 20.4, 28.1, 29.6, 43.8, 47.5, 52.8, 54.1, 110.2, 110.7, 119.2, 121.8, 128.2, 128.4, 128.9, 137.0, 171.0, 172.5.

Preparation of [Au11(PPh3)8Cl3]: NaBH4 (15.2 mg) was dissolved in 2 mL absolute ethanol and slowly added to solution of Au(PPh3)Cl (200 mg) in absolute ethanol (9 mL) over 15 min. After stirring at room temperature for 2 h, the mixture was poured into n-hexane (200 mL) and allowed to stand for 20 h. The solution was then filtered through sintered glass crucible; the brown solid was collected and washed with n-hexane (4×3 mL), CH2Cl2/hexane (1:1, 4×3 mL) and CH2Cl2/n-hexane (3:1, 2 mL). The remaining solid was dissolved in CH2Cl2 and filtrated to remove inorganic impurities. After evaporation of the solvent Au cluster (60 mg) was obtained as a dark brown solid (Shichibu et al. 2005). ESI-HRMS: m/z for [Au11(PPh3)8Cl2]+ Calculated (C144H120Au11Cl2P8) = 4333.2989, found = 4333.3188.

General preparation of fluorescent AuNCs: Ligands [15 eq. of SH compared to Au11(PPh3)8Cl3] were dissolved in HPLC grade methanol and [Au11(PPh3)8Cl3] solution in GR grade DCM was slowly added under nitrogen atmosphere [Methanol: DCM 9:1 in final solution, 3 mL solvent for 5 mg of Au11(PPh3)8Cl3]. The mixture was stirred at room temperature for 48 h, and then evaporated under reduced pressure (at 40°C). The solid product obtained was washed with methanol: DCM (1:1), DCM and finally with diethyl ether until no UV (254 nm) active spot was detected in TLC in the supernatant liquid. The products were then dried under high vacuum and used for further experiments.

UV-Visible spectroscopy:UV-Vis absorption spectra were recorded on Varian CARY 100 Bio UV-Vis spectrophotometer (Varian Inc., USA), with 10 mm quartz cell at 25±0.1°C. For 2-AuNC,3-AuNC; spectra were recorded by dissolving in DMSO and diluting with methanol (>99: 1, methanol: DMSO in final solution). For 3-AuNC,spectra was recorded in 1X PBS buffer (pH 7.4). Before recording the spectra samples were centrifuged to remove un-dissolved particles, if any.

Fluorescence spectroscopy: Fluorescence spectra were recorded on Varian CARY 100 Bio fluorescence spectrophotometer (Varian Inc., USA), with 10 mm quartz cell at 25±0.1°C. For 2-AuNC,3-AuNC; spectra were recorded by dissolving in DMSO and diluting with methanol (>99: 1, methanol: DMSO in final solution). For 3-AuNC spectra was recorded in 1X PBS buffer (pH 7.4). Before recording the spectra samples were centrifuged to remove un-dissolved particles, if any.

FTIR spectroscopy: FTIR analyses were done for solid dry powder of 1, 2, 3, 1-AuNC, 2-AuNC and 3-AuNC after making pellets with KBr on a Bruker Vector 22 FT IR spectrophotometer operating from 400-4000 cm-1.

MALDI mass spectra: MALDI spectra for 1-AuNC and 2-AuNCwere recorded after dissolving the cluster in DMSO:methanol (9:1). In case of 3-AuNC, cluster was dissolved in PBS buffer. Mass spectra were acquired on a time-of-flight mass spectrometer (MALDI-TOF-TOF Autoflex II TOF-TOF, Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser (λ = 337 nm).An external multi-point calibration was carried out before each measurement a standard peptide mixture and a standard protein mixture (depending on the mass range analyzed).Scan accumulation and data processing were performed with FlexAnalysis 3.4 software.Sample preparation:Matrix solutions were freshly prepared: α-cyano-4-hydroxy-cinnamic acid (CHCA) was dissolved to saturation and Sinapinic acid (SA) at 20 mg/mL in a H2O/CH3CN/HCOOH (50/50,1%) solution.Typically, a 1/1 mixture of the sample solution wasmixed with the matrix solution and 0.5 µL of the resulting mixture was deposited on the stainless steel plate.

C. elegans imaging: Escherichia coli (OP50) bacteria were grown overnight at 37°C in 4XYT media containing 3-AuNC. Then this bacterial culture was spread on 35mm Petri dishes with New Nematode Growth Media (NNGM). 50-100 Caenorhabditis elegans, a free living nematode, were placed on each of these plates. For control experiment, same procedure was followed but bacterial culture was grown without 3-AuNC. All these experiments with nematodes were performed at 20°C. For imaging of C. elegans, Zeiss microscope (Imager.M2) was used and pictures of nematodes were taken using Zeiss AxioCam HRm. For taking pictures, nematodes were suspended in 2mM levamisole on a slide and then covered with cover slip.

Same experiment was repeated with GFP expressed C. elegans under ubiquitous promoter.

References:

Barman AK, Verma S (2010) Sunlight mediated disruption of peptide-based soft structures decorated with gold nanoparticles. Chem Commun 46:6992-6994

Shichibu Y, Negishi Y, Tsukuda T, Teranishi T (2005) Large-scale synthesis of thiolated Au25 clusters via ligand exchange reactions of phosphine-stabilized Au11 clusters. J Am Chem Soc 127:13464-13465

ESI-HRMS of Au11(PPh3)8Cl3: (Inset shows isotopic distribution)

1H NMR and 13C NMR spectra of 2:

ESI-HRMS of 2:

1H NMR and 13C NMR spectra of 3:

ESI-HRMS of 3:

Figure S1:1H NMR spectra of a) 2 b) 2-AuNC c) 1 d) 1-AuNC in DMSO-d6.

MALDI mass spectra of 2-AuNC:

Table S1MALDI peak assignments for 2-AuNC

Calculated (m/Z)
(L= 2-3H) / Observed (m/Z)
Au17L5 8177 / 8179
Au18L5 8374 / 8377
Au19L5 8752 / 8754
Au20L5 8768 / 8771
Au21L5 8963 / 8958
Au22L5 9162 / 9154
Au24L5 9556 / 9560
Au20L6 9735 / 9740
Au21L6 9932 / 9934
Au22L6 10128 / 10130
Au23L6 10325 / 10317
Au24L6 10522 / 10521
Au28L6 11310 / 11311
Au31L6 11901 / 11899

MALDI mass spectra of 1-AuNC:

MALDI mass spectra of 3-AuNC:

Table S2MALDI peak assignments for 1-AuNCand 3-AuNC:

1-AuNC / 3-AuNC
Calculated (m/Z) / Observed (m/Z) / Calculated (m/Z) / Observed (m/Z)
Au10S4 (2097.554) / 2097.494 / Au19 S9Na22+ (4076.0927/2 = 2038.046) / 2036.707
Au11S2(2230.576) / 2229.977 / Au10S4 (2097.554) / 2096.955
Au23S62+(4722.0631/2 = 2361.031) / 2361.182 / Au21 S8H2ONa2+ (4433.0745/2 = 2216.537) / 2216.199
Au13S5(2720.426) / 2719.019 / Au22 S4H2O2+ (4479.1630/2 = 2238.081) / 2238.638
Au24S52+(4887.0565/2 = 2443.528) / 2442.714 / Au11 S3H2O (2280.558) / 2280.545
Au28S32+(5610.9797/2 = 2805.489) / 2804.866 / Au21S152+(4615.8787/2 = 2307.939) / 2308.738
Au15S3 (3050.415) / 3050.188 / Au22S10Na 2+ (4675.9746/2 = 2337.98) / 2337.612
Au19S3 (3838.281) / 3838.765 / Au22 S12H2ONa2+ (4757.9293/2 = 2378.964) / 2379.776
Au22S13(4748.901) / 4749.105 / Au12S3(2427.5428) / 2428.195
Au24L2S13 (L= 1-3H) (8191.0383) / 8191.404 / Au12S3(2459.5148) / 2460.231
Au12 S3Na2(2505.494) / 2504.931