A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II

Article abstract of DOI:10.1016/j.bmc.2009.11.048

Etoposide is a widely used anticancer drug that targets topoisomerase II, an essential nuclear enzyme. However, despite the fact that it has been in use and studied for more than 30 years the specific site on the enzyme to which it binds is unknown. In or

Full text of DOI:10.1016/j.bmc.2009.11.048

Bioorganic & Medicinal Chemistry 18 (2010) 830–838
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II
b
b
a
a
Gaik-Lean Chee a, , Jack C. Yalowich , Andrew Bodner , Xing Wu , Brian B. Hasinoff
*
a Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0T5
b Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Etoposide is a widely used anticancer drug that targets topoisomerase II, an essential nuclear enzyme.
However, despite the fact that it has been in use and studied for more than 30 years the specific site
on the enzyme to which it binds is unknown. In order to identify the etoposide binding site(s) on topo-
isomerase II, a diazirine-based photoaffinity etoposide analog probe has been synthesized and its photo-
reactivity and biological activities have been characterized. Upon UV irradiation, the diazirine probe
rapidly produced a highly reactive carbene species that formed covalent adducts containing stable car-
bon-based bonds indicating that it should also be able to form stable covalent adducts with amino acid
residues on topoisomerase II. The human leukemia K562 cell growth and topoisomerase II inhibitory
properties of the diazirine probe suggest that it targets topoisomerase II in a manner similar to etoposide.
The diazirine probe was also shown to act as a topoisomerase II poison through its ability to cause topo-
Received 27 August 2009
Revised 13 November 2009
Accepted 21 November 2009
Available online 27 November 2009
Keywords:
Photoaffinity labeling
Etoposide
Topoisomerase II
Diazirine
isomerase IIa-mediated double-strand cleavage of DNA. Additionally, the diazirine probe significantly
increased protein–DNA covalent complex formation upon photoirradiation of diazirine probe-treated
K562 cells, as compared to etoposide-treated cells. This result suggests that the photoactivated probe
forms a covalent adduct with topoisomerase IIa. In conclusion, the present characterization of the chem-
ical, biochemical, and biological properties of the newly synthesized diazirine-based photoaffinity etopo-
side analog indicates that use of a proteomics mass spectrometry approach will be a tractable strategy for
future identification of the etoposide binding site(s) on topoisomerase II through covalent labeling of
amino acid residues.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
teniposide and etoposide phosphate, have been introduced after
nearly four decades of drug optimization using a ligand-based ap-
Etoposide (Fig. 1A) has been one of the most widely used anti-
cancer drugs in the world in the past three decades.1–3 The cellular
target of etoposide has been identified to be DNA topoisomerase II,
an essential nuclear enzyme required for regulating the topology of
DNA.4–6 The drug acts on topoisomerase II by stabilizing the inter-
mediate topoisomerase II-cleaved DNA covalent complexes (also
referred to as cleavage complexes), converting the enzyme into a
highly potent cellular toxin which triggers irreversible apoptosic
processes.7–9 Even though over two thousand structurally related
analogs have been prepared and numerous studies have been con-
ducted to elucidate the drug–enzyme molecular interaction and
structure–activity requirements for this drug class, the details of
the binding sites are still unknown. This information could be par-
ticularly useful in the design of new etoposide analogs using a
structure-based approach as various X-ray crystal structures of
topoisomerase II are now available.10–15 Structure-based design is
an attractive approach for the epipodophyllotoxin drug class con-
sidering that only two closely related clinically useful analogs,
proach. Teniposide, a more lipophilic analog of etoposide, was
developed as a means to facilitate cellular drug uptake,16,17
whereas etoposide phosphate is a hydrolysable prodrug of etopo-
side that was designed to improve the aqueous solubility of the
drug.3,18
Several techniques have been employed to locate drug binding
sites on proteins. Among them, photoaffinity labeling using a phot-
olabile probe is particularly useful because of the ability of the
probe to photochemically form a covalent adduct with the target
protein. The labeled amino acid residue in the binding site may
then be identified using a proteomics mass spectrometry approach.
Etoposide has been shown to bind to topoisomerase II in the ab-
sence of DNA with an apparent dissociation constant in the low
micromolar range.19,20 This binding ability should allow the use
of photoaffinity labeling as a method to identify the etoposide
binding sites. We previously developed a photoactivatable azido
analogue of etoposide (1 in Fig. 1A) for probing the drug binding
sites on topoisomerase II.21 However, our attempts to identify
the photochemically labeled amino acid residues of topoisomerase
II
a using matrix-assisted laser desorption/ionization mass spec-
* Corresponding author. Tel.: +1 204 272 1589; fax: +1 204 474 7617.
E-mail address: lean_chee@umanitoba.ca (G.-L. Chee).
trometry (MALDI-MS) were unsuccessful, presumably due to the
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.048

G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
831
R
R
A
O
O
4
O
O
H3C
Etoposide
Top-53
O
O
HO
HO
O
O
(CH3)2N
CH3O
N
CH2
4'
CH3
CH3O
OCH3
OH
O
O
O
1
O
HO
HO
B
N3
I
F3C
N
F3C
N
a
O
F3C
CO2H
COCl
2
N
N
NH
N
N
6
7
OH
3
4
5
5/b
N3
NH2
2
Figure 1. (A) Selected structures of 40-demethyl-4b-podophyllotoxin derivatives. (B) The synthetic scheme of diazirine 2; (a) SOCl2, room temperature, and (b) pyridine/ethyl
acetate, room temperature.
loss of the etoposide probe under MALDI conditions (unpublished
B
A
1.5
1.0
results). It is likely that the azido probe formed a drug–enzyme
covalent adduct with a labile nitrogen–heteroatom linkage that
was cleaved at high temperature.22,23
0.15
0.10
2
1
0
140
240
As part of our ongoing effort to identify the etoposide binding
sites on topoisomerase II, we have synthesized a new photo-
affinity etoposide probe containing a 3-trifluoromethyl-3-aryldi-
azirine photophore (2 in Fig. 1A). The diazirine photophores
have gained popularity over the other photoreactive groups due
to their highly desirable properties as photoaffinity probes.24 In
particular, the diazirines undergo rapid photoactivation into
highly reactive carbene species that can even form carbon–carbon
covalent bonds with aliphatic hydrocarbons. The fact that the
photoactivated diazirines produce photoadducts with stable car-
bon-based covalent bonds should overcome the challenges we
previously encountered with azido 1 in identifying the labeled
amino acid residues. In this report, we describe the synthesis,
photochemical reactivity, and biological activities of diazirine 2,
and also demonstrate the potential usefulness of this photo-
affinity probe for identifying the etoposide binding sites on topo-
isomerase II.
0
300
600
0
300
600
Irradiation time (s)
Irradiation time (s)
480
600
80, 100
60
40
C
0.15
0
20
40
20
0
0.10
600
0.05
320
360
400
Wavelength (nm)
250
300
350
400
Wavelength (nm)
Figure 2. UV–vis spectral changes that occurred upon the irradiation of diazirine 2
for increasing periods of time (in s) as indicated. The irradiation was carried out in
methanol at a concentration of 80 lM in a 1-cm silica spectrometer cell at 365 nm
(4.5 mW/cm2 output). The thick and thin lines show the absorbance increase and
decrease in the region of 270–320 nm, respectively. Insets (A) and (B) show the
absorbance changes at 284 and 348 nm, respectively, as a function of irradiation
time. Inset (C) is the expanded plot for the diazirine characteristic region of 320–
400 nm.
2. Results
2.1. Chemical synthesis and photochemical kinetics in organic
solvents
for other diazirines.30 The decrease in absorbance reflects the con-
version of diazirine 2 into other species. While the absorbance at
348 nm decreased, the absorbance at 284 nm increased rapidly
during the first 100 s (Fig. 2A). This has been attributed to the ini-
tial production of a diazo isomer by photochemical rearrangement
of the diazirine moiety, as previously observed for other diazirine
compounds.29,31 Upon further irradiation, the diazo isomer under-
went further photolysis and the absorbance changes was nearly
complete by 600 s. Diazirine 2 also underwent complete photolysis
at 365 nm in about 600 s in ethyl acetate and acetonitrile (data not
shown). The reactions in these two solvents produced similar spec-
tral changes and similar first order kinetics as the photolysis per-
formed in methanol (data not shown).
Photoaffinity etoposide probe, diazirine 2, was prepared from
amine 525 and commercially available benzoic acid 6 in an overall
yield of 41% (Fig. 1B). The photochemical reactions of diazirine 2
were performed to examine the reactivity of 2 upon photoactiva-
tion and to define the reaction conditions required for high-yield
photolabeling of topoisomerase II. The photochemical kinetics of
diazirine 2 in methanol at 365 nm for fixed periods of time was fol-
lowed spectrophotometrically (Fig. 2). The diazirine group of 2
showed a characteristic absorbance peak at about 348 nm, consis-
tent with the spectral properties of other reported 3-trifluoro-
methyl-3-aryldiazirines.26–29 Upon UV irradiation, the absorbance
at 348 nm decreased with a half-life of about 40 s following
approximately first-order kinetics (Fig. 2B) as has been reported

832
G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
2.2. LC–MS/MS analysis of photolysis products produced in
organic solvents
was the result of the loss of 32 amu from [9+H]+, which corre-
sponds to the loss of a methanol molecule (Fig. 5B).
The identity of the most polar component in the chromatogram
shown in Figure 3B is suggested to be either 10 or 11. The actual
structure for this component could not be conclusively determined
by analyzing the MS/MS spectrum shown inFigure 4E because both
10 and 11 have molecular ions with m/z 618. In addition, both
structures could result in two primary fragmentation pathways
that give m/z 616 and 586 as shown in Figure 4E. The loss of 2
and 32 amu from m/z 618 is proposed to be attributable to H2
and O2, giving rise to dioxirane 14b and alkane 14d, respectively
(Fig. 5C). Even though the structure of this photoadduct was not
conclusively determined, it is likely that either 10 or 11 is a cova-
lent adduct resulted from the reaction between the photogenerat-
ed carbene intermediate and dissolved molecular oxygen or water,
respectively.
In summary, the LC–MS/MS study of the photolysis of 2 in
methanol at 365 nm reveals that the photolysis was only partially
complete by 120 s, affording the unreacted diazirine 2, photorear-
rangement isomer 8, and photoadducts 9 and 10/11. However,
continuous irradiation of 2 for 600 s led to complete photolysis
that produced only photoadducts 9 and 10/11 as shown in Figure
3C. Based on our observations on the photolysis of 2 in methanol
and the general photolytic pathway suggested for the 3-trifluoro-
methyl-3-aryldiazirine group,24,31 the photolytic pathway for 2 is
summarized in Figure 3E. Since the diazo isomers are known to
be more efficiently activated at 302 nm to yield carbene intermedi-
ates,34 we also examined the photochemical reactions at both 365
and 302 nm to improve the photolysis efficiency. Photoirradiation
at 365 nm for 90 s followed by 302 nm for 30 s (Fig. 3D) was found
to be nearly as efficient as continuous irradiation at 365 nm for
600 s (Fig. 3C) for producing photoadducts 9 and 10/11.
The photolysis of diazirine 2 in organic solvents was further
examined by analyzing the photoproducts produced using LC–
MS/MS. Photolysis of 2 in methanol at 365 nm for 120 s afforded
four peaks in the total ion chromatogram shown in Figure 3B. Elec-
trospray ionization (ESI) MS/MS analysis (Fig. 4B–E) of these four
peaks was consistent with compounds 2, 8, 9 and 10/11 being pres-
ent (structures in Fig. 4A). High abundances of [M+H]+ and [M+Na]+
were observed in the LC–MS mode for all these compounds, sug-
gesting that they were highly stable under the ESI-MS conditions.
This also implies that the covalent adduct of diazirine 2 and topo-
isomerase II peptide will be stable under our proposed MALDI
experiments. Upon applying moderate collision energy in the
MS/MS mode, the molecular ions of these compounds underwent
minor fragmentations to afford daughter ions that further substan-
tiated the proposed structures (Fig. 4B–E).
The chromatographic peak with the longest retention time in
Figure 3B is assigned to diazirine 2 as it corresponds to the reten-
tion time observed for the unphotolyzed diazirine (Fig. 3A). The
appearance of two major daughter ions, m/z 584 and 383, in the
mass spectrum (Fig. 4B) corresponding to this peak in Figure 3B,
likely resulted from the loss of a molecular nitrogen and the whole
benzoyl amide fragment from 2, respectively (Fig. 5A). The struc-
tures of these daughter ions are proposed to be 14a and 16 as
shown in Figure 5F. Fragment 14a is likely the carbene intermedi-
ate generated thermally under ESI conditions. The structure of 16
has been previous proposed for the MS fragmentations of 40-dem-
ethylpodophyllotoxin derivatives.32 The observation of these two
daughter ions supports the proposal that the compound is diazi-
rine 2.
The compound that eluted immediately before diazirine 2
(Fig. 3B) is proposed to be the photorearrangement isomer, diazo
8. The observation that 8 had a slightly shorter retention time than
2 is consistent with 8 being a more polar compound than 2. In
addition, the MS/MS spectrum corresponding to this peak
(Fig. 4C) shows a similar fragmentation pattern to that of diazirine
2 (Fig. 4B), which is consistent with diazo 8 having the same
molecular weight as and significant structural resemblance to
diazirine 2. These results also demonstrated the ability of 8 to form
a carbene (14a) when thermally activated, as previously reported
for other diazo compounds.33
The photolysis products of diazirine 2 in ethyl acetate and ace-
tonitrile were also examine to further investigate the reactivity of
the carbene intermediate generated from 2 photochemically.
According to the LC–MS/MS analysis (data not shown), the photol-
ysis of diazirine 2 in ethyl acetate and acetonitrile at 365 nm for
600 s afforded two major photoadducts that are proposed to be
12 and 13, respectively. Several other minor photoproducts were
also observed but were uncharacterized due to poor chromato-
graphic separation. Diazirine 2 and diazo 8 were not detected in
the reaction mixture, indicating the completion of the reactions.
High abundances of [M+H]+ and [M+Na]+ in the LC–MS mode were
also observed for 12 and 13 (Fig. 4A). The MS/MS spectrum of pho-
toadduct 12 shows three major daughter ions, m/z 670, 640 and
638, in addition to the molecular ion observed (Fig. 4F). The loss
of 2 amu from 12 suggests that m/z 670 is an H2 elimination prod-
The chromatographic peak with the 4.4 min retention time in
Figure 3B is assigned to photoadduct 9. The primary fragmentation
pathway of 9 also resembled that of 2 and 8, affording two major
daughter ions with m/z 584 and 383 (Fig. 4D). However, m/z 584
10/11 9
8 2
E
9
methanol
A
365 nm
carbene
14a
10/11
B
C
D
O2 or H2O
365 nm
slow
302 nm
fast
2
8
365 nm
3
6
9
12 15
Retention time (min)
Figure 3. LC–MS/MS total ion chromatograms of the photolyzed solution and proposed photolytic pathway of diazirine 2 in methanol. (A) Diazirine 2 before photolysis. (B)
365 nm for 120 s. (C) 365 nm for 600 s. (D) 365 nm for 90 s followed by 302 nm for 30 s. (E) Photolytic pathway of diazirine 2, where 2 is proposed to give rise to a carbene
intermediate (14a in Fig. 5F) and the competing side product diazo 8 that undergoes further photolysis to 14a. Subsequent reactions of 14a with methanol and molecular
oxygen or water produce 9 and 10/11, respectively, as final photolysis products. The chemical structures of 8–11, and 14a are provided in Figures 4 and 5, respectively.

G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
833
A
[M+H]+
(m/z)
[M+Na]+
Molecular
formula
R1
R2
CF3
O
A =
2
NH
C
30H24F3N3O8
N
2
8
612
612
616
618
618
672
634
634
638
640
640
694
A
N
O
O
H24F3N3O8
C30
CF3
O
O
OCH3
OOH
C31H28F3NO9
C30H26F3NO10
C30H26F3NO10
C34H32F3NO10
N+
9
H
H
8
- N
A
O
10
CF3
R2
O
OH
OH
H
11
12
13
R1
CH3O
OCH3
9-13
CH2CO2Et
OH
A
CH2CN
C32H27F3N2O8
H
625
647
B
C
D
100
100
50
0
[2+H]+ 100
612
584
584
584
[8+H]+
612
[9+H]+
616
50
0
50
0
383
383
400
383
200
400
600
200
600
200
400
600
E
100
F
100
G
100
[13+H]+
625
640
383
616
383
670
586
638
[12+H]+
[10/11
+H]+
618
50
0
50
0
50
0
672
383
400
200
400
600
200
600
200
400
600
m/z
Figure 4. Compounds present in the photolyzed solutions of 2 in different organic solvents. (A) Proposed structures, masses of molecular ions observed in the LC–MS mode,
and molecular formula for these compounds. (B)–(G) are MS/MS spectra that correspond to the chromatographic peaks of diazirine 2, diazo 8, 9, 10/11, 12, and 13,
respectively.
-N2
[14a + H]+
(m/z 584)
[16 + H]+
[2/8 + H]+
(m/z 612)
F
A
B
(m/z 383)
O
14
c
R
14
a
R
R
[9 + H]+
(m/z 616)
NH
F3C
H
F3C
-CH3OH
-H2
O
O
..
O
H
F3C
[14b + H]+
[16 + H]+
(m/z 383)
[10/11 + H]+
(m/z 618)
O
F3C
EtO2C
b
d
C
D
O
(m/z 616)
O
[14c + H]+
CH3O
OCH3
-O2
OH
(m/z 586)
O
R
[14d + H]+ -CH3OH [15b + H]+
15
a
[12 + H]+
(m/z 672)
-H2
R
O
O
NH
O
(m/z 670)
(m/z 638)
F3C
O
O
[15a + H]+
[16 + H]+
(m/z 383)
O
O
EtO2C
(m/z 640)
-CH3OH
O
CH3O
OCH3
F3C
EtO2C
OH
:
[13 + H]+
(m/z 625)
[16 + H]+
(m/z 383)
b
CH3O
E
16
O
Figure 5. Proposed MS/MS fragmentation pathways for 2/8, 9, 10/11, 12, and 13, as shown in (A)–(E), respectively. (F) Proposed structures of the major daughter ions
observed in the MS/MS spectra of diazirine 2 and its photoproducts.

834
G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
uct 14d (Fig. 5D and F). Both 12 and 14d could undergo fragmen-
tations through the loss of a methanol molecule to afford fragment
ions m/z 640 and 638, which are proposed to be 15a and 15b,
respectively. The daughter ions observed clearly support the struc-
ture we proposed for 12. Unlike 12, the proposed acetonitrile pho-
As shown in Figure 6C, diazirine 2, tested at 50 and 100
duced the formation of linear DNA to an extent similar to etoposide
tested at 100 M.
lM, in-
l
To further confirm that diazirine 2 acts similarly to etoposide,
we evaluated their effects on the level of covalent complex forma-
tions in intact K562 and K/VP.5 cells. The K/VP.5 cell line is a stable
etoposide-resistant K562 cell line that contains one-fifth the topo-
tolysis product 13 afforded
a simple fragmentation pattern
(Fig. 4G) that showed only the molecular ion m/z 625 and a dom-
inant daughter ion m/z 383 which has already been proposed to
be [16+H]+ (Fig. 5E and F). Our results demonstrated the strong
reactivity of diazirine 2 upon UV irradiation, generating a carbene
intermediate that subsequently formed highly stable carbon–car-
bon covalent bonds with ethyl acetate and acetonitrile.
isomerase II
a
protein content of the parental K562 cells.40 There-
fore, a topoisomerase II poison should produce a lower level of
covalent complexes in K/VP.5 cells than in K562 cells. At tested
concentrations of 50 lM for etoposide and 80 lM for diazirine 2,
protein–DNA covalent complexes were induced to a much lesser
extent in K/VP.5 cells than in K562 cells, as shown in Figure 7A.
These results are also consistent with our observation in the DNA
cleavage assay (Fig. 6C) that diazirine 2 acts similarly to etoposide
as a topoisomerase II poison.
The effects of diazirine 2 and etoposide on the inhibition of
topoisomerase I-mediated relaxation of supercoiled DNA were also
examined. Neither drug inhibited topoisomerase I at a tested con-
2.3. K562 cell growth inhibition
Etoposide has been shown to be cytotoxic to human leukemia
K562 cells with an IC50 in the low micromolar range.35 The growth
inhibitory effect of diazirine 2 was compared to etoposide in K562
cells after a 72 h continuous exposure. Diazirine 2 was 5.2-fold
more cytotoxic than etoposide, with an IC50 of 0.42
to that of etoposide of 2.2 M (Fig. 6A).
lM compared
centration of 20
lM, whereas camptothecin, a topoisomerase I
l
inhibitor, at 50 M resulted in 40% inhibition (data not shown).
l
2.4. Topoisomerase II
topoisomerase I inhibition effects
a
inhibition and poisoning effects and
2.5. Effects of photolysis on drug-induced protein–DNA
covalent complex formation
The cytotoxicity of etoposide is due to its ability to convert
topoisomerase II into a potent cellular poison.36 The enzyme poi-
soning effect is achieved through stabilization of the cleavage com-
plex, which inhibits the ability of the enzyme to religate the
cleaved DNA strands,37 leading to the formation of linear DNA. Eto-
poside-induced stabilization of the cleavage complex also results
in inhibition of the catalytic strand passing activity of topoisomer-
ase II. To determine whether diazirine 2 shares the same mecha-
nism of action as etoposide by targeting topoisomerase II, the
two compounds were compared for their ability to inhibit the cat-
alytic activity of topoisomerase II, induce the formation of linear
DNA, and produce protein–DNA covalent complexes.
The stabilization of the cleavage complex by etoposide is a
reversible process.41 Therefore, if diazirine 2 forms an irreversible
covalent adduct with the cleavage complex, photolysis should in-
crease the extent of cleavage complex formation by shifting the
equilibrium process towards cleavage. Based on this hypothesis,
we examined the effect of UV irradiation on etoposide- and diazi-
rine 2-induced protein–DNA covalent complex formation in intact
K562 cells (Fig. 7B). Incubation of cells with Diazirine 2 (2.5–
20
complex formation. UV irradiation significantly increased the lev-
els of covalent complexes at all diazirine concentrations
(p = 0.004–0.031). In contrast, UV irradiation did not significantly
affect the etoposide (25 M)-induced formation of covalent com-
plexes at 25 M (p = 0.793).
lM) resulted in a concentration dependent increase in covalent
2
The inhibition of topoisomerase II catalytic activity was deter-
mined by the ability of the drug to inhibit enzyme-mediated decat-
enation of kinetoplast plasmid DNA (kDNA) into mini circles of
DNA.38 In the assay, diazirine 2 showed twofold greater potency
l
l
3. Discussion
than etoposide, with an IC50 of 56
side of 114 M (Fig. 6B). The topoisomerase II poisoning effect of
diazirine 2 and etoposide was evaluated in a DNA cleavage assay.39
lM compared to that of etopo-
l
Diazirine 2 is a non-glycoside analog of etoposide and closely
resembles the 4b-benzoylamino derivatives of 40-O-demethyl-4-
A
B
C
35
30
25
20
15
0.8
0.6
0.4
0.2
0.0
NC
LIN
SC
RLX
Etoposide
2
Etoposide
2
0.01 0.1
1
10 100
0.1
1
10 100 1000
[Drug] (μM)
[Drug] (μM)
Figure 6. Comparison of the biological activity of diazirine 2 and etoposide in K562 cells and topoisomerase II assays. (A) The growth inhibitory effects of 2 (s) and etoposide
(d) on K562 cells after a 72 h incubation as measured by an MTS assay. The curve lines are nonlinear least squares fits to a four-parameter logistic equation, and they yield
IC50’s of 0.42 0.06 and 2.20 0.41
smaller than the symbol. (B) The inhibitory effects of 2 (s) and etoposide (d) on the catalytic decatenation activity of topoisomerase II
amount of decatenated kDNA minicircles formed by the action of topoisomerase II on kDNA. The curve solid lines are nonlinear least squares fit to a four-parameter logistic
equation, and they yield IC50’s of 56 14 and 114 53 M for 2 and etoposide, respectively. (C) The effect of 2 and etoposide on the topoisomerase II -mediated cleavage of
supercoiled pBR322 plasmid DNA. The grouping of three fluorescent images from different part of the same gel shows the ability of 2 and etoposide to induce the formation of
linear DNA (LIN) at 100 M in the presence of topoisomerase II (middle two lanes). The left lane of the gel shows that topoisomerase II (+Topo II) relaxed supercoiled DNA
lM for 2 and etoposide, respectively. The values shown are triplicate determinations and where the error bars are not shown, they are
a
. The fluorescence measures the
a
l
a
l
a
a
(SC) to relaxed DNA (RLX) in the absence of drug, whereas the right lane shows the supercoiled pBR322 DNA in the absence of the enzyme and drug (-Topo II). A small amount
of nicked circular DNA (NC) is normally present in the pBR322 DNA.

G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
835
Diazirine 2, which was conveniently synthesized in three steps
from commercially available podophyllotoxin 3 and benzoic acid
6, exhibited the desirable photoaffinity labeling properties de-
scribed above. This conclusion is made on the basis of our findings
obtained in the photochemical kinetic and LC–MS/MS studies. Diaz-
irine 2 underwent facile photolysis in solution, with reaction com-
pleted in 600 s upon continuous irradiation at 365 nm. The
sequential application of irradiation at wavelengths of 365 and then
302 nm, which has also been applied by Hosoya et al. on their diaz-
irine probe,34 significantly shortened the photoirradiation time of 2
for reaching complete photolysis to only 120 s. The increased pho-
tolysis efficiency of 2 by nearly fivefold under this condition should
improve the labeling yield of diazirine 2 on topoisomerase II, which
in turn should enhance the detection of labeled peptide fragments.
The carbene intermediate generated photochemically from diazi-
rine 2 or diazo 8 reacted with methanol and molecular oxygen or
water to form carbon–oxygen photoadducts that were readily de-
tected using LC–MS/MS. The high abundances of the molecular ions
of these photoadducts in ESI-MS mode suggest that the loss of probe
2 via carbon–heteroatom bond cleavage from the labeled peptide
under MALDI-MS conditions should be minimal. The carbene inter-
mediate was also shown to react with ethyl acetate and acetonitrile
to afford photoadducts with highly stable carbon–carbon bonds. The
ability of the carbene to insert into aliphatic C–H bonds is indicative
of the formation of an extremely reactive species, and suggests that
diazirine 2 should be useful as a photoaffinity probe.
Several lines of evidence obtained for non-glycoside etoposide
analogs42–44 support our hypothesis that diazirine 2 interacts with
topoisomerase II in a manner similar to etoposide. The biological
studies we conducted for 2 and etoposide further validate our
hypothesis. Diazirine 2 exhibited five- and twofold greater potency
than etoposide in the K562 cell growth inhibition assay and the
topoisomerase II catalytic inhibition assay, respectively. The
slightly greater potency of diazirine 2 observed in the cell inhibi-
tion assay may be the result of increased cellular drug uptake be-
cause of the higher lipophilicity of the drug compared to
etoposide. Overall, diazirine 2 and etoposide exhibited comparable
levels of potency in these two assays. In addition, diazirine 2 was
able to induce linear DNA formation at levels comparable to that
for etoposide. Diazirine 2 also shared a similar profile as etoposide
in the induction of protein–DNA covalent complexes in K562 and
K/VP.5 cells. Taken together, our results are consistent with diazi-
rine 2 exhibiting similar biological activities as etoposide. Our re-
sults and the fact that diazirine 2 is structurally related to
etoposide strongly support the hypothesis that both drugs interact
with topoisomerase II in a similar manner. The fact that diazirine 2
significantly increased covalent complex formation after the cells
were exposed to UV irradiation strongly suggests that diazirine 2
formed a covalent adduct with topoisomerase II.
A
12
B
12
K562
-UV
K/VP.5
+UV
NS
10
8
10
8
6
6
∗
∗∗
4
∗∗
4
∗∗
2
2
0
0
)
)
)
)
e
e
2
d
M
M
M
µ
d
i
µ
µM
si
o
5 µ
2.
0
0
os
(5
1
p
p
(
(2
(
2
o
t
2
2
2
E
Eto
Figure 7. Comparison of the effects of
2
and etoposide on the formation of
topoisomerase II–DNA covalent complexes in intact cells. Results are shown as
protein–DNA covalent complexes formation induced by etoposide or 2. (A)
Comparison of drug-induced protein–DNA covalent complexes in K562 cells and
the etoposide-resistant K/VP.5 cells containing decreased levels of topoisomerase
IIa. Diazirine 2 at 80 lM and etoposide at 50 lM induced the formation of covalent
complexes to a lesser extent in K/VP.5 cells than in K562 cells. (B) Effect of UV
irradiation on 2- and etoposide-induced formation of covalent complexes in K562
cells. Diazirine
2 (2.5–20 lM), etoposide (25 lM), or DMSO were added to
suspensions of K562 cells then immediately irradiated (or not), as indicated, at
365 nm for 5 min. Cells were then incubated for 60 min at 37 °C in the dark
followed by processing for protein–DNA covalent complex formation as described
in Section 4. Bars represent the mean SEM from nine different experiments
performed on different days. Paired t-tests were performed for each drug
concentration in the presence compared to the absence of UV light. The and
indicate p <0.05 and 0.01, respectively. NS indicates not significant. The results
show that the increase of the levels of drug-induced covalent complexes by UV
irradiation at all four concentrations of diazirine
*
**
2 is statistically significant.
However, UV irradiation did not have any significant effect on the etoposide-
induced formation of covalent complexes.
desoxypodophyllotoxin reported in the literature.42,43 Although
diazirine 2 is structurally different than etoposide, we anticipate
that diazirine 2 will bind to topoisomerase II in a manner similar
to etoposide. In support of this idea, the 4b-benzoylamino deriva-
tives have been shown to be cytotoxic and share similar mecha-
nism of action as etoposide by acting as topoisomerase II
poisons.43 Another non-glycoside analog of etoposide, Top-53
(Fig. 1A), which is also structurally different than etoposide at
the 4b-position, has been shown to compete with etoposide in
binding to topoisomerase II in a competitive binding assay.44 In
addition, the binding of Top-53 to topoisomerase II has been
shown in an NMR spectroscopy study.44 These findings support
further proteomic studies to characterize the binding of diazirine
2 to topoisomerase II.
The 3-trifluoromethyl-3-aryldiazirine group is the photophore
of choice for our photoaffinity etoposide probe for several reasons.
The diazirines are known to be readily photoactivatable to produce
highly reactive carbene species that should enhance the probabil-
ity of labeling drug binding sites and reduce non-specific labeling
caused by migration of the labeling probe from the binding pock-
et.22–24 Another advantage of diazirines is that their photolysis al-
ways gives rise to photoadducts with carbon-based covalent bonds
that should be stable in the subsequent protein sequencing steps
and MALDI-MS experiments. Lastly, the photoactivation of diazi-
rine can be readily achieved with a short photoirradiation time
and at near UV wavelengths (e.g., 365 nm) with less potential to
cause UV-induced protein or cellular damage. Although the diazi-
rine photophore can undergo photorearrangement to afford the
longer lived diazo isomer as a side product, the diazo isomer can
be rapidly converted into a reactive carbene intermediate by a sub-
sequent photolysis at 302 nm.34
In conclusion, our results demonstrate that diazirine 2 should
prove useful for identifying the etoposide binding sites on topoiso-
merase II. The photoaffinity labeling of purified human topoiso-
merase II
a with diazirine 2 and the subsequent identification of
labeled amino acid residues using a proteomics mass spectrometry
approach are underway.
4. Experimental section
4.1. Chemical synthesis of N-[(10S,11S,15R,16R)-16-(4-hydroxy-
3,5-dimethoxyphenyl)-14-oxo-4,6,13-trioxatetracyclo[7.7.-
0.03,7.011,15]hexadeca-1,3(7),8-trien-10-yl]-4-[3-(trifluoro-
methyl)-3H-diazirin-3-yl]benzamide (2)
Reactants 3 and 6 (Fig. 1A and B) were purchased from Ava-
Chem Scientific LLC (San Antonio, TX), and Bachem Americas Inc.

836
G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
(Torrance, CA), respectively. All other reagents were from Sigma–
Aldrich (St. Louis, MO). 1H and 13C NMR spectra were recorded
on a Bruker AM-300 FT instrument. High resolution mass spectrum
(HRMS) was recorded on an Agilent 6210 Time-of-flight LC–MS
instrument. Preparative thin layer chromatography was carried
v) formic acid with a flow rate of 200
was chromatographed on a Varian Pursuit C-18 column (3
l
l/min. The sample (10
ll)
l
m pore
size, 100 ꢀ 2 mm). The ionization mode was ESI positive with N2 at
50 psi as the nebulizer gas. The drying gas was set at 50 psi and
350 °C. The voltages of the needle and shield were 5000 and
80 V, respectively. Positive ions were scanned in the range of
200–760 Da using a scan rate of 15 kDa/s and a mass increment
of 150–2000 Da. The relevant precursor ions were selected for
low energy collision-induced dissociation MS/MS analyses using
helium as the collision gas with a flow rate of 0.8 ml/min and col-
lision voltage in the range of 0.5–2.5 V.
out on Fisher silica gel plates with fluorescent indicator (layer
0
thickness 250 lm, particle size 5–17 lm, pore size 60 ÅA, and plate
dimension 20 ꢀ 20 cm). All reactions involving the diazirine deriv-
atives were carried out under reduced light because of their photo-
sensitivity. Amine 5 was prepared from podophyllotoxin 3 via
azide 4 according to a previously reported procedure.25 The amine
was then reacted with acyl chloride 7,45 which was in turn pre-
pared from 6, to afford diazirine 2 as described in the following
procedure. Carboxylic acid 6 (23 mg, 0.1 mmol) was dissolved in
thionyl chloride (0.2 ml), and the resulting solution was stirred at
room temperature for 20 h. The distillation to remove the excess
thionyl chloride was carefully performed at a reduced pressure of
not lower than 40 mbar for about 20 min at 25 °C to maximize
the removal of thionyl chloride without leading to excessive loss
of the volatile acyl chloride 7.45 The resulting liquid residue was
dissolved in ethyl acetate (0.1 ml) and added dropwise to a stirred
4.4. Cell culture and growth inhibition assay
K562 cells were obtained from American Type Culture Collec-
tion (Rockville, MD). K/VP.5 cells are a 26-fold etoposide-resistant
K562-derived sub-line with decreased levels of topoisomerase II
mRNA and protein.40 These cells were maintained as suspension
cultures in alpha minimum essential medium ( MEM) (Gibco
BRL, Burlington, Canada) containing 2 mM -glutamine and supple-
a
a
L
mented with 10% fetal calf serum (Invitrogen, Burlington, ON, Can-
ada), 20 mM NaHCO3, 20 mM HEPES (Sigma), 100 units/ml
solution of amine
5 (40 mg, 0.1 mmol) and pyridine (23 ll,
0.28 mmol) in ethyl acetate (0.2 ml). After stirring at room temper-
ature for 1 h, the resulting suspension was filtered, concentrated to
about 0.1 ml, and chromatographed on a preparative TLC plate
with 60% ethyl acetate/hexanes. The desired band on the plate
was detected in a dark room briefly under an indirect 254 nm UV
light from a hand-held lamp. The band was scraped out, eluted
with ethyl acetate, and concentrated to afford 2 as a beige solid
(25 mg, 41% yield). 1H NMR (300 MHz, CDCl3) d 2.90 (1H, dd,
J = 4.9, 14.3), 3.03 (1H, m), 3.77 (6H, s), 3.85 (1H, dd, J = 9.5,
10.4), 4.47 (1H, dd, J = 7.6, 9.1), 4.59 (1H, d, J = 4.8), 5.42 (2H, dd,
penicillin G, and 100 lg/ml streptomycin at pH 7.4 in an atmo-
sphere of 5% CO2 and 95% air at 37 °C. For the measurement of
growth inhibition, cells in exponential growth were harvested
and seeded at 6000 cells/well in 96-well plates (100 ll/well). Diaz-
irine 2 and etoposide were dissolved in DMSO and added to the
wells such that the final concentration of DMSO was 0.5% (v/v).
After 72 h incubation, 7 ll of 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
Cell Titer 96Ò AQueous One Solution (Promega, MD, WI) was added
to each well and incubated for a further 3 h. The absorbance was
measured in a Molecular Devices (Menlo Park, CA) plate reader.
The spectrophotometric 96-well plate cell growth inhibition assay
measures the ability of the cells to enzymatically reduce MTS.
Three replicates were measured at each drug concentration, and
the IC50 values and their SEs for growth inhibition were obtained
by fitting the absorbance–drug concentration data to a four-
parameter logistic equation as we previously described.46
J = 5.2, 5.3), 5.98 (2H, AB,
Dd = 6.9, J = 1.1), 6.27 (1H, d, 7.0), 6.31
(2H, s), 6.56 (1H, s), 6.79 (1H, s), 7.27 (2H, d, J = 8.0), 7.81 (2H, d,
J = 8.5); 13C NMR (75.5 MHz, CDCl3) d 28.5 (CN2, q, J = 41), 37.4
(CH), 42.0 (CH), 43.6 (CH), 48.9 (CH), 56.5 (2 ꢀ CH3), 69.1 (CH2),
101.7 (CH2), 107.9 (CH), 108.9 (CH), 110.3 (2xCH), 122.6 (CF3, q,
J = 273) 126.8 (2xCH), 127.6 (2xCH), 128.5 (C), 130.1 (C), 132.9
(C), 133.2 (C), 134.1 (C), 134.2 (C), 146.5 (2xC), 147.8 (C), 148.6
(C), 166.2 (C), 174.2 (C); ESI tandem mass spectrum m/z (relative
intensity) 612 ([M+H]+, 77), 584 (100), 383 (18); HRMS calcd for
[C30H24F3N3O8+H]+ 612.1593, found 612.1590.
4.5. Topoisomerase IIa decatenation and topoisomerase I
relaxation inhibition assays
4.2. Photochemical kinetics in organic solvents
The catalytic inhibition of purified human topoisomerase IIa by
diazirine 2 and etoposide was measured by the ATP-dependent
decatenation of kDNA (Topogen, Columbus, OH) into minicircles
of DNA as we previously described.38 The reaction mixture con-
tained 0.5 mM ATP, 50 mM Tris–HCl (pH 8.0), 120 mM KCl,
The photochemical reactions of diazirine 2 were carried out at
80 lM in selected solvents, as indicated, in 1-cm stoppered silica
cells placed on the surface of the transilluminator in an Alpha Inno-
tech Fluorchem 8900 imaging system (6.2 and 4.5 mW/cm2 at 302
and 365 nm, respectively, at the surface). The solutions were ex-
posed to the UV light at selected wavelengths, as indicated, for
fixed times and the UV–vis spectra were recorded on a Cary 1 spec-
trophotometer (Varian, Mulgarave, Australia).
10 mM MgCl2, 30
irine 2, etoposide, or DMSO (0.5
ase II protein (an amount that gave 80% decatenation). The
lg/ml bovine serum albumin, 40 ng kDNA, diaz-
ll) and 18 ng human topoisomer-
a
enzyme was expressed using a high copy yeast expression vector,
extracted, and purified as we previously described.38 The enzy-
matic reaction was carried out at 37 °C and was terminated by
4.3. LC–MS/MS analysis of photolysis products produced in
organic solvents
the addition of 12
at 8000g at 25 °C for 15 min and 20
added to 180 l of 600-fold diluted PicoGreen dye (Molecular
l
l of 250 mM EDTA. Samples were centrifuged
l
l of the supernatant was
l
Photolyzed diazirine 2 in methanol or acetonitrile, as indicated,
was directly diluted with water to give an 80% organic solvent/
water solution with a concentration of 64 lM (on a reactant basis).
Photolyzed 2 in ethyl acetate was concentrated to dryness and
redissolved in 80% methanol/water to afford the same concentra-
tion. The solutions were then analyzed with a Varian 500-MS LC–
MS/MS ion trap mass spectrometer equipped with 212-LC chroma-
tography equipment using the following analytical conditions. The
isocratic eluent was 60% (v/v) methanol/water containing 0.1% (v/
Probes, Eugene, OR) in a 96-well black plate with a clear bottom.
The fluorescence, which is proportional to the amount of decate-
nated kDNA, was measured in a Fluostar Galaxy (BMG, Durham,
NC) fluorescent plate reader using an excitation wavelength of
485 nm and an emission wavelength of 520 nm. The IC50 values
and their SEs for decatenation inhibition were obtained by fitting
the fluorescence unit-drug concentration data to a four-parameter
logistic equation as we previously described.46 The catalytic inhibi-
tion of topoisomerase I in K562 nuclear extract by diazirine 2 and

G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
837
etoposide was measured using
previously.47
a
procedure we described
tional Institutes of Health grant CA090787 to J.C.Y. and a Canada
Research Chair for Drug Development for B.B.H. We also acknowl-
edge the assistance of Drs. Tom Ward and Rui Zhang for providing
LC–MS/MS technical support and acquiring NMR spectra,
respectively.
4.6. Topoisomerase IIa DNA cleavage assay
Topoisomerase II-cleaved DNA covalent complexes induced by
topoisomerase II poisons such as etoposide may be trapped by rap-
idly denaturing the complexed enzyme with sodium dodecyl sul-
fate (SDS), thus releasing the cleaved DNA as linear DNA.41,48 The
formation of linear DNA was detected by separating the SDS-trea-
ted reaction products using ethidium bromide gel electrophoresis
as described previously.49 The band corresponding to linear DNA
was identified by comparison with that from linear pBR322 DNA
produced by action of the restriction enzyme HindIII acting on a
Supplementary data
Supplementary data (spectroscopic data of diazirine 2 is pro-
vided) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2009.11.048.
References and notes
single site on pBR322 DNA. The 20
l
l cleavage assay reaction mix-
ture39 contained 150 ng topoisomerase II
a
protein, 80 ng pBR322
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Topoisomerase II–DNA covalent complex formation in intact
K562 or K/VP.5 cells, with or without UV irradiation, as indicated,
was measured as we previously described.21 Briefly, mid-log growth
cells were radiolabeled for 24 h with 0.5
l
Ci/ml [methyl-3H]thymi-
dine (0.5 Ci/mmol) and 0.1
l
Ci/ml [14C]leucine (318 mCi/mmol) in
Dulbecco’s modified Eagle’s medium (DMEM) containing 7.5% (v/
v) iron-supplemented calf serum. The cells were pelleted, resus-
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of the cell suspension in 24-well plates was 4.5 mm for all experi-
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DMSO, immediately irradiated (or not) at 365 nm for 5 min using a
UVL-21 hand lamp (0.32 mW/cm2 at 365 nm at 152 mm distance)
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Acknowledgments
This work was supported by institutional grants from the Uni-
versity of Manitoba, Canadian Institutes of Health Research, Na-

838
G.-L. Chee et al. / Bioorg. Med. Chem. 18 (2010) 830–838
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