Cellular Localisation of Antitumoral 6-Alkyl Thymoquinones Revealed by an Alkyne-Azide Click Reaction and the Streptavidin-Biotin System

Article abstract of DOI:10.1002/cbic.201000762

The subcellular distribution and accumulation of thymoquinone 1, a natural anticancer agent, has hitherto been unknown. We prepared 6-(dec-9-ynyl)thymoquinone 3, an alkyne-labelled derivative with anticancer activity similar to that of its parent compound

Full text of DOI:10.1002/cbic.201000762

DOI: 10.1002/cbic.201000762
Cellular Localisation of Antitumoral 6-Alkyl
Thymoquinones Revealed by an Alkyne–Azide Click
Reaction and the Streptavidin–Biotin System
Katharina Effenberger-Neidnicht,[a] Sandra Breyer,[a] Katharina Mahal,[a] Randi Diestel,[b]
Florenz Sasse,[b] and Rainer Schobert*[a]
The subcellular distribution and accumulation of thymoqui-
none 1, a natural anticancer agent, has hitherto been un-
known. We prepared 6-(dec-9-ynyl)thymoquinone 3, an alkyne-
labelled derivative with anticancer activity similar to that of its
parent compound 1. Alkyne 3 was seen, after a Huisgen-type
click reaction with 3-azido-7-hydroxycoumarin, to accumulate
in distinct compartments of the nuclei of PtK2 potoroo kidney
cells, and in adjoining regions that were stained with an anti-
body specific for the Golgi apparatus. In contrast, a biotin-
labelled thymoquinone 4 seemed to accumulate across the
entire cell nucleus upon visualisation with streptavidin; but
this was less easily traceable because of co-staining of other
structures such as mitochondria. In conclusion, for small drug-
like molecules, visualisation by alkyne–azide cycloaddition
seems to be superior to conventional visualisation by the
biotin–streptavidin system.
Introduction
The CuI-mediated 1,3-dipolar cycloaddition (“click reaction”)
between alkyne-labelled proteins and fluorogenic or radio-
labelled azides (to give the corresponding 1,2,3-triazoles) has
been occasionally employed for in vivo visualisation.[1–4] In
comparison, applications of this reaction to the localisation of
low-molecular compounds in cells and biological tissues are
rare.[5] These molecules would benefit highly from an alkyne
label that not only is biologically inert but also induces mini-
mal structural alteration, compared to that with the more
common biotin labels. A particularly small compound of hith-
erto unknown subcellular distribution is thymoquinone (TQ, 1,
Scheme 1), a constituent of the volatile oil of black seed (Ni-
gella sativa). It shows antioxidative, anti-neoplastic and anti-in-
flammatory properties.[6,7] TQ induces apoptosis in cancer cells
in both p53-dependent and p53-independent ways, and it was
found to delay tumour growth by the induction of cell-cycle
arrest in certain xenograft models.[8–12] In a preceding article
we showed that some non-natural 6-alkylthymoquinones, such
as the geranyl derivative 2, differed little from TQ in their effi-
cacy against various tumour cell lines, and in their mode of
apoptosis induction: elevated levels of reactive oxygen species,
a loss of the mitochondrial membrane potential and the acti-
vation of caspases.[13,14] In order to use alkyne–azide cycloaddi-
tion to localise such TQ derivatives in living cells, we prepared
6-(dec-9-ynyl)thymoquinone 3; this features a C10 appendage
and a degree of unsaturation, like analogue 2. We expected 3
to exhibit anticancer effects and affinities to subcellular com-
ponents, similar to those of 6-geranylthymoquinone 2 and of
TQ. The actual visualisation was to be effected by a CuI-cata-
lysed click reaction of the terminal alkyne 3 with 3-azido-7-hy-
droxycoumarin 7 to afford a strongly fluorescent 1,2,3-triazole
8 (Scheme 2). For comparison, we also prepared the C10-teth-
Scheme 1. Reagents and conditions: a) undec-10-ynoic acid, AgNO3,
(NH4)2S2O8, CH3CN/H2O, 1008C, 4 h, 26%; b) 5-bromovaleric acid, AgNO3,
(NH4)2S2O8, CH3CN/H2O, 1008C, 16 h, 13%; c) 1. urotropine, CHCl3, RT, quant.,
2. EtOH, HCl, reflux, quant.; d) biotin, DCC, DMAP, HOBt, CH2Cl2/DMF, RT,
41%.
[a] K. Effenberger-Neidnicht, Dr. S. Breyer, K. Mahal, Prof. R. Schobert
Organic Chemistry Laboratory, University Bayreuth
Universitaetsstrasse 30, 95440 Bayreuth (Germany)
Fax: (+49)921-552671
E-mail: rainer.schobert@uni-bayreuth.de
[b] Dr. R. Diestel, Dr. F. Sasse
Helmholtz Centre for Infection Research (HZI)
Department of Chemical Biology
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
ChemBioChem 2011, 12, 1237 – 1241
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1237

R. Schobert et al.
for the biotin conjugate 4, given its more widely deviating IC50
values. It is worthy of note that neither 3 nor 4 inhibited the
growth of non-malignant human foreskin fibroblasts at con-
centrations below 100 mm.
Next, the click reaction between the alkynylthymoquinone 3
and 3-azido-7-hydroxycoumarin 7 was carried out on a 5 mm
scale under cell-free conditions in a commercial buffer (Click-iT
Cell Reaction Buffer), in order to check its feasibility and to de-
termine the optimum excitation and emission wavelengths for
its intended visualisation within PtK2 potoroo (Potorous tri-
dactylis) kidney epithelial cells (Scheme 2).[23–25] PtK2 cells lend
themselves ideally to this experiment because of their flat
shape. Panel A of Figure 1 shows the results of staining PtK2
cells by incubation first with alkyne 3 (5 mm, 16 h) and then
with azide 7 (5 mm) in Click-iT Cell Reaction Buffer for 30 min.
The blue fluorescence of coupling product 8 is localised to dis-
tinct nuclear regions that might be rich in DNA (e.g., chroma-
tin) or RNA (nucleoli), and in regions adjoining the nucleus.
The latter were stained in control experiments with a dictyo-
some-specific antibody and a secondary antibody conjugated
with Alexa Fluor 594 following the click reaction; so, they
could be the Golgi apparatus or parts of the endoplasmic retic-
ulum (Figure 1B). Figure 1C is an overlay of panel B and a
brightfield image to better indicate the cell boundaries. The
functions of the Golgi apparatus are as diverse as the glycosy-
lation, sorting and secretion of proteins and metabolites of
xenobiotics produced in the endoplasmic reticulum. For the
identification of the true molecular targets of 3, further experi-
ments will be necessary.
Scheme 2. Copper(I)-catalysed 1,3-dipolar azide–alkyne cycloaddition (click
reaction) under cell-free conditions: a) CuSO4, sodium ascorbate (Click-iT Cell
Reaction Buffer Kit), 30 min, RT.
ered biotin conjugate 4 to be visualised via the streptavidin–
biotin system (Scheme 1).[15]
Results and Discussion
Synthesis of labelled 6-alkylthymoquinones 3 and 4
Compound 3 was prepared by a known procedure from TQ,
undec-10-ynoic acid and Ag2S2O8 in aqueous acetonitrile
(Scheme 1).[16,17] For the synthesis of the biotin conjugate 4 the
same protocol was employed to first prepare the bromobutyl
derivative 5 from TQ and w-bromovaleric acid. This bromide
was then converted to the corresponding amine 6 by a De-
lꢁpine reaction with urotropine.[18,19] Finally, amide 4 was ob-
tained from condensation of amine 6 with biotin in the pres-
ence of dicyclohexylcarbodiimide (DCC), 4-(N,N-dimethylami-
no)pyridine (DMAP) and 1-hydroxybenzotriazole (HOBt).[20]
In contrast, the biotin conjugate 4 seemed to accumulate
across the nucleus of PtK2 cells upon incubation at concentra-
tions of 6.5 mm for 24 h (Figure 2). However, visualisation with
streptavidin was less easily traceable because of co-staining of
other structures such as mitochondria; this gave a blurred fluo-
rescent background. Since biotin also plays a critical role in his-
tone modification, the accumulation of 4 in PtK2 nuclei cannot
be interpreted as being solely a consequence of the thymoqui-
none moiety.[26] The actual visualisation was performed by
incubation with a streptavidin–Alexa Fluor 488 conjugate
(10 mgmLÀ1), followed by mounting in ProLong Antifade Gold
that contained DAPI (1 mgmLÀ1) for staining the nuclei. Com-
pared to controls (MeOH, Figure 2A), the cells exposed to 4
(Figure 2B) showed a superposition of the blue DAPI fluores-
cence and the green fluorescence of the streptavidin–biotin
aggregate.
Biological studies
The labelled TQ-derivatives 3 and 4 were first tested by using
the MTT assay for anti-proliferative activity in cells of human
518A2 (melanoma), HL-60 (leukaemia), HT-29 (MRP1-rich colon
carcinoma), KB-V1VBL (cervix carcinoma overexpressing P-gp)
and MCF-7TOPO (BCRP-rich breast adenocarcinoma).[21] As the
measured activities, at least of acetylene 3, lay in the ranges of
those of TQ and the previously tested geranyl derivative 2,[13]
we assumed that the attachment of an alkyne label would not
much alter the cellular uptake and distribution of conjugate 3
when compared to TQ and 2 (Table 1). This might not be true
[a]
Table 1. Inhibitory concentrations IC50 in mm of compounds 1–4 deter-
mined by the MTT assay when applied to various human cancer cells.
Compound/cell line
1
2
3
4
Conclusions
518A2
HL-60
HT-29
KB-V1VBL
MCF-7TOPO
28Æ9[b]
28Æ6[b]
47Æ19[c]
32Æ6[b]
27Æ6[b]
23Æ7[b]
29Æ12[b]
n.m.[d]
24Æ9
34Æ16
3.0Æ0.8
49Æ13
26Æ9
>100
The thymoquinone derivative 3 bears a biologically inert termi-
nal acetylene label that does not significantly alter its polarity
and cytotoxicity compared to analogues without this label,
such as the geranyl conjugate 2. The distribution of compound
3 in living PtK2 was visualised by a click reaction with 3-azido-
7-hydroxycoumarin; this produced the fluorescent 1,2,3-triazol
that accumulated in distinct regions within and adjoining the
nuclei of PtK2 cells. As no molecular target with a strong affini-
ty to acetylene is known within these cellular compartments,
>100
30Æ17
50Æ4
16Æ2[b]
27Æ2[b]
10Æ2
[a] Values are derived from concentration–response curves obtained by
measuring the percentage absorbance of viable cells relative to untreated
controls (100%) after 72 h exposure of 518A2, HL-60, HT-29, KB-V1VBL and
MCF-7TOPO cells to the test compounds. Values represent means of four
independent experiments Æstandard deviations. [b] Values taken from
ref. [13]. [c] Value taken from ref. [22]. [d] Not measured.
1238
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1237 – 1241

Cellular Localisation of Thymoquinones
Figure 1. A) PtK2 cells incubated first with 3 (5 mm, 16 h) then with azide 7 (5 mm, 30 min) in Click-iT Cell Reaction Buffer (blue stain); B) PtK2 cells additionally
stained with a Golgi-selective antibody and a secondary antibody conjugated with Alexa Fluor 594 (red stain); C) overlay of B) with bright field image.
Syntheses
6-(Dec-9-ynyl)thymoqinone (3):
A mixture of thymoquinone 1
(100 mg, 0.61 mmol), 10-undecynoic acid (89 mg, 0.49 mmol),
AgNO3 (13 mg, 0.08 mmol) and CH3CN/H2O (1:1, 15 mL) was stirred
and heated at 1008C while a solution of (NH4)2S2O8 (139 mg,
0.61 mmol) in H2O (0.61 mL) was slowly added. The resulting mix-
ture was refluxed for a further 4 h, then cooled, diluted with H2O,
and extracted with Et2O. The organic phases were washed with
brine and dried over Na2SO4. Volatile solvents were removed under
vacuum, and the residue thus obtained was purified by CC (EtOAc/
cyclohexane, 1:4). Yield: 39 mg (26%) as yellow oil; Rf =0.59 (cyclo-
hexane/toluene, 1:1); 1H NMR (300 MHz, CDCl3): d=1.08 (d, J=
6.9 Hz, 6H), 1.2–1.4 (m, 10H), 1.49 (m, 2H), 1.91 (t, J=2.6 Hz, 1H),
1.98 (s, 3H), 2.15 (dt, J=7.1, 2.6 Hz, 2H), 2.45 (t, J=7.6 Hz, 2H),
3.02 (dsept, J=6.9, 1.2 Hz, 1H), 6.44 (d, J=1.2 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=11.7, 18.3, 21.4, 26.7, 28.4, 28.6, 28.7, 28.9,
29.2, 29.7, 29.8, 29.9, 68.1, 84.7, 129.9, 134.2, 145.3, 154.6, 187.0,
188.5; IR (ATR): nmax =3290, 2928, 2856, 1641, 1612, 1463, 1377,
1306, 1244, 893 cmÀ1; MS (EI, 70 eV): m/z (%): 300 (37) [M]+, 257
(10), 180 (19), 150 (100), 121 (53), 67 (52).
Figure 2. A) PtK2 control cells (MeOH) with the addition of streptavidin–
Alexa Fluor 488 (green) visualises biotin-containing mitochondria; nuclei are
stained with DAPI (blue). B) PtK2 cells incubated with 4 (6.5 mm, 24 h); locali-
sation is visualised by streptavidin–Alexa Fluor 488 (green). Nuclei (chromo-
somes) are stained with DAPI.
we assume that the targeting effect can be attributed to the
thymoquinone core itself. In contrast, the visualisation of the
biotin-labelled thymoquinone 4 by streptavidin was less con-
clusive because of the ubiquitousness of biotin in cells. In
conclusion, for small drug-like molecules, a visualisation based
upon the Huisgen alkyne–azide cycloaddition reaction is more
reliable than conventional visualisation of biotin-labelled com-
pounds by interaction with streptavidin.
6-(4-Bromobutyl)thymoquinone (5): A mixture of thymoquinone 1
(200 mg, 1.22 mmol), w-bromovaleric acid (180 mg, 0.97 mmol),
AgNO3 (20 mg, 1.22 mmol) and CH3CN/H2O 1:1 (15 mL) was heated
at 1008C while a solution of (NH4)2S2O8 (280 mg, 1.22 mmol) in H2O
(0.61 mL) was slowly added. The resulting mixture was refluxed
overnight, then cooled, diluted with H2O, and extracted with Et2O.
The organic phases were washed with brine and dried over
Na2SO4. Volatile solvents were removed under vacuum, and the re-
mainder was purified by CC (EtOAc/cyclohexane, 1:4). Yield: 47 mg
(13%) as a yellow oil; Rf =0.41 (EtOAc/cyclohexane, 1:1); 1H NMR
(300 MHz, CDCl3): d=1.08 (d, J=6.9 Hz, 6H), 1.46 (tt, J=7.9,
8.2 Hz, 2H), 1.59 (m, 2H), 1.99 (s, 3H), 2.49 (t, J=7.9 Hz, 2H), 3.00
(dsept, J=6.9, 1.2 Hz, 1H), 3.65 (t, J=6.4, 2H), 6.44 (d, J=1.2 Hz,
1H); 13C NMR (75 MHz, CDCl3): d=11.7, 21.4, 24.8, 25.8, 26.3, 26.7,
32.5, 130.0, 141.1, 144.9, 154.6, 187.1, 188.4; IR (ATR): nmax =2958,
2934, 2870, 1707, 1645, 1614, 1459, 1377, 1308, 1246, 1060, 1025,
892, 799, 709 cmÀ1; MS (EI, 70 eV): m/z (%): 236 (100), 203 (68), 175
(100), 147 (68).
Experimental Section
Instrumentation and chemicals
Fluorescence microscopy: We used an Axioplan fluorescence micro-
scope (Zeiss, Jena, Germany) equipped with an Axiocam camera,
20ꢂ, 40ꢂ and 63ꢂ objectives, and an XBO150W super pressure
mercury lamp. Data analysis was performed with AxioVision 3.1
software. IR spectra were obtained on a Spectrum One FTIR spec-
trophotometer (PerkinElmer) equipped with an ATR sampling unit.
NMR spectra were obtained from an Avance 300 spectrometer
(Bruker) with chemical shifts (d) given in ppm downfield from
Me4Si. EIMS was performed on a Varian MAT 311A, and microanaly-
ses on a 2400 CHN analyser (PerkinElmer); corrected analyses (with
Æ0.2% for C, H, N) were obtained for 3 and 4. Column chromatog-
raphy (CC) used silica gel 60 (230–400 mesh). All reagent-grade
chemicals were purchased from commercial sources, and solvents
were dried and distilled before use. MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide) was purchased from ABCR
(Karlsruhe, Germany). 3-Azido-7-hydroxycoumarin 7 was prepared
according to a published procedure.[24]
6-(4-Aminobutyl)thymoquinone (6): Bromide 5 (40 mg, 0.13 mmol)
was dissolved in dry CHCl3 and treated with urotropine (19 mg,
0.13 mmol). The resulting mixture was stirred at room temperature
for 8 h, treated with conc. aqueous HCl (2 mL), then stirred and
heated for a further 4 h. It was cooled to ambient temperature,
diluted with H2O, and extracted with EtOAc. The organic phases
were washed with brine and dried over Na2SO4. Volatile solvents
were removed under vacuum, and the residue was purified by CC
(EtOAc/cyclohexane, 1:1). Yield: 30 mg (quant.) as a yellow oil; Rf =
ChemBioChem 2011, 12, 1237 – 1241
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1239

R. Schobert et al.
0.38 (EtOAc/cyclohexane, 1:1); 1H NMR (300 MHz, CDCl3): d=1.08
(d, J=6.9 Hz, 6H), 1.6 (m, 4H), 1.99 (s, 3H), 2.49 (t, J=7.9 Hz, 2H),
3.01 (dsept, J=6.9, 1.2 Hz, 1H), 3.66 (t, J=6.4 Hz, 2H), 6.45 (d, J=
1.2 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=11.8, 21.4, 24.9, 25.8,
26.3, 26.7, 32.5, 130.0, 140.2, 144.9, 154.6, 187.3, 188.4; IR (ATR):
nmax =3378, 2961, 2935, 2873, 1709, 1647, 1612, 1459, 1378, 1308,
1246, 1060, 1025, 892, 799, 708 cmÀ1; MS (EI, 70 eV): m/z (%): 236
(100) [M+1]+, 221 (24), 203 (67), 175 (85), 147 (52), 135 (30), 105
(35), 91 (46), 53 (43), 41 (36).
concentration of 0.05% (HL-60, 518A2, HF) or 0.1% (HT-29, KB-
V1VBL, MCF-7TOPO). After 2 h the precipitate of formazan crystals was
re-dissolved in a 10% solution of sodium dodecylsulfate in DMSO
(containing 0.6% acetic acid in the case of the HL-60 cells). For
adherent cells (518A2, HT-29, KB-V1VBL and MCF-7TOPO), microplates
were swiftly turned to discard the medium before adding the sol-
vent mixture. The microplates were gently shaken in the dark for
30 min, and absorbance at 570 and 630 nm (background) was
measured with an ELISA plate reader. All experiments were carried
out in quadruplicate and the percentage of viable cells was calcu-
lated as the mean ÆSD relative to the controls (100%).
Visualisation via click reaction: PtK2 cells were grown in DMEM
medium (750 mL) in 4-well plates (Nunc) on glass coverslips and in-
cubated overnight with the alkyne 3 (5 mm). The medium was re-
moved and the cells were washed twice with phosphate buffered
saline. The cells were then fixed with 3.7% formalin (15 min), per-
meabilised with 0.1% triton X-100 (5 min) and incubated (30 min,
RT) with 5 mm 3-azido-7-hydroxycoumarine 7 in Click-iT Cell Re-
action Buffer (Molecular Probes, OR, USA) according to the manu-
facturer’s procedure. For staining of the Golgi apparatus the cells
were incubated with a primary mouse anti-Golgi mAb (1:100,
Sigma) for 1 h, then with a secondary Alexa Fluor 594 goat anti-
mouse IgG Ab (1:500, Molecular Probes) for 45 min, and finally
mounted in ProLong Antifade Gold (Molecular Probes) that con-
tained DAPI (1 mgmLÀ1) for staining the nuclei.
N-[4-(5-isopropyl-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)butyl]biotina-
mide (4): A mixture of biotin (52 mg, 0.21 mmol), HOBt (10 mg,
0.06 mmol), dry DMF (20 mL) and DCC (50 mg, 0.23 mmol) was
stirred at room temperature for 3 h, then treated with amine 6
(50 mg, 0.21 mmol) and catalytic quantities of DMAP. Stirring was
continued overnight, then volatile solvents were removed under
vacuum, and the residue thus obtained was purified by CC
(CH2Cl2/MeOH, 12:1). Yield: 40 mg (41%); yellow oil; Rf =0.31
(CH2Cl2/MeOH, 12:1); 1H NMR (300 MHz, CDCl3): d=0.85 (m, 2H),
1.09 (d, J=6.9 Hz, 6H), 1.1–1.7 (m, 8H), 2.00 (s, 3H), 2.02 (m, 2H),
2.2–2.4 (m, 4H), 2.91 (m, 1H), 3.13 (dsept, J=6.9, 1.2 Hz, 1H), 4.07
(t, J=6.4 Hz, 2H), 4.32 (m, 2H), 6.46 (d, J=1.2 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d=11.8, 21.4, 24.9, 25.6, 26.7, 27.9, 28.2, 28.8,
30.9, 32.5, 33.9, 40.5, 55.3, 60.1, 61.7, 130.0, 137.6, 144.7, 154.6,
163.4, 172.4, 187.0, 188.3; IR (ATR): nmax =3326, 2927, 2851, 2111,
1719, 1629, 1571, 1511, 1448, 1344, 1310, 1185, 1045, 891,
759 cmÀ1; MS (EI, 70 eV): m/z (%): 464 (43) [M]+, 227 (100), 218 (47),
166 (17), 97 (38).
Visualisation by the biotin–streptavidin system: PtK2 cells were
grown in DMEM medium (750 mL) in 4-well plates (Nunc) on glass
coverslips, and incubated with the biotin-labelled thymoquinone 4
(6.5 mm) for 24 h. Cells were fixed with 3.7% formalin for 15 min,
permeabilised with 0.1% Triton X-100 for 5 min. They were then
incubated with the streptavidin–Alexa Fluor 488 conjugate
(10 mgmLÀ1, Invitrogen), and mounted in ProLong Antifade Gold
(Molecular Probes) that included DAPI (1 mgmLÀ1) to stain the
nuclei.
Methods
Cell lines and culture conditions: Human HL-60 leukaemia cells were
obtained from the German Collection of Biological Material
(DSMZ), Braunschweig; human melanoma cells 518A2 from the De-
partment of Oncology and Hematology at Martin Luther University,
Halle; KB-V1VBL and MCF-7TOPO cells from the Institute of Pharmacy
at the University Regensburg; and the HT-29 colon carcinoma cells
and human foreskin fibroblasts (HF) from the University Hospital,
Erlangen. HL-60 and HT-29 cells were grown in RPMI-1640 medium
supplemented with 10% foetal calf serum (FCS), penicillin G
Acknowledgements
(100 UmLÀ1),
streptomycin
(100 mgmLÀ1),
amphotericin B
We are grateful to the Deutsche Forschungsgemeinschaft for
financial support (grant Scho 402/8–3).
(0.25 mgmLÀ1) and gentamycin (250 mgmLÀ1; Gibco, Egenstein, Ger-
many). 518A2, HF and KB-V1VBL cells were cultured in Dulbecco’s
modified Eagle medium (DMEM, Gibco) containing 10% FCS, peni-
cillin G (100 IUmLÀ1), streptomycin (100 mgmLÀ1), amphotericin B
(0.25 mgmLÀ1) and gentamycin (250 mgmLÀ1). MCF-7TOPO cells were
grown in Eagle’s minimum essential medium (EMEM, Sigma) sup-
Keywords: alkynes · biotin · click chemistry · fluorescent
probes · thymoquinone
plemented with NaHCO3 (2.2 gLÀ1), sodium pyruvate (110 mgLÀ1
)
[1] Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J.
Am. Chem. Soc. 2003, 125, 3192–3193.
and 5% FCS. PtK2 cells were obtained from the American Type Cul-
ture Collection (ATCC) and cultivated in DMEM medium supple-
mented with 10% FCS. All cells were maintained in a moisture-sa-
turated 5% CO2 atmosphere at 378C in 75 mL culture flasks (Nunc,
Wiesbaden, Germany). They were serially passaged following pro-
tease treatment with 0.05% trypsin/0.02% EDTA (PAA laboratories,
Cçlbe, Germany) or by using cell scrapers. Mycoplasma contamina-
tion was routinely monitored; only mycoplasma-free cultures were
used.
´
[2] W. G. Lewis, L. G. Green, F. Grynszpan, Z. Radic, P. R. Carlier, P. Taylor,
M. G. Finn, K. B. Sharpless, Angew. Chem. 2002, 114, 1095–1099; Angew.
Chem. Int. Ed. 2002, 41, 1053–1057.
[3] K. E. Beatty, J. C. Liu, F. Xie, D. C. Dieterich, E. M. Schuman, Q. Wang,
D. A. Tirrell, Angew. Chem. 2006, 118, 7524–7527; Angew. Chem. Int. Ed.
2006, 45, 7364–7367.
[4] S. Maschauer, J. Einsiedel, R. Haubner, C. Hocke, M. Ocker, H. Hꢃbner, T.
Kuwert, P. Gmeiner, O. Prante, Angew. Chem. 2010, 122, 988–992;
Angew. Chem. Int. Ed. 2010, 49, 976–979.
[5] A. S. Raghavan, H. C. Hang, Drug Discovery Today 2009, 14, 178–184.
[6] R. El Mezayen, M. El Gazzar, M. R. Nicolls, J. C. Marecki, S. C. Dreskin, H.
Nomiyama, Immunol. Lett. 2006, 106, 72–81.
[7] D. R. Worthen, O. A. Ghosheh, P. A. Crooks, Anticancer Res. 1998, 18,
1527–1532.
[8] O. A. Badary, A. M. Gamal El-Din, Cancer Detect. Prev. 2001, 25, 362–368.
[9] H. Gali-Muhtasib, A. Roessner, R. Schneider-Stock, Int. J. Biochem. Cell
Biol. 2006, 38, 1249–1253.
Determination of tumour cell growth (MTT assay): HL-60 cells (5ꢂ
105 mLÀ1), 518A2, HT-29, KB-V1VBL and MCF-7TOPO cells (5ꢂ104 mLÀ1
each) and HF fibroblasts were seeded in 96-well tissue culture
plates and cultured for 24 h. Incubation (5% CO2, 95% humidity,
378C) of the cells following treatment with the test compounds
was continued for 72 h. Blank and solvent controls were treated
identically. MTT in phosphate buffered saline was added to a final
1240
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1237 – 1241

Cellular Localisation of Thymoquinones
[10] H. Gali-Muhtasib, M. Ocker, D. Kuester, S. Krueger, Z. El-Hajj, A. Diestel,
M. Evert, N. El-Najjar, B. Peters, A. Jurjus, A. Roessner, R. Schneider-
Stock, J. Cell. Mol. Med. 2008, 12, 330–342.
[11] H. Gali-Muhtasib, M. Diab-Assaf, C. Boltze, J. Al-Hmaira, R. Hartig, A.
Roessner, R. Schneider-Stock, Int. J. Oncol. 2004, 25, 857–866.
[12] M. A. El-Mahdy, Q. Zhu, Q. E. Wang, G. Wani, A. A. Wani, Int. J. Cancer
2005, 117, 409–417.
[13] S. Breyer, K. Effenberger, R. Schobert, ChemMedChem 2009, 4, 761–768.
[14] K. Effenberger, S. Breyer, R. Schobert, Chem. Biodiversity 2010, 7, 129–
139.
[20] W. Kçnig, R. Geiger, Chem. Ber. 1970, 103, 788–798.
[21] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
[22] K. Effenberger-Neidnicht, R. Schobert, Cancer Chemother. Pharmacol.
2010, 67, 867–874.
[23] R. Huisgen, Angew. Chem. 1963, 75, 742–754; Angew. Chem. Int. Ed.
Engl. 1963, 2, 633–645.
[24] R. Huisgen, Angew. Chem. 1963, 75, 604–637; Angew. Chem. Int. Ed.
Engl. 1963, 2, 565–598.
[25] K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill, Q. Wang, Org.
Lett. 2004, 6, 4603–4606.
[15] G. Gitlin, E. A. Bayer, M. Wilchek, Biochem. J. 1990, 269, 527–530.
[16] N. Jacobsen, K. Torssell, Justus Liebigs Ann. Chem. 1972, 763, 135–147.
[17] R. Jockers, R. D. Schmid, H. Rieger, K. Krohn, Liebigs Ann. Chem. 1991,
315–321.
[26] Y. I. Hassan, J. Zempleni, J. Nutr. 2006, 136, 1763–1765.
[18] M. Delꢁpine, Bull. Soc. Chim. Fr. 1895, 13, 352–361.
ˇ
Received: December 17, 2010
ˇ
´
´
ˇ
[19] N. Blazzevic, D. Kolbah, B. Belin, V. Sunjic, F. Kafjez, Synthesis 1979, 161–
176.
Published online on April 15, 2011
ChemBioChem 2011, 12, 1237 – 1241
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1241