Azide vs Alkyne Functionalization in Pt(II) Complexes for Post-treatment Click Modification: Solid-State Structure, Fluorescent Labeling, and Cellular Fate

Article abstract of DOI:10.1021/jacs.5b09108

Tracking of Pt(II) complexes is of crucial importance toward understanding Pt interactions with cellular biomolecules. Post-treatment fluorescent labeling of functionalized Pt(II)-based agents using the bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddi

Full text of DOI:10.1021/jacs.5b09108

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Article
Azide vs. Alkyne Functionalization in Pt(II) Complexes
for Post-Treatment Click Modification: Solid State
Structure, Fluorescent Labeling, and Cellular Fate
Regina Wirth, Jonathan D. White, Alan D. Moghaddam, Aurora L.
Ginzburg, Lev N. Zakharov, Michael M. Haley, and Victoria J. DeRose
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b09108 • Publication Date (Web): 29 Oct 2015
Downloaded from http://pubs.acs.org on October 29, 2015
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Azideꢀvs.ꢀAlkyneꢀFunctionalizationꢀinꢀPt(II)ꢀComplexesꢀforꢀPost-
TreatmentꢀClickꢀModification:ꢀSolidꢀStateꢀStructure,ꢀꢀꢀFluorescentꢀ
Labeling,ꢀandꢀCellularꢀFateꢀ
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†
,#
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‡
Regina Wirth, Jonathan D. White, Alan D. Moghaddam, Aurora L. Ginzburg, Lev N. Zakharov,
†
,†
Michael M. Haley, and Victoria J. DeRose*
†
Department of Chemistry & Biochemistry and the Institute of Molecular Biology, University of Oregon, Eugene, Oregon
97403-1253, United States
‡
CAMCOR, University of Oregon, 1443 East 13th Ave., Eugene, Oregon 97403, United States
#
These authors contributed equally to this work
Supporting Information
ABSTRACT: Tracking of Pt(II) complexes is of crucial importance towards understanding Pt interactions with cellular bio-
molecules. Post-treatment fluorescent labeling of functionalized Pt(II)-based agents using the bioorthogonal Cu(I)-catalyzed azide-
alkyne cycloaddition (CuAAC) reaction has recently been reported as a promising approach. Here we describe an azide-
2
functionalized Pt(II) complex, cis-[Pt(2-azidobutyl)amido-1,3-propanediamine)Cl ] (1), containing the cis geometry and difunc-
tional reactivity of cisplatin, and present a comparative study with its previously described alkyne-functionalized congener. Single
crystal X-ray diffraction reveals a dramatic change in the solid-state arrangement with exchange of the alkyne for an azide moiety
wherein 1 is dominated by a pseudo-chain of Pt–Pt dimers and antiparallel alignment of the azide substituents, in comparison with a
circular arrangement supported by CH/π(C≡C) interactions in the alkyne version. In vitro studies indicate similar DNA binding and
click reactivity of both congeners observed by fluorescent labeling. Interestingly complex 1 shows in vitro enhanced click reactivity
in comparison to a previously-reported azide-appended Pt(II) complex. Despite their similar behavior in vitro, preliminary in cell-
ulo HeLa studies indicate a superior imaging potential of azide-functionalized 1. Post-treatment fluorescent labeling of 1 observed
by confocal fluorescence microscopy shows nuclear and intense nucleolar localization. These results demonstrate the potential of 1
in different cell line localization studies and for future isolation and purification of Pt-bound targets.
Introduction
Since the
diamminedichloroplatinum(II), was approved by the FDA for
clinical use in 1978, Pt-based therapeutics have become one of
the most widely used and successful treatment options for a
variety of cancers including ovarian, testicular, bladder, non-
small-cell lung carcinomas, and others. Although much pro-
gress has been made in understanding the mechanisms of ac-
tion of Pt compounds as well as their specific cellular targets,
a comprehensive molecular-level understanding of their reac-
Pt has been found to bind to a wide variety of additional nu-
cleophilic targets, including different cellular RNAs, proteins
and sulfur-containing compounds such as glutathione. Despite
a great deal of work, Pt localization in treated cells and bind-
compound
cisplatin,
cis-
ing properties with non-DNA targets, and their consequences,
6,7,15
remain poorly understood.
A better understanding of Pt
1
-4
cellular reactivity is essential in order to design new, more
effective therapeutics and methods of delivery.
In addition to identifying molecular targets of Pt reagents,
a comprehensive identification of all organelle targets is de-
sired. Such an understanding would shed light on apoptotic
and toxicity pathways, as well as differences in efficacy in
dissimilar types of cancers. Highlighting the diversity of po-
tential cellular locales, various studies using atom-based tech-
niques as well as fluorescent tagging have obtained evidence
for cellular Pt accumulation in ribosomes, the nucleus, nucle-
oli, mitochondria, and secretory pathways linked to vesicles,
5-8
tivity is still lacking. It is generally accepted that Pt reagents
are taken up into the cell via passive and active diffusion, then
undergo aquation, the displacement of labile chloride ligands
+
2+
3 2 2 3 3 2 2
by water, forming [Pt(NH ) Cl(OH )] and [Pt(NH ) (OH ) ]
9
,10
species. These highly reactive species form exchange-inert
complexes with purine bases of genomic DNA in addition to
1
1
other biomolecules. In vivo and in vitro studies have unam-
biguously identified cis-1,2-{Pt(NH ) } -d(GpG), 1,2-d(ApG)
3 2
1
6-18
2
+
lysosomes, and Golgi.
Fluorescence detection via confocal microscopy has dis-
tinct advantages of sensitivity and resolution, and can be cou-
pled with existing fluorescent markers for co-localization stud-
ies. An important strategy used to detect Pt in cells comprises
the covalent tethering of fluorescent probes to modified Pt-
coordinated ligands. In one such study, a fluorescein-linked Pt
and 1,3-d(GpNpG) intrastrand and interstrand DNA crosslinks
as products, and these are capable of inhibiting normal tran-
scriptional processes of the cell and initiating apoptotic signal-
1
2,13
ing.
Importantly, only a very small fraction of Pt (less than
1
0% in the case of cisplatin) accumulates within genomic
1
4
DNA. In samples isolated from cells treated with Pt reagents,
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compound was observed to accumulate in the nucleus and completely complementary pair of Pt-click reagents and rho-
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nucleolus after 2-3 h post-treatment, followed by punctate
localization in the cytoplasm after 7 h. A gradual decrease in
total cellular fluorescence was observed over several days,
damine-fluorophore probes. Both compounds 1 and 2 were
designed to mimic the cis geometry and difunctional reactivity
of cisplatin and other Pt(II)-based FDA-approved chemothera-
peutics.
1
7
indicating that Pt actively effluxes from the cells. Other Pt-
fluorescein conjugates have revealed accumulation in the
Golgi and in vesicles, and expression of Cu efflux proteins
1
8
was demonstrated to affect Pt retention. Other important
conclusions regarding the reactivity of Pt compounds have
been determined via modification with fluorescent probes, and
Results and Discussion
1
9
these have been reviewed recently. While fluorophore-linked
Pt probes have been shown to mimic some properties of native
Pt complexes, the physical properties of the large, hydropho-
bic probes and their interactions within the cell are expected to
be quite different from the small, neutral or positively-charged
Compound 1 was synthesized analogously to the prepara-
tion of 2, with the peptide linkage allowing for facile incorpo-
ration of different coupling partners (Figure 2). Therefore di-
tert-butyl (2-aminopropane-1,3-diyl)dicarbamate was coupled
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to 4-azidobutanoic acid with EDC. Deprotection with anhy-
drous HCl followed by platination of [Pt(DMSO )Cl ] yielded
the final product 1 in 16% overall yield. Both complexes 1 and
1
7-20
Pt complexes.
These differences may influence the types
2
2
of interactions that direct localization of the Pt complex, such
as intercalation, hydrophobic, or electrostatic-based interac-
tions. It has been shown, for example, that different amino-
acid-linked cisplatin analogues exhibit altered specificity to
1
95
2 exhibit identical Pt NMR chemical shifts (δ = –2286 ppm,
-DMF; see Supporting Information), indicating no inter- or
d
7
intramolecular interactions between the Pt center and peptide-
2
1
29
ribosomal RNA compared to the parent compound.
linked azide or alkyne groups.
Figure 2. Synthesis of Pt-complex 1. Boc = t-butoxycarbonyl,
EDC
DMSO
=
1-ethyl-3-(3-N,N-dimethylaminopropyl)carbodiimide,
dimethylsulfoxide, DBU 1,8-diazabicyclo-
=
=
[5.4.0]undec-7-ene, DMF = N,N-dimethylformamide.
Crystals of 1 suitable for X-ray diffraction were obtained
from a chilled DMF/H O solution, and the structure obtained
2
Figure 1. (a) Chemical structures of the anticancer drug cisplatin,
with the novel azide-appended 1 and alkyne-appended 2. (b) Re-
action scheme of target-bound 1 undergoing post-binding covalent
click modification to a fluorogenic probe.
confirms that assigned via NMR spectroscopy. The individual
bond lengths and angles around the square planar Pt(II) center
of the azide-containing 1 are very similar to the reported al-
2
6,30
Due to possible undesirable consequences of pre-
attaching probes, fluorescence detection of Pt compounds
through post-binding modifications would be more attractive.
This strategy takes advantage of minimally invasive reactive
handles incorporated onto Pt reagents, followed by conjuga-
tion to fluorescent probes after the Pt compound has bound
irreversibly to biomolecule targets (Figure 1). Using this strat-
egy, we have previously reported use of the Cu-catalyzed az-
kyne-appended 2 (see Supporting Information).
Both com-
pounds form Pt–Pt dimers (Pt…Pt, 3.46 Å in 1) connected in
the crystal structure by hydrogen bonds (Figure 3 and Support-
3
1,32
ing Information).
Each chloride acts as a hydrogen bond
acceptor for the Pt-coordinated amine groups (Cl…N, 2.53 Å,
3
2
2.35 Å in 1) (Figure 3a). In addition, a distance of 2.13 Å
between coordinated amine protons and adjacent carbonyl
oxygen indicates hydrogen bonding (See Supporting Informa-
tion, Figure S3).
2
2
ide-alkyne cycloaddition (CuAAC) click reaction, with
modified Pt(II) complexes containing azide- and alkyne-
Despite the similarities in the solid-state between 1 and 2,
the broader packing arrangement changes drastically upon
exchange of the alkyne with the azide moiety. The arrange-
ment of 2 is primarily dictated by CH/π(C≡C) hydrogen bonds
23-26
reactive “handles”.
We have identified cellular RNAs,
including tRNA, as targets of Pt reagents in vivo, in addition23-t2o7
identifying other nucleotide and protein targets in vitro.
Recently, Bierbach and colleagues have used post-binding
click modification of a monofunctional Pt-acridine hybrid
complex to identify its cellular distribution within the nucleus
26
forming a rare, spoke-type arrangement (Figure 3c). Ar-
rangement of the azide-containing 1 appears to be dictated by
the formation of the Pt dimer, including both Pt–Pt bonding
and hydrogen bonds. The Pt centers are aligned into infinite
1D-zigzag chains with regular alternating distances of 3.46
and 3.98 Å, with a Pt…Pt…Pt bond angle of 159.6º. Interest-
ingly, the azide moieties of adjacent Pt “chains” are aligned
antiparallel and in close proximity to one another, approxi-
mately 3.5 Å apart (Figure 3b). This is in agreement with pre-
2
0b
and nucleoli of lung cancer cells. Herein, we report the de-
sign and use of the new click-capable Pt(II)-azide complex 1
and its cellular distribution in HeLa cells, as observed via con-
focal fluorescence microscopy. The in-cell and in vitro reac-
tivity of 1 is also compared with the previously reported al-
2
6
kyne-modified congener 2, demonstrating the utility of the
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viously reported azide-azide packing distances reported for
well-established parameters (1.12 and 1.28 Å for the terminal
and internal N–N bond distances, respectively, and N≡N–N
bond angle of 174.1º).
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several organo-azides, and may indicate the presence of a
weak dipole-dipole interaction between nitrogen atoms of
3
3,34
3
3
opposing azides. Finally, N–N bond lengths and angles of
the azide moieties themselves are in good agreement with
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Figure 3. (a) Crystal structure arrangement of 1 showing the Pt–Pt dimer formation in a 1D-zigzag chain with a 159.6º angle between the
platinum centers as well as Cl…H–N hydrogen bonding. Selected bond lengths and angles: Pt–Cl 2.315(2) Å, 2.334(3) Å; Pt–N 2.016(8)
Å, 2.045(7) Å, N≡N 1.117(11) Å, N–N 1.280(12) Å, N≡N–N 174.2(11)º. (b) Antiparallel arrangement of the azide moieties of adjacent Pt
chains. (c) Crystal structure arrangement of 2 showing the spoke-type arrangement dictated by CH/π(C≡C) hydrogen bonds. See Support-
ing Information for detailed crystallographic data.
To confirm the ability of 1 to bind known targets of Pt
and subsequently undergo post-binding click modifications, 1
was bound to a short oligonucleotide sequence containing a
peptide-
propanediamine)Cl
utilized by our group for post-treatment fluorescent click la-
and
linker-free
cis-[Pt(2-azido-1,3-
3
7
2
] (5). Compound 5 has been previously
2
5
5
’-GG-3’ pair and subsequently reacted with an alkyne-
beling of nucleotides and proteins in vitro, and rRNA in vivo.
functionalized rhodamine fluorophore (3) (see Supporting
Information) and subjected to dPAGE. Both compounds 1 and
2
undergo facile binding to DNA (18 h, 37 ºC), followed by
near-complete conversion to the rhodamine-labeled click
product (CuSO -ascorbate, 18 h, rt), the latter complex 2 hav-
4
ing been reacted with the analogous azide-functionalized rho-
35
damine fluorophore (4) (Figure 4). Both complexes appear to
bind to DNA in similar yields, which suggests the identity of
the click “handle” (i.e., azide versus alkyne) does not signifi-
cantly affect binding or click reactivity in vitro.
Further in vitro experiments were conducted to obtain
deeper insight into the binding and click reactivity of 1. Since
we now possessed two complementary click-appended Pt(II)
complexes, we were curious if they could be reacted together
to covalently attach two Pt-bound targets. Solutions of 1 and 2
were individually bound to separate 5’-GG-3’ DNA hairpins,
then reacted together under click conditions analogous to the
previously described reactions with rhodamine (24 h, rt). The
appearance of a band of significantly less mobility indicates
the formation of the triazole-linked hairpin DNA dimer (lane
4, Figure 5). This result suggests the potential for these Pt
compounds to be used in nucleotide cross-linking applications
3
6
and other diverse nucleotide modification reactions.
To assess the influence of the peptide linkage of 1 (and 2)
on DNA binding and click reactivity, we compared 1 to the
Figure 4. (a) Reaction scheme of the binding of 1 and 2 to a DNA
hairpin followed by fluorescent click-labeling using complemen-
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tary alkyne- or azide-rhodamines (3, 4). (b) dPAGE experiment
containing rhodamine fluorophore 3 (5 µM, 2h) under CuAAC
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showing the binding of 1 and 2 to a 5’-GG-3’ DNA hairpin (lanes
2 and 3). The click reaction of 1- and 2-bound hairpin with the
complementary rhodamine fluorophores can be observed by the
appearance of a fluorescent band (lanes 4 and 6). Control reac-
tions show no click ligation under Cu-free conditions (lanes 5 and
click conditions (see Supporting Information). Confocal im-
ages of the cells show the highest level of fluorescence in the
nucleoli of the HeLa cells, along with broad localization in the
nucleus (Figure 7b). Importantly, the Cu-free controls show no
fluorescence from nonspecific fluorophore interactions (Figure
7
).
7
f and SI). These results are consistent with previously re-
ported targets of Pt(II) chemotherapeutics. The high accumula-
tion of compound 1 in the nucleoli is in agreement with previ-
1
0
ously identified nuclear DNA adducts of Pt, as well as Pt-
rRNA adducts. rRNA has been shown to bind Pt(II) com-
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plexes using inductively coupled plasma mass spectrometry
24,27
and fluorescent labeling in Saccharomyces cerevisiae.
Pt-
protein adducts may also be present. The nucleolus has also
been identified as a major target using fluorophore pre-
3
9
tethered
Pt(II)
complexes.
Figure 5. (a) Reaction scheme of biomolecule-bound azide 1
clicking to the biomolecule-bound alkyne 2 to form a cross-linked
DNA. (b) dPAGE experiment showing 1 and 2 bound to a 5’-GG-
3’ DNA hairpin (lanes 2 and 3). Lane 4 shows the click reaction
product between 1- and 2-bound DNA hairpins resulting in a tria-
zole-linked DNA dimer (see Supporting Information for complete
conditions).
Figure 6. (a) Reaction scheme of binding of the azide-modified
Pt(II) complexes 1 and 5 to a 5’-GG-3’ DNA hairpin sequence,
followed by subsequent CuAAC with dansyl alkyne fluorophore.
For comparison, 1 and 5 were each bound to a 5’-GG-3’ DNA
hairpin sequence and subjected to click conditions with a dan-
syl alkyne fluorophore (Figure 6a) and analyzed via dPAGE
(
b) dPAGE analysis of binding and fluorophore conjugation of 1
and 5 to DNA hairpin (see Supporting Information for condi-
tions).
(
Figure 6b). Both compounds appear to bind to DNA in simi-
Bierbach and colleagues very recently identified localization
in the nucleolus using an azide-modified Pt-acridine complex
for post-binding fluorescent labeling as well as pre-tethered
Pt-fluorophores, although differences between the two ap-
lar yields (lanes 2 and 3), indicating binding reactivity inde-
pendent of the linker moiety. The click reactivity was evalu-
ated by time-dependent (0.5, 3, or 6 h reaction times) treat-
ment of the Pt-DNA complex with the dansyl-alkyne fluoro-
phore under click conditions. Interestingly, 1-bound DNA is
completely converted to clicked product after only 3 h reaction
time, whereas even after 6 h click reaction time, unreacted
2
0a,b
proaches were noted.
Treatment of HeLa cells using the complementary al-
kyne-containing complex 2 and the rhodamine-azide 4 (25 µM
treatment) shows identical localization (Figure 7d), which
indicates similar cellular binding of both Pt complexes 1 and 2
independent of the click-functionalized moiety. Interestingly,
however, Pt(II)-concentration dependent post-treatment label-
ing in the HeLa cell line shows a dramatic difference in the
observed fluorescent signal between these two complementary
click compounds. Despite the similar localization of 1 and 2,
there is significantly less fluorescent signal following 25 µM
cellular treatment with 2, using identical conditions. HeLa
cells were also treated individually with 5 µM complex 1 and
(
non-clicked) 5-bound DNA is observed (Figure 6, lane 8).
The observed enhanced reactivity of the longer 1 may be due
to increased steric accessibility of the azide moiety once
bound to DNA. Additionally, non-innocence of the peptide
bond participating in the click reaction cannot be ruled out—
for example, Ting and colleagues reported an acceleration of
the CuAAC reaction using organo-azides that contain internal
3
8
Cu(I) chelating moieties.
Encouraged by its apparent enhanced reactivity, we pur-
sued cellular localization studies using the azide-appended 1
and confocal fluorescence microscopy (Figure 7). In initial
studies, HeLa cells were treated with 1 (25 µM, 3h), washed,
fixed and permeabilized, and then labeled with the alkyne-
2
for 3 h followed by click labeling with the complementary
rhodamine fluorophores. At this lower concentration, cells
treated with the alkyne-appended complex 2 showed no de-
tectable fluorescent signal above background, but cells treated
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counterparts may also have an effect on accumulation or reac-
with azide-containing 1 present bright and distinct fluores-
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cence (Figure 7j). Although we have not observed Pt-
catalyzed hydration or hydroamination across the triple bond
in previous experiments using 2, it is well-reported that al-
kyne-containing compounds can undergo these reactions,
which could lead to a decrease in fluorescent signal in-cell.
The hydrophobicity of alkynes in comparison to their azide
tivity in the cell. These promising results show the potential of
compound 1 not only for further localization studies in differ-
ent cancer cell lines, but also for ongoing goals of isolating
and identifying Pt(II)-bound targets using click chemistry.
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Figure 7. Confocal image of fluorescent cellular localization. B shows the fluorescent labeling of HeLa Cells treated with 1 (25 µm, 3h)
and subsequent click with rhodamine alkyne 3. The Cu-free control (f) shows no fluorescence. D shows the fluorescent labeling of HeLa
Cells treated with 2 (25 µm, 3h), after fixation, permeabilization and labeling with the rhodamine azide 4 in the HeLa cell line and Cu-free
control (h). J shows the enlarged image of fluorescent labeling of HeLa Cells treated with 1 (5 µm, 3h) and subsequent click with rho-
damine alkyne 3. The overlay of the DAPI stain and rhodamine labeling is depicted in k.
Conclusions
contrast, the arrangement of the azide analogue is dominated
by Pt–Pt dimer formation and hydrogen bonding forming a
In summary, we have developed a novel azide-containing
Pt(II) complex (1), compared it to its alkyne-containing con-
gener (2), and report here for the first time the post-treatment
fluorescent labeling of a bifunctional cisplatin-based Pt(II)
complex in the HeLa cancer cell line. The exchange of the
alkyne to the azide moiety leads to a completely different ar-
rangement in the solid state, with the alkyne containing 2
forming a circular arrangement dominated by CH/π(C≡C)
hydrogen bonds and secondary Pt–Pt dimer interactions. In
1
D–zig-zag chain. In solution both complexes bind similarly
to a DNA hairpin and show similar click reactivity in post-
binding fluorescent labeling using complementary click-
modified rhodamine fluorophores. Not only were we able to
show the utilization of 1 as a crosslinking agent for larger
biomolecules, but we also demonstrated its faster click reactiv-
ity in comparison to a previously described azide-containing
Pt(II) complex. Finally, we report the first post-treatment fluo-
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rescent labeling of a cisplatin-like bifunctional Pt(II) complex
in HeLa cells, observing Pt(II) localization in the nuclei and
distinctively in the nucleoli. Concentration-dependent localiza-
tion showed stronger fluorescent signals using the azide 1
versus the alkyne 2 Pt-complex. These results show the poten-
tial of the novel compound 1 for further cell-line dependent
localization studies and the isolation of Pt(II)-bound targets.
Tomioka, M.; Goodman, M.; Howell, S. B. Clin. Cancer Res. 2004,
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10, 4661. (d) Jansen, B. A. J.; Wielaard, P.; Kalayda, G. V.; Ferrari,
M.; Molenaar, C.; Tanke, H. J.; Brouwer, J.; Reedijk, J. J. Biol. Inorg.
Chem. 2004, 9, 403. (e) Molenaar, C.; Teuben, J.-M.; Heetebrij, R. J.;
Tanke, H. J.; Reedijk, J. J. Biol. Inorg. Chem. 2000, 5, 655. (f)
Kalayda, G. V.; Jansen, B. A. J.; Molenaar, C.; Wielaard, P.; Tanke,
H. J.; Reedijk, J. J. Biol. Inorg. Chem. 2004, 9, 414. (g) Kalayda, G.
V.; Jansen, B. A. J.; Wielaard, P.; Tanke, H. J.; Reedijk, J. J. Biol.
Inorg. Chem. 2005, 10, 305. (h) Beretta, G. L.; Righetti, S. C.;
Lombardi, L.; Zunino, F.; Perego, P. Ultrastruc. Pathol. 2002, 26,
ASSOCIATEDꢀCONTENTꢀꢀ
3
31. (i) Meijer, C.; Van Luyn, M. J. A.; Nienhuis, E. F.; Blom, N.;
Mulder, N. H.; De Vries, E. G. E. Biochem. Pharmacol. 2001, 61,
73. (j) Khan, M. U. A.; Sadler, P. J. Chem.-Biol. Inter. 1978, 21,
Supporting Information. Text and figures giving methods, ex-
perimental procedures, syntheses, dPAGE data, confocal micros-
copy data and NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org
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227. (k) Berry, J. P.; Galle, P.; Viron, A.; Kacerovská, H.; Macieira-
Coelho, A. Biomed. Pharmacother. 1983, 37, 125. (l) Hall, M. D.;
Hambley, T. W.; Beale, P.; Zhang, M.; Dillon, C. T.; Foran, G. J.;
Stampfl, A. P.; Lai, B. J. Inorg. Biochem. 2003, 96, 141. (m) Hall, M.
D.; Alderden, R. A.; Zhang, M.; Beale, P. J.; Cai, Z.; Lai, B.; Stampfl,
A. P. J.; Hambley, T. W. J. Struct. Biol. 2006, 155, 38. (n) Safaei, R.;
Katano, K.; Larson, B. J.; Samimi, G.; Holzer, A. K.; Naerdemann,
ꢀW.; Tomioka, M.; Goodman, M.; Howell, S. B. Clin. Cancer Res.
AUTHORꢀINFORMATIONꢀ
CorrespondingꢀAuthorꢀ
*derose@uoregon.edu
AuthorꢀContributionsꢀ
2
005, 11, 756.
The manuscript was written through contributions of all authors.
(17) Molenaar, C.; Teuben, J.-M.; Heetebrij, R. J.; Tanke, H. J.;
Reedijk, J. J. Biol. Inorg. Chem. 2000, 5, 655.
18) Safaei, R.; Katano, K.; Larson, B. J.; Samimi, G.; Holzer, A.
(
ACKNOWLEDGMENTꢀꢀ
K.; Naerdemann, ꢀW.; Tomioka, M.; Goodman, M.; Howell, S. B.
Clin. Cancer Res. 2005, 11, 756.
(19) White, J. D.; Haley, M. M.; DeRose, V. J. submitted.
The authors thank the National Science Foundation (CHE-
1
413677 to VJD) for funding.
(
20) (a)Ding, S.; Qiao, X.; Suryadi,J.; Marrs, G. S.; Kucera, G. L.;
REFERENCESꢀꢀ
Bierbach, U. Angew. Chem., Int. Ed. 2013, 52, 3350. (b) Qiao, X.;
Ding, S.; Liu, F.; Kucera, G. L.; Bierbach, U. J. Biol. Inorg. Chem.
014, 19, 415. (c) Wexselblatt, E.; Yavin, E.; Gibson, D. Inorg. Chim.
Acta 2012, 393, 75. (d) Ghosh, B.; Jones, L. H. Med. Chem. Commun.
2014, 5, 247.
(
1) Rozencweig, M.; Von Hoff, D. D.; Slavik, M.; Muggia, F. M.
Ann. Intern. Med. 1977, 112, 100.
2) Harper, B. W.; Krause-Heuer, A. M.; Grant, M. P.; Manohar,
2
(
M.; Garbutcheon- Singh, K. B.; Aldrich-Wright, J. R. Chem.–Eur. J.
2010, 16, 7064.
(
21) Rijal, K.; Bao, X.; Chow, C. S. Chem. Commun. 2014, 50,
918.
22) (a) Kolb, H.C.; Finn, M. G.; Sharpless, K. B. Angew. Chem.,
Int. Ed. 2001, 40, 2004.
23) White, J. D.; Osborn, M. F.; Moghaddam, A. D.; Guzman, L.
E.; Haley, M. M.; DeRose, V. J. J. Am. Chem. Soc. 2013, 135, 11680.
24) Osborn, M. F.; White, J. D.; Haley, M. M.; DeRose, V. J. ACS
Chem. Biol. 2014, 9, 2404.
25) Moghaddam, A. D.; White, J. D.; Cunningham, R. M.; Loes,
A. N.; Haley, M. M.; DeRose, V. J. Dalton Trans. 2015, 44, 3536.
26) White, J. D.; Guzman, L. E.; Zakharov, L. N.; Haley, M. M.;
DeRose, V. J. Angew. Chem., Int. Ed. 2015, 54, 1032.
27) Hostetter, A. A.; Osborn, M. F.; DeRose, V. J. ACS Chem.
Biol. 2012, 7, 218.
28) Ramalingam, K; Raju, N.; Nanjappan, P.; Nowotnik, D. P.
Tetrahedron 1995, 51, 2875.
29) Still, B. M.; Kumar, Aldrich-Wright, J. R.; Price, W. S. Chem.
Soc. Rev. 2007, 36, 665.
30) X-Ray Data for 1: C
3
(
(
3) Dyson, P.J.; Sava, G. Dalton Trans. 2006, 1929.
4) Rosenberg, B.; Renshaw, E.; Van Camp, L.; Hartwick, J.;
(
Drobnik, J. Bacteriol. 1967, 93, 716.
5) Sava, G.; Jaouen, G.; Hillard, E. A.; Bergamo, A. Dalton Trans.
(
(
2
012, 41, 8226.
(
(6) Casini, A.; Reedijk, J. Chem. Sci. 2012, 3, 3135.
(
7) Guggenheim, E. R.; Xu, D.; Zhang, C. X.; Chang, P. V.; Lip-
pard, S. J. Chem. Bio. Chem. 2009, 10, 141.
8) Wexselblatt, E.; Yavin, E.; Gibson, D. Inorg. Chim. Acta 2012,
93, 75.
9) Arnesano F.; Losacco, M.; Natile, G. Eur. J. Inorg. Chem.
2013, 2701.
(
(
(
3
(
(
(
(
(
(
10) Wang, D.; Lippard, S. J. Nat. Rev. 2005, 4, 307.
(
11) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99, 2467.
12) Todd, R. C.; Lippard, S. J. Metallomics 2009, 1, 280.
13) (a) Caradonna, J. P.; Lippard, S. J.; Gait, M. J.; Singh M. J.
(
Am. Chem. Soc. 1982, 104, 5793. (b) Fichtinger-Schepman, A. M. J;
Van der Veer, J. L.; Den Hartog J. H. J; Lohman, P. H. M; Reedijk, J.
Biochemistry 1985, 24, 707. (c) Eastman, A. Biochemistry 1986, 25,
(
7 2 6
H16Cl N OPt, M = 466.25, 0.07 x 0.04 x
0
7
.03 mm, T = 173 K, triclinic, space group P-1, a = 7.330(2) Å, b =
.583(2) Å, c = 11.831(3) Å, α = 85.465(5)°, β = 86.040(5)°, γ =
3
912. (d) Terheggen, P. M. A. B.; Floot, B. G. J.; Scherer, Begg,
A.C.; Fichtinger-Schepman, A. M. J.; Engelse, L. Cancer Res. 1987,
7, 6719.
14) (a) DeConti, R. C.; Toftness, B. R.; Lange, R. C.; Creasey, W.
3
3
c
86.498(5)°, V = 653.1(3) Å , Z = 2, D = 2.371 Mg/m , µ(Mo)= 11.147
–1
4
mm , F(000) = 440, 2θmax = 50.0°, 7931 reflections, 2287 independ-
ent reflections [Rint = 0.0774], R1 = 0.0403, wR2 = 0.0644 and GOF
= 1.011 for 2287 reflections (154 parameters) with I>2σ(I), R1 =
0.0627, wR2 = 0.0704 and GOF = 1.011 for all reflections, max/min
(
A. Cancer Res. 1973, 33, 1310. (b) Akaboshi, M.; Kawai, K.; Ujeno,
Y.; Takada, S.; Miyahara, T. Jpn. J. Cancer Res. 1994, 85, 106. (c)
Akaboshi, M.; Kawai, K.; Maki, H.; Akuta, K.; Ujeno, Y.; Miyahara,
T. Jpn. J. Cancer Res. 1992, 83, 522.
3
residual electron density +1.423/-1.283 eÅ .
(31) Woollins, J. D.; Kelly, P. F. Coord. Chem. Rev. 1985, 65, 115.
(32) Odoko. M; Okabe, N. Acta Crystallogr. Sect. C: Cryst. Struct.
Commun. 2006, 62, m 136
(33) (a) Guo, L.; Thompson C. M.; Twamley B. Acta Crystallogr.
Sect. C: Cryst. Struct. Commun. 2009, 65, 0179. (b) Yang, H.; Carter,
R. G. J. Org. Chem. 2010, 75, 4929. (c) Brady, R. M.; Zhang, M.;
Gable, R.; Norton, R. S.; Baell J. B. Bioorg. Med. Chem. Lett. 2013,
23, 4892. (d) Parsons, S.; Haxton, A.; Flitsch, S.; Wood, P. A., private
(
15) (a) Wisnovsky, S. P.; Wilson, J. J.; Radford, R. J.; Pereira, M.
P.; Chan, M. R.; Laposa, R. R.; Lippard, S. J.; Kelley, S. O. Chem.
Biol. 2013, 20, 1323. (b) Yu, F.; Megyesi, J.; Price, P. M. Am. J.
Physiol. Renal Physiol. 2008, 295, F44-F52.
(
16) Inter alia: (a) Gemba, M.; Nakatani, E.; Teramoto, M.; Na-
kano, S. Toxicol. Lett. 1987, 38, 291. (b) Samimi, G.; Katano, K.;
Holzer, A. K.; Safaei, R.; Howell, S. B. Mol. Pharmacol. 2004, 66,
2
5. (c) Samimi, G.; Safaei, R.; Katano, K.; Holzer, A. K.; Rochdi, M.;
6
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Journal of the American Chemical Society
communication to the Cambridge Crystallographic Data Centre,
2004, DOI:10.5517/cc88jrs.
34) (a) Braese, S.; Gil, C.; Knepper, K.; Zimmermann, V. Angew.
Chem., Int. Ed. 2005, 44, 5188. (b) Chen, F-F.; Wang, F. Molecules
009, 14, 2656.
35) Baier, G.; Siebert, J. M.; Landfester, K.; Musyanovych, A.
(39)(a) Liang, X. J.; Shen, D. W.; Chen, K. G.; Wincovitch, S. M.;
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Garfield, S. H.; Gottesman, M. M. J. Cell. Physiol. 2005, 202, 635.
(b) Colombo, A; Fiorini, F.; Septiadi, D.; Dragonetti, C.; Nisic, F.;
Valore, A.; Roberto, D.; Mauro,M.; DeCola,L. Dalton Trans., 2015,
44, 8478. (c) Shen, C.; Harris, B. D. W.; Dawson, L. L.; Charles, K.
A.; Hambley, T. W.; New. E. J. Chem. Commun., 2015, 51, 6312. (d)
Wu, S.; Zhu, C.; Zhang, C.; Yu, Z.; He, W.; He, Y.; Li, Y.; Wand, J.;
Guo, Z. Inorg. Chem. 2011, 50, 11847.
(
2
(
Macromolecules 2012, 45, 3419.
(36) Chapman, E. G.; DeRose, V. J. J. Am. Chem. Soc. 2012, 134,
2
56.
(40) Belluco, U; Bertani, R.; Michelin, R. A.; Mozzon, M. J. Or-
ganomet. Chem. 2000, 600, 37.
(
(
37) Urankar, D.; Kosmrlj, J. Inorg. Chim. Acta 2010, 363, 3817.
38) Uttamapinant, C.; Tangpeerachaikul, A.: Grecian, S.; Clarke,
S.; Singh, U.; Slade, P.; Gee, K. R.; Ting, A. Y. Angew. Chem., Int.
Ed. 2012, 51, 5852.
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