An unusual ligand coordination gives rise to a new family of rhodium metalloinsertors with improved selectivity and potency

Article abstract of DOI:10.1021/ja5072064

Rhodium metalloinsertors are octahedral complexes that bind DNA mismatches with high affinity and specificity and exhibit unique cell-selective cytotoxicity, targeting mismatch repair (MMR)-deficient cells over MMR-proficient cells. Here we describe a new generation of metalloinsertors with enhanced biological potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both δ- and -[Rh(chrysi)(phen)(DPE)]²⁺ (where chrysi =5,6 chrysenequinone diimmine, phen =1,10-phenanthroline, and DPE = 1,1-di(pyridine-2-yl)ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the mismatch site. We ascribe this difference to the higher pK_a of the coordinated immine of the chrysi ligand in these complexes, so that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than cisplatin and N-methyl-N'-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective cytotoxicity for MMR-deficient versus proficient colorectal cancer cells.

Full text of DOI:10.1021/ja5072064

Article
pubs.acs.org/JACS
An Unusual Ligand Coordination Gives Rise to a New Family of
Rhodium Metalloinsertors with Improved Selectivity and Potency
Alexis C. Komor and Jacqueline K. Barton*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena California 91125, United States
*
S Supporting Information
ABSTRACT: Rhodium metalloinsertors are octahedral com-
plexes that bind DNA mismatches with high affinity and
specificity and exhibit unique cell-selective cytotoxicity,
targeting mismatch repair (MMR)-deficient cells over MMR-
proficient cells. Here we describe a new generation of
metalloinsertors with enhanced biological potency and
selectivity, in which the complexes show Rh−O coordination.
In particular, it has been found that both Δ- and Λ-
2
+
[
Rh(chrysi)(phen)(DPE)] (where chrysi =5,6 chrysenequi-
none diimmine, phen =1,10-phenanthroline, and DPE = 1,1-
di(pyridine-2-yl)ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This
is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the
mismatch site. We ascribe this difference to the higher pK of the coordinated immine of the chrysi ligand in these complexes, so
a
that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the
presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment.
Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays;
indeed, all are more potent than cisplatin and N-methyl-N′-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating
chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective
cytotoxicity for MMR-deficient versus proficient colorectal cancer cells.
INTRODUCTION
on the development of rhodium metalloinsertors (Figure 1),
which bind mismatches in duplex DNA with high specificity,
preferentially targeting thermodynamically destabilized mis-
■
Since the successful application of cisplatin (cis-diamminedi-
chloroplatinum) as an anticancer drug, the field of inorganic
medicinal chemistry has undergone a revolution.
years, the field focused on the development of more potent
analogues (second- and third-generation derivatives), leading to
the FDA approval of two additional cis-platinum(II) complexes,
carboplatin and oxaliplatin. Cisplatin and carboplatin, in
particular, have been highly successful in the treatment of a
variety of cancers, including testicular, ovarian, cervical, and
8,9
1
−4
matches over matched base pairs by a factor of over 1000.
For many
The binding mode of these complexes to mismatched DNA
was subsequently crystallographically determined; the wide
aromatic chrysi (chrysi = 5,6 chrysenequinone diimmine)
ligand inserts into the DNA from the minor groove and ejects
both mismatched bases in a binding mode termed metal-
5
1
0,11
loinsertion.
Ejection of the mismatched bases results in a
6
large lesion that we have suggested may serve as a unique target
within the cell.
non-small cell lung cancers. However, these treatments are
often associated with severe side effects and a buildup of
resistance. These issues have led to a shift in the design of
chemotherapeutics; researchers have begun to focus on a new
design strategy where the chemotherapeutic interacts with a
Mismatches in genomic DNA arise naturally as a
consequence of replication, but, if left uncorrected, can lead
1
2,13
to mutations.
The mismatch repair (MMR) pathway serves
7
as a checkpoint to increase the fidelity of DNA replication
specific biological target found predominantly in cancer cells.
14
∼
1000 fold. Importantly, deficiencies in the mismatch repair
The proposed mechanism of action of classical platinum-
based chemotherapeutics is the formation of covalent DNA
adducts, followed by cellular processing of these lesions,
machinery have been associated with several types of cancer as
well as increased resistance to classical chemotherapeutics such
as cisplatin as well as commonly used DNA-alkylating
6
ultimately leading to apoptosis. The synthesis of new-
15
therapeutics. Therefore, the development of a targeted
therapy for MMR-deficient cancers would be invaluable in
the clinic. Due to the unique DNA mismatch-binding
generation classical therapeutics with enhanced DNA-binding
properties in order to increase cytotoxicity has been extensively
explored. However, the design and synthesis of therapeutics
which bind to specific DNA lesions that are more prevalent in
cancer cells than healthy cells may represent a targeted strategy
for new chemotherapy. In particular, our laboratory has focused
Received: July 16, 2014
Published: September 25, 2014
©
2014 American Chemical Society
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Article
Figure 2; the DPE ligand was discovered to coordinate to the
rhodium center via one pyridine ring and the alcohol oxygen, in
contrast to all other previously reported metalloinsertors, which
coordinate via two pyridine nitrogens (Figure S2). It was
hypothesized that this new ligand coordination environment
was responsible for the enhanced biological activity of this
compound, and we thus sought to synthesize a family of
compounds inspired by this new ligand scaffold.
In this report, we describe the discovery of an unusual ligand
coordination for the rhodium metalloinsertor [Rh(chrysi)-
(
phen)(DPE)]2+ and subsequent development of a family of
compounds based on this new type of coordination environ-
ment. The entire family of compounds has been found to
possess enhanced potency and cell selectivity in our cellular
assays. In fact, all compounds studied are more potent than the
FDA-approved chemotherapeutic cisplatin. All compounds
have binding affinities for CC mismatches that range from
6
−1
2
.6 to 5.5 × 10 M , comparable to previous metalloinsertors.
However, the amount of intracellular rhodium required for
optimal biological activity for this family of compounds is more
than five times lower than that of previously reported
metalloinsertors, thus making them potent in the nanomolar
concentration range. Importantly, all of the complexes exhibit a
highly selective cytotoxicity for cells that are deficient versus
proficient in MMR.
EXPERIMENTAL SECTION
■
Materials. Commercially available chemicals were used as received.
All organic reagents and Sephadex ion-exchange resin were obtained
from Sigma-Aldrich unless otherwise noted. Sep-pak C18 solid-phase
extraction (SPE) cartridges were purchased from Waters Chemical Co.
(
Milford, MA). Media and supplements were purchased from
Invitrogen (Carlsbad, CA). Bromodeoxyuridine (BrdU), antibodies,
buffers, peroxidase substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), and acidified lysis buffer (10% SDS in
1
0 mM HCl) solution were purchased in kit format from Roche
Molecular biochemical (Mannheim, Germany).
Figure 1. Chemical structures of metalloinsertors and binding affinities
for oligonucleotides containing CC mismatches. All DNA binding
Oligonucleotide Synthesis and Purification. Oligonucleotides
were synthesized using standard phosphoramidite chemistry at IDT
DNA (Coralville, IA) and purified by HPLC using a C18 reverse-phase
column (Varian, Inc.) on a Hewlett-Packard 1100 HPLC.
Quantification was performed on a Cary 100 Bio UV−vis
affinities were measured in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer
i
on the duplex 36-mer with the sequence 3′-GCG ATG CAG ATA
TAC CTA CTA GGA TTC ACT GTC ATG-5′ on one strand
(
underline denotes the mismatch) in a competition assay through
spectrophotometer using extinction coefficients at 260 nm (ε
)
60
2
3+
photocleavage by [Rh(bpy) chrysi] . Samples were irradiated and
2
estimated for single-stranded DNA.
electrophoresed through a 20% denaturing PAGE gel, and the percent
of DNA cleavage at each concentration was plotted as a function of log
Synthesis and Characterization of Ligands and Metal
3
+
Complexes. The complexes [Rh(bpy) (chrysi)] and [Rh(HDPA) -
2
2
B
(chrysi)]3 were prepared according to published procedures.
+
19,20
[
Rh]. The data were fitted to a sigmoidal curve, and K values were
determined by calculating the concentration of rhodium at the
inflection points of the curve and solving simultaneous equilibria.
[
Rh(chrysi)(phen)(NH ) ]Cl . According to an adaptation of
3
2
3
2
1
previously described procedures, [Rh(chrysi)(phen)(NH ) ]Cl was
3 2 3
prepared. A 1 L round-bottomed flask was charged with
Rh(NH ) (phen)][OTf] (0.500 g, 0.626 mmol), chrysene-5,6-
[
3
4
3
properties of rhodium metalloinsertors and the structural
perturbation to DNA, their biological properties in MMR-
deficient cells were investigated. The compounds have been
found to inhibit growth and induce necrosis selectively in
MMR-deficient colorectal cancer cells over MMR-proficient
dione (0.162 g, 0.626 mmol), and 1:9 H O:MeCN was added (400
2
mL). A 1 M solution of NaOH (1.5 mL) was added to the orange
solution. Over the next hour, the solution changed color from orange
to red, after which time a 1 M solution of HCl (1.5 mL) was added to
quench the reaction. The MeCN was evaporated in vacuo, and the
resulting red solution was loaded onto a SPE cartridge, eluted with
25% acetonitrile in 0.1% TFA(aq), and lyophilized to give a red solid.
The chloride salt can be obtained from a Sephadex QAE anion
1
6,17
cells.
Recently in our laboratory, a family of 10 metalloinsertors
with varying lipophilicites yet similar mismatch binding
affinities was synthesized. Their abilities to preferentially target
MMR-deficient cells over MMR-proficient cells were found to
exchange column equilibrated with 0.1 M MgCl . Yield: 0.536 g, 94%.
2
1
H NMR (300 MHz, d -DMSO): δ 13.59 (s, 1H); 13.43 (s, 1H); 9.56
6
1
8
(d, J = 5.3 Hz, 1 H); 9.16 (m, 2H); 8.93 (d, J = 8.3 Hz, 1H); 8.77 (d, J
vary dramatically. One metalloinsertor in particular,
=
5.3 Hz, 1H); 8.35−8.57 (m, 7H); 8.23 (d, J = 8.3 Hz, 1H); 7.99 (m,
2
+
[
(
Rh(chrysi)(phen)(DPE)] (Figure 1, DPE = 1,1-di-
pyridine-2-yl)ethan-1-ol), was found to possess enhanced
2
3
H); 7.83 (m, 2H); 7.54 (t, J = 7.6 Hz, 1H); 4.95 (s, 3H); 4.79 (s,
H). ESI-MS (cation): m/z calcd, 571.13 (M − 2H ); obs., 570.9.
+
potency and selectivity compared to the others. Crystallization
of the compound revealed the solid-state structure shown in
1-R-1-(Pyrid-2-yl) Ethanol (R = Pyridine, Phenyl, Methyl,
Hexyl; DPE, PPE, PPO, PyOctanol). According to an adaptation of a
1
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Figure 2. Structure of a new metalloinsertor, illustrating the coordination and ligand buckling. Left: X-ray crystal structure of
Rh(chrysi)(phen)(DPE)]Cl . Displacement of ellipsoids is drawn at 50% probability. For clarity, chloride and hydrogen atoms (except immine
[
2
protons) have been omitted. Top right: Side view. Note the severe distortion of the planarity of the chrysi ligand. Bottom right: View showing the
steric clash responsible for the buckling of the chrysi ligand. Both hydrogens are shown as van der Waals surfaces.
2
2
previously described procedure, 1-R-1-(pyrid-2-yl) ethanol (R =
pyridine, phenyl, methyl, hexyl; DPE, PPE, PPO, PyOctanol) were
synthesized. The appropriate ketone py(CO)(L) (8.3 mmol) was
dissolved in dry diethyl ether (100 mL) in an oven-dried 250 mL
Schlenk flask under Ar. The solution was cooled to −78 °C, and MeLi
[Rh(chrysi)(phen)(L)]Cl2 (L = DPE, PPE, PPO, PyOctanol).
18
According to an adaptation of a previously described method,
[Rh(chrysi)(phen)(L)]Cl (L = DPE, PPE, PPO, PyOctanol) were
prepared. [Rh(chrysi)(phen)(NH ]TFA (62.3 mg, 0.092 mmol)
and L (0.138 mmol) were dissolved in 1:12 H O:EtOH (90 mL). The
2
)
3
2
3
2
resulting red solution was heated to 98 °C and allowed to reflux for 18
(
12.9 mL of a 1.6 M solution) was added slowly over 15 min. The
resulting yellow solution was allowed to stir at −78 °C for 1 h and
then warmed to ambient temperature. Next, saturated NH Cl (aq) (30
mL) was added to quench the reaction, and the resulting solution was
extracted with EtOAc (3 × 75 mL) and dried over Na SO , and the
solvent evaporated in vacuo. The crude product was purified via flash
chromatography (SiO , 1:1 EtOAc:CH Cl for L = Me and hexyl, 1:3
h. The resulting solution was dried in vacuo, redissolved in H O (10
2
mL), filtered, and purified via flash chromatography (C −SiO , 17:3
18
2
4
0
(
.1% TFA (aq): MeCN). All compounds but [Rh(chrysi)(phen)-
PyOctanol)]2 were synthesized as a mixture of diasteriomers; only
+
2
4
2+
the diasteriomers of [Rh(chrysi)(phen)(DPE)] were resolvable via
HPLC (17:3 0.1% TFA (aq): MeCN to 1:1 0.1% TFA (aq): MeCN
gradient over 45 min). The chloride salt can be obtained from a
Sephadex QAE anion exchange column equilibrated with 0.1 M
MgCl2.
2
2
2
EtOAc:CH Cl for L = Ph) to afford a light-yellow oil.
2
2
,1-Di(pyridine-2-yl)ethan-1-ol (DPE). Yield: 100%. 1H NMR
CDCl , 300 MHz): δ 8.57 (d, J = 5.1 Hz, 2H); 7.92 (d, J = 6.9
1
(
3
[
Rh(chrysi)(phen)(DPE)]Cl . Yield: 30%. ESI-MS (cation): m/z
2
Hz, 2H); 7.80 (m, 2H); 7.27 (m, 2H); 6.60 (s, 1H); 2.08 (s, 3H). ESI-
MS (cation): m/z calcd, 201.09 (M + H ); obs., 201.0.
-Phenyl-1-(pyridine-2-yl)ethan-1-ol (PPE). Yield: 97%. H NMR
CDCl , 300 MHz): δ 8.52 (d of m, J = 5.1 Hz, 1 H); 7.65 (t of d, J =
.8 Hz, 1.8 Hz, 1 H); 7.48 (m, 2H); 7.31 (m, 3H); 7.17−7.26 (m,
H); 5.85 (s, 1H); 1.94 (s, 3H).
-(Pyridine-2-yl)propan-2-ol (PPO). Yield: 55% H NMR NMR
CDCl , 300 MHz): δ 8.52 (d of m, J = 4.8 Hz, 1 H); 7.71 (t of m, J =
+
2+
calcd, 737.15 (M − H ), 369.1 (M ); obs., 737, 369. UV−vis (H O,
pH 7): 272 nm (102,100 M cm ), 303 nm (35,400 M , cm ), 440
nm (10,600 M , cm ). H NMR (CD CN, 500 MHz): δ 15.10 (s, 1
+
2
−1
−1
−1
−1
1
1
−1
−1
1
3
(
7
2
3
H); 11.30 (s, 1 H); 9.62 (d, J = 5.5 Hz, 1H); 8.98 (d, J = 7.0 Hz, 1H);
8
.92 (d, J = 8.0 Hz, 1H); 8.88 (m, 2H); 8.36 (m, 4H); 8.27 (d, J = 8.5
Hz, 1H); 8.21 (m, 1H); 8.13 (d, J = 8.5 Hz, 1H); 8.00 (m, 2H); 7.94
d, J = 8.5 Hz, 1H); 7.73−7.78 (m, 4H); 7.62 (t, J = 8.0 Hz, 1H); 7.57
(d, J = 8.0 Hz, 1H); 7.50 (t, J = 7.0 Hz, 1H); 7.32 (d, J = 6.5 Hz, 1H);
.14 (m, 1H); 6.95 (t, J = 7.5 Hz, 1H); 6.33 (t, J = 6.0 Hz, 1H); 1.98
s, 3H). Crystals suitable for X-ray diffraction were obtained from
1
2
(
(
7
1
3
.8 Hz, 1 H); 7.38 (d of m, J = 8.1 Hz, 1H); 7.21 (t of m, J = 6.2 Hz,
7
(
H); 5.08 (s, 1H); 1.54 (s, 6H).
2
-(Pyridine-2-yl)octan-2-ol (PyOctanol). Yield: 35% 1H NMR
vapor diffusion of diethyl ether into a concentrated solution of
NMR (CDCl , 300 MHz): δ 8.51 (d of m, J = 6.0 Hz, 1 H); 7.71 (t of
m, J = 9.0 Hz, 1 H); 7.32 (d of m, J = 9.0 Hz, 1H); 7.20 (t of m, J = 6.0
Hz, 1H); 5.18 (s, 1H); 1.78 (m, 2H); 1.51 (s, 3H); 1.19 (m, 6H); 0.82
[Rh(chrysi)(phen)(DPE)]Cl dissolved in ethanol.
3
2
[Rh(chrysi)(phen)(PPE)]Cl . Yield: 80%. ESI-MS (cation): m/z
2
+
2+
calcd, 736.2 (M − 1H ), 368.6 (M ); obs., 736.0, 368.8. UV−vis
(H O, pH 7): 270 nm (165,800 M cm ), 300 nm (56,300 M
2
−1 −1 −1
(
m, 5H).
1
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Article
−1
−1
−1
1
cm ), 430 nm (16,100 M cm ). H NMR (CD CN, 600 MHz): δ
from three separate experiments were pooled to determine average
3
1
5.45 (s, 1 H); 14.38 (s, 1 H); 11.76 (s, 1 H); 11.08 (s, 1 H); 9.71 (d,
J = 5.2 Hz, 1H); 9.49 (d, J = 5.2 Hz, 1H); 9.12 (d, J = 5.6 Hz, 1H);
.99 (d, J = 9.0 Hz, 1H); 8.94 (m, 2H); 8.88 (m, 3H); 8.81 (d, J = 8.0
Hz, 1H); 8.46 (d, J = 8.0 Hz, 1H); 8.35−8.39 (m, 4H); 8.29−8.33 (m,
H); 8.20−8.25 (m, 4H); 8.14 (t, J = 8.0 Hz, 1H); 8.10 (m, 2H); 8.03
m, 1H); 7.99 (m, 1H); 7.92 (d, J = 7.5 Hz, 1H); 7.89 (d, J = 8.5 Hz,
pK values.
a
In addition, 25 μM solutions of 1:1 [Rh(chrysi)(phen)-
2
+
2+
8
(DPE)] :DNA, [Rh(chrysi)(phen)(PPE)] :DNA, [Rh(chrysi)-
2+
3+
(phen)(PPO)] :DNA, or [Rh(HDPA) (chrysi)] :DNA (3 mL, in
100 mM NaCl, 20 mM NaP , pH 7.1 buffer) were prepared and
2
3
i
(
1
absorption-spectra measured on a Cary 100 Bio UV−vis spectropho-
tometer. The DNA hairpin 5′-GGCAGGCATGGCTTTTTGC-
CATCCCTGCC-3′ (underline denotes the CC mismatch) was used.
Photocleavage Competition Titrations. The oligonucleotide 3′-
GCG ATG CAG ATA TAC CTA CTA GGA TTC ACT GTC ATG-
H); 7.83 (m, 1H); 7.68−7.80 (m, 5H); 7.64 (m, 1H); 7.55−7.60 (m,
4
H); 7.52 (m, 2H); 7.40 (d, J = 5.7 Hz, 1H); 7.28−7.34 (m, 4H); 7.23
(
t, J = 6.6 Hz, 1H); 7.16 (m, 3H); 6.61 (t, J = 6.6 Hz, 2H); 6.33 (t, J =
6.6 Hz, 1H); 2.49 (s, 3H); 2.13 (s, 3H) (1:1 mixture of diasteriomers).
32
32
Crystals suitable for X-ray diffraction were obtained from vapor
diffusion of diethyl ether into a concentrated solution of [Rh(chrysi)-
5′ was P-labeled at the 5′-end by incubating DNA with P-ATP and
polynucleotide kinase (PNK) at 37 °C for 2 h, followed by purification
using gel electrophoresis. A small amount of the labeled DNA (<1% of
the total amount of DNA) was added to 2 μM unlabeled DNA and its
corresponding unlabeled complement (with a CC mismatch
incorporated at the underlined site) in 100 mM NaCl, 20 mM NaPi,
pH 7.1 buffer. The duplex DNA was annealed by heating at 90 °C for
10 min and cooling slowly to ambient temperature over a period of 2
h. Solutions of non-photocleaving rhodium complex ranging from
nanomolar to micromolar concentration as well as a 4 μM
(
phen)(PPE)]Cl dissolved in methanol.
2
[
Rh(chrysi)(phen)(PPO)]Cl . Yield: 40%. ESI-MS (cation): m/z
2
+
2+
calcd, 674.1 (M − 1H ), 337.6 (M ); obs., 674.0, 337.7. UV−vis
−
1
−1
−1
(
H O, pH 7): 270 nm (122,400 M cm ), 300 nm (41,600 M
2
−
1
−1
−1
1
cm ), 430 nm (12,300 M cm ). H NMR (CD CN, 500 MHz): δ
3
13.29 (br s, 1.7 H); 11.68 (br s, 1 H); 9.61 (d, J = 5.2 Hz, 1H); 9.54
(
(
(
d, J = 5.2 Hz, 1.7H); 9.09 (d, J = 5.5 Hz, 1H); 8.93 (m, 5.4H); 8.88
m, 2.7H); 8.30−8.42 (m, 12.5H); 8.26 (m, 1H); 8.23 (m, 1.7H); 8.14
m, 4.4H); 7.93−8.04 (m, 11.5H); 7.74−7.85 (m, 5.4H); 7.55 (m,
3+
[Rh(bpy) (chrysi)] solution were made in Milli-Q water. Annealed
2
3
+
4
.4H); 7.49 (t, J = 8.0 Hz, 1H); 7.21 (m, 2H); 7.13 (m, 1H); 7.09 (m,
2 μM DNA (10 μL), 4 μM [Rh(bpy) (chrysi)] (5 μL), and 5 μL of
2
2
.7H); 2.00 (s, 3H); 1.96 (s, 5.1H); 1.67 (s, 3H); 1.66 (s, 5.1H);
non-photocleaving Rh solution at each concentration were mixed in a
microcentrifuge tube and incubated at 37 °C for 10 min. A light
control (ØRh), in which the DNA was mixed with 10 μL of water and
irradiated, and a dark control (Øhν), in which the DNA was mixed
with the highest concentration of rhodium complex without
irradiation, were also prepared. The samples were then irradiated on
an Oriel (Darmstadt, Germany) 1000 W Hg/Xe solar simulator (340−
440 nm) for 15 min. The samples were dried and electrophoresed in a
20% denaturing polyacrylamide gel. The gel was then exposed to a
phosphor screen, and the relative amounts of DNA in each band were
quantitated by phosphorimagery (ImageQuant).
(
1:1.7 mixture of diasteriomers). Crystals suitable for X-ray diffraction
were obtained from vapor diffusion of diethyl ether into a
concentrated solution of [Rh(chrysi)(phen)(PPO)]Cl dissolved in
isopropanol.
2
[
Rh(chrysi)(phen)(PyOctanol)]Cl . Yield: 10%. ESI-MS (cation):
2
+
2+
1
m/z calcd 744.2 (M − 1H ), 372.6 (M ); obs. 744.1, 372.8). H
NMR (CD CN, 500 MHz): δ 15.00 (s, 1 H); 12.80 (s, 1 H); 9.55 (d, J
3
=
5.0 Hz, 1H); 9.12 (d, J = 8.0 Hz, 1H); 9.09 (d, J = 5.5 Hz, 1H); 8.94
(
d, J = 8.9 Hz, 1H); 8.88 (d, J = 8.5 Hz, 1H); 8.85 (d, J = 8.5 Hz, 1H);
8.32−8.44 (m, 5H); 8.17 (m, 2H); 8.08 (m, 1H); 7.91 (t, J = 7.0 Hz,
1H); 7.86 (t, J = 8.0 Hz, 1H); 7.80 (m, 2H); 7.54 (t, J = 7.5 Hz, 1H);
7.41 (d, J = 8.0 Hz, 1H); 7.31 (d, J = 6.0 Hz, 1H); 7.05 (t, J = 7.0 Hz,
1H); 1.74 (s, 3H); 1.55 (m, 2H); 0.71−0.96 (m, 11H).
Multiple Sequence Contexts. The oligonucleotide hairpins of
the sequences 5′ -GGCAGTXCTGGCTTTTTGCCAGYACTGCC-
32
3′ (XY = CC, CA, CT, AA) were P-labeled at the 5′-end as described
above. Samples of labeled DNA were combined with unlabeled carrier
and annealed as described. Solutions of Δ- and Λ-[Rh(chrysi)(phen)-
(DPE)]2 ranging from 0.5 μM to 2 mM as well as either 4 μM (CC
and CT mismatches) or 20 μM (CA and AA mismatches)
Crystals of [Rh(HDPA) (chrysi)]Cl (one chrysi immine deproto-
2
2
nated) suitable for X-ray diffraction were obtained from vapor
diffusion of diethyl ether into a concentrated solution of
+
[
Rh(HDPA) (chrysi)]Cl dissolved in ethanol.
2 2
3
+
Enantiomeric Separation. A 2 mM solution (1.5 mL) of
[Rh(bpy) (chrysi)] solutions were made in Milli-Q water. Annealed
2
Rh(chrysi)(phen)(DPE)]2 was injected, 30 μL at a time, onto an
+
2 μM DNA (10 μL), 4 or 20 μM [Rh(bpy) (chrysi)] (5 μL), and 5
3+
[
2
2
+
Astec CYCLOBOND I 2000 Chiral HPLC Column that was heated to
0 °C. An isocratic method of 50% acetonitrile, 50% 100 mM KPF6
μL of Δ- or Λ-[Rh(chrysi)(phen)(DPE)] solution at each
concentration was mixed in a microcentrifuge tube and incubated at
37 °C for 10 min. Samples were then irradiated and electrophoresed as
described above.
4
was used to separate the two enantiomers. An automatic fraction
collector was used to collect each peak separately. The resulting dilute
solutions were loaded onto a SPE cartridge and rinsed with copious
amounts of 0.1% TFA(aq). The SPE cartridge was eluted with 10%
acetonitrile in 0.1% TFA(aq). The chloride salts were obtained from a
Sephadex QAE anion exchange column equilibrated with 0.1 M
MgCl . Circular dichroism spectra were taken on an Aviv 62DS
spectropolarimeter in a 1 mm path length cell.
X-ray Structure Determination. Details of the structure
determinations and refinements for all structures are provided in
Supporting Information.
Binding Constant Determination. The fraction of DNA cleaved
in each lane on the gel (see Figure S5 for a typical autoradiogram) was
normalized and plotted against the log of the concentration of
rhodium complex. The data were fit to a sigmoidal curve using
OriginPro 6.1 (Figure S6). The resulting midpoint value (i.e., the log
of [rhodium complex] at the inflection point of the curve) was
converted to units of concentration ([Rh50%]). The binding and
dissociation constants of the non-photocleaving complex were
determined by solving simultaneous equlibria involving DNA,
2
3+
Metalloinsertor pH Titrations. Solutions (25 μM) of
[Rh(bpy) (chrysi)] , and the complex in question in Mathematica
2
Rh(chrysi)(phen)(DPE)]2 , [Rh(chrysi)(phen)(PPE)]
+
2+
,
6.0. The data from at least three photocleavage titrations were
averaged for each metal complex to give an average binding affinity.
Covalent DNA Binding Assay. A 6.0 μM solution (250 μL) of
the DNA hairpin 5′-GGCAGGCATGGCTTTTTGC-
CATCCCTGCC-3′ (underline denotes the CC mismatch) in either
[
[
[
2
+
3 +
Rh(chrysi)(phen)(PPO)]
,
[Rh(bpy) (chrysi)]
,
or
2
3+
Rh(HDPA) (chrysi)] (3 mL, in 0.1 M NaCl) were prepared, and
2
absorption spectra measured on a Cary 100 Bio UV−vis
spectrophotometer. The pH of the solutions and their blanks were
adjusted (and monitored by an internal electrode) from approximately
in 100 mM NaCl, 20 mM NaP , pH 7.1 or 100 mM NaCl, 20 mM
i
4
.5 to 10.5 and back via titration with either 6 mM NaOH or 10 mM
NaP , 5 mM glutathione (to mimic the reducing environment of the
i
3
+
HCl. After each acid or base addition, an absorption spectrum was
taken. A single wavelength was selected for each metal complex where
a large change was observed over the course of the titration. The
absorbance at this wavelength was then plotted as a function of pH to
generate a titration curve. The pKa of each metal complex was
determined from the inflection point of this sigmoidal curve. The data
cell) was added to a 6.0 μM solution of [Rh(bpy) (chrysi)] ,
2
3+
2+
[Rh(HDPA) (chrysi)] , [Rh(chrysi)(phen)(PPE)] , or H O (250
2
2
μL). The resulting solution was allowed to incubate at 37 °C for 30
min, followed by a 10 min incubation at 90 °C. A 3.0 M solution of
NaOAc (50 μL) was added, followed by EtOH (1.5 mL) in order to
precipitate the DNA. The resulting solution was vortexed and
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incubated on dry ice for 1.5 h, after which time it was spun at 14,000
rpm for 12 min. The supernatant was discarded, and the pellet
dissolved in water (100 μL) and EtOH precipitated out again. The
supernatant was discarded, and the pellet dissolved in water (500 μL).
An electronic absorption (UV−vis) spectrum was then taken of the
resulting solution on a Cary 100 Bio UV−vis spectrophotometer.
RESULTS
■
Synthesis and Characterization of Compounds. Single
crystals of [Rh(chrysi)(phen)(DPE)]Cl were grown from a
2
vapor diffusion of diethyl ether into a concentrated solution of
the complex in ethanol. The solved structure revealed the DPE
ligand to coordinate via the oxygen instead of the second
pyridine nitrogen. In order to assess the generality of this
coordination environment and to determine if the solid-state
2+
Circular Dichroism Study of Δ/Λ-[Rh(chrysi)(phen)(DPE)]
Bound to Mismatched DNA. 50 μM solutions of the DNA hairpin
′-GGCAGGCATGGCTTTTTGCCATCCCTGCC-3′ (underline
denotes the CC mismatch) and Δ- and Λ-[Rh(chrysi)(phen)-
5
DPE)]2 (200 μL in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer)
+
structure of [Rh(chrysi)(phen)(DPE)]Cl is the same as its
2
(
i
solution structure, we synthesized a phenyl (PPE) derivative
and a methyl (PPO) derivative (Figure 1). Both ligands are
notable in their lack of a second nitrogen to coordinate to the
rhodium center. These new compounds can be synthesized via
analogous methods and in reasonable yields. Single crystals
grown of both compounds confirmed the generality of this
unusual ligand coordination (Figures S3 and S4).
These compounds were also designed to ascertain the
importance of both the ligand coordination environment (the
presence of a Rh−O bond) and the bulky “dangling” pyridine
group with regards to the enhanced potency and cell-selective
activity of the parent compound. An additional compound with
a greasy hexyl group appended to this ligand scaffold was also
synthesized to assess the effects of lipophilicity on the biological
activity of this family of compounds.
were prepared, and CD spectra were taken on an Aviv 62DS
spectropolarimeter in a 1 mm path length cell. Each metal complex
solution (100 μL) was then added to 100 μL of the DNA solution, and
CD spectra were immediately recorded. After 30 min, additional CD
spectra were recorded to confirm that no changes in the spectra
occurred.
Cell Culture. HCT116N and HCT116O cells were grown in RPMI
medium 1640 supplemented with 10% FBS; 2 mM L-glutamine; 0.1
mM non-essential amino acids; 1 mM sodium pyruvate; 100 units/mL
penicillin; 100 μg/mL streptomycin; and 400 μg/mL Geneticin
(
G418). Cells were grown in tissue culture flasks (Corning Costar,
Acton, MA) at 37 °C under 5% CO and humidified atmosphere.
2
Cellular Proliferation ELISA. HCT116N and HCT116O cells
were plated in 96-well plates at 2000 cells/well and allowed 24 h to
adhere. The cells were then incubated with either rhodium or the
appropriate chemotherapeutic (cisplatin or MNNG) for the
concentration and durations specified. After 24 h, the chemo-
therapeutic-containing media was replaced with fresh media, and the
cells were grown for the remainder of the 72 h period. Cells were
labeled with BrdU 24 h before analysis. The BrdU incorporation was
The acidity constants of the immine protons in [Rh(chrysi)-
(
[
(
phen)(DPE)]2+, [Rh(chrysi)(phen)(PPE)] , and
2+
2+
Rh(chrysi)(phen)(PPO)] as compared to [Rh(HDPA) -
2
chrysi)]3+ and [Rh(bpy) (chrysi)] were determined by pH
3+
2
3
quantified by antibody assay according to established procedures.
2
Cellular proliferation was expressed as the ratio of the amount of BrdU
incorporated by the treated cells to that of the untreated cells.
MTT Cytotoxicity Assay. Cytotoxicity assays were performed as
spectroscopic titrations. It has previously been demonstrated
that the visible absorbance changes which occur as the pH of a
rhodium complex solution is titrated can be used for the
24
27
described in the literature. HCT116N and HCT116O cells were
plated in 96-well plates at 50,000 cells/well and incubated with
rhodium for the durations specified. After rhodium incubation, cells
were labeled with MTT for 4 h at 37 °C under 5% CO2 and
humidified atmosphere. The resulting formazan crystals were dissolved
with solubilizing reagent purchased from Roche according to the
manufacturer’s instructions. The dissolved formazan was quantified as
the absorbance at 570 nm minus the background absorbance at 690
nm. Percent viability was determined as the ratio of the amount of
formazan in the treated cells to that of the untreated cells.
determination of the pK values of the compounds. As the pH
a
of the solution increases from 4.5 to 11, the band centered near
4
40 nm (which corresponds to a charge-transfer transition
between the Rh center and the chrysi ligand) undergoes a blue
shift (Figures 3 and S7−S10). When the absorbance values at
the initial maximum are plotted as a function of pH, a titration
curve can be constructed, and the pK value of each metal
a
complex can be determined from the inflection point of the
curve (Figures S7−10, insets). The pK values for these
a
Cell Death Mode Flow Cytometry Assay. Cell death was
complexes are given in Table 1. These values reflect differences
in equilibrium constants of almost 2 orders of magnitude, likely
due to the negative charge of the alkoxide ligands as compared
to HDPA and bpy.
25
characterized by a dye exclusion assay. After 24, 48-, or 72 h
incubation with rhodium, cells were harvested from adherent culture
by trypsinization, washed with cold PBS, and centrifuged at 2,000 rpm
for 5 min. The resultant pellets were resuspended in PBS to a
6
All three crystallographically characterized compounds are
synthesized as a mixture of two diastereomers (see Exper-
imental Section), but only the diastereomers of [Rh(chrysi)-
(phen)(DPE)]2+ were resolvable via HPLC. The enantiomers
concentration of 10 cells/mL and stained with propidium iodide to a
final concentration of 1 μg/mL and YO-PRO-1 to a final
concentration of 50 nM for 30 min prior to analysis by flow cytometry.
Assay for Whole-Cell Rhodium Levels. HCT116O cells were
6
plated at 1 × 10 cells/well in a 6-well plate. The cells were allowed 24
h to adhere, then treated with 10 μM [Rh(HDPA) (chrysi)]Cl , 5 μM
2
3
Table 1. pK Values for All Compounds As Determined by
a
[
0
Rh(chrysi)(phen)(DPE)]Cl , 1 μM [Rh(chrysi)(phen)(PPE)]Cl , or
a
2
2
Spectrophotometric Titration
.5 μM [Rh(chrysi)(phen)(PPO)]Cl . After 24 h, the media was
2
decanted, the cell monolayer washed with 3 mL PBS, and the cells
lysed with 800 μL of 1% SDS. The cell lysate was sonicated on a
Qsonica Ultrasonic processor for 10 s at 20% amplitude. 750 μL of the
b
compound
pKa
[
[
[
[
Rh(bpy) (chrysi)]3
+/2+
5.6 ± 0.2
7.0 ± 0.5
8.7 ± 0.2
8.9 ± 0.4
8.3 ± 0.3
2
Rh(HDPA) (chrysi)]3
+/2+
2
lysate was then combined with 750 μL of a 2% HNO (v/v) solution,
3
Rh(chrysi)(phen)(DPE)]2
+/1+
while the remainder of the lysate was quantified for protein by a
26
2+/1+
Rh(chrysi)(phen)(PPE)]
bicinchoninic assay (BCA). The 1% HNO solution was analyzed for
3
2+/1+
rhodium content on a Thermo X Series II ICP-MS unit. Rhodium
counts were normalized to the amount of protein determined from the
BCA analysis (to obtain ng [Rhodium]/mg [protein] values).
Standard errors for three independent experiments are shown. The
experiment was repeated with HCT116N cells to verify similar uptake
of rhodium by the two cell lines.
[Rh(chrysi)(phen)(PPO)]
a
Titrations were performed at ambient temperature in 0.1 M NaCl
solutions. Reported errors were found from repeated trials.
3+
[Rh(bpy) (chrysi)] pKa matches with the previously reported
value of 5.2 ± 0.2. All compounds were used as racemic mixtures.
2
27 b
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2+
2+
Figure 3. Top: pH titration of [Rh(chrysi)(phen)(DPE)] . Shown are absorption spectra of a 25 μM solution of [Rh(chrysi)(phen)(DPE)] as
the pH changes from 5.6 (red) to 10.2 (purple). The black arrows exhibit the direction in which the various bands change as the pH increases.
(
Inset) The absorbance at 440 nm was plotted as a function of pH and fit to a sigmoidal curve. The pK was determined from the inflection point of
a
2+
3+
this curve. Bottom: Absorption spectra of [Rh(chrysi)(phen)(DPE)] (left) and [Rh(bpy) (chrysi)] (right) bound to mismatched DNA. Shown
2
in blue are absorption spectra of 25 μM solutions of [Rh(chrysi)(phen)(DPE)]2 and [Rh(bpy) (chrysi)] with 25 μM of the DNA hairpin 5′-
+
3+
2
GGCAGGCATGGCTTTTTGCCATCCCTGCC-3′ (underline denotes the CC mismatch) in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer. The
i
absorption spectra of the species retaining the immine protons are shown in green, those of the deprotonated species are in red, and those of the
compounds in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer with no DNA are shown in purple.
i
of [Rh(chrysi)(phen)(DPE)]2+ were separated and character-
ized via CD spectroscopy (Figure S11). HPLC analysis of the
purified fractions confirmed >95% ee of the Δ isomer and
retain both immine protons upon DNA binding (Figure S12),
3+
while [Rh(HDPA) (chrysi)] appears to be a mixture of
2
protonated and deprotonated species when bound to
mismatched DNA (Figure S12).
The new family of complexes does not promote DNA
cleavage upon irradiation, and, as such, their binding affinities
∼90% ee of the Λ isomer (data not shown). These enantiomers
are stable in aqueous solution for up to 1 month; no
interconversion of the enantiomers is observed in either neutral
buffer or buffer with 5 mM glutathione.
were determined through binding competition titrations with 1
Binding of Complexes to Oligonucleotides Contain-
3+
μM rac-[Rh(bpy) (chrysi)] , which does cleave DNA upon
irradiation.
2
ing Single Base Mismatches. To ascertain the protonation
8,9,28
Titrations were performed with a 36-mer DNA
2+
states of the chrysi ligands of [Rh(chrysi)(phen)(DPE)] ,
duplex containing a CC mismatch (underlined): 3′-GCG ATG
CAG ATA TAC CTA CTA GGA TTC ACT GTC ATG-5′. A
representative photocleavage titration can be found in Figure
S5. The degree of photocleavage can be plotted against the
log([Rh]) and fit to a sigmoidal curve (Figure S6). On the basis
2
+
2+
[
[
Rh(chrysi)(phen)(PPE)] , [Rh(chrysi)(phen)(PPO)] ,
3
+
3+
Rh(HDPA) (chrysi)] , and [Rh(bpy) (chrysi)] upon mis-
2
2
matched DNA binding, mismatched DNA was added to all
compounds in a 1:1 ratio and absorption spectra recorded. As
can be seen in Figure 3, [Rh(chrysi)(phen)(DPE)]2+ binds to
3+
of the binding constant of [Rh(bpy) (chrysi)] , the binding
2
mismatched DNA with retention of both immine protons. Due
to the extent of hypochromicity and red-shifting of the charge-
transfer band, it is clear that the complex binds strongly to the
constants of all subsequent complexes are then determined by
solving simultaneous equilibria at the inflection point of the
photocleavage titration curve. The results are shown in Figure
1. All racemic mixtures of compounds exhibit similar binding
mismatched DNA. In contrast, the chrysi ligand of [Rh(bpy) -
2
chrysi)]3 is deprotonated upon mismatched DNA binding.
+
(
6
−1
affinities, varying from 2.6 to 5.5 × 10 M . Most interesting,
however, is the observation that the two enantiomers of
[Rh(chrysi)(phen)(DPE)]2+ bind to mismatched DNA with
Again, hypochromicity and red-shifting of the CT band show a
strong binding of the complex to mismatched DNA. This result
is consistent with the various crystal structures of
3
+
10,11
6
6
−1
[
Rh(bpy) (chrysi)] bound to different DNA mismatches.
the same affinity (6.0 × 10 and 5.7 × 10 M for Δ and Λ,
2
3
+
The other Rh−O containing compounds [Rh(chrysi)(phen)-
respectively). This is in direct contrast to [Rh(bpy) (chrysi)] ,
in which only the Δ isomer binds to mismatched DNA.
2
PPE)]2 and [Rh(chrysi)(phen)(PPO)] were also found to
+
2+
8
(
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2
+
3+
Table 2. Thermodynamic Binding Constants of Δ- and Λ-[Rh(chrysi)(phen)(DPE)] and Δ-[Rh(bpy) (chrysi)] at Varied
2
Mismatched Sites
a
Δ (× 10 M−1)
6
Λ (× 10 M−1)
6
[Rh(bpy) (chrysi)] (× 10 M−1)b
3+
6
c
mismatch
destabilization (kcal/mol)
2
CC
CT
CA
AA
1.1
1.1
1.0
2.4
3.5
2.4
4.6
2.0
1.6
2.15
2.0
0.55
0.47
0.23
1.4
0.8
a
DNA binding affinities were measured in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer on the 29-mer hairpins 5′-GGCAGTXCTGGCTTTTTGC-
i
CAGYACTGCC-3′ (XY = CC, CT, CA, or AA, underline denotes the mismatch) in a competition assay through photocleavage by
3+
b
c
[
Rh(bpy) chrysi] . Binding constants all have standard errors within 10%. Mismatch thermodynamic values found from ref 29.
2
2+
Figure 4. CD spectra of Δ- and Λ-[Rh(chrysi)(phen)(DPE)] bound to mismatched DNA. Shown are spectra of 25 μM solutions of Δ- and Λ-
Rh(chrysi)(phen)(DPE)]2 in 100 mM NaCl, 20 mM NaP , pH 7.1 buffer bound to the DNA hairpin 5′-GGCAGGCATGGCTTTTTGC-
+
[
i
CATCCCTGCC-3′ (underline denotes the CC mismatch), and the DNA hairpin alone (Δ in blue, Λ in red, DNA alone in gray).
Due to the drastic differences in mismatched DNA binding
each other, even after 30 min of incubation (the average time
frame of a competition gel titration, wherein both enantiomers
bind to DNA). This confirms that the enantiomers bind to
mismatched DNA without racemization.
2
+
characterization between [Rh(chrysi)(phen)(DPE)] and
3
+
[
Rh(bpy) (chrysi)] , binding affinities of both enantiomers
2
of the DPE complex at a variety of DNA mismatches were
The ability of [Rh(chrysi)(phen)(PPE)]2+ to bind covalently
to DNA was also assessed by UV−vis spectroscopy. In the
event that the Rh−O bond is labile, covalent DNA binding is a
possibility. In order to determine if covalent binding occurs,
determined. It has been reported that the mismatch-specific
3
+
DNA binding affinities of Δ-[Rh(bpy) (chrysi)] correlate
2
28
with the thermodynamic destabilization of the mismatch.
This correlation is due to the unique binding mode of
metalloinsertion where the mismatched base pair is completely
3
+
3+
H O, [Rh(bpy) (chrysi)] , [Rh(HDPA) (chrysi)] , or
2
2
2
10
2+
ejected from the double helix upon metal complex binding.
[Rh(chrysi)(phen)(PPE)] was incubated with mismatched
DNA under either neutral conditions (phosphate buffer) or a
reducing environment (5 mM gluthathione in phosphate
buffer) for 30 min. The DNA was subsequently denatured to
release any non-covalently associated rhodium complex and
precipitated out of solution. Covalent DNA binding would
result in a characteristic rhodium metalloinsertor CT band near
400 nm, which is not present in any of the samples.
Furthermore, the absorbance at 260 nm of the various samples
confirmed comparable yields of DNA precipitation. Thus, UV−
vis spectra of the various samples of precipitated DNA revealed
no covalent DNA binding by any of the three complexes
(Figure S13) under either neutral or reducing conditions
Inhibition of Cellular Proliferation using an Enzyme-
Linked Immunosorbent Assay (ELISA). An ELISA for DNA
synthesis was used to quantify the effects of the new family of
metalloinsertors on the proliferation of HCT116N cells
(MMR-proficient) and HCT116O cells (MMR-deficient)
Therefore, four different DNA hairpins of the form 5′-
GGCAGTXCTGGCTTTTTGCCAGYACTGCC-3′ (XY =
CC, CA, CT, AA) were synthesized, and the binding affinities
2
+
of Δ- and Λ-[Rh(chrysi)(phen)(DPE)] determined as
described above. The results, along with mismatch thermody-
29
namic destabilization values, are shown in Table 2; the
binding affinities of the Δ enantiomer correlate with the
6
−1
thermodynamic destabilization of the mismatch (1.1 × 10 M
6
−1
for a CC mismatch versus 0.23 × 10
M
for an AA6
mismatch), while those of the Λ enantiomer do not (1.09 × 10
−
1
6
−1
M
for a CC mismatch versus 3.54 × 10 M for an AA
mismatch). Binding competition gels between both Δ- and Λ-
2
+
3+
[
Rh(chrysi)(phen)(DPE)] and 4 μM [Rh(bpy) (phi)] (a
2
non-sequence specific intercalator that binds at all sites in the
duplex) exhibit no binding at matched sites up to 1:15 DNA:
2
+
[
Rh(chrysi)(phen)(DPE)] (data not shown), eliminating the
possibility of non-mismatch-specific binding by either enan-
tiomer.
3
+
(Figure 5). The compound [Rh(HDPA) (chrysi)] was
2
In order to ascertain if racemization of the two enantiomers
occurs upon DNA binding, CD spectra were taken of the two
enantiomers before and after addition to mismatched DNA. As
can be seen in Figure 4, the CD spectra of the two
metalloinsertor-mismatched DNA complexes are distinct from
included as a control with which to compare the potencies
and cell-selectivities of the new compounds. Cisplatin and
methylnitronitrosoguanidine (MNNG) were also included as
prototypical, FDA-approved chemotherapeutic agents that
exhibit decreased effectiveness against MMR-deficient cancer
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2
+
2+
2+
Figure 5. Inhibitory effects of rac-[Rh(chrysi)(phen)(DPE)] , rac-[Rh(chrysi)(phen)(PPE)] , rac-[Rh(chrysi)(phen)(PPO)] , rac-[Rh-
3+
(
HDPA) (chrysi)] , MNNG, and cisplatin. Shown are plots of BrdU incorporation (a measure of DNA synthesis and therefore cellular
2
proliferation) normalized to the BrdU incorporation of untreated cells as a function of rhodium concentration. Standard error bars for five trials are
shown.
1
5
cells. Incubations were performed for 24 h, after which time
the medium containing the chemotherapeutic was replaced
with fresh medium, and the cells were grown for the remainder
of the 72 h period. The extent of cellular proliferation is
expressed as the ratio of BrdU incorporated by the treated cells
as compared to untreated controls. We define differential
inhibition as the difference in %BrdU incorporation between
the HCT116N and HCT116O cells. The results are shown in
Figure 5.
(our previously most potent compound) to 3 μM for
2+
[Rh(chrysi)(phen)(DPE)] , 1 μM for [Rh(chrysi)(phen)-
2+
2+
(PPE)] , and 300 nM for [Rh(chrysi)(phen)(PPO)] . These
compounds are also more potent than the FDA-approved
chemotherapeutics in these assay conditions, which require 4
and 6 μM for optimal biological activity of MNNG and
3
0
cisplatin, respectively. In addition, the enhanced cell-
selectivity that was observed previously with [Rh(chrysi)-
(phen)(DPE)]2+ is conserved across the entire family. At their
optimal concentrations, their differential inhibitions are 55 ±
2%, 53 ± 7%, and 62 ± 2% for the DPE, PPE, and PPO
derivatives, respectively. Note that any cell selectivity by
cisplatin or MNNG favors the MMR-proficient cells. This is
Clearly, the new family of complexes has significantly
enhanced potency as compared to the earlier generation
metalloinsertor; concentrations required for optimal differential
3+
activity are reduced from 10 μM for [Rh(HDPA) (chrysi)]
2
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2
+
2+
2+
Figure 6. Differential cytotoxicities of rac-[Rh(chrysi)(phen)(DPE)] , rac-[Rh(chrysi)(phen)(PPE)] , rac-[Rh(chrysi)(phen)(PPO)] , and rac-
[
2+
4
Rh(chrysi)(phen)(PyOctanol)] . HCT116N (green) and HCT116O (red) cells were plated in 96-well format at densities of 5 × 10 cells/well
and treated with the concentrations of rhodium metalloinsertors indicated. After 72 h, the cells were labeled with MTT for 4 h.
characteristic of the DNA-binding therapeutics and leads to
instance of a metalloinsertor with a lipophilic ancillary ligand
that retains the cell-selective activity unique to our compounds.
The cell-selective cytotoxicities of the two enantiomers of
[Rh(chrysi)(phen)(DPE)]2+ were also assessed (Figure S14).
Just as both enantiomers bind with equal affinity to DNA
mismatches, both enantiomers display similar cell-selectivity;
their optimal differential cytotoxicities are 78 ± 1% and 75 ±
31
significant buildup of drug resistance in the clinic. The family
of metalloinsertors instead shows activity preferentially in the
MMR-deficient cells. This selectivity is unique to all active
metalloinsertors, but this new generation of complexes exhibits
levels of selectivity and potency previously unseen.
MTT Cytotoxicity Assay. The cytotoxic effects of all
24
2
% for Δ and Λ, respectively, although the Λ enantiomer
complexes were determined by MTT assay. Briefly, reduction
of the MTT reagent by metabolically active cells leads to the
production of formazan, which can then be dissolved in
acidified SDS to produce a characteristic absorbance at 570 nm.
Thus, this absorbance reflects the amount of metabolically
active cells in each sample. HCT116N and HCT116O cells
were plated and treated with the various rhodium complexes at
the concentrations indicated in Figure 6 for 72 h. Percent
viability is defined as the ratio of the amount of formazan in the
treated cells to that in the untreated cells, and differential
cytotoxicity is defined as the difference between the percent
viabilities of the two cell lines. The results are shown in Figure
requires a higher concentration. This is again in direct contrast
3
+
to [Rh(bpy) (chrysi)] , where only the Δ isomer displays
2
32
differential biological activity.
ICP-MS Assay for Whole-Cell Rhodium Levels.
HCT116O and HCT116N cells were treated with each
rhodium complex at the concentrations indicated in Figure
S15 for 24 h. Whole cell lysates were subsequently analyzed for
rhodium levels by ICP-MS and normalized to protein content
(
Figure S15). These Rh complex concentrations correspond to
those necessary for optimal biological activity in the 24 h
ELISA experiment for the four different complexes. Therefore,
the intracellular rhodium concentrations in this experiment
reflect the amount of rhodium in the cell that is required for an
optimal biological response. It is striking that the new family of
compounds requires significantly less intracellular rhodium to
6. All four compounds in this new family display differential
cytotoxicity in excess of 50%, and three compounds exhibit
maximal activity at nanomolar concentrations. Specifically, the
differential cytotoxicities and optimal concentrations of
elicit a biological response than the prototypical [Rh(HDPA) -
2
2
+
2+
(
chrysi)]3+ compound; specifically, the new compounds exhibit
[
[
(
Rh(chrysi)(phen)(DPE)] , [Rh(chrysi)(phen)(PPE)] ,
2
+
3
+
Rh(chrysi)(phen)(PPO)] , and [Rh(chrysi)(phen)-
comparable biological activity to [Rh(HDPA) (chrysi)] with
only 20 ± 2% ([Rh(chrysi)(phen)(DPE)] ), 15 ± 5%
2
PyOctanol)]2 are 76 ± 2% at 6 μM, 66 ± 2% at 720 nM,
+
2
+
2
+
66 ± 3% at 320 nM, and 55 ± 3% at 480 nM, respectively. This
([Rh(chrysi)(phen)(PPE)] ), or 13 ± 2% ([Rh(chrysi)-
2+
is an increase in cytotoxic potency of almost 2 orders of
(phen)(PPO)] ) of the amount of intracellular rhodium.
Mode of Cell Death. The mode of cell death upon
rhodium treatment was characterized through a dye-exclusion
3+
magnitude as compared to [Rh(HDPA) (chrysi)] (25 μM
2
required for optimal cytotoxicity). Moreover, this is the only
1
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25
flow cytometry assay. The assay differentiates between live
cells, dead cells, and cells undergoing apoptosis or necrosis
through concurrent staining with propidium iodide (a dead-
cell-permeable dye) and YO-PRO-1 (an apoptotic-cell-perme-
able dye). By plotting the fluorescence of the YO-PRO-1
channel against the PI channel, a pattern emerges. Healthy cells
are seen in the lower lefthand corner of the plot as they
incorporate neither dye. Apoptotic cells exhibit higher YO-
PRO-1 fluorescence but still exclude propidium iodide, placing
them in the upper lefthand quadrant of the pattern. Conversly,
necrotic cells admit propidium iodide but not YO-PRO-1 and
are thus in the lower righthand quadrant. Dead cells admit both
dyes and are therefore seen in the upper righthand quadrant of
the image. Upon flow cytometry analysis, cells can be classified
as live, apoptotic, necrotic, or dead by defining regions in the
fluorescence plane corresponding to each category.
The HCT116N and HCT116O cell lines were incubated
2
+
with 0−0.5 μM of [Rh(chrysi)(phen)(PPO)] for 72 h. After
harvesting the cells and staining with both PI and YO-PRO-1,
the cells were analyzed by flow cytometry to obtain raw
fluorescence data. Representative data for 200 nM rhodium
treatment for 72 h are shown in Figure 7. YO-PRO-1
fluorescence is shown on the y-axis, and PI fluorescence is
shown on the x-axis. The raw data were analyzed by gating the
fluorescence events into one of four categories. Figure 7 also
shows histograms of live, apoptotic, necrotic, and dead cells for
the HCT116N and HCT116O cell lines based on the flow
cytometry. Rhodium treatment was either 0.2 or 0.5 μM
2
+
[
[
Rh(chrysi)(phen)(PPO)]
for 72 h. As with
3
+
17
2+
Rh(HDPA) chrysi] , [Rh(chrysi)(phen)(PPO)] treat-
2
ment induces necrosis preferentially in the MMR-deficient
HCT116O cell line; the number of necrotic cells increases from
1
.2 ± 0.1% to 13 ± 2% after treatment with 0.2 μM
2+
[
Rh(chrysi)(phen)(PPO)] . There is no significant change in
the percentage of cells in the apoptotic region in either cell line
following metalloinsertor treatment (26 ± 7% vs 25 ± 7% for
the HCT116N cell line, and 10 ± 6% vs 17 ± 7% for the
HCT116O cell line). The effect of rhodium treatment is
significantly more pronounced in the MMR-deficient
HCT116O cell line, in which live cells drop from 82 ± 7%
to 28 ± 6% after treatment with 0.2 μM [Rh(chrysi)(phen)-
PPO)]2 versus the MMR-proficient HCT116N cell line,
+
(
which shows no decrease in live cells (57 ± 9% versus 57 ±
2+
7
%) after treatment with 0.2 μM [Rh(chrysi)(phen)(PPO)] .
DISCUSSION
■
A new family of rhodium complexes containing Rh−O
coordination have been prepared, and all show higher potency
and selectivity for MMR-deficient cells compared to earlier
rhodium metalloinsertors. The DPE ligand was originally
designed to coordinate to the Rh center via the two pyridine
rings, as per previously synthesized metalloinsertors. The solid-
state structure of [Rh(chrysi)(phen)(DPE)]2+ revealed the true
coordination to include an axial Rh−O bond. The driving force
for this ligand coordination may be the formation of a five-
membered ring, as opposed to the six-membered ring that
would form if the second pyridine ring coordinated. This
family of complexes offers new possibilities for binding DNA
mismatches and for providing potent targets for cellular
processing.
Figure 7. Flow cytometry assay of cell death mechanism. (Top)
HCT116N and HCT116O cells were treated with 0.2 μM
2+
[
Rh(chrysi)(phen)(PPO)] for 72 h. The color scale represents the
number of cells, with blue indicating fewer cells at a given pair of
fluorescence levels, and orange representing a greater number of cells
at a given pair of fluorescence levels. Rhodium treatment causes cells
to move away from the origin, along the necrotic pathway (lower
branch of pattern). The effect is more pronounced in the HCT116O
33
cell line. (Bottom) HCT116N and HCT116O cells were treated with
+
0
.2 or 0.5 μM [Rh(chrysi)(phen)(PPO)]2 for 72 h. Rhodium
treatment causes a sharp decrease in the live population of the
HCT116O cell line with a corresponding increase in the necrotic and
dead cell populations. Less of an effect is seen in the HCT116N cell
2
+
Characterization in Solution and Binding to Mis-
matched DNA. Spectrophotometric titrations reveal that the
acidity of the chrysi immine proton of the new complexes
line. Thus, these data are consistent with [Rh(chrysi)(phen)(PPO)]
preferentially inducing necrosis in the MMR-deficient HCT116O cell
line.
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Article
(
[
(
pK ’s of 8.3 to 8.9) vary significantly from that of
(DPE)]2+ were separated by HPLC. In the case of a labile Rh−
a
3
+
27
Rh(bpy) (chrysi)] (pKa of 5.2) and [Rh(HDPA)2-
O bond, racemization of the enantiomers would be expected to
2
chrysi)]3 (pK of 7.0). This difference is most likely due to
+
occur over time. However, samples of both enantiomers in
34
a
the negative charge on the DPE ligand; a more basic
environment is required to deprotonate the 2+ [Rh(chrysi)-
(
species. Furthermore, in the case of [Rh(bpy) (chrysi)] , the
chrysi immine proton must be deprotonated in order for the
aqueous solution in light exhibited no racemization over a
month, as confirmed by chiral HPLC and CD. Furthermore,
the two enantiomers do not racemize upon DNA binding
(Figure 4), and, in fact, exhibit distinct CD spectra when bound
to DNA, providing direct evidence that both enantiomers are
stable and bind individually to mismatches.
phen)(DPE)]2 species than the 3+ [Rh(HDPA) (chrysi)]
+
3+
2
3+
2
1
0,11
molecule to bind to mismatched DNA.
When both immine
protons are retained, the chrysi ligand is not planar (Figures 2
and 8) due to steric clashing, which, effectively prevents full π-
Do the complexes bind to mismatched DNA through
metalloinsertion? Both the Δ and Λ isomers of [Rh(chrysi)-
(
phen)(DPE)]2+ bind with equally high specificity for
mismatched DNA and with similar binding affinities. For Δ-
[
Rh(chrysi)(phen)(DPE)]2+ we also see a correlation between
binding affinity and destabilization of the mismatch, and this
provides strong support for binding through metalloinsertion
for the Δ-isomers. The fact that the DNA mismatch binding
2+
affinities of Λ-[Rh(chrysi)(phen)(DPE)] do not correlate
with the destabilization of the mismatch is surprising. It may be
the case that buckling by the chrysi ligand coupled with steric
clashes by the Λ-isomer with the helix means that full insertion
may not occur, and instead there may be some twisting of the
complex within the site. More structural characterization is
clearly required here.
Biological Effects of the Complexes in Cells. Most
important is the high biological activity of the new generation
of complexes coupled with their high selectivity for MMR-
deficient cells. As can be seen from Figures 5 and 6, all four
complexes exhibit the MMR-deficient cell-selective activity that
is unique to rhodium metalloinsertors. Indeed, appending
additional steric bulk off the back of the complexes does not
interfere with the cellular response to the rhodium-DNA lesion
2
+
Figure 8. Possible models of Δ-[Rh(chrysi)(phen)(DPE)] bound to
mismatched DNA. Top left: The crystal structure of Δ-[Rh(chrysi)-
+
(
phen)(DPE)]2 has been modeled into the crystal structure of Δ-
3+
[
Rh(bpy) (chrysi)] bound to an AC mismatch by overlaying the
2
chrysi ligands to maximize π-stacking between the chrysi ligand and
the adjacent base pairs. As can be seen in green, there is a great deal of
steric clashing between the DPE ancillary ligand and the DNA. Top
associated with mismatch binding. The fact that [Rh(chrysi)-
2+
(
phen)(PyOctanol)] retains its cell selectivity is also
2
+
right: The crystal structure of Δ-[Rh(chrysi)(phen)(DPE)] has been
surprising given our recent results that the more lipophilic
3
+
modeled into the crystal structure of Δ-[Rh(bpy) (chrysi)] bound to
2
metalloinsertors localize to the mitochondria, which in effect
an AC mismatch by rotating the compound to minimize steric clash
between the ancillary ligands and the DNA, resulting in a reduced
amount of π-stacking between the chrysi ligand and the adjacent base
18
abolishes their cell selectivity. One possibility is that the
ancillary PyOctanol ligand dissociates either prior to or
following cellular uptake. Again, our results showing the lack
2+
pairs. Bottom right: Superposition of Δ-[Rh(chrysi)(phen)(DPE)]
2
+
3+
(
blue) and Δ-[Rh(bpy) (chrysi)] (red) in this model. As can be
of racemization of the [Rh(chrysi)(phen)(DPE)] isomers
over a month in a reducing aqueous environment argue against
2
seen, only two rings of the chrysi ligand of the DPE complex are now
2
+
situated for π-stacking in the duplex. Bottom left: Superposition of the
chrysi ligands of Δ-[Rh(chrysi)(phen)(DPE)] (blue) and Δ-
Rh(bpy) (chrysi)] (red). When both immine protons are retained,
that explanation. Both Δ- and Λ-[Rh(chrysi)(phen)(DPE)]
2
+
display comparable cell-selective activity in the MTT assay.
This result implies similar Rh-DNA lesions being formed for
the two enantiomers; if the enantiomers were binding to DNA
using markedly different binding modes, their biological
activities would vary greatly.
3+
[
2
the chrysi ligand buckles and is no longer planar (blue).
stacking with the base pairs adjacent to the mismatched site
upon DNA binding. Despite this fact, the entire family of
complexes appears to bind to mismatched DNA without
deprotonation (Figures 3 and S12). Additionally, substantial
hypochromicity is observed upon DNA binding, confirming
that there must be significant π-stacking between the DNA and
the chrysi ligand. These observations suggest a different
conformation for mismatch binding with this new family of
We also assessed cellular uptake of the family and compared
3+
them to that of [Rh(HDPA)
complexes require from 7.5- to 5-fold less intracellular rhodium
as compared to [Rh(HDPA)
chrysi]3+ to evoke the same
chrysi] . The new family of
2
2
cellular response. This may be an indication that the Rh-DNA
lesion being formed is more readily recognized by the cellular
machinery, and thus fewer lesions are required for cytotoxicity.
Furthermore, the three complexes with Rh−O bonds exhibit
similar intracellular rhodium concentrations; they vary <1.5-
fold from each other. This is despite a 10-fold difference in the
rhodium concentration of the media between [Rh(chrysi)-
3
+
complexes as compared to [Rh(bpy) (chrysi)] .
2
We also explored the possibility of covalent binding through
displacement of the Rh−O bond and substitution of a DNA
base as a ligand. The ensuing covalent lesion could be a potent
6
2+
2+
target for the machinery of the cell. However, even under an
(phen)(DPE)] and [Rh(chrysi)(phen)(PPO)] . This clearly
demonstrates that the latter compound has more efficient
uptake than the parental DPE compound.
extremely reducing environment (5 mM glutathione), we find
no evidence of covalent binding of the complex to DNA.
In order to further characterize the compounds and their
interactions with DNA, the enantiomers of [Rh(chrysi)(phen)-
Finally, we evaluated the mode of cell death caused by
treatment of HCT116 cells with the new complex [Rh(chrysi)-
1
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Journal of the American Chemical Society
phen)(PPO)]2+ as a representative member of the new family.
Article
(
CONCLUSIONS
■
The admission of the dead-cell stain propidium iodide by the
HCT116 cell lines upon rhodium treatment reveals that cell
death proceeds through a necrotic, rather than apoptotic
Here we have described a family of rhodium metalloinsertors
with a new coordination containing a Rh−O bond axial to the
inserting chrysi ligand. This new ligand coordination has been
found to lead to enhanced potency and selectivity toward
MMR-deficient cancer cells. Indeed, the new complexes appear
to be significantly more selective and also more potent than
classic chemotherapeutics. The complexes share a higher pKa
of the chrysi ligand, leading to the absence of immine
deprotonation and thus the buckling of the inserting ligand
within the mismatched site. For many years, the focus of
bioinorganic chemists has been on the preparation of more
potent analogues of cisplatin that bind covalently to DNA
3
+
pathway, similar to [Rh(HDPA) chrysi] treatment (Figure
7
2
1
7
). If there is a different Rh-DNA lesion being formed by the
new family of complexes, then it is not different enough to
evoke a significantly altered global cellular response. Certainly,
the enhanced potency and selectivity of these new complexes
make them suitable candidates for the next step in chemo-
therapeutic development.
30
Models for DNA Insertion by the Complexes. Given the
potency of the complexes, it becomes important to explore how
they might be oriented on the DNA helix to provide a target for
cellular responses. Figure 8 compares two different proposed
3,4
bases.
However, focus has recently shifted toward the
preparation of chemotherapeutics that are more selective than
cisplatin owing to design strategies where the complex interacts
with a specific biological target found prominently in cancer
2
+
models of Δ-[Rh(chrysi)(phen)(DPE)] bound to a mis-
matched site in DNA. Preserving the DNA conformation from
3
+
7
the crystal structure of [Rh(bpy) (chrysi)] bound to an AC
cells. This new generation of metalloinsertors provides for
2
1
0
mismatch, in one model, we simply overlaid the chrysi ligand
non-covalent binding to a specific cellular target, DNA
mismatches, with high potency and selectively in cells deficient
in MMR. This new generation of complexes thus provides an
exciting new platform for therapeutic development.
2
+
of Δ-[Rh(chrysi)(phen)(DPE)] with that of Δ-[Rh(bpy) -
2
(
chrysi)]3 when bound to mismatched DNA. In the other
+
2
+
model, Δ-[Rh(chrysi)(phen)(DPE)] has been flipped and
rotated to alleviate the steric clashing between the two ancillary
ligands and the DNA, yet retain π-stacking between two rings
of the chrysi ligand and the adjacent base pairs. Figure 8 shows
that in the first model, there is the potential for significant steric
clashing between the dangling pyridine group of Δ-[Rh-
ASSOCIATED CONTENT
Supporting Information
■
*
S
Crystallographic data, representative binding affinity determi-
nation, pH titrations, enantiomeric characterization, further
DNA binding characterization, and cytotoxicity of the two
enantiomers as well as cellular uptake data. This material is
available free of charge via the Internet at http://pubs.acs.org.
2
+
(
chrysi)(phen)(DPE)] and the DNA backbone (shown in
green). This steric clash is partially alleviated when the DPE
complex is exchanged for the PPO complex and exacerbated
2
+
when exchanged for Λ-[Rh(chrysi)(phen)(DPE)] (not
shown). Given the similarity in binding affinities of the various
complexes, it is clear that this cannot be the correct binding
conformation for this new family. In the second model, there is
no steric clashing between the ancillary ligands and the DNA.
This model, in which π-stacking between the chrysi ligand and
the base pairs adjacent to the mismatch is reduced in order to
circumvent this steric clashing, is consistent with all our results.
Due to the presence of both chrysi immine protons upon DNA
binding, it is not possible for the chrysi ligand to π-stack with the
adjacent base pairs using all four rings. This in turn allows for
more flexibility in the placement of the metal complex in the
minor groove, thus permitting positioning of the complexes in
the minor groove without any steric clashing. This reduced
stacking may account for the ∼5-fold lower binding affinity of
AUTHOR INFORMATION
Corresponding Author
jkbarton@caltech.edu
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work from the NIH (GM33309) is
gratefully acknowledged. We also thank the National Science
Foundation for a Graduate Research Fellowship to A.C.K. This
project benefitted from the use of instrumentation made
available by the Caltech Environmental Analysis Center.
Lawrence Henling and Dr. Michael K. Takase (Caltech) are
gratefully acknowledged for X-ray crystallographic structural
determination. The Bruker KAPPA APEXII X-ray diffractom-
eter was purchased via an NSF CRIF:MU award to the
California Institute of Technology (CHE-0639094). The
Caltech catalysis center is gratefully acknowledged for
assistance with enantiomeric separation. We thank R. Diamond
for expertise in flow cytometry.
2
+
Δ-[Rh(chrysi)(phen)(DPE)] for mismatched DNA as
3+
35
compared to Δ-[Rh(bpy) (chrysi)] .
2
It is also important to consider the orientation of the ejected
bases in the mismatch-bound complexes. Indeed these bases
may be the target for the high cellular response. Significant
differences in positioning of the ejected bases are evident when
3
+
we compare the crystal structures of [Rh(bpy) (chrysi)] and
2
2
+
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■
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to the wall of the minor groove; perhaps the ejected bases
further stabilize that positioning similar to what is seen for the
Ru complex. Understanding these structural aspects will require
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