Supplementary Information
Ubiquinone-quantum dot bioconjugates for in vitro
and intracellular complex I sensing
Wei Ma1†, Li-Xia Qin1†, Feng-Tao Liu2, Zhen Gu1, Jian Wang2, Zhi Gang Pan3, Tony D. James4 & YiTao Long1*
1State
Key Laboratory of Bioreactor Engineering & Department of Chemistry, East China University of
Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China, 2Department of
Neurology, Huashan Hospital Affiliated to Fudan University, 12 Wulumuqi Zhong Road, Shanghai,
200040, P. R. China, 3Department of Medicine, Zhongshan Hospital Affiliated to Fudan University, 180
Fenglin Road, Shanghai, 200032, P. R. China, 4Department of Chemistry, University of Bath, Claverton
Down, Bath, BA2 7AY, UK.
Correspondence and requests for materials should be addressed to Y.T.L. (ytlong@ecust.edu.cn)
†These
authors contributed equally to this work.
Table of Contents
S1. Experimental section
S2. Result and Discussion
S3. Synthetic procedures of surface-capping QnNS ligands
S4. References
1
S1. Experimental section
Material. All analytical grade reagents were purchased from Sigma-Aldrich. The rotenone was
purchased from Sigma-Aldrich. Water soluble CdSe/ZnS core-shell QDs coating with surface functional
carboxylic acid group (emission maxima centered at ~550 nm) were purchased from Ocean NanoTech,
LLC. All electrodes for electrochemical experiments were purchased from Shanghai Chenhua Co., Ltd.,
China. N2 (99.998%, prepurified) was obtained from Cryogenic Gases (Detroit, MI). All chemical
reagents for synthesis were of analytical grade, obtained from commercial suppliers, and used without
further purification unless otherwise noted. Millipore water (Milli-Q system) purified to a resistivity of
18.2 MΩ·cm with a UHQ II system (Elga) was used to prepare all the solutions. Phosphate buffer
solution (PBS) was prepared using Na2HPO4 and NaH2PO4. The electrolyte solutions were prepared
with Millipore water and deaerated by purging with N2 for 5 min before experiment.
Apparatus. Fluorescence emission and excitation spectra were obtained with a Shimadzu Cary Eclipse
(Varian) fluorometer. A Xenon lamp light source (LE-XM500, LEO Photonics tech Co. Ltd. China) was
used. The cell was irradiated with a Xenon lamp operated at 500 W, through a home-made light
chopper. The shutter chopping frequency was controlled manually. The pH value of a solution was
measured by a PHS-3C (Switzerland Mettler Toledo Delta 320 pH meter). Desktop multi-function
centrifugal ultrafilters were used as received (Eppendorf-5430, St. Co. Germany). A CHI660
electrochemical workstation (Shanghai Chenhua Co., Ltd., China) equipped with a stirring machine (CH
Instruments Inc). All fluorescence images of cellular QDs were acquired with the same parameters with
a wide-field inverted fluorescence microscopy (Nikon-Ti, Co. Ltd. Japan) using a 60× 1.2 NA objective.
Preparation for the functionalised CdSe/ZnS QDs. The same size of 5 μM CdSe/ZnS QDs were
incubated with the same amount of 1 mM QnNS in 0.2 M PBS of pH 8.0 and shaken vigorously for 30
min controllably self-assembled onto CdSe/ZnS QDs to form QnNS-QDs bioconjugates. After adding
acetone with a volume ratio of 1:1 and the solution was centrifuged at 7840 rpm for 20 min, resulting in
a pellet of CdSe/ZnS nanocrystals to remove supernatant, and purified for three times. The obtained
QDs bioconjugates were dissolved in PBS and waiting for the various tests.
Electrochemistry. A standard jacketed three-electrode cell was used for electrochemistry. A Pt
electrode and saturated calomel electrode (SCE) electrode were used as counter electrode and reference
electrode, respectively. Electrochemistry measurements of QnNS and QnNS-modified CdSe/ZnS QDs
were carried out at glassy carbon electrode in 0.2 M PBS solution of pH 8.0. For all electrochemical
experiments, oxygen was removed by bubbling N2 through the solution prior to making measurements.
The temperature was controlled by using 25°C circulating water bath through the outer cell jacket.
In-situ UV-vis spectroelectrochemical experiments. A quartz cell featured platinum mesh working
electrode (Alfa Aesar, MA, USA, 99.9%, 52 mesh, open area = 62.7%, wire diameter = 0.102 mm) and
2
salt bridge to a platinum wire counter electrode and a Ag/AgCl reference electrode via an Ocean Optics
DT-minutesi-2 halogen resource and USB2000+ spectrometer. The mesh electrode was aligned such
that the UV-vis beam passed through the centre of the working electrode. Potentials during the UV-vis
spectral measurement were controlled by CHI 1232A electrochemical workstation (Shanghai Chenhua
Co., Ltd., China). Incident laser light was positioned in such a manner that the laser-electrode junction
was the same as the UV-vis beam-electrode junction. In the process of tracing in vitro complex I,
NADH was added to Q5NS-functionalised QDs PBS solution in the thin layer quartz cell (optical path
length is 0.4 ± 0.05 mm). To the above quartz cell, complex I was added to the top of the cuvette very
slowly to minimize mixing. As the enzyme and NADH diffused into the beam path the conversion of
ubiquinone to ubiquinol was assessed by the absorption spectra change in the ubiquinone and NADH.
All prepared solutions were deoxygenated prior to each experiment and the cell was kept under a N2
atmosphere throughout the experiment.
S2. Results and Discussion
Figure S1 | Absorption spectra of oxidized QnNS-functionalised CdSe/ZnS QDs (red line) and reduced
HQnS-functionalised CdSe/ZnS QDs (black line).
In situ UV-vis spectroelectrochemical experiments were employed in order to confirm the presence of
a reduced ubiquinol capping layer on QDs’ surface. Before reduction, Q2NS, Q5NS and Q10NS-QDs
exhibited intense absorptions at 260, 263 and 267 nm, and the relative weak and broad absorptions at
515, 510 and 510 nm which are indications of the π-π* and n-π* electronic transitions of the ubiquinone
rings, respectively (see Fig. S1, red line). Upon applying a potential of -0.30 V vs. SCE for 50, 55 and 60
s, the absorption bands were replaced by a new strong absorbance at 285, 290 and 300 nm, which are
assigned to the π-π* absorption spectra of ubiquinone converting to ubiquinol (see Fig. S1, black line).
Re-applying an oxidizing potential of 0.10 V vs. SCE for 50, 55 and 60 s, the absorption band of
ubiquinol disappeared while the characteristic absorptions of ubiquinone at 260, 263 and 267 nm
3
returned (see Fig. S1, red line). The spectroelectrochemical results show that transformation of surface
capping layer between ubiquinone and ubiquinol can be modulated effectively by electrochemistry.
Figure S2 | Time-dependent fluorescence changes upon the interaction of (a) Q1S, (b) Q5S and (c)
Q10S-functionalised CdSe/ZnS QDs with complex I (4.9 U) in the presence of 10 mM NADH. The
insets illustrate the increase of fluorescence (∆F) as a function of incubation time. All measurements
were performed in a 0.2 M deaerated PBS solution of pH 8.0.
4
Figure S3 | Cyclic voltammetry of 0.5 mM free QnNS on glassy carbon electrode in PBS buffer solution
of pH 8.0 at a scan rate of 100 mV·s−1. (a) Q2NS; (b) Q5NS; (c):Q10NS. SCE is saturated calomel
electrode.
Figure S4 | Cytotoxicity of Q2NS-QDs labelled SH-SY5Y cells after 24 hr of exposure to Q2NS-QDs at
concentrations ranging from 3.75 to 200 μg·ml-1 for different rotenone damaged SH-SY5Y cells.
5
S3. Synthetic procedures of surface-capping QnNS ligands.
All reactions were monitored by thin layer chromatography (TLC) using silica-coated plates and
visualizing under UV light. THF was distilled from sodium benzophenone ketyl. Proton magnetic
resonance (1H NMR) spectra were recorded on Bruker FT (500MHz) NMR spectrometers at 25°C.
Proton chemical shifts are expressed in parts per million (ppm, δ scale) and are referenced to residual
protium in the NMR solvent (CHCl3, δ 7.26). Data are represented as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet). Carbon nuclear magnetic resonance spectra (13C NMR)
were recorded on Bruker FT (126 MHz) NMR spectrometers at 25°C. Carbon chemical shifts are
expressed in parts per million (ppm, δ scale) and are referenced to the carbon resonances of the NMR
solvent (CDCl3, δ 77.0). Mass spectra (TOF MS EI+) were recorded under electron impact.
Scheme S1 | Synthesis of 6-(2’-propynylamino) ubiquinone 1.
6-(2’-propynylamino) ubiquinone (1): To a stirred solution of coenzyme Q0 (1.82 g, 10 mmol, 1.0
equiv) in MeOH (5 ml) was added dropwise of 2-propynylamine (0.28 g, 5 mmol, 0.5 equiv) in 10 ml of
MeOH over 10 min. The reaction mixture was stirred for 4 hr at room temperature1. After 4 hr, the
reaction mixture was then added with H2O (5 ml) and the biphasic reaction mixture was extracted with
EtOAc (3 × 5 ml). The combined organic layers were washed with brine (15 ml), dried (MgSO4), and
concentrated. Flash column chromatography gave terminal alkyne-substituted ubiquinone 1 (1.05 g, 4.5
mmol, 45% yield) as a purple solid. 1H NMR (500.0 MHz, CDCl3): δ 5.65 (br s, 1H, -NH-), 4.20 (d, 2H,
-CH2-), 4.08 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 2.34 (s, H, -C≡ CH), 2.08 (s, 3H, -CH3); 13C NMR
(126 MHz, CDCl3): δ 183.5, 181.5, 147.0, 142.0, 140.5, 109.0, 80.0, 73.5, 61.5, 35.5, 10.5; TOF MS
EI+: 235.1, Found: 235.1.
6
Figure S5 | 1H NMR spectrum of 6-(2’-propynylamino) ubiquinone 1 recorded in CDCl3
Figure S6 | 13C NMR spectrum of 6-(2’-propynylamino) ubiquinone 1 recorded in CDCl3
Figure S7 | Mass spectum (EI) of 6-(2’-propynylamino) ubiquinone 1
7
Scheme S2 | Synthesis of bis-n-azidoalkyl disulfide 4a, 4b and 4c.
2-(Thioacetoxy)-1-bromoethane (2a): A solution of 1, 2-dibromoethane (3.73 g, 20 mmol, 1.0 equiv)
and potassium thioacetate (1.14 g, 10 mmol, 0.5 equiv) in anhydrous THF (50 ml) was refluxed for 10
hr under N2 atmosphere2. After cooling to room temperature, the KBr was removed by vacuum
filtration. The solvent in the filtrate was removed under reduced pressure in a rotary evaporator. The
residue was purified with column chromatography to give 2a (2.74g, 15 mmol, 75% yield) as a colorless
oil. 1H NMR (500.0 MHz, CDCl3): δ 3.42 (t, 2H, -CH2Br), 3.27 (t, 2H, -SCH2-), 2.35 (s, 3H, -COCH3).
Figure S8 | 1H NMR spectrum of 2-(thioacetoxy)-1-bromoethane 2a recorded in CDCl3
5-(Thioacetoxy)-1-bromopentane (2b): Synthetic step of 2b is similar to afford 2a. 1H NMR (500.0
MHz, CDCl3): δ 3.40 (t, 2H, -CH2Br), 2.88 (t, 2H, -SCH2-), 2.30 (s, 3H, -COCH3), 1.87 (quint, 2H, CH2-), 1.48-1.65 (m, 4H, 2×-CH2-).
8
Figure S9 | 1H NMR spectrum of 5-(thioacetoxy)-1-bromopentane 2b recorded in CDCl3
10-(Thioacetoxy)-1-bromodecane (2c): Synthetic step of 2c is similar to afford 2a. 1H NMR (500.0
MHz, CDCl3): δ 3.42 (t, 2H, -CH2Br), 2.85 (t, 2H, -SCH2-), 2.30 (s, 3H, -COCH3), 1.82 (quint, 2H, CH2-), 1.58 (quint, 2H, -CH2-), 1.25-1.45 (m, 12H, 6×-CH2-).
Figure S10 | 1H NMR spectrum of 10-(thioacetoxy)-1-bromodecane 2c recorded in CDCl3
2-(Thioacetoxy)-1-azidoethane (3a): The compound 2a (1.82 g, 10 mmol, 1.0 equiv) was added to a
solution of sodium azide (5.85 g, 90 mmol, 9.0 equiv) in dimethylformamide (DMF, 100 ml). The
mixture was heated to 60°C for 10 hr3. The salt NaBr was filtered off, DMF was removed under vacuum
and the resulting crude mixture was dissolved in CH2Cl2 and washed with brine (3 × 50 ml), dried
(MgSO4), and concentrated. Flash column chromatography gave 3a (1.09 g, 7.5 mmol, 75% yield) as a
9
light yellow oil. 1H NMR (500.0 MHz, CDCl3): δ 3.45 (t, 2H, -CH2S-), 3.05 (t, 2H, -NCH2-), 2.30 (s,
3H, -COCH3).
Figure S11 | 1H NMR spectrum of 2-(thioacetoxy)-1-azidoethane 3a recorded in CDCl3
5-(Thioacetoxy)-1-azidopentane (3b): Synthetic step of 3b is similar to afford 3a. 1H NMR (500.0
MHz, CDCl3): δ 3.27 (t, 2H, -CH2S-), 2.86 (t, 2H, -NCH2-), 2.38 (s, 3H, -COCH3), 1.39-1.60 (m, 6H, 3
×-CH2-).
Figure S12 | 1H NMR spectrum of 5-(thioacetoxy)-1-azidopentane 3b recorded in CDCl3
10
10-(Thioacetoxy)-1-azidodecane (3c): Synthetic step of 3c is similar to afford 3a. 1H NMR (500.0
MHz, CDCl3): δ 3.28 (t, 2H, -CH2S-), 2.85 (t, 2H, -NCH2-), 2.33 (s, 3H, -COCH3), 1.55-1.62 (m, 4H, 2
× -CH2-), 1.25-1.42 (m, 12H, 6 × -CH2-).
Figure S13 | 1H NMR spectrum of 10-(thioacetoxy)-1-azidodecane 3c recorded in CDCl3
Bis-2-azidoethane disulfide (4a): A solution of 3a (0.73 g, 5.0 mmol), methanol (40 ml), and 10 ml of
concentrated HCl (35 wt % in water) was heated at 45°C for 3 hr under the protection of nitrogen4. The
mixture was diluted with water and was extracted with methylene chloride (3 × 40 ml). The combined
organic layers were washed with brine (150 ml), dried (MgSO4). The extract was evaporated to get 2azido ethanethiol. The obtained crude product was used in the next reaction without further purification.
To a stirred solution of 2-azido ethanethiol (5.0mmol, 1.0 equiv) in EtOAc (15 ml) was added I2 (12.5
mg, 0.05 mmol, 0.01 equiv) and 30% H2O2 (0.6 ml, 5.0mmol, 1.0 equiv) and the mixture was stirred at
room temperature for 1 hr5. Saturated aqueous Na2S2O3 solution (75 ml) was added, and the resulting
mixture was extracted with EtOAc (3 × 50 ml). The combined organic phases were washed with brine
(50 ml) and dried (MgSO4). The solvent was evaporated, and the residue was purified by silica gel
column chromatography to afford 4a (0.18 g, 42%) as an light yellow oil. 1H NMR (500.0 MHz,
CDCl3): δ 2.74 (t, 2H, -CH2N-) 3.26 (t, 2H, -CH2S-); TOF MS EI+: 204.0, Found: 204.0;
11
Figure S14 | 1H NMR spectrum of bis-2-azidoethane disulfide 4a recorded in CDCl3
Figure S15 | Mass spectum (EI) of bis-2-azidoethane disulfide 4a
Bis-5-azidopentane disulfide (4b): Synthetic step of 4b is similar to afford 4a. 1H NMR (500.0 MHz,
CDCl3): δ3.25 (t, 2H, -CH2S-), 2.68 (quint, 2H, -CH2S-CH2-), 1.45-1.75 (m, 6H, 3 × -CH2-); TOF MS
EI+: 288.1, Found: 288.1.
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Figure S16 | 1H NMR spectrum of bis-5-azidopentane disulfide 4b recorded in CDCl3
Figure S17 | Mass spectum (EI) of bis-5-azidopentane disulfide 4b
Bis-10-azidodecane disulfide (4c): Synthetic step of 4c is similar to afford 4a. 1H NMR (500.0 MHz,
CDCl3): δ3.27 (t, 2H, -CH2S-), 2.69 (quint, 2H, -CH2S-CH2-), 1.57-1.70 (m, 4H, 2 × -CH2-), 1.27-1.45
(m, 6H, 3 × -CH2-); TOF MS EI+: 428.3, Found: 428.3.
13
Figure S18 | 1H NMR spectrum of bis-10-azidodecane disulfide 4c recorded in CDCl3
Figure S19 | Mass spectum (EI) of bis-10-azidodecane disulfide 4c
Scheme S3 | Synthesis of ubiquinone-terminated disulphides QnNS.
Ubiquinone-terminated disulphides (QnNS): The copper(II) complex was prepared by adding
copper(II) sulfate pentahydrate to a DMSO solution of the tris-(benzyltriazolylmethyl)amine (TBTA).
The overall volume in each test tube was 2 ml (DMSO: H2O = 2:1), containing a solution of alkyne 1
(1.0 equiv) and azide 4 (1.0 equiv). Sodium ascorbate (0.04 equiv) of freshly prepared 1 M solution in
water was added, followed by the addition of copper(II) sulfate TBTA (0.01 equiv)6. The resulting
reaction was vigorously stirred for 30 min at room temperature. The reaction mixture was extracted with
EtOAc (3 × 5 ml). The combined organic layers were washed with brine (15 ml), dried (MgSO4), and
concentrated. Flash column chromatography gave target compounds QnNS.
S4. References
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Chem. 29, 1792-1795 (1986).
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(S2) Porter-Peden, L., Kamper, S. G., Wal, M. V.,Blankespoor, R. & Sinniah, K. Estimating Kinetic and
Thermodynamic Parameters from Single Molecule Enzyme-Inhibitor Interactions. Langmuir 24, 1155611561 (2008)
(S3) Sommer W. J. & Weck, M. Facile functionalization of gold nanoparticles via microwave-assisted
1, 3 dipolar cycloaddition. Langmuir 23, 11991-11995 (2007).
(S4) Hong, H. G. & Wonchoul, P. Electrochemical characteristics of hydroquinone-terminated selfassembled monolayers on gold. Langmuir 17, 2485-2492 (2001).
(S5) Kirihara, M., Asai, Y., Ogawa, S., Noguchi, T., Hatano, A. & Hirai, Y. A mild and environmentally
benign oxidation of thiols to disulfides. Synthesis 21, 3286-3289 (2007).
(S6) Chan, T. R., Hilgraf, R., Sharpless, K. B. & Fokin, V. V. Polytriazoles as copper (I)-stabilizing
ligands in catalysis. Org. Lett. 6, 2853-2855 (2004).
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