Supporting Information · The column used for separation was a Jupiter ProteoAXIA Packed 4µ 90Å 250 x 21.2 mm, flow: 20 mL/min, eluents: H. 2. O + 0.1% TFA (A), CH. 3. CN + 0.1%

S1

Supporting Information

Light-triggered thiol-exchange on gold nanoparticles at low micromolar concentrations in water

Christian Franceschini, Paolo Scrimin* and Leonard J. Prins*

Department of Chemical Sciences, University of Padova Via Marzolo 1, 35131 Padova, Italy

S2

Contents Page

1. Materials and instrumentation……………………………………………………………………………….. 3

2. Synthesis and characterization of peptides………………………………………………………………… 4

2a. Synthesis and characterization of Ac-C(Coum)-OH 3.......…………………………………………… 10 3. Characterisation data for Au NP 1....................................................................................................... 15 4. Methods……………………………...……………………………………………………………….………… 17

4a. Surface saturation concentrations (SSCs) (Figure 2a)………………………….…………………….. 17

4b. Displacement studies (Figures 2b+4c)………………………………………………………………..... 17

4c. Kinetic studies of the displacement process (Figure 2c)…………………………………………..... 17

5. Ultrafiltration experiments…………………………………………………………………………………….. 18

6. Compound structures and masses (Figure 3)…………………………………………............................. 19

7. Fluorescent displacement of (Ac-CW-OH)2 by probe A…………………………………………………… 20 8. Concentration of Ac-CW-OH and the thiol 2 in the dialysate as a function of the amount of Ac-CW-OH

added to Au NP 1.……………………………………………………………………………………………… 21 9. Impurity at the SIM-channel of thiol 2 originating from the membrane…………………………………… 23 10. Fluorescence titration of probe A before and after exposure of Au NP 1 to Ac-CW-OH……………… 24 11. Light-triggered decay of compound Ac-C(Coum)-OH (3) ……………………………………………… 25 12. Binding of Ac-C(Coum)-OH to Au NP 1…………………………………………………………………… 27 13. Fluorescence spectra of Au NP 1 + probe B + Ac-C(Coum)-OH before and after irradiation.......... 28 14. Fluorescence emission of Ac-C(Coum)-OH in the presence of Au NP 1............................................. 29 15. Influence of ionic strength on the binding of Ac-CWD-OH to Au NP 1................................................ 30 16. Exchange of thiol 2 by Ac-C-NH2 ....................................................................................................... 31

17. Experimental details for the Figures in the manuscript…………………………………………………… 32

S3

1. Materials and instrumentation The synthesis and characterization of Au NP 1 has been described elsewhere 1 Stock solutions were

conserved at 4° C in mQ water. The concentration of trimethylammonium head groups was determined

by 1H-NMR in D2O using pyrazine as internal standard. 2’ Deoxy-3’-O-(N’-methylanthraniloyl)-adenosine-5’-

triphosphate (probe A) was purchased from Biolog Life Science Institute and used as received. The synthesis and characterization of probe B has been described previously. 2 All commercially available

reagents and solvents were used without further purification.

HPLC purifications were performed on a preparative HPLC Shimadzu LC-8A equipped with a Shimadzu

SPD-20A UV detector. The column used for separation was a Jupiter Proteo AXIA Packed 4µ 90Å 250 x

21.2 mm, flow: 20 mL/min, eluents: H2O + 0.1% TFA (A), CH3CN + 0.1% TFA (B), gradient: 0-30 min 5-95%

B, λdet = 220 or 380 nm.

Aqueous phases were concentrated at reduced pressure using a Genevac EZ-2 Plus centrifuge.

The UHPLC/MS analysis were performed using an Agilent 1290 Infinity UHPLC equipped with diode array

and ESI-MS detector. The chromatographic column used was an Agilent RRHD Zorbax Eclipse Plus C18

(2.1x150 mm 1.8 micron), eluents: H2O + 0.1% HCOOH (A), CH3CN + 0.1% HCOOH (B).

Peptide concentrations were determined both by weight and by absorbance using a Varian Cary 100 UV/Vis

spectrophotometer equipped with thermostatted multiple cell holders, The following molar extinction

coefficients were used: ɛ355 (MANT) = 5800 M-1cm-1 (for A), ɛ280 (tryptophan) = 5500 M-1cm-1 (for Trp-containing peptides) and ɛ450 (C343) = 45000 M-1cm-1 (for B) in water at pH 7.0.

Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer equipped

with a thermostatted cell holder.

Ultrafiltration experiments were performed using Vivaspin 500 centrifugal concentrators (PES membrane

with 10 KDa MWCO).

1H-NMR spectra were recorded at 298 K using a Bruker AVIII 500 spectrometer equipped with a 5 mm BBI z

gradient probe operating at 500.13 MHz. Chemical shifts are reported in ppm using residual solvent CDCl3

(7.26 ppm) or D2O (4.79 ppm) for calibration.

TEM images were recorded on a Jeol 300 PX electron microscope. Dynamic light scattering was performed on a Malvern Zetasizer Nano-S instrument. UV-Visible spectra were recorded on a Varian Cary50 Biospectrophotometer equipped with thermostatted multiple cell holders.

1 G.Pieters, A.Cazzolaro, R.Bonomi, L.J.Prins, Chem. Commun. 2012, 48, 1916-1918 2 C.Pezzato, B.Lee, K.Severin, L.J.Prins, Chem. Commun. 2013, 49, 469-471

S4

2. Synthesis and characterization of peptides The synthesis of all peptides was performed manually in SPPS reaction vessels starting from Wang resin (0.6 mmol/gr; 100-200 mesh) using standard Fmoc-chemistry and DIC/HOBt, HBTU or HATU as coupling

agents. The final cleavage was performed using 5 mL of a mixture of TFA : TIS-triisopropylsilane : H2O

(95:2.5:2.5). After TFA cleavage, the peptides were precipitated using Et2O and purified with preparative RP-

HPLC (Jupiter Proteo AXIA Packed 4µ 90Å 250 x 21.2 mm, flow: 20 mL/min, eluents: H2O + 0.1% TFA (A),

CH3CN + 0.1% TFA (B), gradient: 0-30 min 5-95% B, λdet = 220 or 380 nm. The purified products were

analyzed with UHPLC/MS (Agilent Zorbax Eclipse Plus C18 2.1 x 150 mm, 1.8 micron), flow 0.4 mL/min,

eluents H2O + 0.1% HCOOH (A), CH3CN + 0.1% HCOOH (B), gradient: 0-5 min, 2-52% (B), 0-10 min, 5-

95% (B), T = 40 °C, λdet = 226-280-380 nm.

The stock solution concentrations were determined by UV-Vis spectroscopy using the following molar

extinction coefficient: ɛ280 (tryptophan) = 5500 M-1cm-1 (for Trp-containing peptides) and ɛ450 (C343) = 45000 M-

1cm-1 (for B) in water at pH 7.0.

The exact mass of Ac-C(Coum)-OH (3) was determined by Mariner ESI-TOF spectrometer (Perceptive

BioSystems) in positive (M+H) ion mode.

The cysteine containing peptides was stored at -20 °C in 10 mM CH3COONa buffer at pH 4.7.

The disulfide peptide (Ac-CW-OH)2 was obtained by stirring the monomer Ac-CW-OH overnight in a 0.1 M

solution of NaOH in mQ water in an open vessel at room temperature. The quantitative dimerization was

confirmed by UHPLC.

UHPLC chromatograms and MS spectra for all peptides are shown in Figures S1-S10

S5

Ac-CWD-OH

Figure S1. RP-UHPLC (Agilent RRHD Zorbax Eclipse Plus C18; 2-52% (B) in 5 min; 280 nm): 3.83 min.

Figure S2. MS (ESI+, ACN + HCOOH 0,1%): m/z = 465.1 ([M+H]+, calcd. 465.1399); 929.2 (2M+H)+; 951.2

(2M+Na)+.

-20

0

20

40

60

80

100

120

140

160

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Abso

rban

ce a

t 280

nm

(A.U

.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000 1250 1500 1750 2000

(%) I

nten

sity

Mass (m/z)

465.

1 46

7.1

466.

2 929.

2 95

1.2

S6

Ac-CW-OH


Figure S4. MS (ESI+, ACN + HCOOH 0,1%): m/z = 350.1 ([M+H]+, calcd. 350.1130); 699.3 (2M+H)+.

-200

0

200

400

600

800

1000

1200

1400

1600

1800

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Abso

rban

ce a

t 280

nm

(A.U

.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000

(%) I

nten

sity

Mass (m/z)

350.

1

699.

3

S7

(Ac-CW-OH)2


Figure S6. MS (ESI+, ACN + HCOOH 0,1%): m/z = 697.3 ([M+H]+, calcd. 697.2070).

-2000

200400600800

100012001400160018002000

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Abso

rban

ce a

t 280

nm

(A.U

.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000 1250 1500 1750 2000

(%) I

nten

sity

Mass (m/z)

697.

3 69

8.3

S8

Ac-WD-OH


.


-100

0

100

200

300

400

500

600

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Abso

rban

ce a

t 280

nm

(A.U

.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000

(%) I

nten

sity

Mass (m/z)

362.

1 36

3.1

723.

2

S9

Ac-C(tBut)WD-OH



-100

0

100

200

300

400

500

600

0 1 2 3 4 5 6 7 8 9 10

Rela

tive

Abso

rban

ce a

t 280

nm

(A.U

.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000 1250 1500 1750 2000

(%) I

nten

sity

Mass (m/z)

521.

2

1041

.4

S10

2a. Synthesis and characterization of Ac-C(Coum)-OH 3 The synthesis of Ac-C(Coum)-OH is based on literature procedures. 3

7-Diethylamino-4-formylcoumarin (1):

O ON

OH

O ON

7-Diethylamino-4-MethylCoumarin

SeO2

1,4-Dioxane, Rfx 6h

1

Procedure: Commercially available 7-Diethylamino-4-methylcoumarin (2 gr, 8,64 mmol, 1 eq.) was dissolved in 20 mL

1,4-dioxane by heating, SeO2 (1,15 gr, 10,37 mmol, 1.2 eq.) was added and the reaction mixture was

refluxed overnight. The hot mixture was filtered with celite to remove black Se and the filtrate was

concentrated in vacuo. The residue was dissolved in EtOAc and extracted with K2CO3 to remove carboxylic

acids. Then, Na2S2O5 was added to form the aldehyde bisulfate adduct. The pH of the aqueous layer was

adjusted to 10 using K2CO3 to release the aldehyde, which was then extracted with EtOAc. The organic

layer was dried over Na2SO4 and concentrated in vacuo to afford an orange-brown solid in 16 % yield,

(317,1 mg).4 Rf (TLC) 0,68 (AcOEt/EtPt 3:1).

1H-NMR: (δ ppm, CDCl3, 500 MHz): 1.19 (t, J=6.8 Hz, 6H); 3.43 (q, J=7.5 Hz, 4H); 6,44 (s,1H); 6.51 (s, 1H);

6.62 (d, J=10 Hz, 1H); 8.30 (d, J=10 Hz, 1H); 10.0 (s, 1H).

MS (ESI+, ACN + HCOOH 0,1%): m/z = 246.2 ([M+H]+, calcd. 246.1085).

3 N.Kotzur, B.Briand, M.Beyermann, V.Hagen, J. Am. Chem. Soc. 2009, 131, 16927-16931. 4 B.M.Peek, G.T.Ross, S.W.Edwards, G.J.Meyer, T.J.Meyer, B.W.Erickson, Int. J. Peptide Protein Res. 38, 1991, 114-123

S11

7-Diethylamino-4-(hydroxymethyl)coumarin (2):

O ON

OH

O ON

OH

NaBH4

MeOH, 25°C, 2h

1 2

Procedure: NaBH4 (83,2 mg, 2,2 mmol, 1.7 eq.) was added to aldehyde 1 (317,1 mg, 1,3 mmol) in MeOH, and the

mixture was stirred at RT for 2 h. The reaction mixture was diluted with H2O, acidified (pH 5) with HCl 1N

and extracted with 3 x 50 mL EtOAc. The combined organic layers were dried over Na2SO4 and

concentrated in vacuo to afford a yellow solid in 94% yield (296,7 mg). Rf (TLC) 0,50 (EtOAc/EtPt 3:1).

1H-NMR: (δ ppm, CDCl3, 500 MHz): 1.19 (t, J=6.8 Hz, 6H); 2.00 (s,1H); 3.42 (q, J=7.5 Hz, 4H); 4.84 (s, 2H);

6.32 (s, 1H); 6.62 (s, 1H); 6,69 (d, J=8,2 Hz, 1H); 7.36 (d, J=8.8 Hz, 1H).

MS (ESI+, ACN + HCOOH 0,1%): m/z = 248.2 ([M+H]+, calcd. 248.1242).

S12

[7-Diethylamino-coumarin-4-yl] methyl 4’-nitrophenyl carbonate (4):

O ON

OH

2

O ON

O

O

O

NO2

4

O

NO2O

Cl

EtN/Pr2, DCM, 25°C, 2h

Procedure: EtN/Pr2 (418 µL, 2,4 mmol, 2 eq.) and 4-nitrophenyl chloroformate (338,62 mg, 1,68 mmol, 1,4 eq. ) were

added to a solution of 2 (296,7 mg, 1,2 mmol) in 10 mL DCM. The mixture was stirred at RT overnight. The

crude mixture was used as such for the next reaction step.

[[7-Diethylamino-coumarin-4-yl] methoxycarbonyl]]-N-Acetyl-(L)-cysteine (3): Ac-C(Coum)-OH 3

O ON

O

O

O

NO2

4

O ON

O

O

SNH

COOH

3

N,Ac-(L)-Cys-OH

EtN/Pr2, DCM, 25°C, 20 h

O

Procedure: To a solution of [7-diethylamino-coumarin-4-yl] methyl 4’-nitrophenyl carbonate 4 (494,8 mg, 1,2 mmol) in DCM (10 mL) was added a solution of N,Ac-(L)-Cys-OH (293,7 mg, 1,8 mmol, 1,5 eq.) in DCM and EtN/Pr2

(313 µL, 1,8 mmol, 1,5 eq.). The reaction was stirred overnight at RT° and then evaporated. The crude

product was dissolved in AcOEt and extract with K2CO3. The acqueous layer was acidified with HCl 1M and

extract with AcOEt. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to

afford yellow solid. Further purification by preparative RP-HPLC using 20-60% (B) in (A), 0-20 min, afforded

3 that was used in the SPPS synthesis.

1H-NMR: (δ ppm, CDCl3, 500 MHz): 1.19 (t, J=7.0 Hz, 6H); 2.01 (s, 3H); 3.36 (dd, J=14.5 Hz, 1H); 3.43 (q,

J=6.9 Hz, 4H); 4.93 (dd, J=11.5 Hz, 1H); 5.26 (d, J=15.3 Hz, 1H); 5.48 (d, J=15.3 Hz, 1H); 6.14 (s, 1H); 6.61

(s, 1H); 6.72 (d, J=8.9 Hz, 1H); 7.31 (d, J=9.0 Hz; 1H); 7.49 (d, J=7.2 Hz, 1H).

S13

RP-UHPLC:


UV-Vis spectra:

Figure S12. UV-Vis spectrum in EtOH.

-500

0

500

1000

1500

2000

2500

3000

3500

0 2 4 6 8 10

mAU

Time (min)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

220 250 280 310 340 370 400 430 460 490

Abs

Wavelength

S14

Figure S13. MS (ESI, +, ACN + HCOOH 0,1%): m/z = 437.1340 ([M+H]+, calcd. 437.1377).

0.00E+00

1.00E+04

2.00E+04

3.00E+04

4.00E+04

5.00E+04

6.00E+04

150 250 350 450 550 650 750

% In

tens

ity

Mass (m/z)

438.1328

437.1378

439.1336 440.1355

S15

3. Characterisation data for Au NP 1

Even though the synthesis and characterization of Au NP 1 has already been described elsewhere5, we

provide here TEM, UV-vis and DLS data to demonstrate the reproducibility of their synthesis.

Figure S14. TEM analysis of Au NP 1.

Figure S15. UV-vis absorption spectrum of Au NP 1 (150 µM), [HEPES]=10mM, pH 7.0.

5 G.Pieters, A.Cazzolaro, R.Bonomi, L.J.Prins, Chem. Commun. 2012, 48, 1916-1918

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

200 300 400 500 600 700 800

Abso

rban

ce

Wavelength

S16

Figure S16: DLS of Au NP 1 in [HEPES]=10 mM, pH 7.0.

S17

4. Methods

4a. Surface saturation concentrations (SSCs) (Figure 2a) Fluorescence titrations were performed by adding increasing amounts of the fluorescent molecule to 3 mL of

an aqueous solution of Au NP 1 ([head group] ≈ 150 µM) in HEPES (10 mM, pH 7.0, 10% ACN, 37 °C) and measuring the fluorescence intensity after each addition (λex=280 nm, λem=360 nm, slits: 5/10 nm). The

SSCs were determined via extrapolation of the linear part of the curves of the respective titrations (Table

S1).

Table S1. The SSCs of fluorescent peptides

Peptide

SSC (µM)

Ac-CWD-OH 22.50 Ac-CW-OH 23.58 Ac-WD-OH 10.44

Ac-C(tBut)WD-OH 9.85 4b. Displacement studies (Figures 2b+4c) The displacement experiments were performed by adding consecutive amounts of peptides (up to 80 µM) to

3 mL of an aqueous solution of Au NP 1 ([head groups] ≈ 150 µM) and A (10 μM) in HEPES (10 mM, pH 7.0, 10% ACN, 37°). The following instrument settings were used: λex = 350 nm, λem = 450 nm, slits = 5/10 nm. The light-triggered displacement experiment was performed by first adding consecutive amounts of Ac-

C(Coum)-OH 3 (up to 30 µM) to a 3 mL buffered aqueous solution of Au NP 1 [≈150 µM head groups] and B (10 μM) in HEPES (10 mM, pH 7.0, 10% ACN, 37°). After a concentration of 30 μM of 3 had been reached the cuvette was irradiated at 365 nm for 15 minutes after which the fluorescence intensity at 493 nm was

measured. The following instrument settings were used: λex = 450 nm, λem = 493 nm, slits = 2.5/5 nm.

4c. Kinetic studies of the displacement process (Figure 2c) The rate of the exchange process was followed by adding 50 µM of either Ac-CWD-OH, Ac-CW-OH or Ac-C-

OH to a 3 mL aqueous buffered solution of Au NP 1 [≈150 µM head groups] and A (10 µM) in HEPES (10

mM, pH 7.0, 10% ACN, 37°C). After addition, the fluorescence intensity was measured for 40 minutes. The

following instrument settings were used: λex = 350 nm, λem = 450 nm, slits = 5/10 nm).

S18

5. Ultrafiltration experiments

Ultrafiltration experiments were performed using Vivaspin 500 filter units having a 10 kDa MWCO PES-

membrane (filtrations were performed for 90 sec at 12000 rpm for each sample leading to around 250 μl of

dialysate). The dialysates were subsequently analyzed by LC-MS in Single Ion Mode following the SIM

channels of (Ac-CW-OH)2 (m/z=350.1), Ac-CW-OH (m/z=697.2) and trimethylammonium thiol (m/z=205.2).

The following conditions were used: flow 0.4 mL/min, eluents: H2O + 0.1% HCOOH (A), ACN + 0.1%

HCOOH (B), gradient: 0-5 min 2-52% (B). The final quantification of the concentration of the molecules was

obtained by comparing the signal intensities with the respective calibration curves.

The chromatograms reported in Figure 3 of the manuscript were obtained by filtrating solutions of (a) 5 µM

(Ac-CW-OH)2 or (b) 10 µM Ac-CW-OH in 500 µL buffered aqueous solution (10 mM HEPES, pH 7.0, 10%

ACN) containing I) 5 equivalent of probe A, II) 5 equivalent of probe A and Au NP 1 [≈150 µM head groups], and III) a solution in which 5 equivalent of probe A was added after addition of 5 µM (Ac-CW-OH)2 or (b) 10 µM Ac-CW-OH to Au NP 1.

Solutions were typically filtrated 10 minutes after preparation. Each experiment was performed in triplicate.

The chromatograms reported in Figure 3 of the manuscript are representative examples.

S19

6. Compound structures and masses (Figure 3)

SH

HN

ONH

OOH

O

NH

Ac-CW-OH

S

NHO

HNO

OHO

NH

S

HNO

NHO

HOO

NH

(Ac-CW-OH)2m/z: 697,2 m/z: 350,1

SS

HN

O

HN

OOH

NH

ON

HSN

m/z: 552,2

m/z: 205,2

SNS

N

m/z: 407,3

Heterodimer

Trimethylammonium Thiol (Trimethylammonium Thiol)2

S20

7. Fluorescence displacement of (Ac-CW-OH)2 by probe A

Figure S14. Amount of displaced (Ac-CW-OH)2 as a function of the amount of probe A added to a solution of Au NP 1 ([head groups] ≈ 150 µM] and (Ac-CW-OH)2 (5µM) in HEPES (10 mM, pH 7.0, 10% ACN, 37°C). Instrument settings: λex = 280 nm, λem = 360 nm, slits = 5/10 nm. The amount of displaced (Ac-CW-OH)2 after the addition of 25 μM of A was calculated by dividing the

measured fluorescence intensity by the expected fluorescence intensity for a full release of (Ac-CW-OH)2.

The latter value was obtained using the slope of the linear part of the surface saturation profile.

0

50

100

0 10 20 30 40 50

% (A

c-CW

-OH)

2 disp

lace

d

[Probe A] (µM)

S21

8. Concentration of Ac-CW-OH and thiol 2 in the dialysate as a function of the amount of Ac-CW-OH added to Au NP 1.

A series of samples were prepared containing containing Au NP 1 [≈150 µM head groups] and different

amounts of Ac-CW-OH (5, 10, 15, 25, 30, 40 and 50 µM) in HEPES (10 mM, pH 7.0, 10% ACN). To these

solutions probe A (50 µM) was added after 10 minutes, and next the samples were filtered using the procedure described above. The dialysates were analyzed by UHPLC using ES-MS in SIM mode. Flow 0.4

mL/min, eluents: H2O + 0.1% HCOOH (A), ACN + 0.1% HCOOH (B), gradient: 0-5 min 2-52% (B).

Concentrations were determined using calibration curves.

Figure S15. Concentrations of Ac-CW-OH (blue circles) and thiol 2 (green circles) in the dialysate as a function of the concentration of Ac-CW-OH. The dotted lines are trendlines.

Figure S16. UHPLC calibration curve for Ac-CW-OH.

0

5

10

15

20

25

0 10 20 30 40 50 60

[Ac-

CW-O

H] (o

) and

[Thi

ol 2

] (o)

(µM

)

[Ac-CW-OH] (µM)

Ac-CW-OH Trimethylammonium Thiol

y = 89658x + 51982 R² = 0.9971

0.E+00

5.E+05

1.E+06

2.E+06

2.E+06

3.E+06

3.E+06

4.E+06

4.E+06

5.E+06

5.E+06

0 10 20 30 40 50 60

SIM

Area

Inte

nsity

[Ac-CW-OH] (µM)

S22

Figure S17. UHPLC calibration curve for thiol 2.

y = 43086x + 92803 R² = 0.9712

0.E+00

5.E+05

1.E+06

2.E+06

2.E+06

3.E+06

0 10 20 30 40 50 60

SIM

Are

a In

tens

ity

Thiol 2 (µM)

S23

9. Impurity at the SIM-channel of thiol 2 originating from the membrane

The peaks marked with an asterisk in Figure 3 of the manuscript originate from an (unidentified) impurity as

evidenced by the chromatogram of the dialysate of a solution containing just HEPES (10 mM).

Figure S18. Chromatogram (detection at the thiol 2 SIM channel) of the dialysate of a filtered solution of an aqueous solution of HEPES (10 mM, pH = 7.0, 10% ACN) (red line). LC-MS conditions: flow 0.4 mL/min, eluents: H2O + 0.1% HCOOH (A), ACN + 0.1% HCOOH (B), gradient: 0-5 min 2-52% (B).

0.E+00

1.E+04

2.E+04

3.E+04

4.E+04

5.E+04

6.E+04

7.E+04

8.E+04

0 1 2 3 4 5 6 7 8 9 10

(m/z

=205

.2)

Time (min)

S24

10. Fluorescence titration of probe A before and after exposure of Au NP 1 to Ac-CW-OH Probe A was titrated to Au NP 1 ([head groups] ≈150 µM) after exposure of Au NP 1 to Ac-CW-OH (23.5 μM). Comparison of the obtained profile with the one obtained from a titration of A to Au NP 1 (without Ac-CW-OH) shows that after insertion of Ac-CW-OH in the monolayer A the SSC of probe A is strongly

diminished.

Figure S19. Fluorescence intensity as a function of the amount of probe A added to a solution of Au NP 1 [≈150 µM head groups] (red circles) or Au NP 1 [≈150 µM head groups] and Ac-CW-OH (23.5 µM) (blue circles) in HEPES (10 mM, pH = 7.0, 10% ACN, 37°C). Instrument settings: λex = 350 nm, λem = 450 nm, slits = 5/10 nm.

0

200

400

600

800

1000

0 10 20 30 40 50

FI 45

0 nm

(a.u

.)

[probe A] (µM)

After Exchangewith Ac-CW-OH(SSCs)

StandardConditions

S25

11. Light-triggered decay of compound Ac-C(Coum)-OH (3) A solution of Ac-C(Coum)-OH 3 (50 μM) in HEPES (10 mM, pH 7.0, 10% ACN) was irradiated at 365 nm. At

regular intervals samples were taken and analyzed by UHPLC (both DAD and SIM detection, 2-72% (B) in 5

min, λdet = 400 nm). Ac-C(Coum)-OH completely had disappeared after 15 minutes of irradiation, which was

accompanied by the formation of CoumOH and Ac-C-OH (in 1:1 ratio) and the thioether side product (see

next section). The quantification of the concentrations of 3 and CoumOH using respective calibration curves

showed a 60% yield which is in accordance with the literature.

Figure S20. Concentrations of Ac-C(Coum)-OH and Coum-OH as a function of the irradiation time at 365 nm monitored by a DAD at 385 nm (a) or by MS in SIM mode (b). Repetition of the experiment irradiating at 400 nm showed a more efficient decomposition of 3.

Figure S21. Concentrations of Ac-C(Coum)-OH and Coum-OH as a function of the irradiation time at 400 nm.

0

10

20

30

40

50

60

0 5 10 15 20

[Ac-

C(Co

um)-O

H] a

nd [C

oum

OH]

(µM

)

Time (min)

Ac-C(Coum)-OH

CoumOH

0

10

20

30

40

50

60

0 5 10 15 20[Ac-

C(Co

um)-O

H] a

nd [C

oum

OH]

(µM

)

Time (min)

Ac-C(Coum)-OH

CoumOH

0

10

20

30

40

50

60

0 5 10 15 20

[Ac-

C(Co

um)-O

H] a

nd [C

oum

OH]

(µM

)

Time (min)

Ac-C(Coum)-OH at 400 nm

CoumOH at 400 nm

a) b)

S26

Formation of the thioether side-product was confirmed by LC-MS (Figure Y)

O ON

S OHNH

O

O

Figure S22. Thioether side-product structure.

Figure S23. UHPLC profile of the reaction mixture after irradiation of Ac-C(Coum)-OH 3.

Figure S24. ESI-MS spectra of the reaction mixture after irradiation of Ac-C(Coum)-OH 3 at 365 nm.

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7 8 9 10

Rela

tvie

Abs

orba

nce

at 3

85 n

m (A

.U.)

Time (min)

0

20

40

60

80

100

120

0 250 500 750 1000 1250 1500

(%) I

nten

sity

Mass (m/z)

Ac-C(Coum)-OH

CoumOH

Thioether

248.

2

393.

2

437.

1

S27

12. Binding of Ac-C(Coum)-OH 3 to Au NP 1

Figure S25. Fluorescence intensity as a function of the amount of probe 3 added to a solution of Au NP 1 [≈150 µM head groups] in HEPES (10 mM, HEPES pH 7.0, 10% ACN, 37°C). Instrument settings: λex = 385 nm, λem = 495 nm, slits = 10/20 nm.

0

200

400

600

800

1000

0 5 10 15 20

FI 49

5 nm

(a.u

.)

[Ac-C(Coum)-OH] (µM)

S28

13. Fluorescence spectra of Au NP 1 + probe B + Ac-C(Coum)-OH before and after irradiation

Figure S26. Fluorescence emission spectra before and after irradiation of a mixture of Au NP 1, probe B, and Ac-C(Coum)-OH. Experimental conditions: Au NP 1 ([head groups] ≈ 150 µM); 3 mL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN); 10 µM probe B; 30 µM; Ac-C(Coum)-OH 58 µM; T=37°C; λex = 450 nm, λem = 493 nm, slits = 2.5/5 nm. Irradiation conditions as indicated in the Figure.

0

100

200

300

400

500

450 500 550

F.I.

493

nm(a

.u.)

Wavelength (nm)

AuNP 1 + probe B

Au NP 1 + probe B + Ac-C(Coum)-OH

after 5 min irradiation at 365 nm




S29

14. Fluorescence emission of Ac-C(Coum)-OH in the presence of Au NP 1

Even in the absence of binding, the presence of Au NP 1 causes a decrease in fluorescence intensity of fluorophores cause by the absorption of light by the gold nanoparticles. This is illustrated by the titration of Au NP 1 to Ac-C(Coum)-OH (Figure S26). However, this effect is much weaker compared to the fluorescence quenching upon binding of fluorophores to the surface of Au NP 1 (see for example Figure 2a in the manuscript).

Figure S27. Fluorescence emission of Ac-C(Coum)-OH in the presence of increasing amounts of Au NP 1.

0

100

200

300

400

500

450 460 470 480 490 500 510 520 530 540 550

F.I.

493

nm (a

.u.)

Wavelength

Ac-C(Coum)-OH [5uM]

+ Au NP 1 [4,4 uM]

+ Au NP 1 [8.87uM]

+ Au NP 1 [13.28uM]

+ Au NP 1 [17.68uM]

+ Au NP 1 [22.06uM]

+ Au NP 1 [26.43uM]

+ Au NP 1 [30.79uM]

+ Au NP 1 [35.13uM]

+ Au NP 1 [39.45uM]

+ Au NP 1 [43.77uM]

+ Au NP 1 [48.06uM]

+ Au NP 1 [50.64uM]

S30

15. Influence of ionic strength on the binding of Ac-CWD-OH to Au NP 1

The SSC of Ac-CWD-OH on Au NP 1 is not affected by the presence of 1 mM of NaCl (Figure S28).

Figure S28. Fluorescence titrations of Ac-CWD-OH to Au NP 1 in the presence (red) or absence (black) of 1 mM NaCl. Au NP 1 ([head group] ≈ 150 µM), 3 mL aqueous buffered solution (10 mM HEPES, pH 7.0; 10% ACN, T=37°C). λex=280 nm, λem=360 nm, slits = 5/10 nm.

0

100

200

0 10 20 30 40 50

F.I.

360

nm (a

.u.)

Peptide (µM)

S31

16. Exchange of thiol 2 by Ac-C-NH2

The displacement of thiol 2 by neutral thiol Ac-C-NH2 was investigated also by LC/MS after ultracentrifugation. Comparison of the obtained intensities with those measured using Ac-CW-OH at the

same concentration (50 μM) confirms the inefficient displacement of thiol 2 by Ac-C-NH2.

Figure S29. Ultrafiltration experiments followed by LC/MS upon the addition to Ac-CW-OH (black) or Ac-C-

NH2 to Au NP 1. Au NP 1 ([head groups] ≈ 150 µM); 500 µL aqueous buffered solution (10 mM HEPES pH

7.0; 10% ACN); 50 µM Ac-CW-OH or 50 µM Ac-C-NH2;LC-MS method: flow 0.4 mL/min, eluents: H2O +

0.1% HCOOH (A), ACN + 0.1% HCOOH (B), gradient: 0-10 min 5-95% (B).

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

0 1 2 3 4 5 6 7 8 9 10

SIM

Inte

nsity

Time (min)

C8 Displaced by Ac-CW-OH

C8 Displaced by Ac-C-NH2*

S32

17. Experimental details for the Figures in the manuscript.

Figure 2a: Au NP 1 ([head group] ≈ 150 µM), 3 mL aqueous buffered solution (10 mM HEPES, pH 7.0; 10% ACN, T=37°C). λex=280 nm, λem=360 nm, slits = 5/10 nm.

Figure 2b: Au NP 1 ([head group] ≈ 150 µM); 3 mL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN, T=37°C); 10 µM probe A; λex = 350 nm, λem = 450 nm, slits = 5/10 nm.

Figure 2c: Au NP 1 ([head group] ≈ 150 µM); 3 mL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN, T=37°C); 10 µM probe A; λex = 350 nm, λem = 450 nm, slits = 5/10 nm. Figure 3: Au NP 1 ([head group] ≈ 150 µM); 500 µL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN); 25 µM probe A to 5 µM (Ac-CW-OH)2; 50 µM probe A to 10 µM Ac-CW-OH. Ultrafiltration at 12’000

rpm for 90 sec.

LC-MS method: flow 0.4 mL/min, eluents: H2O + 0.1% HCOOH (A), ACN + 0.1% HCOOH (B), gradient: 0-5

min 2-52% (B).

Figure 4c: Au NP 1 ([head groups] ≈ 150 µM); 3 mL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN); 10 µM probe B; 30 µM Ac-C(Coum)-OH 58 µM; N-Ac-Cys-OH. T=37°C; λex = 450 nm, λem = 493 nm, slits = 2.5/5 nm. Irradiation conditions: 5’ at 365 nm, (time 0-20 min).

Figure 4d: Au NP 1 ([head groups] ≈ 150 µM); 500 µL aqueous buffered solution (10 mM HEPES pH 7.0; 10% ACN); 50 µM Ac-C(Coum)-OH; 50 µM N,Ac-Cys-OH; Ultrafiltration at 12’000 rpm for 90 sec.

LC-MS method: flow 0.4 mL/min, eluents: H2O + 0.1% HCOOH (A), ACN + 0.1% HCOOH (B), gradient: 0-10

min 5-95% (B).

Documents

Supporting Information · The column used for separation was a Jupiter ProteoAXIA Packed 4µ 90Å 250 x 21.2 mm, flow: 20 mL/min, eluents: H. 2. O + 0.1% TFA (A), CH. 3. CN + 0.1%