Structure-activity relationship studies on rhodamine B-based fluorogenic probes and their activation by anticancer platinum(II) compounds

Article abstract of DOI:10.1016/j.jinorgbio.2015.10.002

Fluorescence microscopy has emerged as an attractive technique for imaging intracellular Pt species arising from exposure to clinical anticancer drugs such as cisplatin. A rhodamine-B based fluorogenic probe termed Rho-DDTC can be activated selectively in the presence of Pt(II) compounds, and possesses the ability to discriminate Pt(II) species from Pt(IV) carboxylate prodrug complexes, thereby providing a unique platform to investigate the reduction of these Pt(IV) complexes after cell entry. In this report, we seek to establish the mechanism of activation of Rho-DDTC through a structure-activity relationship study on its structural analogues.

Full text of DOI:10.1016/j.jinorgbio.2015.10.002

JIB-09817; No of Pages 7
Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Structure–activity relationship studies on rhodamine B-based fluorogenic probes and
their activation by anticancer platinum(II) compounds
Jun Xiang Ong a, Jian Yu Yap a,b, Siew Qi Yap a, Wee Han Ang a,b,
⁎
a
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
b
NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescence microscopy has emerged as an attractive technique for imaging intracellular Pt species arising
from exposure to clinical anticancer drugs such as cisplatin. A rhodamine-B based fluorogenic probe termed
Rho-DDTC can be activated selectively in the presence of Pt(II) compounds, and possesses the ability to discrim-
inate Pt(II) species from Pt(IV) carboxylate prodrug complexes, thereby providing a unique platform to investi-
gate the reduction of these Pt(IV) complexes after cell entry. In this report, we seek to establish the mechanism of
activation of Rho-DDTC through a structure–activity relationship study on its structural analogues.
© 2015 Published by Elsevier Inc.
Received 15 May 2015
Received in revised form 2 October 2015
Accepted 5 October 2015
Available online xxxx
Keywords:
Fluorogenic probes
Platinum drugs
SAR studies
Platinum(IV) prodrugs
Cisplatin
1. Introduction
these Pt-based compounds are processed at the cellular level. Several
techniques have been developed to meet this need, centered on deter-
Pt(II) drugs, namely cisplatin, carboplatin and oxaliplatin are impor-
tant anticancer compounds used in combination chemotherapy to treat
a variety of different malignancies including testicular, ovarian, lung and
colorectal cancers (Fig. 1) [1–2]. However, their efficacies are limited by
their high toxicity, severe side-effects and occurrences of drug resis-
tance [3–4]. In the context of addressing some of these limitations,
Pt(IV) carboxylate complexes have emerged as potential candidates as
they are native prodrugs to established Pt(II) agents (Fig. 1). These
Pt(IV) prodrug complexes are kinetically inert by virtue of their low-
spin d6 electronic configuration and saturated coordination sphere,
but they can be activated to release cytotoxic Pt(II) species upon reduc-
tion with concomitant loss of axial carboxylate ligands [5–7]. Conse-
quently, there is a significant body of work directed at exploiting this
Pt(IV) scaffold towards developing them as selective Pt anticancer
agents with improved therapeutic profiles [8–10].
mining the elemental Pt composition and the spectroscopic profiles of
different Pt oxidation states. An important example would be the use
of X-ray adsorption near edge spectroscopy (XANES) to quantitate
levels of Pt(IV) and Pt(II) species in tissue samples treated with Pt(IV)
prodrugs to investigate intracellular reduction of these prodrugs
occurred after cell entry [11]. However, these techniques are generally
inaccessible because they require a synchrotron X-ray source and
cannot be applied to living cells owing to their destructive nature.
Fluorescence microscopy has emerged as the most effective tool
for imaging intracellular Pt species due to its good optical resolution,
compatibility with biological systems and availability in most research
facilities. However, the technique necessitated the use of a fluorophore
as a reporter and consequently, there have been prior attempts to tag
fluorescent functional groups to Pt(II)/Pt(IV) scaffolds [12–19]. The
main drawback of this system is that the structural modification of the
Pt complexes being investigated with bulky organic fluorophores po-
tentially changes the pharmacophore and alters their uptake properties.
To circumvent this limitation, our group recently reported a fluorogenic
probe based on a rhodamine B (RhoB) fluorophore designed to activate
selectively in the presence of Pt(II) compounds such as cisplatin [20].
The probe possesses the ability to discriminate Pt(II) species from
their parental Pt(IV) prodrugs, thereby providing a platform to investi-
gate the reduction of these Pt(IV) complexes after cell entry. Using
confocal microscopy, we showed that Pt(IV) carboxylate prodrug com-
plexes are reduced after cell entry and that there was a difference in
intracellular Pt distribution between Pt(IV) and Pt(II) complexes. We
There is currently a lack of imaging tools capable of directly visualiz-
ing the uptake and accumulation of these compounds in cancer cells
that would allow investigators to elucidate the mechanisms by which
Abbreviations: XANES, X-ray adsorption near edge spectroscopy; ESI-MS, Electro-
spray ionization mass spectrometry; UV–vis, Ultraviolet–visible; RhoB, rhodamine B;
NaDDTC, sodium diethyldithiocarbamate; DDTC, diethyldithiocarbamate; HEPES, 2-[4-
(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; DMF, dimethylformamide; DMSO,
dimethylsulfoxide; NEt3, triethylamine; EtOH, ethanol.
⁎
Corresponding author at: Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543, Singapore.
E-mail address: chmawh@nus.edu.sg (W.H. Ang).
http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002
0162-0134/© 2015 Published by Elsevier Inc.
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

2
J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 1. FDA-approved Pt(II) drugs and representative Pt(IV) carboxylate prodrug complex.
therefore seek to understand the mechanism of probe activation from a
structural standpoint, through a structure–activity relationship study
centered on a rhodamine B-based fluorogenic probe termed Rho-
DDTC (Scheme 1), with a view to improve its efficacy towards Pt(II)
detection.
spectrometry were obtained on a Thermo Finnigan MAT ESI-MS system.
UV–vis spectra were recorded on a Shimadzu UV-1800 UV–vis spectro-
photometer. Fluorescence spectra were recorded on a Biotek Synergy
H1 hybrid multimode plate reader.
2.2. Synthesis of RhoB probes (see Scheme 1 for structures and compound
abbreviations)
2. Experimental section
2.1. Materials and instrumentation
2.2.1. Synthesis of Rho-OC2
Rho-OH (220 mg, 0.48 mmol) was dissolved in DMF (2 mL) and was
added NEt3 (671 μL, 4.81 mmol) and 1,2-dibromoethane (829 μL, 9.62
mmol). The reaction mixture was heated at 60 °C for 4 h. The solvent
was removed and the residue taken up in CH2Cl2 (15 mL). The organic
mixture was washed with water (10 mL) and NaHCO3 (10 mL), and
dried over Na2SO4. The solvent was removed and the crude residue
purified by column chromatography (1:3 v/v ethyl acetate:hexane) to
yield intermediate compound Rho-OC2-Br. Yield: 199 mg (73%, Rf =
0.45). Rho-OC2-Br (199 mg, 0.35 mmol) was treated with sodium
All reagents were purchased from commercial vendors and used
without purification. Satraplatin, JM118 and oxaliplatin were purchased
from Merlin Chemicals Ltd. Cisplatin [21], Pt(en)Cl2 [22] and oxoplatin
[23] were prepared in accordance with literature procedures. RhoB
hydroxamate (Rho-OH) was synthesized according to literature
methods [24]. 1H NMR spectra were recorded on a Bruker Avance
300 MHz model. Chemical shifts are reported in parts per million rela-
tive to residual solvent peaks. Electro-spray ionization mass
Scheme 1. Synthetic route of RhoB fluorescent probes.
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
diethyldithiocarbamate (NaDDTC) (159 mg, 0.70 mmol) in DMF (2 mL)
at r.t. for 12 h. The solvent was removed and the residue was taken up in
CH2Cl2 (15 mL). The organic mixture was washed with water (10 mL)
and brine (10 mL), and dried over Na2SO4. The solvent was removed
and the crude residue purified by column chromatography (1:3 v/v
ethyl acetate:hexane) to yield the product Rho-OC2 as white solid.
Crystals suitable for single crystal X-ray diffraction analyses were
grown via vapor diffusion of diethyl ether into a concentrated CHCl3 so-
lution. Yield: 204 mg (92%, Rf = 0.25). 1H NMR (300 MHz, CDCl3): δ 7.92
(1H, d, J = 5.7 Hz), 7.47 (2H, m), 7.08 (1H, d, J = 5.7 Hz), 6.53–6.56 (2H,
m), 6.32–6.38 (4H, m), 3.94–3.98 (4H, m), 3.71 (2H, q, J = 6.9 Hz), 3.40
(2H, t, J = 6.0 Hz), 3.34 (8H, q, J = 6.9 Hz), 1.15–1.29 (18H, m). ESI-MS:
m/z 633.2 [M + H]+; 655.1 [M + Na]+. Anal. calcd. for C35H44N4O3S2: C
66.42, H 7.01, N 8.85. Found: C 66.15, H 6.64, N 8.87.
4.45 (2H, t, J = 7.2 Hz), 3.63 (2H, t, J = 7.2 Hz), 3.33 (8H, q, J =
7.2 Hz), 1.16 (12H, t, J = 7.2 Hz). ESI-MS: m/z 580.1 [M + H]+. Anal.
calcd. for C30H34BrN3O2S: C 62.06, H 5.90, N 7.24. Found: C 61.98, H
6.02, N 7.03.
2.2.6. Synthesis of Rho-C2
RhoB·HCl (1 g, 2 mmol) was refluxed in excess POCl3 (10 mL) for
18 h, after which the solvent was removed to give dark violet-red oil.
The crude acid chloride was dissolved in CH3CN (15 mL), then added
2-bromoethylamine hydrobromide (1 g, 5 mmol) and triethylamine
(1.5 mL). The reaction mixture was stirred at r.t. for 24 h, the solvent re-
moved and the organic layer extracted with CH2Cl2 (3 × 20 mL). The or-
ganic portions were combined and dried over Na2SO4. The solvent was
removed and the crude residue purified by column chromatography
(1:3 v/v ethyl acetate:hexane) to give the intermediate compound
Rho-C2-Br as white solid. Yield: 350 mg (32.0%, Rf = 0.50). Rho-C2-
Br (300 mg, 0.547 mmol) and NaDDTC (246.8 mg, 1.09 mmol) was dis-
solved in DMF and the reaction mixture was stirred at r.t. for 12 h. The
solvent was removed and the residue taken up in CH2Cl2 (10 mL). The
organic mixture was washed with water (10 mL) and brine (10 mL)
and dried over Na2SO4. The solvent was removed and the crude residue
purified by column chromatography (1:3 v/v ethyl acetate:hexane) to
give the product Rho-C2 as a white solid. Yield: 311 mg (92.3%, Rf =
0.30). Crystals suitable for single crystal X-ray diffraction analyses
were grown via slow evaporation from a concentrated CH3CN/CH2Cl2
solution. 1H NMR (300 MHz, CDCl3): δ 7.90 (1H, d, J = 5.7 Hz), 7.43
(2H, m), 7.07 (1H, d, J = 6.0 Hz), 6.29–6.47 (6H, m), 3.91 (2H, q, J =
6.6 Hz), 3.62 (2H, q, J = 6.9 Hz), 3.47 (2H, t, J = 6.9 Hz), 3.32 (8H, q,
J = 7.2 Hz), 3.11 (2H, t, J = 5.7 Hz), 1.14–1.66 (18H, m). ESI-MS: m/z
617.3 [M + H]+; 639.3 [M + Na]+. Anal. calcd. for C35H44N4O2S2: C
68.15, H 7.19, N 9.08. Found: C 68.09, H 7.03, N 8.90.
2.2.2. Synthesis of Rho-OC3
Rho-OH (200 mg, 0.44 mmol), NEt3 (610 μL, 4.37 mmol) and 1,3-
dibromopropane (887 μL, 8.74 mmol) were treated in accordance
with the procedure for Rho-OC2 to yield intermediate Rho-OC3-Br as
white solid. Yield: 189 mg (75%, Rf = 0.45). Rho-OC3-Br (189 mg,
0.33 mmol) was reacted with NaDDTC (149 mg, 0.66 mmol) as de-
scribed in the same procedure to give the product Rho-OC3 as white
solid. Yield: 197 mg (93.1%, Rf = 0.25). Crystals suitable for single crys-
tal X-ray diffraction analyses were grown via slow evaporation from a
concentrated CH3CN/CH2Cl2 solution. 1H NMR (300 MHz, CDCl3): δ
7.92 (1H, d, J = 5.5 Hz), 7.49 (2H, m), 7.09 (1H, d, J = 5.4Hz), 6.54–
6.57 (2H, m), 6.34–6.43 (4H, m), 3.98 (2H, q, J = 6.3 Hz), 3.79 (2H, t,
J = 5.7 Hz), 3.66 (2H, t, J = 6.9 Hz), 3.35 (8H, q, J = 6.9 Hz), 3.21 (2H,
t, J = 6.9 Hz), 1.83 (2H, t, J = 7.2 Hz), 1.16–1.59 (18H, m). ESI-MS:
m/z 647.2 [M + H]+; 669.2 [M + Na]+. Anal. calcd. for C36H46N4O3S2:
C 66.84, H 7.17, N 8.66. Found: C 66.77, H 6.83, N 8.46.
2.2.3. Synthesis of Rho-OC4
2.2.7. Synthesis of Rho-C3
Rho-OH (150 mg, 0.33 mmol), NEt3 (457 μL, 3.28 mmol) and 1,4-
dibromobutane (780 μL, 6.56 mmol) were treated in accordance with
the procedure for Rho-OC2 to yield intermediate Rho-OC4-Br as
white solid. Yield: 153 mg (78%, Rf = 0.50). Rho-OC4-Br (153 mg,
0.26 mmol) was reacted with NaDDTC (116 mg, 0.52 mmol) as de-
scribed in the same procedure to give the product Rho-OC4 as white
solid. Yield: 154 mg (91%, Rf = 0.30). 1H NMR (300 MHz, CDCl3): δ
7.91 (1H, d, J = 5.3 Hz), 7.47 (2H, m), 7.09 (1H, d, J = 5.7 Hz), 6.50–
6.52 (2H, m), 6.29–6.41 (4H, m), 3.99 (2H, q, J = 6.6 Hz), 3.73–3.75
(4H, m), 3.34 (8H, q, J = 6.3 Hz), 3.09 (2H, t, J = 6.6 Hz), 1.14–1.24
(22H, m). ESI-MS: m/z 661.3 [M + H]+; 683.2 [M + Na]+. Anal. calcd.
for C37H48N4O3S2: C 67.24, H 7.32, N 8.48. Found: C 67.07, H 6.96, N 8.41.
The intermediate Rho-C3-Br was prepared in accordance with the
procedure for Rho-C2 using 3-bromopropylamine hydrobromide
(1.1 g, 5 mmol) to yield a white solid. Yield: 340 mg (30.3%, Rf =
0.50). Rho-C3-Br (300 mg, 0.535 mmol) was reacted with NaDDTC
(246.3 mg, 1.07 mmol) as described in the same procedure to yield
Rho-C3 as white solid. Yield: 316 mg (93.8%, Rf = 0.30). Crystals suit-
able for single crystal X-ray diffraction analyses were grown via slow
evaporation from a concentrated CH3CN/CH2Cl2 solution. 1H NMR
(300 MHz, CDCl3): δ 7.89 (1H, d, J = 5.4 Hz), 7.42 (2H, m), 7.07 (1H,
d, J = 5.7 Hz), 6.30–6.45 (6H, m), 3.96 (2H, q, J = 6.6 Hz), 3.68 (2H, q,
J = 7.2 Hz), 3.34 (8H, q, J = 6.9 Hz), 3.23 (2H, t, J = 7.2 Hz), 3.05 (2H,
t, J = 6.9 Hz), 1.59 (2H, m), 1.17–1.26 (18H, m). ESI-MS: m/z 631.4
[M + H]+; 653.4 [M + Na]+. Anal. calcd. for C36H46N4O2S2: C 68.54, H
7.35, N 8.88. Found: C 68.50, H 6.97, N 8.82.
2.2.4. Synthesis of Rho(S)-OC2
Rho-OC2 (100 mg, 0.16 mmol) and Lawesson's reagent (68 mg,
0.17 mmol) were suspended in benzene (8 mL) and refluxed for 2 h.
CH2Cl2 (10 mL) was added and the reaction mixture was washed with
water (2 × 10 mL). The organic portion was dried over Na2SO4, the sol-
vent removed and the crude residue purified by column chromatogra-
phy (1:4 v/v ethyl acetate:hexane) to give the product as white solid.
Yield: 90.0 mg (85.7%, Rf = 0.55). 1H NMR (300 MHz, CDCl3): δ 7.88
(1H, d, J = 6.4 Hz), 7.33 (2H, m), 7.03 (1H, d, J = 6.8 Hz), 6.77–6.79
(2H, m), 6.31 (4H, m), 4.46 (2H, t, J = 6.4 Hz), 4.03 (2H, q, J = 6.8 Hz),
3.72–3.75 (4H, m), 3.33 (8H, q, J = 6.8 Hz), 1.27 (6H, t, J = 7.2 Hz),
1.16 (12H, t, J = 6.8 Hz). ESI-MS: m/z 649.1 [M + H]+. Anal. calcd. for
2.2.8. Synthesis of Rho-C4
The intermediate Rho-C4-Br was prepared in accordance with the
procedure for Rho-C2 using 4-bromobutylamine hydrobromide (1.2 g,
5 mmol) to yield a white solid. Yield: 332 mg (28.9%, Rf = 0.55). Rho-
C4-Br (300 mg, 0.522 mmol) was reacted with NaDDTC (239.4 mg,
1.04 mmol) as described in the same procedure to yield Rho-C4 as
white solid. Yield: 305 mg (90.8%, Rf = 0.35). 1H NMR (300 MHz,
CDCl3): δ 7.90 (1H, d, J = 5.4 Hz), 7.43 (2H, m), 7.07 (1H, d, J =
5.7 Hz), 6.35–6.48 (6H, m), 3.98 (2H, q, J = 6.9 Hz), 3.68 (2H, q, J =
7.2 Hz), 3.35 (8H, q, J = 6.9 Hz), 3.16 (2H, t, J = 7.2 Hz), 3.02 (2H, t,
J = 7.5 Hz), 1.47 (2H, m), 1.16–1.26 (20H, m). ESI-MS: m/z 645.3
[M + H]+; 667.3 [M + Na]+. Anal. calcd. for C37H48N4O2S2: C 68.91, H
7.50, N 8.69. Found: C 68.67, H 7.67, N 8.57.
C35H44N4O2S3: C 64.78, H 6.83, N 8.63. Found: C 64.40, H 6.94, N 8.23.
2.2.5. Synthesis of Rho(S)-OC2-Br
Rho-OC2-Br (100 mg, 0.18 mmol) and Lawesson's reagent (72 mg,
0.18 mmol) was reacted in accordance with the earlier procedure to
yield Rho(S)-OC2-Br as a white solid. Yield: 81.1 mg (78.5%, Rf =
0.75). 1H NMR (300 MHz, CDCl3): δ 7.85 (1H, d, J = 6.0 Hz), 7.35
(2H, m), 7.04 (1H, d, J = 6.3 Hz), 6.73–6.78 (2H, m), 6.32 (4H, m),
2.3. Fluorescence determination
All RhoB probes were prepared as 1 mM stock solutions in DMSO.
Pt(II) complexes were prepared as 1 mM stock solutions in H2O and
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

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J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Pt(IV) complexes as 1 mM stock solutions in DMSO. Fluorescence exper-
iments were carried out by incubating RhoB probes (20 μM) with plat-
inum complexes (100 μM) at 37 °C. NaCl, KCl, MgCl2, NiCl2, CuSO4, ZnCl2,
FeSO4, FeCl3 and K2PtCl4 were used as the source of metal ions and pre-
pared as 10 mM stock solutions in water. Selectivity of Rho(S)-OC2 to-
wards various metal ions was carried out by incubating Rho(S)-OC2
(20 μM) with metal ions (20 μM) at 25 °C. Selectivity of Rho(S)-OC2 to-
wards various metal ions at physiological conditions was carried out at
closed-ring spirolactams (Fig. 2). The core RhoB and DDTC structures
remained intact, with key bond distances and angles largely the same.
The key difference is the separation of the DDTC recognition motif
from the spirolactam ring due to variations in linker length. The
straight-line separation between DDTC and spirolactam through-
space, measured from N3 to S1, were determined to be 4.071 Å,
5.230 Å, 5.006 Å and 5.731 Å for Rho-C2, Rho-C3, Rho-OC2 and
Rho-OC3, respectively, while the cumulative through-bond distances
from N3 to S1 were 4.782 Å, 6.295 Å, 6.151 Å and 7.673 Å (Table 1).
The solid-state structures for Rho-C3 and Rho-OC2 are remarkably sim-
ilar with differences in the N3–S1 separation arising from shorter N–O/
C–O bond lengths in Rho-OC2 due to the hydroxamate linkage, com-
pared to N–C/C–C bond lengths in Rho-C3.
37 °C. Job's plot was carried out with a total [Rho(S)-OC2] and [Pt2+
]
concentration of 50 μM by varying the mole fraction of Pt2+ from 0 to
1. Fluorescence responses of Rho(S)-OC2 and Rho-OC2 towards cisplat-
in were carried out by incubating the probes (20 μM) with cisplatin
(100 μM) at 37 °C and fluorescence emission measured at 20 min inter-
vals over a period of 3 h. For all experiments, HEPES buffer (10 mM,
pH 7.4, 30% v/v EtOH) was used.
3.2. Structure–activity relationship studies
2.4. Determination of solid-state structure
We hypothesized that recognition of cisplatin by Rho-DDTC probe
proceeded via a 2-step activation process culminating in spiro-ring
opening. First, the DDTC recognition motif reacted with cisplatin
resulting in mono-substitution by the thiocarbonyl group. The
thiocarbonyl group on the DDTC recognition motif labilises the trans-
ligand via trans-effect which resulted in subsequent reactivity with
the spirolactam on RhoB. This reaction relieved the ring strain via
spiro-ring opening and enabled fluorescence turn-on. To establish the
validity of this hypothesis, we prepared structurally analogous Rho-
DDTC probes with varying linker chain lengths namely Rho-OC2,
Rho-OC3 and Rho-OC4. We reasoned that increasing linker length
would prevent cooperativity between the DDTC recognition motif and
the fluorophore, eventually leading to reduced probe activation.
RhoB probes were treated with cisplatin (5× eq.) in HEPES buffer
(10 mM, pH 7.4, 30% v/v EtOH). A marked decrease in both fluorescence
and absorbance were observed for Rho-OC3 and Rho-OC4 comprising
4-atom and 5-atom linker lengths between DDTC and spirolactam, re-
spectively, compared to prototypical Rho-OC2 which has 3-atom link-
age (Figs. 3, S1). Similar trends were observed when JM118 (5×
eq.) was utilized. JM118 is the Pt(II) congener of Pt(IV) prodrug can-
didate satraplatin, currently undergoing Phase III clinical trials for
hormone-refractory prostate cancer. The results indicated that the
probes were poorly activated in the presence of cisplatin due to the
increased length of the linker, validating our hypothesis. Thus after
initial binding of cisplatin to the probe through DDTC, subsequent
nucleophilic attack on Pt by the spirolactam motif was less likely
since the extended linker chain would put it out of reach from the
spirolactam. In addition, the difference in probe activation also sug-
gested that spiro-ring opening was preceded by DDTC recognition
of cisplatin, not vice-versa. Otherwise, the three probes would have
displayed similar levels of reactivity by virtue of their common
RhoB core structures.
We replaced the hydroxamate group on the probe with the amide
with a view to tune their reactivity towards Pt complexes and to further
simplify the synthesis. A marked reduction in fluorescence turn-on was
observed for Rho-C3 compared to Rho-OC2, even though both com-
prised 3-atom linkers, suggesting that the hydroxamate O-atom played
a crucial role in the ring opening reaction. This was presumably due
to the electron-donating O atom stabilizing the formation of the
carboxyimidate group following complexation and ring-opening. In its
absence, spirolactam Rho-C3 preferentially adopted the amide func-
tionality and was therefore less susceptible towards ring-opening
(Fig. S10). Intriguingly, low levels of probe activation were observed
when the linker length was reduced i.e. Rho-C2 with 2-atom linker.
This was because the short linker length would exert a considerable
strain on ring formation with cisplatin bound to both DDTC and RhoB
leading to an unfavorable binding conformation. In keeping with Rho-
OC3, Rho-C4 exhibited lower probe activation compared to Rho-C3
due to increased separation between the recognition motif and the
spirolactam fluorophore. All in all, the results pointed to the linker
X-ray data for Rho-C2, Rho-OC2 and Rho-OC3 were collected with a
Bruker AXS SMART APEX diffractometer equipped with a CCD camera
using Mo Kα radiation while Rho-C3 was measured on a Bruker AXS
D8 Venture diffractometer equipped with a Photon 100 CMOS active
pixel sensor detector using Cu Kα radiation. All data were collected at
100(2) K with the SMART suite of programs. Data were processed and
corrected for Lorentz and polarization effects using SAINT software
[25], and for absorption effects using the SADABS software [26]. Struc-
tural solution and refinement were then carried out using the
SHELXTL suite of programs. Structure was solved using Direct methods
[27] and subsequent differences Fourier maps, then refined by least
squares procedures on weighted F2 values using the SHELXL-version
2014/6 [28] included in WinGX system programs for Windows [29].
All non-hydrogen atoms were assigned anisotropic displacement pa-
rameters. All H atoms were put at calculated positions using the riding
model. The detailed crystallographic data is provided in Table S1.
3. Results and discussion
3.1. Synthesis and characterization
The series of compounds were synthesized as shown in Scheme 1
from RhoB as the starting material. RhoB was treated sequentially
with POCl3 and NH2OH to obtain Rho-OH. Linkers with varying chain
lengths were installed by the reaction of Rho-OH with different
dibromoalkanes in the presence of triethylamine as the base. These in-
termediates were finally treated with NaDDTC to give the desired com-
pounds Rho-OC2, Rho-OC3 and Rho-OC4. Treatment of Rho-OC2 with
Lawesson's reagent in benzene afforded Rho(S)-OC2. As the C_S
double bond is weak, Rho(S)-OC2 is expected to exist predominately
as tautomer B (Fig. S9) [30–31]. Prevalence of the thioether tautomer
in solution was consistent with the detection of a single species in 1
H
NMR and the downfield shift of the methylene resonances on the linker
motif, indicating a change in chemical environment. Furthermore, the IR
spectrum of Rho(S)-OC2 also exhibited a distinct C_N stretch at
1613.5 cm−1 (Fig. S20). Reaction of RhoB with POCl3 followed by 2-
bromoethylamine, 3-bromopropylamine and 4-bromobutylamine, re-
spectively in the presence of triethylamine, yielded bromo-terminated
Rho-B amides which were subsequently treated with NaDDTC to yield
Rho-C2, Rho-C3 and Rho-C4. All the final compounds were analyzed
and fully characterized by 1H NMR, ESI-MS as well as X-ray crystallo-
graphic analyses.
Single crystals of Rho-C2, Rho-C3 and Rho-OC3 suitable for X-ray
diffraction studies were obtained from slow evaporation of concentrat-
ed CH3CN/CH2Cl2 solutions of the compounds whereas single crystals of
Rho-OC2 were grown by vapor diffusion of diethyl ether into a concen-
trated CHCl3 solution. The solid-state structures of Rho-C2, Rho-C3,
Rho-OC2 and Rho-OC3 revealed that these compounds exist as
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
5
Fig. 2. Molecular representation of selected RhoB probes; atoms are represented as thermal ellipsoids at 50% probability. Hydrogen atoms are omitted for clarity.
length being an important parameter for the efficacy of this class of
fluorogenic probes with a 3-atom linkage being the optimal separation.
component would enhance the overall reactivity of the probe. We rea-
soned that a soft S-atom at the fluorophore would bind more strongly
with the soft Pt metal centre in the second nucleophilic attack by the
spirolactam, thereby promoting spiro-ring opening and consequently,
increasing fluorescence turn-on. Therefore, Rho-OC2 was converted
quantitatively to Rho(S)-OC2 using the Lawesson's reagent with con-
comitant conversion to its thioether tautomer. Encouragingly, Rho(S)-
OC2 exhibited ca. 4-fold increase in fluorescence compared to Rho-
OC2 when treated with cisplatin under the same reaction conditions
(Figs. 4, S2), with a slight shift in λmax. This is accompanied by higher
UV–vis absorbance levels with a more intense color change which
could be observed visually (Fig. 5). A more rapid probe response was
also noted with fluorescence turn-on registered within 20 min of reac-
tion time for Rho(S)-OC2 compared to Rho-OC2 which required at
least 120 min (Fig. S4). This significantly-enhanced sensitivity and
reactivity towards cisplatin could make it an ideal fluorogenic probe
for real-time imaging of Pt drugs within living cells. A similar trend in
the fluorescence and absorbance properties was also observed when
the probes were treated with JM118.
3.3. Thionylation of spirolactam to enhance reactivity
In order to increase levels of probe activation, we investigated
whether introducing sulfur-based functional groups at the spirolactam
3.4. Selectivity of thionylated probe Rho(S)-OC2
We proceeded to establish whether the DDTC recognition motif was
essential for probe activation in this tautomeric thioether form of
Rho(S)-OC2, since there was a distinct possibility that probe activation
would proceed via reaction of thioether with cisplatin, potentially
Fig. 3. Fluorescence responses (λex = 500 nm, λem = 595 nm) of RhoB compounds
(20 μM) after treatment with cisplatin and JM118 in HEPES buffer (10 mM, pH 7.4, 30%
v/v EtOH).
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

6
J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 4. Fluorescence responses (λex = 500 nm, λem = 595 nm) of Rho-OC2, Rho(S)-OC2
and Rho(S)-OC2-Br (20 μM) after treatment with cisplatin and JM118 in HEPES buffer
(10 mM, pH 7.4, 30% v/v EtOH).
Fig. 6. Fluorescence spectra (λex = 500 nm, λem = 595 nm) of Rho(S)-OC2 (20 μM) after
treatment with Pt(II) and Pt(IV) complexes in HEPES buffer (10 mM, pH 7.4, 30% v/v
EtOH).
rendering the probe unselective. Therefore, intermediate Rho-OC2-Br,
which contained the 3-atom linker but without the DDTC recognition
motif, was treated with Lawesson's reagent to yield Rho(S)-OC2-Br as
a negative control. When exposed to cisplatin under the same condi-
tions, Rho(S)-OC2-Br only yielded basal fluorescence values indicating
that there was no reactivity (Fig. 4), thereby pointing out that initial
DDTC binding was essential for the probe activation of Rho(S)-OC2.
We concluded that the mechanism of action involved in the activation
of Rho(S)-OC2 by cisplatin was akin to that for Rho-OC2, with a recog-
nition step for selective Pt-binding followed by spiro-ring opening
(Fig. S11). Indeed despite its enhanced reactivity, the selectivity of
Rho(S)-OC2 towards Pt2+ was high compared to other metal ions
such as Na+, K+, Cu2+, Ni2+, Zn2+, Fe2+ and Fe3+ which play impor-
tant roles in biological systems (Fig. S6). Analysis of a Job's plot indicated
maximum fluorescence intensity at a mole fraction of 0.5, which
suggests that Rho(S)-OC2 interacted with Pt2+ in a 1:1 ratio (Fig. S5).
Selectivity for cisplatin was high at elevated concentrations of other
physiologically-relevant metal ions i.e. Na+, K+ and Mg2+ (15 mM)
making it a suitable intracellular probe (Fig. S7). Linear fluorescence
turn-on response to the probe was observed at clinically-relevant cis-
platin concentrations between 20–100μM (Fig. S8).
center, having a more reactive thiospirolactam would not result in acti-
vation of the probe. In the absence of DDTC binding to Pt metal center,
the probe was not activated. Lastly, we also investigated the ability of
Rho(S)-OC2 to distinguish between Pt(II) drugs and their parental
Pt(IV) complexes (Fig. 6). Both oxoplatin and satraplatin which are
based on cisplatin and JM118 templates respectively did not trigger
any fluorescence turn on. This clearly shows that the probe is able to dif-
ferentiate between Pt(II) and Pt(IV) metal centers which makes it an
ideal tool to study the reduction of Pt(IV) prodrugs in biological sys-
tems. Pt(IV) prodrugs are known to undergo intracellular reduction to
generate Pt(II) species to release its cytotoxic effects on cancer cells.
With the rapid activation of Rho(S)-OC2 by Pt(II) species, it would be
feasible to adopt this probe to study real-time reduction of Pt(IV)
prodrugs to Pt(II) species within living cells.
4. Conclusion
With this more reactive Rho(S)-OC2 probe in hand, we investigated
whether it could be activated in the presence of Pt(II) complexes
containing chelating amine ligands which was previously shown not
to activate Rho-OC2 due to the stable amine chelate. Treatment of
Rho(S)-OC2 with Pt(en)Cl2 and oxaliplatin showed no activation of
the probe as indicated by baseline fluorescence signals (Fig. 6). Since
the recognition motif remains unchanged in this novel compound and
the initial step has been determined to be binding of DDTC to Pt metal
A series of RhoB fluorescent probes were synthesized to study the
mechanism of action for the activation of these probes in the presence
of Pt(II) drugs such as cisplatin and JM118. The probe Rho-OC2 un-
dergoes a two-step activation process involving binding of the substrate
to the recognition motif, followed by reactivity with the RhoB
spirolactam as proposed previously. Extending or contracting the dis-
tances between the DDTC recognition motif and the fluorophore led to
a decrease in probe activation, indicating that they are both co-
operatively involved. Thionylation of the spirolactam functional group
led to formation of the thioether tautomer which significantly enhanced
the reactivity of Rho(S)-OC2 towards Pt(II) complexes; presumably by
promoting nucleophilic attack on the Pt metal center in the second
step. The high selectivity of the probe was preserved as Rho(S)-OC2
could effectively distinguish between Pt(IV) prodrug complexes and
their Pt(II) products, making it an ideal tool to study the reduction of
Pt(IV) prodrugs within living cells.
Acknowledgments
Fig. 5. Color changes of RhoB probes (20 μM) after treatment with cisplatin in HEPES buffer
(10 mM, pH 7.4, 30% v/v EtOH). Only Rho-OC2 and Rho(S)-OC2 displayed visible color
changes after incubation with cisplatin.
Financial support from the Ministry of Education (R143-000-572-
112) and the National University of Singapore is gratefully acknowledged.
Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002

J.X. Ong et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
7
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Please cite this article as: J.X. Ong, et al., Structure–activity relationship studies on rhodamine B-based fluorogenic probes and their activation by
anticancer platinum(II) compounds, J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.10.002